Proteostasis and Disease: From Basic Mechanisms to Clinics (Advances in Experimental Medicine and Biology, 1233) 3030382656, 9783030382650

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Advances in Experimental Medicine and Biology 1233

Rosa Barrio James D. Sutherland Manuel S. Rodriguez   Editors

Proteostasis and Disease From Basic Mechanisms to Clinics

Advances in Experimental Medicine and Biology Volume 1233 Series Editors Wim E. Crusio, CNRS and University of Bordeaux UMR 5287, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, Pessac Cedex, France John D. Lambris, University of Pennsylvania, Philadelphia, PA, USA Heinfried H. Radeke, Clinic of the Goethe University Frankfurt Main, Institute of Pharmacology & Toxicology, Frankfurt am Main, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), Journal Citation Reports/Science Edition, Science Citation Index Expanded (SciSearch, Web of Science), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. More information about this series at http://www.springer.com/series/5584

Rosa Barrio • James D. Sutherland • Manuel S. Rodriguez Editors

Proteostasis and Disease From Basic Mechanisms to Clinics

Editors Rosa Barrio CIC bioGUNE, Basque Research and Technology Alliance (BRTA) Derio, Spain

James D. Sutherland CIC bioGUNE, Basque Research and Technology Alliance (BRTA) Derio, Spain

Manuel S. Rodriguez Centre Pierre Potier, ITAV Toulouse Cedex 1, France

ISSN 0065-2598 ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-030-38265-0 ISBN 978-3-030-38266-7 (eBook) https://doi.org/10.1007/978-3-030-38266-7 # Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Protein homeostasis (proteostasis) is critical for maintaining all cellular functions. Multiple mechanisms are interconnected to preserve the equilibrium of proteins, starting with their synthesis, followed by the performance of their various functions, and finishing with their degradation. When one or more of those mechanisms fail, the functional capacity of the cell is affected, resulting in diverse pathologies depending on the cell type or the affected pathway. Post-translational modifications including phosphorylation, acetylation, or modifications by members of the ubiquitin family play an important role in maintaining protein equilibrium and are indispensable to maintain cellular health and ability to respond to stress. In this book, the focus is on the post-translational modifications by ubiquitin and ubiquitin-like proteins (UbLs). Ubiquitin serves as a signal to control the activation of multiple intracellular signaling pathways, such as cell proliferation by altering the activity of crucial cellular factors (cyclins, JunB, and p53). Changes to UbL-mediated regulation can lead to gain or loss of function of multiple pathways. To address dysregulation of activity or stability mediated by UbLs, most current therapies target the enzymes necessary for adding UbLs to substrates (known as E1 activators, E2 conjugases, or E3 ligases) or the demodifying isopeptidase enzymes that cleave and recycle UbLs. Chapters in this book are dedicated to the various processes and diseases caused or influenced by proteostasis disequilibrium, such as cancer and neurodegeneration, as well as infectious, developmental, and rare diseases. Finally, the effect of diet on proteostasis modulation is addressed. Proteostasis disequilibrium in cancer occurs in some of the most aggressive tumors, such as those seen in pancreas, lung, and prostate cancer, but also some of the most common hematologic disorders like acute myeloid leukemia, multiple myeloma, or mantle cell lymphoma. Historically, the first mechanisms studied to understand protein equilibrium were intracellular proteolytic pathways such as the ubiquitin-proteasome systems (UPS) and the autophagy-lysosome (ALS) pathway. For this reason, the first inhibitors developed were directed at the proteasome or autophagy. Bortezomib, ixazomib, and carfilzomib are examples of proteasome inhibitors that have been approved for clinical use, in all cases for multiple myeloma. Whether other cancer types respond to these therapies is under investigation. Furthermore, natural or acquired resistance was observed in some patients. While the UPS is an effective target for drug development, a better understanding of v

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how resistance arises is crucial. The search for drugs that target other aspects of the UbL and autophagy cycles may yield new candidates to expand the range of treatable cancers and suggest new combination therapies to increase efficacy and suppress resistance. As in cancer, neurodegenerative diseases show a strong link with proteostasis disequilibrium. The formation of protein aggregates is the origin of the tauopathies, a consequence of the accumulation of the protein Tau. Tauopathies include Alzheimer disease (AD), Pick’s disease, and frontotemporal dementia, among other pathologies. AD is the most common neurological disorder. Accumulation of Amyloid-β (Aβ) and hyperphosphorylation of Tau are hallmarks of this disease, and the dysregulation of UPS and autophagy pathways is implicated in this accumulation. In the case of Parkinson’s disease (PD), misfolded alpha-synuclein (aSyn) accumulates and is thought to be a causative event in the disorder. Furthermore, degradation machineries decline with aging, contributing to the decay of proteostasis and health deterioration. Other rare neurodegenerative disorders, like Machado–Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3), are also characterized by abnormal protein aggregates. Specifically, a polyglutamine repeat expansion in ataxin-3, a deubiquitylation enzyme, leads to aggregates and is at the heart of cellular stress. Infection, inflammation, and developmental disorders depend on the proteostasis pathways. For instance, infection by viruses triggers type I interferon (IFN), as well as modification of target proteins by SUMO, the small ubiquitin-like modifier. Furthermore, some viruses exploit the host SUMOylation machinery for their own benefit. During cardiac intercellular communication, degradation of connexin 43 has been associated with cardiac disorders. Extracellular vesicles have been implicated in inflammation, angiogenesis, and fibrosis. Autophagy might be cardioprotective or detrimental depending on the stage of the disease. Development is a finely tuned process that relies heavily on protein homeostasis. Dysregulation of developmental processes is the origin of many rare diseases. The UPS is involved in the assembly and disassembly of cilia, a cellular antenna necessary for cell signaling. In the retina, photoreception occurs in a highly specialized neurosensory cilium. Mutations in UPS genes cause cilia malfunction, involved in syndromic and non-syndromic inherited retinal disorders. Mutations in E3 ligases involved in ubiquitination can also cause rare diseases. For instance, mutations in the tripartite motif (TRIM) family of ligases are the origin of Opitz G/BBB syndrome, X-linked intellectual disability, anencephaly, Charcot–Marie–Tooth, or limb girdle muscular dystrophy, among other rare diseases, unveiling the importance of this family of enzymes during development and physiology. In recent years, an explosion of new information about the role of UbLs, the proteasome, and autophagy components in cellular events, coupled with molecular and biochemical dissection of the underlying complexity, will inform the development of new, more specific compounds that target these pathways. Interestingly, dietary natural compounds have been identified as UPS modulators and exhibit anti-aging and anti-aggregation properties. With more than 600 E3 ligases and 100 isopeptidases regulating the UbL cycles in

Preface

Preface

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humans, many targets are still open to exploration for diseases and disorders linked to dysregulated proteostasis. Proteolysis targeting chimeras (PROTACs) are designed to target particular proteins linked to disease for degradation. They are an emerging class of drugs that are indebted to the study of E3 ligases and the proteasome, generating new hope for undruggable targets. A promising future lies ahead as new insights are revealed through studies of the UbL and autophagy pathways. Derio, Spain Derio, Spain Toulouse Cedex 1, France

Rosa Barrio James D. Sutherland Manuel S. Rodriguez

Acknowledgments

We are grateful to the members of the scientific community interested in ubiquitin family. Thanks to them this is a vibrant and open field where newcomers can feel welcome. The contributors and editors of this book were members of the PROTEOSTASIS network, European Cooperation in Science and Technology COST program (http://cost-proteostasis.eu/). The network gathered together 274 members representing 30 countries. RB and JDS acknowledge the support of public finding entities for their research: BFU2017-84653-P (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), and SAF2017-90900-REDT (UBIRed Program) (Spain). RB, JS, and MSR are part of the UbiCODE project European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 765445. MSR is also funded by LASSERLAB-EUROPE grant number 654148, the Institut National du Cancer, France (PLBIO16-251), the Occitanie region REPERE and Pre-maturation programs (France), and CONACyT-SRE grant 0280365 (Mexico). We apologize to those researchers who could not be included in this book due to space restrictions.

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Contents

Part I

Cancer

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Ubiquitin-Regulated Cell Proliferation and Cancer . . . . . . . . Beatriz Pérez-Benavente, Alihamze Fathinajafabadi Nasresfahani, and Rosa Farràs

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pierre Gâtel, Marc Piechaczyk, and Guillaume Bossis

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The Proteasome System in Health and Disease . . . . . . . . . . . . Olivier Coux, Barbara A. Zieba, and Silke Meiners

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Proteostasis Dysregulation in Pancreatic Cancer . . . . . . . . . . 101 Leena Arpalahti, Caj Haglund, and Carina I. Holmberg

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Divergent Modulation of Proteostasis in Prostate Cancer . . . . 117 Petek Ballar Kirmizibayrak, Burcu Erbaykent-Tepedelen, Oguz Gozen, and Yalcin Erzurumlu

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Resistance to the Proteasome Inhibitors: Lessons from Multiple Myeloma and Mantle Cell Lymphoma . . . . . . 153 Maria Gonzalez-Santamarta, Grégoire Quinet, Diana Reyes-Garau, Brigitte Sola, Gaël Roué, and Manuel S. Rodriguez

Part II

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Neurodegeneration

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Altered Proteostasis in Neurodegenerative Tauopathies . . . . . 177 Katerina Papanikolopoulou and Efthimios M. C. Skoulakis

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The Ubiquitin System in Alzheimer’s Disease . . . . . . . . . . . . . 195 Lee D. Harris, Sarah Jasem, and Julien D. F. Licchesi

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The Interplay Between Proteostasis Systems and Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Diana F. Lázaro and Tiago F. Outeiro

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides . . . . . . . . . . . . . . . 237 Nico P. Dantuma and Laura K. Herzog xi

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Contents

Part III

Infection, Inflammation and Developmental Disorders

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SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Ahmed El Motiam, Santiago Vidal, Rocío Seoane, Yanis H. Bouzaher, José González-Santamaría, and Carmen Rivas

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The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication . . . . . . . . . . . . . . . . . . . . . . . . . 279 Daniela Batista-Almeida, Tania Martins-Marques, Teresa Ribeiro-Rodrigues, and Henrique Girao

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By the Tips of Your Cilia: Ciliogenesis in the Retina and the Ubiquitin-Proteasome System . . . . . . . . . . . . . . . . . . 303 Vasileios Toulis and Gemma Marfany

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TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders . . . . . 311 Germana Meroni

Part IV 15

Diet

We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products . . . . . . . . . . . . . . . . . 329 Eleni Panagiotidou and Niki Chondrogianni

Part I Cancer

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Ubiquitin-Regulated Cell Proliferation and Cancer Beatriz Pérez-Benavente, Alihamze Fathinajafabadi Nasresfahani, and Rosa Farràs

Abstract

Ubiquitin ligases (E3) play a crucial role in the regulation of different cellular processes such as proliferation and differentiation via recognition, interaction, and ubiquitination of key cellular proteins in a spatial and temporal regulated manner. The type of ubiquitin chain formed determines the fate of the substrates. The ubiquitinated substrates can be degraded by the proteasome, display altered subcellular localization, or can suffer modifications on their interaction with functional protein complexes. Deregulation of E3 activities is frequently found in various human pathologies, including cancer. The illegitimated or accelerated degradation of oncosuppressive proteins or, inversely, the abnormally high accumulation of oncoproteins, contributes to cell proliferation and transformation. Anomalies in protein abundance may be related to mutations that alter the direct or indirect recognition of proteins by the E3 enzymes or alterations in the level of expression or activity of ubiquitin ligases. Through a few examples, we illustrate here the complexity and diversity Authors “Beatriz Pérez-Benavente” and “Alihamze Fathinajafabadi Nasresfahani” contributed equally for this chapter. B. Pérez-Benavente · A. F. Nasresfahani · R. Farràs (*) Oncogenic Signaling Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain e-mail: [email protected]

of the molecular mechanisms related to protein ubiquitination involved in cell cycle regulation. We will discuss the role of ubiquitin-dependent degradation mediated by the proteasome, the role of non-proteolytic ubiquitination during cell cycle progression, and the consequences of this deregulation on cellular transformation. Finally, we will highlight the novel opportunities that arise from these studies for therapeutic intervention. Keywords

Ubiquitin · Eukaryotic ubiquitin conjugation · E3 ligases · Ubiquitin-dependent degradation · Non-proteolytic ubiquitination · Cell cycle · Cancer

1.1

Introduction to Cell Cycle Regulation

Alterations in the molecular mechanisms regulating cell cycle progression may result in uncontrolled cell proliferation. Cell cycle comprises a set of molecular events that gives rise to the division of one cell into two identical daughter cells. It is composed of two main periods: mitosis (M) and interphase, the latter being, in turn, divided into three phases: G1, S, and G2. During G1, the gap between mitosis and S phase (synthesis phase), the cell increases in size and synthesizes proteins and RNAs required for DNA synthesis. This is an important

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_1

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regulatory period in which cells must decide whether they continue into the cell cycle or they enter into a quiescent state (G0), a decision that is strongly affected by the presence of mitogenic factors [1]. In S phase the duplication of DNA occurs. It is followed by the G2 phase, the period in which the cell prepares for mitosis. Finally, in mitosis the duplicated chromosomes are segregated and the cell is divided into two daughter cells (cytokinesis). The progression along the cell cycle is controlled by heterodimeric protein kinases composed of regulatory subunits, called Cyclins, and catalytic subunits, Cyclin-dependent kinases (CDKs). Several CDK–Cyclin complexes act during different cell cycle periods activating or inactivating specific substrates to allow the sequential progression of cell cycle processes. Thus, CDK4 and CDK6 bind to Cyclin D in G1 controlling cell cycle entry, CDK2–Cyclin E is responsible for the G1/S transition, CDK2– Cyclin A and CDK1–Cyclin A are involved in S phase and G2/M transition, respectively, and CDK1–Cyclin B promotes mitotic entry (Fig. 1.1) [2]. The activity of the different CDK–Cyclin complexes is regulated by several mechanisms. First, it depends on the levels of the Cyclins during the cell cycle, which are regulated by the balance between their synthesis and their degradation by the ubiquitin–proteasome system (UPS). CDK–Cyclin activity is also regulated by phosphorylation/dephosphorylation of some residues of the CDKs [3]. To be fully active CDKs must be phosphorylated in a residue close to the catalytic site by the CDK-activating kinase complex (CAK), formed by the association of CDK7 with Cyclin H and Mat1. However, phosphorylations on other residues may have an inhibitory role on CDK activity. For instance, phosphorylations on Thr-14 and Tyr-15 mediated by the Wee1 and Myt1 kinases inhibit CDK activity and dephosphorylations by members of the phosphatase family Cdc25 are necessary to activate CDK complexes. A third level of CDK– Cyclin activity regulation is mediated by the binding of Cyclin-dependent kinase inhibitors (CKIs), whose abundance is also regulated by

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degradation by the UPS. Seven CKIs have been described that bind to CDK–Cyclin complexes: p16INK4a, p15INK4b, p18INK4c and p19INK4d, from INK4 family, that specifically bind complexes containing CDK4 and CDK6, and p21Cip1, p27Kip1 and p57Kip2, members of the CIP/KIP family, that are general inhibitors of CDK–Cyclin complexes. It is worth mentioning that the UPS is not only involved in the degradation of the Cyclins and the CKIs but also in their synthesis through the regulation of the levels and/or activity of transcription factors stimulating or repressing their gene expression. In early G1, and also in quiescent cells (G0), CDK activity is minimal due to low levels of Cyclins and the presence of CKIs. After mitogenic stimulation Cyclin D is synthetized, it binds to CDK4/6 and the active CDK4/6–Cyclin D complex phosphorylates retinoblastoma protein (pRb) releasing it from its inhibitory binding to E2F transcription factors. Once activated, E2F stimulates the expression of its target genes that include Cyclin E, Cyclin A, and other proteins required for S phase. Cyclin E binds to CDK2 and phosphorylates pRb contributing to E2F activation and the transition through restriction point. From this moment the cell is able to complete the cycle without any additional stimuli; the different CDK–Cyclin complexes are activated in a sequential order controlling progression through the different cell cycle phases [1, 4]. Any error occurring during DNA replication or chromosome segregation may give rise to genomic instability and, ultimately, to the development of cancer. To prevent this scenario, the cell has several checkpoints operating during the cell cycle to verify that one phase has been properly completed before continuing to the next one (Fig. 1.1). The DNA damage checkpoint activates a signaling cascade, in which p53 protein plays a pivotal role by inhibiting CDK–Cyclin complexes and blocking the cell cycle in G1, S, and G2/M until the DNA repair mechanisms correct the DNA lesion. In case of unrepairable DNA damage, the apoptotic pathway is activated. The unreplicated DNA checkpoint blocks mitotic entry by inhibition of mitotic CDK–Cyclin complexes if DNA is not accurately replicated.

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Ubiquitin-Regulated Cell Proliferation and Cancer

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Fig. 1.1 Ubiquitin-mediated proteolysis in the control of cell cycle progression. Cyclin-dependent kinases are the driving force in cell cycle progression. They interact with the regulatory subunits Cyclins to form several CDK– Cyclin complexes that act sequentially during cell cycle. Ubiquitin–proteasome system plays an important role in cell cycle by controlling the abundance of CDK activity modulators. The main ubiquitin ligases acting in cell cycle are the SCF and APC/C complexes. SCF bound to the F-box proteins FBXW7, Skp2, or β-TrCP targets substrates for degradation from late G1 to early mitosis and contributes to cell cycle arrest during DNA damage

checkpoint. APC/C sequentially associates with its coactivators Cdc20 and Cdh1 controlling mitosis progression and G1 establishment. Abbreviations: APC/C Anaphase-promoting complex/cyclosome, Cdc20 Cell division cycle-20, Cdc25A Cell division cycle-25A, Cdh1 Cadherin-1, CDK Cyclin-dependent kinase, Emi1 Early mitotic inhibitor 1, FBXW7 F-boxWD repeat domain-containing-7, PLK-1 Polo like kinase 1, SCF SKP1-cullin-1-F-box, Skp2 S-phase kinase-associated protein-2, β-TrCP β-transducin repeat-containing protein, UbcH10 Ubiquitin conjugating enzyme E2 C (Ube2C), Ube2S Ubiquitin conjugating enzyme E2 S

Finally, the spindle-assembly checkpoint halts the cell cycle at metaphase if the chromosomes are not properly assembled to the mitotic spindle by inhibiting one of the main components of the UPS acting in the cell cycle, the APC/CCdc20 [2, 5].

SCF (SKP1-cullin-1-F-box) and APC/C (anaphase-promoting complex/cyclosome). SCF is composed of three invariable components: the adaptor protein SKP1 (S-phase kinase associated protein-1), the scaffold protein CUL1 and the RING-finger protein RBX1, which recruits the E2, and a variable component, the F-box protein, which is responsible for substrate recognition. The F-box protein interacts, on one side, to the adaptor protein SKP1 through its F-box motif, allowing the recruitment of the other E3 complex components, and, on the other site, to the substrate to be ubiquitinated through a specific interaction motif. Depending on the interaction motif, the F-box proteins classify into FBXWs, containing WD40 domains, FBXLs, owing leucine-rich domains, and FBXOs, with

1.1.1

Regulation of CDK–Cyclin Activity by the UPS

The proteasomal degradation of key components of cell cycle machinery is essential to ensure the unidirectional and irreversible progression through the cell cycle [6]. Two multimeric complexes of ubiquitin ligases from the CullinRING E3 ligases family play a central role in the timing degradation of multiple cell cycle proteins:

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other domains. The main F-box proteins involved in cell cycle regulation are F-box WD repeat domain-containing-7 (FBXW7) and β-transducin repeat-containing protein (β-TrCP), belonging to the FBXWs subgroup, and S-phase kinaseassociated protein-2 (Skp2), from the FBXL family [7, 8]. Interestingly, most substrates of SCF complex must be phosphorylated on phosphodegron motifs by one or several kinases in order to be recognized by their F-box proteins, linking the degradation process to specific signaling events [9]. APC/C is a large complex composed of at least 14 different subunits among which are the scaffold cullin-like protein Apc2, the RING-finger protein Apc11, which binds E2 conjugating enzyme, and a coactivator unit that, in addition to activate the complex, is responsible for substrate recognition. Two coactivator units play a fundamental role in cell cycle regulation: Cell division cycle-20 (Cdc20) and Cadherin-1 (Cdh1). Unlike SCF complex, substrate recognition by APC/C does not require phosphorylation on the substrate; the WD40 domains of Cdc20 and Cdh1 bind to specific motifs on the primary structure of the substrate, mainly D-box (destruction-box motif) or KEN-box [10, 11]. APC/C assembles preferentially atypical Lys11-linked chains to its substrates to promote proteasome degradation and mitotic exit. This process involves the E2 UbcH10 (Ube2C) to initiate chain formation and the E2 Ube2S responsible for chain extension [12–18]. UbcH10 together with Ube2S generate K11/K48 branched chains that enhance the efficiency of proteasomedependent proteolysis of the ubiquitinated substrate [19]. UbcH10 is also a cell cycle-regulated protein. Its levels peak during mitosis and decrease as cells enter G1. At the end of G1 it is recognized by APC/CCdh1 for its proteasomal degradation [20, 21]. Interestingly, SCF and APC/C regulate each other during cell cycle progression (see text below) [22]. Although SCF and APC/C are the two main E3 complexes involved in cell cycle regulation, there are other ubiquitin ligases targeting important cell cycle regulators. For instance, p53, the genome gatekeeper, is targeted

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to proteasomal degradation by Mdm2, a single subunit RING E3 [23], and by the HECT E3 ARF-BP1 [24]. The CKI p27Kip1 is a substrate of the RING E3 KPC [25] besides being a SCFSkp2 substrate [26]. As noted in these two examples, more than one E3 may target the same protein for proteasomal degradation.

1.1.2

SCF in Cell Cycle Control

SCF is mainly involved in the degradation of cell cycle substrates from late G1 to the onset of mitosis. Depending on the variable F-box subunit, the SCF complex targets different positive and negative cell cycle regulators (Fig. 1.1). During G1 phase, the presence of the CDK inhibitors p21Cip1 and p27Kip1 keeps CDK1 and CDK2 complexes inactive, preventing the premature onset of S phase and mitosis. In late G1, SCFβ-TrCP targets Cdh1 for ubiquitin-mediated proteolysis contributing to the inactivation of the APC/CCdh1 complex and the accumulation of the APC/CCdh1 substrate Skp2 [6]. The subsequent assembly of the SCFSkp2 complex results in the degradation of p21Cip1 and p27Kip1. This event, together with the E2F-mediated synthesis of Cyclin E, Cyclin A, and the Cdc25A phosphatase, leads to the activation of CDK2–Cyclin E and CDK2–Cyclin A complexes required for S phase entry and progression [22]. As cell progress through S phase, Cyclin E is phosphorylated by several kinases, including autophosphorylation by the CDK2– Cyclin E complex, before being ubiquitinated by the SCFFBXW7 complex and degraded by the proteasome [27]. pRb plays an important role in G1 arrest not only by inhibiting E2F transcriptional activity until its phosphorylation by CDK complexes releases E2F, but also by influencing the activity of SCFSkp2 and APC/CCdh1 complexes. On one hand, pRb interacts with Skp2 interfering with SCFSkp2-mediated p27Kip1 degradation [28] and, on the other hand, pRb binds to APC/CCdh1 and Skp2 contributing to APC/CCdh1-mediated Skp2 degradation and leading, consequently, to p27Kip1

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Ubiquitin-Regulated Cell Proliferation and Cancer

stabilization [29]. pRb is degraded by the proteasome [30, 31]. The E3 ubiquitin ligase Mdm2 binds selectively to the hypophosphorylated form of pRb and prevents its interaction with E2F complexes, thereby stimulating E2F transcriptional activity and cell growth [32]. Mdm2–pRb interaction increases both ubiquitin-dependent [33] and ubiquitin-independent pRb degradation [34]. SCFβ-TrCP influences both positively and negatively the activity of CDK complexes. In S phase and early G2, it targets Cdc25A for degradation, decreasing the amount of the phosphatase in charge of removing CDK inhibitory phosphorylations and therefore, attenuating CDK activity. In contrast, in late G2, SCFβ-TrCP promotes the complete activation of CDK1 complexes involved in G2 and M phases by mediating the degradation of the inhibitory kinase Wee1 [35]. Another SCFβ-TrCP target is the APC/C inhibitor Emi1, which is phosphorylated in early mitosis by CDK1–Cyclin B and PLK-1 before being recognized by the E3 complex and degraded by the proteasome [22]. β-TrCP substrates contain a destruction motif, DSGXXS, which is recognized by β-TrCP after phosphorylation of serines by specific kinases [33].

1.1.3

SCF in DNA Damage

The SCFβ-TrCP-mediated Cdc25A degradation is enhanced upon DNA damage, when the checkpoint kinases Chk1 and Chk2 phosphorylate Cdc25A increasing its proteasomal degradation and decreasing CDK activity to prevent cell cycle progression before DNA is repaired [36]. In addition, SCFβ-TrCP contributes to cell cycle arrest after DNA damage by triggering the proteasomal degradation of the ubiquitin ligase Mdm2, the major negative regulator of p53, after its phosphorylation by Casein kinase-1 (CK1) (Fig. 1.1) [37]. Under normal cell growth conditions, Mdm2 induces the ubiquitin-dependent degradation of p53 and inhibits P53 transcription, resulting in negative feedback on both p53 activity and abundance [23]. In response to genotoxic lesions,

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intracellular signaling cascades are activated and lead to stabilization and accumulation of p53. P53-dependent activation or suppression of specific target genes triggers G1/S or G2/M arrest and the repair of DNA lesions or, in case of too severe lesions, the induction of apoptosis. Phosphorylations of p53 play a very important role in controlling its stability (for review see [38]). Several kinases are activated by genotoxic stresses, such as ATM, ATR, DNA-PK, Chk1 and Chk2 checkpoint kinases, JNK and p38 [39, 40]. Activation of ATM/ATR kinases leads to phosphorylation of p53 on Ser15, and activation of Chk1 and Chk2 kinases phosphorylate p53 on Ser20. These phosphorylations prevent the recruitment of Mdm2 by p53. Phosphorylation of Mdm2 at Ser395 by ATM also contributes to the inhibition of the p53/Mdm2 interaction [41]. Inhibition of Mdm2 interaction with p53, causes p53 activation, stabilization, and the transcription of p53 target genes, such as CDKN1A encoding the CKI p21Cip1 protein. In response to DNA damage, Casein kinase-1 (CK1) also phosphorylates Mdm2, targeting it for degradation by SCFβ-TrCP in late G1 to allow p53 to accumulate [37]. p53 activation may also be due to exacerbated mitogenic stimulation through activation of oncogenes (e.g., Ras, c-Myc, E2F1). This involves the synthesis of the p14ARF tumor suppressor protein. Interaction of p14ARF with Mdm2 sequesters Mdm2 in the nucleolus, thus preventing Mdm2 binding with p53. In addition, the expression of p14ARF stabilizes p53, whereas p53 represses the expression of p14ARF [42]. If the p53-dependent apoptotic program is not committed, once the DNA lesions are repaired or the oncogenic signal is decreased, Mdm2 interacts with and ubiquitinates p53 targeting it for degradation by the proteasome, allowing cell cycle progression. Other E3 ligases, such as Pihr2 and Cop1, may also induce p53 ubiquitination. p53 controls the transcription of their respective genes, establishing a feedback loop. It is not yet clear whether Mdm2, Pihr2, and Cop1 have partially redundant functions or whether they are differentially activated [43]. Chk1 also phosphorylates Cdc25A priming it for phosphorylation by CK1 and ubiquitination

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by SCFβ-TrCP [44–49]. Degradation of Cdc25A leads to inhibition of CDK1 and CDK2 activity, causing delayed S-phase and G2/M progression. During recovery from replication stress or DNA damage-induced cell cycle arrest in G2/M, Chk1 activity is gradually diminished, concomitantly with the reduction of Claspin levels, leading to activation of CDK1. Phosphorylation of Cdc25A by CDK1–cyclin complexes inhibits its degradation by SCFβ-TrCP. After PLK-1 phosphorylation, both Wee1 and Claspin become targets of SCFβ-TrCP -mediated degradation [50–53]. This is critical to terminate the checkpoint, enabling mitotic entry.

1.1.4

APC/C and the Regulation of Mitosis

Depending on the activator subunit, Cdc20 or Cdh1, APC/C mediates the proteasomal degradation of different substrates from mitosis to the end of G1 phase, playing a central role in controlling metaphase to anaphase transition, mitotic exit, and G1 establishment [10] (Fig. 1.1). APC/C activity is regulated by the association with the pseudo-substrate Emi1 that keeps APC/C inactive from late G1 to early mitosis, when SCFβ-TrCP mediates Emi1 proteasomal degradation [54]. APC/CCdc20 is active from prometaphase to anaphase, being instrumental for anaphase onset and mitotic exit [55]. Phosphorylation of APC/C mediated by CDK1 complexes allows Cdc20 binding, while the APC/CCdh1 complex remains inactive because of Cdh1 phosphorylation. APC/CCdc20 is essential for chromosome segregation as it targets Securin for degradation. Securin inhibits Separase, the enzyme that disrupts Cohesin complex, giving rise to sister chromatids separation. The activity of APC/CCdc20 is regulated by the spindle-assembly checkpoint: it is inhibited by proteins such as Mad2 and BubR1 that remain active until all kinetochores are correctly attached to microtubules, so preventing premature Separase degradation and anaphase onset [56–60]. APC/CCdc20 also targets Cyclin A and Cyclin B for degradation decreasing the activity of CDK1–Cyclin A and CDK1–Cyclin

B complexes. This reduction in CDK1 complexes activity contributes to Cdh1 dephosphorylation allowing the binding to the APC/C core and the assembly of the active E3 complex. APC/CCdh1 contributes to mitotic exit and is a key player in the establishment and maintenance of G1 phase. Among its multiple targets in mitosis are Cyclin B, the mitotic kinases PLK-1 and Aurora A, and Cdc25A phosphatase, leading to CDK1 inactivation and mitotic exit [6, 22]. APC/ CCdh1 negatively regulates APC/CCdc20 and SCFSkp2 by targeting Cdc20 and the F-box protein Skp2 for degradation. The inactivation of the S phase promoter SCFSkp2 at the beginning of G1 by APC/CCdh1 avoids premature degradation of the CKIs p21Cip1 and p27Kip1 [61, 62]. Several mechanisms lead to the inactivation of the APC/CCdh1 complex at the end of G1. The coactivator subunit Cdh1 is targeted for proteasomal degradation by SCFβ-TrCP and by APC/CCdh1 itself, which also triggers the degradation of its E2s enzymes UbcH10 and Ube2S [15, 20, 63, 64]. Additionally, phosphorylation of Cdh1 by CDK2 leads to its dissociation from the APC/C core and the E2F-induced accumulation of Emi1 further inhibits APC/C [65].

1.2

Non-proteolytic Protein Ubiquitination in the Control of Cell Cycle Progression

In spite of the importance of UPS-mediated degradation of cell cycle regulators, there has recently been a remarkable increase in the number of examples of non-degradative protein ubiquitination of proteins involved in the control of cell cycle, including substrate monoubiquitination and Lys63 polyubiquitination. In many cases these ubiquitinated proteins are deubiquitinated by specific proteases called deubiquitinating enzymes (DUBs) to reverse the process and to control the duration of the signal [66, 67]. In this section we highlight some of the more relevant cases of these non-proteolytic ubiquitination during cell cycle progression, indicating their protein substrates, the function of their ubiquitinated and/or deubiquitinated

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forms and their relevance in the control of cell cycle progression. In addition, we will mention the E3 ligase responsible for each ubiquitination and the DUBs associated to these processes when they are known.

1.2.1

Cell Signaling and Non-proteolytic Protein Ubiquitination in G1

Mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-kinase (PI3K)-Serine/ threonine protein kinase AKT-mammalian target of rapamycin mTOR (PI3K/AKT/mTOR) signaling pathways regulate cell growth and proliferation. Indeed, sustained signaling due to constitutive activation of these pathways is frequent in cancer. Components of these pathways are regulated by ubiquitin-dependent proteolysis [68]. Besides, and not less important, non-proteolytic ubiquitination is also essential for activation of these pathways [68]. Assembly of mTOR complex 1 (mTORC1), one of the core components of the PI3K/AKT/mTOR signaling pathway, is dependent on the Lys63 polyubiquitination of mTOR. This ubiquitin linkage is mediated by the E3 ligase Tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), which enhances the interaction of mTOR with the other protein components of mTORC1 to obtain an active complex (Fig. 1.2a: 1) [69]. In addition, Lys63 polyubiquitination of the Target of rapamycin complex subunit LST8 (GβL), a component of both mTORC1 and mTORC2, by the E3 ligase tumor necrosis factor (TNF)-receptor-associated factor 2 (TRAF2) prevents its interaction with mSIN1 (MAPKAP1), a component of mTORC2, and its association with mTORC2 to restrict the formation of an active mTORC2 complex. Deubiquitination of GβL by OTU domaincontaining protein 7B (OTUD7B) restores the interaction between GβL and mSIN1 and the formation of an active mTORC2 complex (Fig. 1.2a: 2). Alteration of GβL ubiquitination is associated to several types of cancers including melanoma, where the ubiquitination site of GβL is mutated, or lung cancer, where OTUD7B is amplified

9

[70]. AKT1 and AKT2 are regulated by Lys63-linked polyubiquitination mediated by SCFSkp2. This specific ubiquitination promotes AKT interaction with the epidermal growth factor receptor family ErbB and its activation (Fig. 1.2a: 3) [71]. Non-proteolytic ubiquitination also regulates MAPK signaling pathway, for example by the mono- and di-ubiquitination of KRAS and NRAS by RAB5 GDP/GTP exchange factor (RABEX5) E3 ligase. This induces their localization and retention in endosomes which prevents activation of their signaling cascades [72]. Inversely, the DUB OTU domaincontaining ubiquitin aldehyde binding protein 1 (OTUB1) prevents RAS ubiquitination leading to increase of RAS proliferative signaling (Fig. 1.2a: 4) [73].

1.2.2

Non-proteolytic Protein Ubiquitination During G1/S to G2 Transition

Non-proteolytic protein ubiquitination plays an important role in the G1/S transition. For example, to prevent premature chromatin recombination in G1, Partner and localizer of BRCA2 protein (PALB2) is monoubiquitinated by the Cullin3-RING E3 ubiquitin ligase (CRL3) associated with the substrate-specific adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), thus preventing the interaction of PALB2 with Breast cancer type 1 susceptibility protein BRCA1 [74]. This interaction is necessary for sister chromatid homologous recombination during S/G2 transition. In S phase, ubiquitination of PALB2 is reversed by the deubiquitinating enzyme Ubiquitin carboxyl-terminal hydrolase 11 (USP11) to reestablish the interaction between PALBP2 and BRCA1, allowing sister chromatid homologous recombination (Fig. 1.2a: 5) [74]. Non-proteolytic ubiquitination during S phase promotes unperturbed DNA replication by controlling changes in the chromatin structure while the DNA replication fork is advancing. During DNA replication key proteins, such as histones, have to be removed from nucleosomes and then be

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Fig. 1.2 Non-proteolytic ubiquitination in the control of cell cycle progression. Representation of non-proteolytic mono- and polyubiquitination, and deubiquitination of proteins during G1 phase (a), S phase (b) and G2 phase (c). Modifications in protein–protein interaction and/or intracellular localization of ubiquitinated/deubiquitinated substrates and its biological function are shown in each case. Abbreviations: BRCA1 Breast cancer type 1 susceptibility protein, CLR3-KEAP1 Cullin3-RING E3 ubiquitin ligase (CLR3) associated with substrate-specific adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), CLR3-KLHL18 Cullin3-RING E3 ubiquitin ligase (CRL3) associated with substrate-specific adaptor protein Kelch-like protein 18 (KLHL18), CLR4-WDR23 Cullin3RING E3 ubiquitin ligase (CRL4) associated with substrate-specific adaptor protein DDB1- and CUL4-

associated factor 11 homolog (WDR23), ERLIN2 ERmicrotubule-binding protein, GβL Target of rapamycin complex subunit LST8, mSIN1 Target of rapamycin complex 2 subunit MAPKAP1, KRAS GTPase KRas, mTORC1 mTOR complex 1, NRAS GTPase NRas, OTUB1 OUT domain-containing ubiquitin aldehyde binding protein 1, OTUD7B OTU domain-containing protein 7B, PALB2 Partner and localizer of BRCA2, RABEX5 RAB5 GDP/GTP Exchange factor, SCF-Skp2 F-box containing complex E3 ubiquitin ligase (SCF) associated with substrate-specific adaptor protein S-phase kinaseassociated protein 2 (Skp2), SLBP Stem-loop binding protein, TOP2A Topoisomerase IIα, TRAF2 TNF receptorassociated factor 2, TRAF6 TNF receptor-associated factor 6, USP10 Ubiquitin carboxyl-terminal hydrolase 10, Usp16 Ubiquitin-specific peptidase 16

delivered again in a semiconservative way onto new synthesized DNA strands. In this context, it is very important to synthesize enough quantity of histones to convert duplicated DNA to functional nucleosomes. This is provided by an enhanced

maturation of histone mRNAs by the processing factor Stem-loop binding protein (SLBP) that induces translation of these mRNAs. SLBP expression is regulated during S phase, increasing more than tenfold during the latter part of G1.

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Fig. 1.2 (continued)

Monoubiquitination of SLBP by CRL4WDR23 E3 ligase is necessary for its activation and binding to histone mRNAs to process them (Fig. 1.2b: 1) [75]. Once histone mRNAs are maturated, they are translated by polysome machinery to provide sufficient amount of histone proteins during DNA replication. After completion of its specific function in S phase, SLBP is polyubiquitinated and degraded by the proteasome in a Cyclin F-dependent manner. This limits the synthesis of histones exclusively to S phase and controls genome stability [76]. Once histones are synthesized they are placed onto the newly

replicated DNA. Non-degradative multimonoubiquitination of Histone H3 by Cullin4 E3 ligase enhances specifically H3 deposition onto newly synthesized DNA to promote nucleosome assembly (Fig. 1.2b: 2) [77, 78]. When the whole chromatin is correctly replicated the cells enter to G2 phase where they continue growing by producing macromolecules that are necessary for cell division in mitosis. In this phase non-proteolytic protein ubiquitination is important for a correct onset of mitosis. For instance, Cullin3-RING E3 ubiquitin ligase (CRL3) associated with the substrate-specific

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adaptor protein Kelch-like protein 18 (KLHL18) polyubiquitinates the fraction of Aurora A that is localized in the centrosomes to activate mitotic entry [79]. It has been suggested that non-degradative Aurora A polyubiquitination is necessary to regulate its substrate binding or kinase activity (Fig. 1.2c: 1) [79]. Another example is the Lys63 polyubiquitination of Topoisomerase IIα (TOP2A) by the E3 ubiquitin ligase RNF168. This ubiquitin linkage is necessary for the binding of TOP2A to chromatin to mediate chromatin decatenation and condensation. This process is reversed by deubiquitination of TOP2A that prevents binding of TOP2A to chromatin (Fig. 1.2c: 2) [80]. TOP2A is also modified by non-proteolytic polyubiquitination in a BRCA1-dependent manner, thus enhancing its decatenation activity [81]. The E3 ligase RNF168 has a key role in DNA damaged repair and is involved in different types of cancer, being an important regulator of genome integrity. RNF168 ubiquitinates histone H2A at the Doble Strand Breaks (DBS) of damaged DNA to induce the association of repair machinery proteins such as BRCA1 to the damaged site [82].

1.2.3

Non-proteolytic Ubiquitination in Mitosis

Non-proteolytic ubiquitination is an important regulator in kinetochore and mitotic spindle assembly and also in the correct segregation of sister chromatids during mitosis. Chromosomal Passenger Complex (CPC) is an essential protein complex in mitosis that controls chromosome alignment, segregation, and cytokinesis. This complex is formed by Aurora B kinase, Inner centromere protein (INCENP), Survivin and Boreallin. The whole complex assembly activates the kinase activity of Aurora B that is necessary for the correct function of the complex [83]. CPC subcellular localization and function is regulated during mitosis by differential non-proteolytic ubiquitination of its components. For example, this complex binds to centromeres during prometaphase to regulate kinetochore assembly

and the correct chromosome alignment. At the beginning of chromosome alignment, the junction between kinetochore and microtubules is random and prone to errors, and thus CPC activity is crucial for detachment of incorrect unions. In this regard, Lys63 polyubiquitination of Survivin by Ubiquitin recognition factor in ER-associated degradation protein 1 (Ufd1) promotes the association of CPC to centromeres. Once the connections between kinetochore and microtubules are correctly established, Survivin polyubiquitinated on Lys63 is deubiquitinated by the Probable ubiquitin carboxyl-terminal hydrolase FAF-X (hFAM) to dissociate CPC from centromeres (Fig. 1.3a) [84]. On the other hand, during anaphase, CPC is localized to the spindle midzone microtubules to ensure fidelity of chromosome segregation. Non-proteolytic ubiquitination of Aurora B, mediated by BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase associated with the substratespecific adaptor protein Kelch-like protein 21 (KLHL21), promotes the association of CPC to the spindle midzone microtubules [85]. The interaction of ubiquitinated Aurora B and the Ubiquitin-associated and SH3 domain-containing protein B (UBASH3B), localized previously in microtubules, is important to target the whole CPC to spindle midzone microtubules and control the correct sister chromatid segregation (Fig. 1.3b) [86]. Non-proteolytic ubiquitination also controls the correct alignment of chromosomes toward mitotic spindle in prometaphase to obtain a suitable sister chromatid segregation during mitosis. Deubiquitination and monoubiquitination of Polo-Like Kinase 1 (PLK-1) plays an important role in this alignment. During early mitosis a high portion of monoubiquitinated PLK-1 is deubiquitinated by Ubiquitin-specific peptidase 16 (Usp16) which promotes its localization to kinetochore where it interacts with the Mitotic spindle checkpoint protein BUBR1. This interaction regulates the junction between kinetochore and microtubules that is necessary for a correct chromosome alignment. In this process the initial phosphorylation of Usp16 by CDK1–Cyclin B1 is necessary to enhance the binding of Usp16 to

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Fig. 1.3 Non-proteolytic ubiquitination in the control of mitosis progression. Representation of non-proteolytic mono- and polyubiquitination, and deubiquitination of proteins during mitosis. Modifications in protein–protein interaction and/or intracellular localization of ubiquitinated/deubiquitinated substrates and its biological function are shown in each case. Abbreviations: BCRKLHL21 BTB-CUL3-RBX E3 ubiquitin ligase (BCR) associated with substrate-specific adaptor protein Kelchlike protein 21 (KLHL21); BRISC, BRCC36 isopeptidase, BubR1 Mitotic spindle checkpoint protein BUBR1, CDK1 Cyclin-dependent kinase 1, CENP-A Centromeric protein A, CPC Chromosomal passenger complex,

CYLD Ubiquitin carboxyl-terminal hydrolase CYLD, Dvl Disheveled protein, hFAM Probable ubiquitin carboxylterminal hydrolase FAF-X, HJURP Holliday junction recognition protein, INCENP Inner centromere protein, MAP Microtubules associated proteins, NuMA Nuclear mitotic apparatus protein, PLK-1 Polo-like kinase 1, RBX1COPS8 Cullin-4A type E3 ubiquitin ligase (RBX1) with substrate-specific adaptor protein COP9 signalosome complex subunit 8 (COPS8), Ufd1 Ubiquitin recognition factor in ER-associated degradation protein 1, UBASH3B Ubiquitin-associated and SH3 domain-containing protein B

PLK-1 and then a second phosphorylation of Usp16 by PLK-1 allows its ubiquitination. During metaphase, once the chromosomes are correctly aligned, PLK-1 is monoubiquitinated by CUL3-KLHL22 E3 ligase to promote its dissociation from the kinetochore (Fig. 1.3c) [87, 88]. Orientation of the cell division plane with respect to the cell cortex is a critical step in mitosis, which determines cell fate, function,

and tissue organization. In this regard, Lys63 polyubiquitination of Disheveled protein (Dvl) plays an important role to localize polyubiquitinated Dvl into the cell cortex which prevents the binding of expanding astral microtubules to the cell cortex. However, during early mitosis Dvl is deubiquitinated by the Ubiquitin carboxyl-terminal hydrolase CYLD to permit the expansion of astral microtubules toward the cell cortex. In this process the

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Fig. 1.3 (continued)

interaction of deubiquitinated Dvl, located in the cell cortex, with the Nuclear mitotic apparatus (NuMA)/Dynein complex, located in the astral microtubules, is necessary for the binding of expanding astral microtubules to the cell cortex. The connection between astral microtubules and cell cortex is crucial to obtain a correct orientation of the aligned chromosomes with respect to the cell cortex (Fig. 1.3d) [89]. CYLD is considered a tumor suppressor protein. Mutations in CYLD promote several types of tumor development including familial cylindromatosis and multiple familial trichoepithelioma. It is also involved in other cancers including hepatocellular carcinomas, multiple myeloma, and melanoma [90]. CYLD deficiency enhances cell proliferation due to enhanced proliferative signaling mediated by Nuclear factor kappa-B (NF-κB) and Wnt/β-catenin pathways [91, 92].

Another case of non-degradative ubiquitination/deubiquitination during mitosis is Lys63 polyubiquitination of NuMA. This protein is an important member of the spindle assembly factors (SAFs) that regulates a proper assembly of bipolar mitotic spindle necessary for the correct distribution of chromosomes to each daughter cell. During mitosis, Lys63 polyubiquitinated NuMA interacts with Dynein and Importin-ß to form a complex that sequesters NuMA and prevents its association to the mitotic spindle. Otherwise, deubiquitination of NuMA by the deubiquitinating complex BRCC36 isopeptidase (BRISC) inhibits the interaction of NuMA with Dynein and Importin-ß. This deubiquitination enhances the binding of NuMA to the mitotic spindle to promote its correct assembly (Fig. 1.3e) [93]. It should be noted that BRCC36, a subunit of BRISC, is overexpressed in many types of human cancer suggesting that

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uncontrolled Lys63 deubiquitination of its substrates enhances cell proliferation [94]. Another example of non-degradative ubiquitination is Lys63 polyubiquitination of Cyclin B1 enhanced by ER–microtubule-binding protein (ERLIN2) during mitosis. Cyclin B protein reaches its maximum level of expression in mitosis to bind to microtubules by its interaction with Microtubule-associated proteins (MAPs). In microtubules, CDK1–Cyclin B1 activity controls cell cycle progression in early mitosis. Polyubiquitination of Cyclin B1 by an unknown E3 ligase enhances its localization to the microtubules. This new localization of Cyclin B inhibits its degradation and facilitates its interaction with protein partners in this compartment (Fig. 1.3f) [95]. Centromere-specific protein composition is an important factor in the kinetochore assembly and chromosome segregation during mitosis. One of the most important proteins that specifically binds to centromeres during late telophase is a centromere-specific histone H3 variant called Histone H3-like centromeric protein A (CENPA). The interaction of CENP-A with the histone chaperone Holliday junction recognition protein (HJURP) is required for the correct localization of CENP-A to centromeres. This interaction and the localization of CENP-A to centromeres are promoted by monoubiquitination of CENP-A at Lys121 by Cullin-4A type E3 ubiquitin ligase RBX1 with the substrate-specific adaptor protein COP9 signalosome complex subunit 8 (COPS8) (Fig. 1.3g) [96].

1.3

Deregulation of Ubiquitin-Mediated Proteolysis in Cancer

There is increasing evidence that uncontrolled proteolysis of cell cycle regulators contributes to the formation of tumors and it has been observed to occur in different types of cancer. Some ubiquitination enzymes may be oncogenic if their specific function is to degrade tumor suppressors (such as p53, pRB, p27, p21). On the other hand, loss of activity of the ubiquitin–

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proteasome system can lead to the exacerbated stability of positive oncogenic regulators, such as Cyclins, c-Myc, c-Jun and β-catenin, among others. Deregulation of E3s activity could be caused by epigenetic and genetic mechanisms or, as a consequence of altered posttranslational mechanisms [97]. A better understanding of the mechanisms that regulate the function and activity of E3s in tumorigenesis will help to identify new prognostic markers and will allow the development of new therapeutic strategies against cancer. In this section we highlight the role of the E3 ligases SCF and APC/C in regulating the abundance of cell cycle regulators that are associated with human cancers.

1.3.1

pRb Degradation and Cancer

The retinoblastoma (RB) gene was the first tumor suppressor gene to be identified [98]. In humans, the pRb protein is encoded by the RB gene located on the 13q14 chromosomal locus. Mutation of the two alleles of this gene leads to the development of retinoblastoma, a tumor of the retina. pRb is repressed or mutated in more than 70% of human tumors [99]. As we explained above, pRb induces G1 arrest via inhibition of the transcriptional activity of E2F complexes and by controlling CKI p27 stability through interaction with SCFSkp2 and APC/CCdh1. In human tumors, loss of pRb function may be caused by hyperphosphorylation of pRb linked to overexpression of CDK4 or Cyclin D, deletion or mutation of the RB locus (e.g., retinoblastoma and small cell lung cancer) and sequestration or accelerated degradation of pRb, thus preventing association and inhibition of E2F transcriptional complexes (for review, see [99]). Loss of pRb also affects SCFSkp2 and APC/CCdh1 activity. Therefore, elucidation of the effects of pRb loss in cancer development is a major challenge. In some tumors, pRb degradation by the proteasome may be also induced by the expression of viral oncoproteins (e.g., cervical cancer) or by expression of proto-oncogenes (e.g., hepatocellular carcinomas). Human papillomaviruses (HPV) are small DNA viruses that infect epithelial cells

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and, depending on their subtype, are associated with benign diseases or uterine cancers. Of the 30 subtypes of HPV associated with anogenital diseases, two of them, HPV16 and 18, are responsible for the development of cervical cancer. These two viruses encode E6 and E7 viral oncoproteins, which interact with p53 and pRb proteins, respectively [100–102]. E7 contains the pRb-binding motif LxCxE and its interaction with pRb increases ubiquitin–proteasome dependent degradation of pRb [103, 104]. Another oncoprotein, Gankyrin, is involved in the destabilization of pRb in liver cancer. Gankyrin is involved in cellular growth, proliferation, and invasion, and it contributes to oval cellmediated liver regeneration and cell cycle progression (for review see [105]). It is a small protein with an ankyrin repeat domain and a LxCxE pRb binding domain, and it is frequently overexpressed in hepatocellular carcinoma. It interacts with the S6b subunit (Rpt3) of the proteasome 19S regulator complex and with CDK4 to form a Gankyrin-CDK4–Cyclin D ternary complex [106]. Gankyrin inhibits pRb activity by competing with the CKI p16INK4a for interaction with CDK4, leading to hyperphosphorylation of pRb and an increase in the transcriptional activity of E2F complexes, thus promoting cell cycle progression [107]. Additionally, phosphorylation of pRb induced by Gankyrin accelerates its degradation by the proteasome [108]. Interestingly, Gankyrin also interacts with Mdm2, which facilitates the ubiquitination and degradation of p53 [109].

1.3.2

SCFb-TrCP, Mdm2, and DNA Damage

SCFβ-TrCP regulates Mdm2, a negative regulator of the p53 tumor suppressor [37, 110]. As mentioned above, p53 has a crucial role in the response to genotoxic stress [111, 112]. Without p53 activity, cells containing damaged DNA continue to divide and accumulate genetic abnormalities that can lead to cancer development. Indeed, its functional inactivation or negative regulation occurs in most human cancers

[113, 114]. In many cases, the p53 loss of function is linked to mutations in the gene following exposure to carcinogens. In other cases, such as in some human sarcomas, loss of wild-type p53 function is associated with exacerbated protein degradation via genomic amplification of the MDM2 locus resulting in overexpression of the Mdm2 protein [115]. It is interesting to note that in human tumors mutations on the p53 gene and amplification of the MDM2 locus are never found simultaneously. Since both proteins are part of a self-regulatory loop, a change in the level of expression of only one of the two partners is enough to induce carcinogenesis. In other cases, the increase in Mdm2 may be due to an increase in gene transcription. A polymorphism at position 309 in the intron promoter region of MDM2 has been identified. It increases the affinity for the transcription factor Sp1, which leads to an increase in mRNA and protein levels [116]. This polymorphism is associated with elevated levels of Mdm2 in renal cell carcinoma, normal esophageal tissue, and chronic B-cell lymphocyte leukemia [117]. In certain cancers (glioblastomas, neuroblastoma, astrocytomas, squamous oral carcinomas, etc.), the deletion of the INK4β-ARF locus leads to the alteration of the p14ARF–Mdm2–p53 pathway, accounting for inactivation of the p53 pathway [42, 118]. In cervical cancer, linked to HPV16 and HPV18 high-risk papillomavirus infection, p53 protein is destabilized by another mechanism. These small DNA viruses encode the E6 oncoprotein that interacts with the cellular HECT E3 ligase named E6-AP, which is a HECT E3 ligase, leading to an efficient ubiquitination and proteasomal degradation of p53 [119]. HPV vaccines have recently been developed to prevent the development of cervical cancer, which appears several years after the viral infection [120].

1.3.3

SCFb-TrCP and Colorectal Cancer

Numerous studies indicate that β-TrCP has oncogenic function in some tissues. High levels of β-TrCP and constitutive activation of the

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NF-κB pathway are characteristic of pancreas, colon and liver tumors [121–123]. The activation of the NF-κB transcriptional complex seems to be linked to the exacerbated ubiquitindependent degradation by SCFβ-TrCP of its specific IκBα inhibitor, thus inducing NF-κB nuclear translocation and activation of its target genes [124]. In the case of colorectal cancers, deregulation of Wnt mitogenic pathway leads to an increase in β-catenin abundance [125, 126]. β-catenin, apart from being involved in cell adhesion by its interaction with E-cadherin [127], is a transcription factor that activates the expression of genes involved in cell proliferation, such as CCND1 and MYC [128–130]. The progressive accumulation of nuclear β-catenin functioning as a transcription factor negatively regulates Wnt pathway, causing the loss of control of cell proliferation, differentiation and migration and has been described as the initial event that triggers colon tumorigenesis. Wnt/β-catenin signaling pathway involves the phosphorylation, ubiquitination and subsequent SCFβ-TrCP-dependent degradation of β-catenin in the cytoplasm. This complex consists of the central scaffold protein Axin, the Adenomatous polyposis coli (APC) protein and the kinases Glycogen synthase kinase-3 alpha/beta (GSK3) and Casein kinase-1 (CK1). Mutations in the APC tumor suppressor gene lead to an increase in β-catenin abundance [131–133]. This type of abnormality is characteristic of familial adenomatous polyposis (FAP), an inherited autosomal disorder characterized by a predisposition to colon cancer. In more than 90% of cases, the mutations in APC lead to cleavage of the carboxy-terminal part of APC, which prevents the recruitment of β-catenin in the APC/Axine/ CK1/GSK3β complex. Mutations in APC gene or CTNNB1 gene encoding β-catenin are common in sporadic colorectal cancers. These mutations inhibit β-catenin degradation by numerous means including impaired ubiquitination [134]. High levels of β-catenin are also observed, among others, in hepatocarcinomas and malignant melanomas and are also due to a stabilization of β-catenin

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linked to mutations affecting APC, Axin or β-catenin [8].

1.3.4

SCFSkp2, Cell Cycle Progression, and Cancer

Cell cycle progression is regulated by SCFSkp2 that mediates ubiquitination of a number of CDK inhibitors, of which the best studied is the tumor suppressor p27Kip1 [26, 135–137]. p27 is a negative regulator of CDK2–Cyclin E and CDK2–Cyclin A complexes whose activities are necessary for progression in phase S. In G0 and beginning of G1, the synthesis and stability of CKI p27 are maximal, then degradation of p27 by SCFSkp2 allows progression from G1 to S phase [138, 139] (for reviews, see [8, 139]). In humans, downregulation of p27 expression is observed in about 50% of carcinomas and correlates with aggressive tumors and poor prognosis. The decrease in p27 abundance is related to active degradation of the protein and may be related to exacerbated phosphorylation of p27 on the Tyr88 residue thus stimulating phosphorylation of Thr187 by CDK2 and its binding to Skp2. Depending on the types of cancer, several mechanisms may lead to Tyr88 phosphorylation, such as activation of the Src pathway following oncogenic activation of tyrosine kinase receptors (RTK) such as ERBB2, which is amplified in a number of breast as well as in different epithelial tumors, or activation of Lyn kinases, Abl, Lck and Fyn, in lymphomas and other hematopoietic cancers [139]. The accelerated degradation of p27 can also be related to an increase in Skp2 expression level. High levels of Skp2, associated with low levels of p27, are found in lymphomas, breast, prostate, colon, lung, stomach and head and neck cancers [139]. This increase may be due to a chromosomal amplification encompassing the locus containing the gene coding for Skp2 (this is the case, for example, in a number of small cell lung cancers [140]) or a deletion of the locus containing the gene encoding Phosphatase and tensin homolog (PTEN) tumor suppressor in a number of other cancers. Indeed, Skp2 levels are

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inversely correlated with those of p27 and PTEN [141] and the data suggest that PTEN might repress Skp2 expression. In addition, PTEN negatively regulates Skp2 assembly in the SCFSkp2 complex and this effect is induced via a decrease in Cul1 neddylation, a critical event for the association of Skp2 with Cul1. Thus, in the absence of PTEN, the expression of Skp2 is increased and Cul1 is efficiently neddylated, which allows the recruitment of Skp2 in the SCFSkp2 complex and the accelerated degradation of p27 [142]. Overexpression of CUL1 also promotes proliferation through p27 degradation and has been correlated with reduced survival [143– 146]. Additional data support the notion that overexpression of Skp2 plays an active role in oncogenesis: (1) Skp2 cooperates with H-ras in murine primary fibroblasts and induces tumor formation in nude mice [147], (2) double transgenic mice co-expressing Skp2 and N-Ras in the T-lineage develop T-cell lymphomas, three times more than the mice expressing the activated N-Ras transgene alone [136, 148], and (3) co-expression of Skp2 and myristoylated Akt1 in the mouse liver results in hepatocellular carcinoma (HCC) [149]. Conversely, Skp2 knockout in mice, with deficiency of Rb1 or p53, or both, inhibits tumorigenesis due, at least in part, to the increased abundance of p27Kip1 [150–153]. The high expression of Cks1, a Skp2 co-factor in the SCFSkp2 complex, also has oncogenic potential. A high abundance of Cks1 has been observed in breast tumors [154] and colorectal carcinomas [154, 155] and correlates with low abundance of p27, with aggressive tumors and poor prognosis. All of these data highlight the proto-oncogenic character of Skp2 and qualifies it as a promising candidate target for cancer treatment.

1.3.5

SCFFBXW7 and Cancer

FBXW7 is an F-box protein with WD40 repeats domains which functions as the substrate recognition component of the SCF E3 ubiquitin ligase, and regulates the abundance of a series of proteins that play a central role in cell division, cell growth,

and differentiation. The majority of its substrates are oncoproteins such as Cyclin E, c-Myc, c-Jun, Notch, mTOR, Mcl-1 and Aurora A that are involved in a wide range of human cancers [156]. According to this, FBXW7 is considered a tumor suppressor and is one of the most commonly deregulated proteins of the UPS in human cancer. In fact, mutations in the FBXW7 gene have been identified in lymphoma, ovarian, breast, and colorectal cancers [156]. A common feature of the substrates recognized by FBXW7 is that they have in their protein sequence a recognition motif known as CPD (consensus-phospho-degron). Phosphorylation of this motif activates the signal for substrate recognition by FBXW7 [27]. This allows for additional cellular pathways to converge and regulate substrate phosphorylation/degradation. Glycogen synthase kinase 3 beta (GSK3β) is commonly the kinase phosphorylating the targets for FBXW7-mediated degradation. In addition, homodimerization of the FBXW7 protein can increase the binding to its substrates, especially for those with weak phosphodegrons [157]. The FBXW7 gene is located in the 4q32 region, a chromosomal region frequently altered in cancer cells. Approximately 6% of primary human tumors contain mutations in FBXW7. Thirty percent of the mutations were found in cholangiocarcinoma and T cell acute lymphoblastic leukemias (T-ALL) while pancreas, gastric and colon carcinomas, as well as prostate and endometrial cancer showed a frequency of mutations in the range of 4–15%. Seventy-five percent of the mutations are point mutations that cause substitutions in key amino acids and affect the binding of the substrate; the rest of the mutations cause stop codons that produce truncated proteins and also eliminates the interactions with the substrates. Therefore, inactivation of the FBXW7 protein causes the stabilization of its oncogenic substrates. For example, FBXW7 mutations that lead to increased Cyclin E protein levels have been associated with several malignant diseases such as in breast and colorectal cancers and its constitutive expression leads to genomic instability [158, 159]. The expression of proto-oncogene c-Myc is also increased in many malignant tumors.

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Sequential phosphorylation of c-Myc in the CPD, at Ser62, by extracellular signal-regulated kinases, and at Thr58, by Glycogen synthase kinase-3β (GSK3β), induces its recognition by SCFFBXW7 to target it for degradation. Mutations in the Thr58 of c-Myc increase the stability and activity of the protein in Burkitt’s lymphoma [160–163]. The deubiquitinating enzyme USP28 counteracts the activity of FBXW7 by removing ubiquitin from its substrates, including c-Myc, resulting in their stabilization [164, 165]. In a colorectal mouse model, depletion of USP28 resulted in the accelerated degradation of c-Myc, c-Jun and Notch and promoted tumor cell differentiation accompanied by decreased proliferation [166]. SCFSkp2 also regulates the ubiquitindependent proteolysis of c-Myc and concomitantly stimulates c-Myc transcriptional activity [167–169]. The hepatitis B virus (HBV) X protein induces stabilization of Myc by inhibiting its SCFSkp2-mediated ubiquitination and contributes to the increased risk of developing hepatocellular carcinoma following chronic HBV infection [170, 171]. Mutations that cause the stabilization of the protein in 50% of human T-ALLs leukemias have also been described in the CPD region of Notch [172]. AP-1 transcription complex is another target of FBXW7. Constitutive activation of AP-1 family members, c-Jun and JunB, due to altered degradation by SCFFBXW7 contributes to tumor development [173–175].

1.3.6

SCFFBXW7, MCL-1, and Apoptosis

Myeloid cell leukemia 1 (MCL-1) is an antiapoptotic member of BCL-2 family proteins. It prevents apoptosis by sequestering proapoptotic proteins BAK/BAX, thereby allowing cells to maintain homeostasis. MCL-1 is targeted for degradation by several E3s regulating apoptosis via a number of distinct pathways [31, 144, 176–179]. In mitosis, after prolonged mitotic arrest, MCL-1 is phosphorylated and ubiquitinated by APC/CCdc20 [180] and SCFFBXW7 [177] for degradation.

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MCL-1 is also ubiquitinated by SCFFBXW7 after phosphorylation of its CPD by GSK3β. Degradation of MCL-1 by SCFFBXW7 occurs following overexpression of the oncoproteins Myc, c-Jun, or Notch [179]. Therefore, loss of SCFFBXW7 activity leads to cell survival due to increased levels of its substrates and confers resistance to apoptosis by upregulated MCL-1 expression. Aberrant levels of MCL-1 are linked to cancer progression and to resistance to cancer treatments. Amplification and overexpression of MCL-1 have been reported in various human tumors, including hematological malignancies and solid tumors (e.g., non-small cell lung cancer, breast, ovarian, prostate, and pancreatic cancers) [181, 182]. Lack of SCFFBXW7 activity in cancer cells treated with anti-tubulin drugs confers resistance to apotosis by accumulation of MCL-1 levels [182].

1.3.7

APC/C in Cancer

APC/C complex is fundamental in the regulation of the cell cycle and in the control of genomic stability; therefore, its deregulation has an important impact in cancer. Mutations have been identified in several subunits of APC/C in colon tumors and cancer cell lines [183]. The F-box protein Emi1, an inhibitor of APC/C activity, is overexpressed in human lymphomas, carcinomas, adenocarcinomas, and melanomas, leading to the accumulation of APC/C substrates such as Cyclins, Aurora A, and Securin causing rereplication or endoreduplication. In cells with inactive p53, overexpression of Emi1 causes polyploidy, genomic instability and premature cell proliferation [10, 184]. Overexpression of the Cdc20 APC subunit has been observed in oral squamous cell carcinoma cell lines and in primary brain and neck tumors. Overexpression of Cdc20 is associated with premature anaphase resulting in chromosomal instability [185]. The APC E2 conjugating enzyme UbcH10 is overexpressed in many human cancer types including lung, prostate, breast, bladder, ovarian, uterine, thyroid, esophageal, and gastric carcinomas and is associated with tumor

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B. Pérez-Benavente et al.

progression [186–190]. Overexpression of UbcH10 in mouse embryonic fibroblasts (MEFs) induces premature degradation of Cyclin B, mitotic abnormalities and aneuploidy, and accelerates tumor formation in the UBCH10 transgenic mouse [190]. Depletion of UbcH10 in human colorectal cancer cell lines inhibits cancer cell growth [191–193].

1.4

Targeted Therapy of the Ubiquitin–Proteasome System in Cancer

The increasing recognition and understanding of the dysregulated E3s activities in cell proliferation and cancer suggest that they constitute therapeutic targets of great interest. Through the few examples described in this chapter, we have illustrated different levels of alteration that can lead to carcinogenesis. Currently, two therapeutical strategies are being used to address the control of ubiquitination and proteasome degradation for cancer treatment: (1) the development of proteasome inhibitors and (2) the development of inhibitors targeting specifically some ubiquitinating enzymes (E3s and DUBs). Given the pleiotropic role of the proteasome, it was not clear that the first strategy to inhibit proteasomal function could have a specific role on tumor cells to be used in cancer therapies. However, the FDA-approved proteasome inhibitor Bortezomib (marketed as Velcade by Millenium Pharmaceuticals) has been shown to be effective in the treatment of multiple myeloma [194]. Bortezomib is a reversible inhibitor of the chymotrypsin-like activity of the 20S proteasome. Inhibition of the proteasome in cancer cells results in activation of apoptosis and decreased expression of anti-apoptotic proteins. It induces stabilization of IκB, a suppressor of NF-κB signaling [195], and accumulation of p27Kip1 and p53 tumor suppressors [196]. Other inhibitors targeting the proteolytic activities of the proteasome are under development, such as Carfilzomib (PR-171, developed by Proteolyx), a synthetic analog of epoxomycin, which irreversibly and specifically inhibits the chymotrypsin-

like activities of the proteasome. Studies on human xenograft models have shown that PR-171 has a stronger antitumor effect than Bortezomib [197] as it induces responses in bortezomib-resistant multiple myeloma [198]. The second strategy is to develop compounds that specifically target ubiquitinating enzymes by (i) repressing their expression, (ii) altering their subcellular localization, (iii) inhibiting their dimerization, (iv) selectively blocking their catalytic activities or (v) inhibiting their enzyme–substrate interactions [199, 200]. This has led to the development of E3 drugs such as inhibitors of APC/C [182, 201], Mdm2 [202, 203], Skp2 [204, 205], SPOP [206], and cIAP [199, 207], being some of them under evaluation in clinical trials. For example, molecules inhibiting the interaction of p53 with Mdm2 [208] have been developed, such as Nutlins that have been identified by high throughput screening analysis. These compounds bind to Mdm2 disrupting its interaction with p53. Nutlins induce accumulation of p53 and inhibit cell growth in osteosarcomas and colon carcinomas cell lines in a p53-dependent manner. Nutlins are also effective in vivo by oral administration on mouse xenograft cancer models being able to reduce tumor growth without significant toxicity in healthy tissues [209]. RITA [2,5-bis (5-hydroxymethylthienyl) furan] is another molecule that regulates Mdm2-p53 interaction. RITA binds to the N-terminus of p53 and induces a conformational change that prevents interaction with Mdm2, stabilizing p53. This compound induces apoptosis of tumor cells without significantly affecting normal cells. p53 thus stabilized by RITA remains transcriptionally active. Treatment with RITA leads, in a dose-dependent manner, to a decrease in xenograft tumor growth in nude mice [210]. Since RITA binds p53 but not Mdm2, it might inhibit other interactions of p53. Other examples are the molecules targeting MCL-1 in cancers with inactive SCFFBXW7. In this context, to overcome resistance to cancer treatments, small molecules have been developed to act as BCL2 homology-3 mimetics, to release BAK/BAX sequestered by MCL-1 proteins. These molecules restore the apoptotic pathway in cancer cell lines and mouse experimental models [211, 212]. Also,

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the use of the proteolysis-targeting chimera (PROTAC) technology to induce selective intracellular proteolysis by the use of bifunctional molecules that guide E3 enzymes to degrade oncogenic substrates has great potential [213, 214]. Another way to interfere with the ubiquitin– proteasome system is to target the ubiquitin deconjugation system by inhibiting deubiquitination enzymes. For example, many efforts are being made to identify USP7 inhibitors. Indeed, USP7 deubiquitinates, among others, Mdm2 and the inactivation of USP7 leads to the accumulation of p53. Synthetic USP7 inhibitors have been developed and one of the molecules (HBX41,108) stabilizes p53 in vitro, leads to p53 activation, inhibits cell growth, and induces apoptosis in the absence of genotoxic stress in colon cancer cell lines [215]. Recently developed selective small-molecule USP7 inhibitors (GNE-6640 and GNE-6776) attenuate ubiquitin binding and inhibit USP7 deubiquitinase activity inducing tumor cell death and enhanced cytotoxicity with chemotherapeutic agents and targeted compounds [216]. In another study, specific inhibitors of USP7 (FT671 and FT827) have been identified and show their ability to inhibit tumor growth [217]. The deubiquitinase USP28 is another potential cancer drug target. USP28 stabilizes oncogenic factors, including c-Myc, c-Jun, and Notch. Deletion of USP28 in a mouse model for colorectal cancer hampered tumor formation and promoted tumor cell differentiation accompanied by decreased proliferation due to the stabilization of oncogenic proteins, such as c-Myc [166]. However, to date no DUB inhibitors have entered clinical trials [218]. Other drugs targeting other potential targets of the UPS are currently under study (for review, see [219–221]). The development of new molecules capable of modulating the UPS activity as an anticancer therapeutic strategy has great potential for the future. However, it is a complex task due to the complexity of E3s regulation and function. Indeed, E3s can act as tumor suppressor or oncogene depending on their substrate and cell context. At the same time, posttranslational modifications influence E3s activity and their

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substrate recognition, and there exists overlapping of substrate specificities between SCF and APC/C complexes. Finally, there is a lack of information about specificity and function for every E3s, DUBs and their substrates. Therefore, understanding the structure of the E3s and the mechanisms that regulate their activities is crucial to develop more specific molecules that disrupt specific interactions in a cellular context and holds promise for potent and less toxic drug action.

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer Pierre Gâtel, Marc Piechaczyk, and Guillaume Bossis

Abstract

Keywords

Ubiquitin defines a family of approximately 20 peptidic posttranslational modifiers collectively called the Ubiquitin-like (UbLs). They are conjugated to thousands of proteins, modifying their function and fate in many ways. Dysregulation of these modifications has been implicated in a variety of pathologies, in particular cancer. Ubiquitin, SUMO (-1 to -3), and Nedd8 are the best-characterized UbLs. They have been involved in the regulation of the activity and/or the stability of diverse components of various oncogenic or tumor suppressor pathways. Moreover, the dysregulation of enzymes responsible for their conjugation/deconjugation has also been associated with tumorigenesis and cancer resistance to therapies. The UbL system therefore constitutes an attractive target for developing novel anticancer therapeutic strategies. Here, we review the roles and dysregulations of Ubiquitin, SUMO, and Nedd8 pathways in tumorigenesis, as well as recent advances in the identification of small molecules targeting their conjugating machineries for potential application in the fight against cancer.

Ubiquitin · SUMO · Nedd8 · Cancer

P. Gâtel · M. Piechaczyk · G. Bossis (*) Equipe Labellisée Ligue Contre le Cancer, IGMM, Univ Montpellier, CNRS, Montpellier, France e-mail: [email protected]

2.1

Introduction

Ubiquitin is the founding member of a polypeptide family of approximately 20 protein posttranslational modifiers [1]. For the sake of simplicity, these, together with Ubiquitin, will be called the Ubiquitin-likes, or UbLs, hereafter. Among them, Ubiquitin, Nedd8, and the three members of the SUMO family (SUMO-1 to -3) are at the heart of this review. UbLs are small globular polypeptides of 8–12 kDa. They share low sequence similarity but high structural identity with one α-helix and five β-sheets (β-grasp fold) followed by a C-terminal tail [2]. In most cases, they are covalently conjugated to proteins via formation of an isopeptide bond between their C-terminal glycine and the ε-ΝΗ2 group of lysines from substrates. Nevertheless, other, quantitatively minor, conjugations of Ubiquitin to other amino acid residues or at the N-terminus of proteins have been described. The mechanisms of conjugation are very similar amongst the UbLs even though each one of them is transferred to protein substrates using specific sets of enzymes. Yet, certain of these enzymes can intervene in the conjugation of more than one UbL type under certain conditions (Table 2.1). As UbL conjugation/deconjugation processes have been described extensively in a number of reviews

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_2

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Table 2.1 Ubiquitin-like modifiers: conjugation/deconjugation enzymes and main functions

Modifier Ubiquitin

Homology with ubiquitin 100

SUMO1-5

15

NEDD8

58

ISG15

27

FUB1

36

FAT10 URM1

27 17

UBL5

25

UFM1

23

ATG8 ATG12 MAP1LC3A MAP1LC3B MAP1LC3C GABARAP GABARAPL1 GABARAPL2 GABARAPL3

12 9 13 10 8 12 14

E1 UBE1, UBA6 SAE1/ SAE2 NAE1/ UBA3 UBE1L

E2 38

E3 >600

Protease ~100

UBC9

>15

~10

UBC12, UBE2F UBCH8

>10

CSN5, NEDP1

HERC5, EFP, HHARI

USP18

UBA6 UBA4

USE2

UBA5

UFC1

ATG7 ATG7 ATG7

ATG3 ATG10 ATG3

0

UFL1

ATG12, ATG5, ATG16L

[3, 4], they will only be addressed briefly below. UbLs are first activated by UbL-activating enzymes called E1s. These use ATP to form a thioester bond between the UbL C-terminal glycine and their catalytic cysteine. The C-terminal glycine is then trans-thiolated, allowing the UbL to be transferred onto the catalytic cysteine of UbL-conjugating enzymes called E2s. Although E1s and E2s can, in some cases, be sufficient to conjugate certain UbLs on target proteins, they most often require a third factor called E3 [5, 6]. More than 600 E3s have been proposed for Ubiquitin, but much less for the other UbLs. Certain E3s can be full-blown enzymes. This is the case of the E3 Ubiquitin ligases from the HECT family, which harbor catalytic cysteines forming thioester bonds with the Ubiquitin C-terminal glycine before transfer of Ubiquitin onto protein

UFSP1, UFSP2 ATG4 0 ATG4A, ATG4B, ATG4C, ATG4D

Functions Multiples, proteasomal degradation Protein/protein interaction, regulation of transcription Cullins activation, cell cycle Immune response, response to stress Unknown, immune response regulation Proteasomal degradation tRNA thiolation, oxidative stress response RNA splicing, cell polarization Hematopoiesis, NF-κB regulation Autophagy Autophagy Autophagosomes formation

substrates. However, in most cases, E3s function as mere adaptors between E2s and substrates to confer reaction selectivity. Certain UbLs can be conjugated to themselves via the formation of isopeptide bonds between their C-terminal glycines and certain of their own lysines. This is especially true for ubiquitin, which can form chains involving each one of its seven lysines (K6, K11, K27, K29, K33, K48, K63). These chains can be homotypic or heterotypic due to the multiplicity of conjugatable lysines on Ubiquitin [7]. Mixed chains between different UbLs can also be formed. The bestknown ones are those between Ubiquitin and SUMO or Nedd8 [8]. Importantly, UbL conjugation is reversible and highly dynamic with most substrates being constantly modified and demodified. Deconjugation is carried out by isopeptidases, which cleave the isopeptide bonds

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer

between UbLs and target lysines. This allows UbLs, which are highly stable polypeptides, to be recycled and reconjugated to other proteins. Some isopeptidases are also involved in the proteolytic maturation of UbLs, which are synthetized in the form of precursors displaying extra amino acids at their C-termini. Similar to E3s, isopeptidases show substrate specificity or, at least, preference for particular chain linkages [9]. Concerning the SUMO pathway, deSUMOylases, such as SENP6 and SENP7, preferentially cleave SUMO-2 chains, while others, such as SENP-1 and SENP-2, rather deconjugate SUMO bound to target proteins [10]. Some deSUMOylases such as SENP-3, SENP-5, and USPL1 have preference for SUMO2 over SUMO-1 [11, 12]. The consequences of UbL conjugation are numerous. They depend on the UbL type, possibly the nature of UbL chains formed and, obviously, the substrate. As they have been reviewed extensively elsewhere [3, 13–15], only the main physiological roles of Ubiquitylation, SUMOylation, and Neddylation are considered hereafter. The biological outcomes of Ubiquitin conjugation are highly dependent on the chain linkage types, which, due to their diversity and complexity, create the so-called “Ubiquitin code” [14]. The most abundant and best-characterized Ubiquitin chains are long K48-linked ones (>4 Ubiquitins). They constitute a protein degradation signal recognized by the 26S proteasome, which is the main cell proteolytic machinery [16–18]. This discovery led Avram Hershko, Irwin Rose, and Aaron Ciechanover to be awarded the Nobel Prize in 2004. It is, however, important to keep in mind that K48-linked Ubiquitin chains can also be involved in signaling events and transcription regulation not involving protein destruction [19– 21]. K63-linked chains are best known as involved in protein–protein interactions, signaling, inflammatory response, DNA repair, and ribosomal function [8, 22, 23]. K11 chains were shown to play important roles in cell cycle regulation and the activation of the NK-κB pathway [24, 25]. Moreover, Ubiquitin can also form linear head-to-tail chains when its C-terminal glycine is linked to the N-terminal methionine of another Ubiquitin [26, 27]. Such chains are formed by the LUBAC

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complex and play key roles in immune signaling [28]. Finally, Ubiquitin can also be conjugated to protein substrates as monomers, sometimes at multiple sites, to regulate transcription, DNA repair or membrane receptor internalization and possibly degradation [29–31]. More than 6000 SUMOylated proteins have been identified recently thanks to proteome-wide mass spectrometry approaches [32, 33]. SUMOylation modifies the surface of target proteins and, thereby, alter their function and fate. In particular, SUMO can recruit SUMO-interacting motifs (SIM)-bearing proteins. However, only a handful of such effectors of SUMOylation have been identified so far. This is, for example, the case of the SUMO-targeted Ubiquitin ligases (StUBLs). These proteins, which include RNF4 [34, 35] and Arkadia/RNF111 [36] harbor multiple SIMs recognizing poly-SUMO2 chains irrespectively of the substrate they are bound to. As the interaction between SUMO and SIMs is of low affinity, SUMOylation most often stabilizes an already existing interaction rather than promotes a new one. This is typically illustrated by the binding of the DNA helicase Srs2 to SUMOmodified PCNA [37]. Albeit SUMO has been involved in many cellular processes, its bestdescribed functions are nuclear, consistently with a higher accumulation of SUMOylated proteins in the nucleus. In particular, SUMOylation plays key roles in DNA damage repair through the modification of critical proteins involved in this process [38]. SUMO also modifies a high number of proteins involved in gene expression (transcription factors, co-regulators, histones, transcription machinery) and participates in the regulation of transcription [39–43]. SUMOylation often concerns protein complexes comprising multiple SUMOylatable subunits. In this case, the biological outcomes are usually thought to result from SUMOylation of the complex irrespective of the SUMOylation site or of the SUMOylated proteins within the complexes [44]. SUMOylation is highly regulated by stresses [45]. Some stresses affect limited number of SUMO substrates, while others can alter the activity of the whole pathway by affecting SUMO-conjugatingand/or SUMO-deconjugating enzymes. For example, upon proteotoxic stress induced by heat shock, SUMO-2 conjugation is quantitatively rewired to

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chromatin-bound proteins [46], which prevents protein aggregation and targets them for degradation by the Ubiquitin–proteasome system [47]. Oxidative stress is also a critical regulator of SUMOylation through its ability to induce the reversible inactivation of the SUMO E1 and E2 via the formation of a disulfide bond between their catalytic cysteines [48]. This redox regulation of SUMOylation participates in the activation of ATM kinase and is required for proper DNA damage response [49]. Reactive Oxygen species have also been involved in the regulation of specific SUMO E3 and isopeptidases [50]. Nedd8 (Neural precursor cell-expressed developmentally downregulated 8) is the closest kin of Ubiquitin, as they share 60% of homology. Cullins, which are key components of the family of the multimeric cullin RING Ubiquitin ligases (CRL) are, by far, the most abundant Neddylated proteins. Cycles of Neddylation/deNeddylation are required for their Ubiquitin ligase activity [51]. Nedd8 is also conjugated to many non-cullin substrates [15]. These include transcription factors, such as p53 [52], TAp73 [53] and E2F1 [54, 55], as well as the VHL (Von Hippel–Lindau) [56], BCA3 (Breast Cancer-Associated protein 3) [57], the chemokine receptor CXCR5 [58], and several ribosomal proteins [59–62]. In these cases, Neddylation is involved in their localization, stabilization, or regulation of their interaction with partners [15]. Similarly to SUMOylation, Neddylation has also been implicated in response to proteotoxic stresses [63].

2.2

Oncogenic and Tumor Suppressor Pathways Are Controlled by UbLs

UbLs, through the variety of proteins they conjugate, are involved in each one of the “Hallmarks of cancer,” as defined by Hanahan and Weinberg [64]. For reasons of space, it is impossible to summarize here all cancer-relevant pathways regulated by UbLs. The reader is, therefore, referred to recent comprehensive reviews on this subject [65, 66]. Below, we will focus only on pathways that are controlled by at least two UbLs.

2.2.1

The p53 Pathway

The tumor suppressor protein p53 is certainly the best-studied transcription factor in cancer where its major cell protection functions are most often, if not always, lost. Physiologically, p53 participates in multiple cellular functions. They non-exhaustively include regulation of cell cycle and death, senescence, autophagy, DNA damage repair, and metabolism. p53 is mutated in approximately 50% of tumors, where its mutations can be associated with oncogenic gains of function. In most of the other tumors, either the p53 gene is deleted or its activity, or that of its protein product, is inhibited following a diversity of mechanisms, which results in inability to control cell proliferation or to induce apoptosis or senescence [67]. The first and best-characterized p53 modification by UbLs is Ubiquitylation [68]. The main cellular E3 ligase for p53 is the MDM2 protein, which maintains low p53 levels under basal conditions via K48-linked chain Ubiquitylation and subsequent proteasomal degradation [69, 70]. Upon genotoxic stress, for example, this Ubiquitylation is arrested, permitting p53 to accumulate and to exert its cell protection functions. On the contrary, upon hyperactivity or amplification of the pro-oncogenic MDM2 gene, p53 is continuously maintained at a low level, favoring tumorigenesis [71]. A similar pro-oncogenic p53 inactivation process occurs in human papilloma virus (HPV) 16/18-infected cervix epithelial cells where the p53-interacting viral protein E6 recruits the cellular E6AP HECT E3 ligase [72, 73]. Interestingly, MDM2 can also mono-Ubiquitylate p53, which entails nuclear export and, thereby, inhibition of transcriptional activity [74]. Such a cytosolic export also impacts other p53 functions, including inhibition of autophagy and induction of apoptosis [75]. Other E3 Ubiquitin ligases such as MSL2 [76] and WWP1 [77] were shown to target p53 to the cytosol without, however, affecting its proteasomal degradation. Finally, the E3 ligase E4F1 was reported to Ubiquitylate p53 on chromatin. E4F1 increases p53’s ability to activate specific transcriptional programs related to cell cycle arrest without affecting its degradation [20] (Fig. 2.1)

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer

p53 activity can also be controlled by Neddylation, which involves the E3 ligases activities of MDM2 [52], as well as that of FBOX11 [78]. Neddylation occurs on three lysines (370, 372, 373) and reduces p53 transcriptional activity [52, 78–80]. In addition, p53 Neddylation was shown to limit its Ubiquitinmediated nuclear export [79]. Finally, p53 can also undergo modification by SUMO on its lysine 386 [81–83]. However, the role of this SUMOylation is still debated and might depend on the cellular context [84]. Indeed, p53 SUMOylation was initially described to increase its transcriptional activity [81, 82]. However, other studies showed that SUMOylation is involved in neither p53 localization nor transcriptional activity [85]. SUMOylation was also suggested to regulate p53 subcellular localization. For example, SUMOylation of mouse p53 was shown to be required for nuclear accumulation

33

and enhanced stability in granulosa cells [86]. However, and contrasting with the latter observation, androgen-mediated SUMOylation of p53 was suggested to be important for export of p53 to the cytosol [87] (Fig. 2.1).

2.2.2

The NF-kB Pathway

The NF-κB pathway is overactive in a vast majority of cancers where it is thought to participate in cancer cell resistance to apoptosis and sustained proliferation. This is especially true in hematological malignancies, where the function of various components of this pathway can be altered, notably by oncogenic mutations or rearrangements/ translocations [88]. Under basal physiological conditions, the NF-κB transcription factor is maintained latent in the cytoplasm through physical interaction

Ub p53

S p53

Cytosol Ub Ub Nucleus

Ub

Ub

?

MDM2 MSL2 WWP1

MDM2 Pirh2 COP1 p53

p53

N8

MDM2 FBOX11

E4F1 p53

Proteasomal degradation

Fig. 2.1 Regulation of p53 by UbL. N8 Nedd8, Ub Ubiquitin, S SUMO

DNA repair Apoptosis Cell cycle arrest Senescence Autophagy...

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P. Gâtel et al.

with its IκBα inhibitor [89]. Ubiquitylation is involved in its activation at several steps. A typical example of NF-κB pathway activation is as follows. In response to an appropriate extracellular stimulus, the RIPK1 (receptor-interacting serine/threonine protein kinase 1) kinase, is modified with non-proteolysis-inducing linear Ubiquitin chains by the LUBAC complex and K11- and K63-linked chains by the cIAP1 (cellular inhibitor of apoptosis protein-1) Ring domain-bearing Ubiquitin E3 factor. These Ubiquitylations serve as a platform for recruiting a downstream kinase effector complex (made up of TAK1, TAB2/3, IKKγ/NEMO), which activates another kinase complex (IKK complex made up of IKKα and IKKβ). Finally, the latter phosphorylates IkBα, which triggers its subsequent K48-linked Ubiquitylation and is followed by proteasomal degradation. The NF-κB transcription factor is, thereby, released and can then enter the nucleus to activate its target genes [24]. In the early days of the SUMO field, it was discovered that SUMOylation of IκBα competes with Ubiquitylation by targeting the same lysine residue (Lys 210 [90]. SUMOylation is also involved in the regulation of NEMO activity. In particular, NEMO gets SUMOylated upon genotoxic stress. This leads to its addressing to the nucleus and subsequent ATM-dependent Ubiquitylation and activation of the IKK complex in the cytoplasm [91]. Finally, it has been suggested that NEMO is Neddylated, which inhibits the NF-κB pathway [92]. Neddylation was also involved in the regulation of NF-κB-dependent transcription through modification of BCA3, one of its partners in chromatin. BCA3 Neddylation recruits the deacetylase SIRT1 and, thereby, was proposed to repress transcription of NF-κB target genes [57].

2.2.3

The TGF-b Pathway

The TGF-β (Transforming Growth Factor β) pathway generally exerts tumor suppressor activity in normal or premalignant cells but, on the contrary, often promotes tumorigenesis at later stages, including metastasis. Depending on the cell/

tumor type, it can be involved in the regulation of cell proliferation, apoptosis, epithelial–mesenchymal transition (EMT), and cell migration [93]. In the canonical pathway, the binding of TGF-β to its cell membrane receptor (made up of two subunits, TβR-I and TβR-II) initiates a cascade of intracellular phosphorylation events. Among the first phosphorylated proteins are Smad-2 and Smad-3, which are transcription factors maintained latent in the cytoplasm in the absence of TGF-β receptor activation. Phosphorylated Smad-2 and -3 then assemble with the Smad-4 protein to form a trimeric complex that translocates into the nucleus where it binds to DNA and stimulates the expression of TGF-β target genes [94]. Interestingly, Ubls control this pathway at multiple and intermingled levels [95]. More specifically, Smad-7 is a cytoplasmic TGF-β-induced negative regulator of the pathway that can recruit Smurf-1 and Smurf2 (Smad-specific E3 Ubiquitin ligase 1 and 2), two Ubiquitin ligases of the HECT family. These can Ubiquitylate TβR-I, leading to its degradation and, thereby, inducing a negative feedback loop on the pathway [96, 97]. TβR-I Ubiquitylation, and consequently its degradation, can, however, be antagonized by the deUbiquitylase Usp15, which associates with Smurf-2, and act as a positive regulator of the pathway [98]. Interestingly, Smurf-1 and -2 are positively regulated by Neddylation via particular mechanisms. Indeed, Nedd8 is, first, transferred from the Nedd8conjugating E2 enzyme Ubc12 to the catalytic cysteine of Smurf-1 via the formation of a thioester bond. It is, then, transferred to lysines of Smurf-1. This Neddylation increases the recruitment of Ubiquitin E2(s) and, thereby activates the Ubiquitylation of Smurf-1 protein substrates [99]. Noteworthy, Smurf-1 and -2 were also shown to bind non-covalently to Nedd8, which also contributes to increasing their Ubiquitin ligase activity [100]. Complexifying the picture, SUMO can also enter into the game at different levels, as diverse components of the TGF-β pathway can undergo SUMOylation with implications in cancer [101]. For example, SUMOylation of TβR-I increases the activation of Smad-3 with, as a

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer

biological consequence, enhancement of invasion and metastasis by Ras-transformed cells [102] and suppression of EMT in bladder cancer cells [103]. Smurf-2 is also SUMOylated (on lysines 26 and 369) thanks to the SUMO ligase PIAS3, which increases its Ubiquitin ligase activity and, hence, the degradation of TβR-I. In this case, important outcomes of TβR-I proteolysis consist of decreased TGF-β-induced cell proliferation and reduced invasion by breast cancer cells [104, 105]. Transcription factors downstream of the TGF-β pathway are also SUMOylated with sometimes antagonistic effects. For example, SnoN [106] and Sip1/Zeb2 [107] SUMOylations repress the pathway whereas those of Snail [108] and Slug [109] activate it. Finally, non-covalent SUMO binding might also be involved in the regulation of the TGF-β pathway. As a matter of fact, the already mentioned SUMO-targeted Ubiquitin ligase Arkadia/RNF111 can be recruited via its cluster of SIMs (i.e., most probably due to binding to SUMO-2 chains) to TGF-β pathway-target genes with, as a result, antagonization of the Polycomb repressor complex at the level of their regulatory domains [110].

variety of cellular activities that include responses to stresses and viral infections, as well as control of cell death, senescence, or DNA repair. It has long been known that they are disrupted by the PML–RARα fusion protein in APL cells [115], which is essential for oncogenesis. Interestingly, the combination of arsenic trioxyde and retinoic acid has been shown efficient at curing APL patients. Indeed, it consists of the first successful oncogenic protein-targeted therapy that has been described [116]. In brief, the drug combination entails the polySUMOylation of PML–RARα, which is followed by the recruitment of the StUbl RNF4 and, hence, its Ubiquitylation and degradation by the proteasome [34, 35]. An important consequence of PML–RARα destruction is not only induction of cancer cell differentiation into short-lived granulocytes but also abrogation of cancer cell self-renewal through the reformation of PML nuclear bodies and subsequent p53 pathway activation [117].

2.3 2.3.1

2.2.4

PML–RARa in Acute Promyelocytic Leukemias

Acute Promyelocytic Leukemias (APL) are a minor subtype (2/3 of a 322 patient cohort with intra-hepatic cholangiocarcinoma, which was associated with higher global protein Neddylation and tumor progression [263]. Finally, the Jab1/CSN5 protein, which is responsible for deNeddylation of the members of the CRL family of Ubiquitin E3 ligases (see above), was found overexpressed in numerous cancers (breast cancer, ovarian cancer, hepatocellular carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, etc.) and associated with adverse prognosis [251].

2.3.4

Dysregulations of the SUMO Pathway in Cancer

Increasing evidence suggests that both SUMOconjugation and -deconjugation machineries are dysregulated in various cancers (Table 2.2). Yet, the contributions of these alterations to tumorigenesis have not often been established formally in most cases [65]. Interestingly, the level of Uba2, the catalytic subunit of the SUMO-activating E1 enzyme, is increased in colorectal cancer tissues, the highest Uba2 expression being associated with both the highest colorectal cancer stages and the poorest prognosis [242]. The SUMOconjugating enzyme Ubc9 is also overexpressed in many cancers. This is the case of hepatocellular

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer

carcinomas, where its overexpression participates in the resistance to chemotherapies [245]. HyperSUMOylation has also been described in Myc oncogene-driven lymphomas. This results from a strong transcriptional activation of the expression of most enzymes of the SUMO pathway and is essential for tumorigenesis [264]. Using synthetic lethality screens, it was also shown that Myc-overexpressing breast cancer cells are highly dependent on a functional SUMO pathway for growth and survival [265], pointing to a novel therapeutic window through targeting SUMOylation enzymes in this cancer type. Finally, deSUMOylases of the SENP family are either upregulated or downregulated in cancers. SENP1 is, for example, overexpressed in prostate cancer, where it promotes tumor formation and metastasis [266] and downregulated in osteosarcomas, which is important for the maintenance of cancer stem cells [236].

2.4

Targeting UbLs: New Perspectives in Cancer Treatment

Considering their critical roles and their widespread dysregulations in cancer, UbL pathways have emerged as promising therapeutic targets. Considerable efforts have consequently been made worldwide to develop strategies to inhibit their enzyme components [267]. We will focus here on the strategies used to target E1, E2, and E3 factors. For information on the targeting of deconjugating enzymes, the reader is referred to recent review [268, 269].

2.4.1

E1 Inhibitors

2.4.1.1 Ubiquitin E1 Inhibitors PYR-41 and PYZD-4409 were the first described inhibitors of an Ubiquitin-activating E1 enzyme. They are based on a pyrazolidine cycle and were identified during chemical library screenings [270, 271]. They bind to UBE1 and inhibit the formation of the thioester bond with Ubiquitin. Whether they inactivate UBA6, the second Ubiquitin E1, has however not been determined

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yet. PYZD-4409 was shown to induce apoptosis of Acute Myeloid Leukemia cells, with minimal toxicity for normal hematopoietic cells. It also displayed antitumor activity in vivo in mice xenografted with human AMLs [270]. A more potent inhibitor of UBE1, TAK-243 (also known as MLN7243), was generated recently by Takeda Pharmaceuticals [272]. It forms an adduct with Ubiquitin and inhibits UBE1 in the nanomolar range. When used on cell lines, it leads to cell cycle arrest, induction of ER stress, and impaired DNA damage response. Interestingly, TAK-243 showed (1) antitumor activity in vivo in immunodeficient mice subcutaneously grafted with various human tumor cell lines [272] and (2) antileukemic activity on primary human AML cells both in vitro and in vivo after xenografting to immunodeficient mice (PDX) [273]. Finally, in a phase I dose-escalation clinical trial (clinicaltrial.gov identifier: NCT02045095) involving 29 patients with advanced solid tumors, it however entailed serious adverse events in more than 1/3 of the individuals treated. Its in vivo efficacy could be demonstrated by immunohistochemistry using antibodies directed to either polyUbiquitin chains or Ubiquitylated-histone H2B (uH2B; which is the second most Ubiquitylated proteins in mammalian cells after Ubiquitylated H2A). A second phase I trial is scheduled to start soon with patients undergoing relapse or suffering from hematological malignancies refractory to standard chemotherapies (clinicaltrial.gov identifier: NCT03816319).

2.4.1.2 Nedd8 E1 Inhibitors MLN4924, also called TAK-924 or pevonedistat for its clinical form, is the first mechanism-based inhibitor of a UbL E1 enzyme that was designed by the Millenium-Takeda company. As TAK-243, MLK4924 is an ATP-competitive inhibitor of the Nedd8-activating E1 enzyme NAE. It forms a covalent adduct with Nedd8, which is catalyzed by NAE. The adduct cannot be transferred to Nedd8 E2s, blocking the activity of the E1, including in vivo [274]. Initial experiments showed that the treatment of immunocompromised mice xenografted with HCT-116

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colon cancer cells with MLN4924 led to increased DNA damage in cancer cells and limited tumor growth [275]. MLN4924 was then shown to have promising antitumor activity in various preclinical cancer models, including patient-derived xenografts (PDX). Several phase I clinical trials in cancer patients have shown it is well tolerated [276–278] and various phase II trials have now been launched, in particular to treat hematological malignancies. MLN4924 was also shown to synergize, both in vitro and in vivo, with genotoxic drugs such as Cytarabine [279] or the demethylating agent Azacytidine [280] in AMLs. A phase Ib clinical trial in elderly patients unfit for conventional chemotherapies suggests a potential clinical benefit for the combination of Azacytidine and Pevonedistat [281]. A randomized phase III trial involving 450 patients with AML, CML (Chronic Myelomonocytic Leukemia), or MDS (Myelodysplastic syndrome) is now ongoing to prove the efficacy of this combination on a large scale (clinicaltrial.gov identifier: NCT03268954).

2.4.1.3 SUMO E1 Inhibitors Ginkgolic acid was identified as an inhibitor of the SUMO-activating E1 enzyme during a screening using botanical extracts. This molecule, and its anacardic acid derivative, were shown to bind to the E1 and to inhibit the transfer of SUMO from the E1 to the E2 [282]. Anacardic acid was shown to inhibit cell division, to induce apoptosis, and/or to inhibit migration of various cancer cell lines and primary samples [283–287]. It was also shown to limit tumor growth in mice xenografted with human KG1a AML cells [283]. However, anacardic acid is not a potent SUMOylation inhibitors, as it requires concentrations above 25 μM to inhibit SUMOylation when used on cultured cells [282] and its use in preclinical models is unfortunately limited by its very poor solubility. Moreover, anacardic acid has been shown to inhibit various other enzymes, the best characterized one being the Histone Acetyl Transferase p300 [288]. Recently, Takeda Pharmaceutical has developed ML-792, a mechanism-based inhibitor of the SUMO E1 with nanomolar potency

P. Gâtel et al.

[289]. Similarly to the other UbL E1 inhibitors developed by Takeda Pharmaceutical, it forms a covalent adduct with SUMO that is catalyzed by the SUMO E1. Treatment of cell lines with this inhibitor induces strong mitotic defects, which leads to their apoptosis. Interestingly, ML-792 preferentially affects the proliferation and viability of cancer cells overexpressing the Myc oncogene in vitro [289].

2.4.2

E2 Inhibitors

Few inhibitors inhibiting UbL-conjugating E2 enzymes have been discovered so far and none of them is used in clinical trials yet. The NSC697923 molecule was originally identified in a screen for molecules inhibiting the NF-κB pathway. It actually binds to the catalytic cysteine of the Ubc13-Uev1A Ubiquitin E2 that catalyzes the formation of K63-linked polyUbiquitin chains. Thereby, it prevents the formation of the thioester bond with Ubiquitin [290, 291]. This inhibitor limits the proliferation of diffuse large B-cell lymphoma cells in vitro [290]. BAY 11-7082, which was also initially reported as an inhibitor of the NF-κB pathway, was subsequently shown to inhibit K63-linked polyUbiquitin chains formation by targeting the catalytic cysteines of Ubc13 and UbcH7 [292]. CC0651 is an allosteric inhibitor of the CDC34 Ubiquitin E2, which is associated with Ubiquitylation by cullin-RING ligases. It prevents the discharge of Ubiquitin to acceptor lysines on the target proteins. In particular, this molecule leads to an accumulation of the CDK inhibitor p27kip1, a target of the SCF complex, which contributes to decreased cancer cell lines proliferation [293, 294]. Using a virtual screening, the triazine analog SMI#9 was identified as an inhibitor of the Ubiquitin E2 Rad6B via binding to its catalytic site [295]. This molecule was shown to enhance cancer cell sensitivity to platinum-based drugs, including in vivo [296]. Small molecules targeting the SUMO E2 Ubc9 have also been identified in various screens. Spectomycin B1, which was originally identified as an antibiotic, directly binds to Ubc9 and

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer

prevents formation of the Ubc9-SUMO thioester bond. It inhibits ERα-regulated gene expression and growth of ERα-positive breast cancer cell lines [297, 298]. The flavonoid derivative 2-D08 was found to inhibit the transfer of SUMO from Ubc9 to target proteins [299]. Moreover, 2-D08 also sensitized non-promyelocytic AML cells to retinoids-induced differentiation and death both in vitro and in vivo [300]. Unfortunately, all of these SUMO E2 inhibitors have low potency and poor solubility, which prevents their use in therapy.

2.4.3

E3 Inhibitors

Inhibiting E1 or E2 enzymes affects the activity of the whole UbL pathway, which may entail deleterious effects on normal, noncancerous cells. Targeting E3s is consequently considered as a more specific and, potentially, less toxic approach in living individuals. To date, no molecule targeting SUMO or Nedd8 E3s has been identified. By contrast, many molecules targeting Ubiquitin E3s have been discovered [301], as illustrated below in the case of MDM2 and IAPs.

2.4.3.1 MDM2 Inhibitors As mentioned previously, MDM2 is physiologically responsible for the Ubiquitylation of the tumor suppressor p53, but is overexpressed in numerous cancers, preventing p53 pathway activation, in particular in case of genotoxic insults. Inhibiting MDM2 pharmacologically therefore constitutes an intense research area with several molecules in preclinical development and others already in clinical trials [302]. Nutlins are imidazoline compounds, which compete with p53 for the binding to MDM2. Such a competition restores the p53 pathway via inhibiting p53 degradation and induces cell cycle arrest and apoptosis of cancer cell lines both in vitro and in vivo [303]. RG7112, a member of the Nutlin family, was the first MDM2 inhibitor to be used in phase I clinical trials, in particular for liposarcoma- [304] and hematological malignancies-presenting patients [305]. This compound was shown to efficiently activate p53 in these tumors and a

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fraction of patients showed a clinical response. However, therapy-related adverse events were observed in most of treated individuals. Quite similarly to Nutlins, MI-219 mimics p53 primary structure motifs, binds to MDM2, and prevents its association with p53. It permits robust inhibition of tumor growth in vivo without affecting normal tissues in mouse cancer models [306]. AMG-232 is also to be added to the list of small molecules inhibiting the p53-MDM2 interaction [307]. This molecule, which can be administered orally and is highly potent, is currently tested in phase I and II clinical trials [308]. Another emerging approach to target the p53/MDM2 interaction is the use of stapled peptides. These peptides mimic α-helixes through side-chain crosslinking between nonnatural amino acids introduced in the peptide during synthesis [309]. ALRN-6924 is a stapled peptide that efficiently disrupts the p53/MDM2 interaction, activates p53-dependent transcriptional programs, and shows a robust antileukemic activity in mouse preclinical models [310]. Phase I/II trials are ongoing in patients with solid tumors and hematological malignancies (clinicaltrial.gov identifier: NCT02264613, NCT02909972).

2.4.3.2 IAP Inhibitors As presented above, IAP/BIRC family members are Ubiquitin E3 ligases, which inhibit apoptosis by preventing, directly or indirectly, the activation of Caspase-3 and -9. SMAC is a mitochondrial antagonist of IAPs binding to their BIR domain and, thereby, preventing their activation. SMAC-mimetics mimicking the N-terminal residues of SMAC induce the dimerization of IAPs, which is followed by auto-Ubiquitylation and subsequent degradation [311]. These molecules were shown to have antitumor activity as single agents or when combined with cytotoxic agents in various preclinical models. For example, Birinapan is a bivalent SMAC mimetic that was shown to activate RIPK-1-dependent apoptosis in relapsed and refractory Acute Lymphoblastic Leukemias (ALL) and to efficiently limit tumor growth in vivo [312]. It was also shown to synergize with various drugs, including the DNA-demethylating agent 5-azacytidine in AMLs [313]. A phase I clinical trial showed that

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it is well tolerated and leads to an important reduction of cIAP1 and the activation of cell death pathways in the tumors and PBMCs ( peripheral blood mononuclear cells) from the treated patients [314]. Similar results were obtained with LCL161, another SMAC mimetic under clinical development [315].

2.5

Conclusion

In conclusion, SUMO, Nedd8, and Ubiquitin play key roles in the control of essential cellular pathways and functions that are often dysregulated in cancer and/or participate to cancer response to therapies. Moreover, enzymes of SUMO, Nedd8, and Ubiquitin pathways are also dysregulated in many tumor types. They therefore constitute attractive therapeutic targets. Intense efforts by academic and industry laboratories have recently been made to discover small pharmacological agents targeting them. A number of these are now being tested in early phase clinical trials and others are about to enter clinical testing. This might pave the way to better cancer treatment. Acknowledgments We are grateful to all members of the “Oncogenesis and Immunotherapy” group of IGMM for support and fruitful discussions. Funding was provided by the CNRS, Ligue Nationale contre le Cancer (Programme Equipe Labellisée), INCA (ROSAML), Association Laurette Fugain (contract ALF-2017/02), the Fondation ARC (to PG), the Fédération Leucémie Espoir, The EpiGenMed Labex, and the ANR under the “Investissements d’avenir” programme (ANR-16-IDEX0006). Conflict of Interest The authors declare no conflict of interest.

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Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer

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The Proteasome System in Health and Disease Olivier Coux, Barbara A. Zieba, and Silke Meiners

Abstract

The proteasome is involved in the regulation of all cellular pathways and consequently plays a central role in the control of cellular homeostasis. Together with its regulators, it is at the frontline, both as an actor and as a target, in human health and when homeostasis is disturbed in disease. In this review, we aim to provide an overview of the many levels at which the functions of the proteasome and its regulators can be regulated to cope with cellular needs or are altered in pathological conditions. Keywords

Proteasome · 19S complex · PA28 · PA200 · PI31 · Disease

O. Coux (*) Centre de Recherche de Biologie cellulaire de Montpellier (CRBM), CNRS UMR 5237, Université de Montpellier, Montpellier, France e-mail: [email protected] B. A. Zieba Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany S. Meiners Comprehensive Pneumology Center (CPC), University Hospital, Helmholtz Zentrum Muenchen and LudwigMaximilians University, Munich, Germany

3.1

Introduction

The proteasome is a key actor of homeostasis, both at cellular and organism levels, due to its multiple roles in timely and spatially controlled selective protein degradation. After the success of its inhibitor bortezomib as an anticancer drug, which revealed the potential of therapeutic targeting of the proteasome to treat pathologies, a large effort is made worldwide to learn how to manipulate its activities in order to improve human health [1]. However, what is often overlooked is that “proteasome” is a generic term, which covers in reality multiple molecular entities that share a common proteolytic core, the 20S proteasome or 20S core particle (20S CP) but differ by the regulators that bind to it and control its activities. It is thus justified to consider the proteasome as a complex system made of building blocks, as schematically illustrated in Fig. 3.1. Even though this is not formally demonstrated for all of them, it is likely that each regulator serves in the degradation of specific set(s) of substrates, either by recruiting them to the 20S proteasome or by directing the latter to specific locations or pathways. Consequently, it is most likely that the organization into building blocks allows the system to cope with the extreme diversity of its substrates, and to rapidly adjust to cellular needs in a timely and spatially controlled manner [2]. The best known form of the proteasome is the so-called 26S proteasome, which is the central

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_3

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component of the ubiquitin–proteasome system (UPS). Thanks to its 19S regulator (or regulatory particle, RP), this form degrades mainly proteins that have been tagged with polyubiquitin chains, as well as some non-ubiquitylated proteins, in an ATP-dependent manner. The main role of ATP hydrolysis by the 19S complex is to provide energy to unfold the substrates and inject them into the 20S core. However, other forms in which the 20S proteasome is bound to other regulators exist in the cell. Three of them, namely PA28αβ, PA28γ, and PA200, are classically described as activators. Their exact roles and mechanisms of action are still quite obscure. They are believed to mostly function in ATP- and ubiquitinindependent proteolysis by the 20S proteasome. However, they are also parts of so-called “hybrid 26S proteasomes” which are capable of degrading ubiquitylated proteins. In addition to these regulators, PI31 is another protein able to bind to the 20S proteasome, but its functions in proteolysis remain unclear. Other proteins such as ECM29, ubiquitylation/deubiquitylation enzymes, or protein chaperones are found associated to different forms of the proteasome and regulate their functions. Finally, “free” 20S proteasome represents a non-negligible part of the total proteasome population, which might have a particularly important role in the degradation of oxidized and unfolded cellular proteins. Overall, there is thus not one but many proteasomes that together represent a cornucopia of potential drug targets for highly selective drug intervention (Fig. 3.1). As the central component of the ubiquitin– proteasome system (UPS), the proteasome is a critical player in the regulation of most (in fact most likely all) cellular processes, especially since it contributes to the control of the expression patterns (i.e., tissue distribution, temporal windows, and intracellular/local levels of expression) of most regulatory proteins through selective degradation, and to the quality control of the proteome through elimination of supernumerary, abnormal, damaged, or aggregating proteins. These central roles of the proteasome system in cellular proteostasis imply that any of its dysfunctions or misregulations can lead to

O. Coux et al.

pathological imbalance of cellular homeostasis, and that, conversely, artificially manipulating its activity or specificity might help to answer a plethora of therapeutic or bioengineering needs. The latter notion is the conceptual basis for a huge effort that is pursued worldwide to develop drugs targeting the proteasome. Indeed overall proteasome inhibition can be useful to impair growth of cells heavily dependent on proteasome activity, such as hyperproliferative cells, cells dependent on pathways intimately linked to UPS activity (e.g., NF-κB signaling) or cells facing a huge load of abnormal proteins to eliminate [3]. These features are often found in cancer cells, rendering them usually more sensitive to proteasome inhibition than their normal counterparts. The first efforts to manipulate proteasome activity have thus been directed to the development of inhibitors of the catalytic sites of the 20S core proteasome, and have led to novel anticancer drugs currently used in the clinic against multiple myeloma and mantle cell lymphoma [4]. On the other hand, proteasome activation might be beneficial in diseases, in which the degradative capacity of the proteasome becomes limiting, making it unable to effectively contain accumulation of abnormal and/or aggregating proteins, such as for example in certain neurodegenerative disorders or in stressinduced pathophysiological conditions [5]. Much less progress has been made in this direction, however, compared to proteasome inhibition. Other therapeutic interventions within the UPS involve the targeting of upstream mechanisms of proteome surveillance and of substrate selection such as for example ubiquitin-ligases and deubiquitinating enzymes. In fact, many researches are currently pursued at all levels to better understand the molecular bases of the degradation of specific proteins, and to explore various strategies to manipulate them for better wellbeing. Exciting progress, which will not be covered in this overview, is presently made in manipulating the ubiquitylation machinery for therapeutic and bioengineering purposes [6, 7]. In recent years, many reviews have summarized the current knowledge on the links between proteasome function and diseases. In this

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The Proteasome System in Health and Disease

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Fig. 3.1 The proteasome system: schematic of different forms of the proteasome

chapter, our aim is to provide an additional point of view by also describing what is known on a usually little covered issue, namely the implication of and the therapeutic potential of the targeting of the different regulators of the proteasome in health and disease.

3.2

20S Proteasomes: Structure and Regulation

Proteasome activity can be modulated in many ways by natural as well as artificial cues. This includes exchange of 20S proteasome subunits, swapping of regulators, posttranslational modifications, chemical modulation of its catalytic activities, and changes in localization. It must be stressed, however, that it is still enigmatic in most cases how these changes in activity relate to defined changes in specific intracellular functions. Proteasome activities are usually quantified using artificial fluorogenic peptides that are very convenient to use but have little to do with genuine, endogenous proteasome substrates, as both their small size and their lack

of secondary and higher order structures make them very distant mimics of folded proteins. One striking example of the difficulty to interpret changes in proteasome activities was the observation that in some cultured cells where the amount of proteasome is apparently not limiting, up to 80% of the chymotrypsin-like activity of the proteasome, measured against peptides, can be inhibited without clear effect on the degradation of a protein substrate [8]. In addition, it is very likely that changes in its peptidase activities do not impact similarly on all proteasome substrates, depending both on their amino acid composition/ sequence and of the upstream cues that target them to degradation. It is therefore impossible, in many if not most cases, to draw simple and direct conclusions regarding the impact of subtle changes in proteasome activity on the stability of a specific protein substrate. Nevertheless, modulating proteasome activity remains an active field of research. We present below a condensed overview of the different regulatory layers that fine-tune proteasome functions, which illustrate the many possible levels at which these functions can be altered or modulated.

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3.2.1

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Alternative Composition of the 20S Proteasome

All 20S proteasome subunits originate from a unique ancestor originating from a hypothetical precursor present in LUCA, the Last Universal Common Ancestor [9]. Through successive duplications, the ancestral gene evolved in two distinct families, α and β, themselves organized in seven subfamilies, leading to the generic α7β7β7α7 architecture of eukaryotic 20S proteasome [9–11]. Within this 4-rings structure, the catalytic sites are borne by three out of seven of the β subunits, namely β1 (caspase-like activity), β2 (trypsin-like activity), and β5 (chymotrypsinlike activity), and are enclosed in the internal chamber formed by the β-rings. Extensive sequencing of proteasomal cDNAs during the 1990s showed the existence of several isoforms for many subunits resulting from alternative splicing or alternative transcription starts. In most cases these isoforms differ by small peptide insertions/deletions [12], the consequences of which have not yet been explored. In the same period, two proteasome genes were found to reside in the class II region of the MHC, and to be inducible by cytokines such as interferon γ (IFNγ) that activate immune responses. A third gene also inducible by IFNγ was later found outside the MHC locus. These three genes (PSMB9, PSMB10, and PSMB8) turned out to encode active proteasomal β subunits (now called β1i, β2i, and β5i, respectively—i for inducible), that replace the constitutive active subunits (β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5), respectively) upon IFNγ (but also IFNα, IFNβ, or TNFα) induction [13, 14]. This discovery in turn led to the observation that the proteasome system is the major source for peptide antigens presented at the cell surface to the immune system by the MHC class I molecules, and that the βi-containing 20S proteasome, often referred to as immunoproteasome, is a form of the proteasome specialized in the production of peptides usually efficiently pipelined for MHC class I-mediated presentation to immune cells [14]. Of note however, βi subunits are constitutively (i.e., in a

IFNγ-independent manner) expressed in lymphoid tissues [14], and various cell types express certain but not all of these βi subunits at different levels [15, 16], indicating that the role of these subunits in proteasome functions is not restricted to antigen processing [14]. More recently, a new β5 gene, PSMB11 or β5t (for thymus-specific β5 family member) has been shown to be specifically expressed in the thymus [17], more precisely in cortical thymic epithelial cells, under the control of the Foxn1 transcription factor. It is incorporated into the 20S proteasome together with the β1i and β2i subunits, defining a novel form of the complex referred to as the thymoproteasome, which plays a key role in thymic positive selection of CD8+ T cells [18]. Similarly, it was found that an isoform of the α4 subunit, called α4s for spermatid/sperm-specific α4 subunit (PSMA8), is expressed exclusively in mammalian testis, assembling into a spermspecific form of the 20S proteasome (spermatoproteasome) that appears to be mainly associated to the proteasome regulator PA200 [19]. Finally, even though the biological relevance of these forms is unclear, it is worth noting that the α4 subunit can replace the α3 subunit, leading to proteasomes in which the α-ring possesses two α4 and no α3 subunit [20], and that some 20S proteasome subunits can assemble into noncanonical complexes [21]. The plasticity of the 20S proteasome underlines the existence of a functional diversity within its population that is not well understood yet. Even if this point is not ascertained yet, it is likely that different forms of the 20S proteasome coexist in the same cell, and even that a single 20S proteasome can be built of distinct α- or β-rings. At the molecular level, one can imagine that the diversity of 20S complexes allows finetuning of its functions through modulation of its affinity for specific substrates and/or specific regulators/cofactors. Indeed, proteomics analyses indicate that the immunoproteasome has higher affinity for PA28αβ, but reduced affinity for PI31, compared to the constitutive proteasome [22]. How far this conclusion can be extended with respect to physiological and diseased

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conditions, however, remains presently unclear. Nevertheless, this diversity of proteasome complexes represents an entry point for strategies aiming at inhibiting only subpopulations of the proteasome, to improve specificity and/or efficiency and decrease toxicity. As mentioned below, this entry point is already exploited to design proteasome inhibitors able to hit an inducible β subunit but not its constitutive counterpart, or vice-versa.

3.2.2

Expression and Assembly of the 20S Proteasome

The proteasome is an abundant protein complex. For a long time, it was believed that regulation of the expression of its subunits was not an important issue, the model being that they were synthesized in excess and that only those being integrated into functional complexes were stabilized, whereas those not integrated were rapidly degraded. With time, this view turned out to be too simplistic. RNA interference experiments in Drosophila cells showed that transient deficit in specific subunits was associated with the increased expression of other subunits [23]. Further research demonstrated that proteasomes could be rate limiting under certain conditions, particularly upon stress, and that indeed cells respond to proteasome shortage by inducing the coordinated transcription of all proteasome genes [24, 25]. To date, several transcription factors have been implicated in the control of proteasomal gene expression (reviewed in [26]). However, it is now well established that the transcription factor Nrf1 (nuclear factor erythroidderived 2—related factor 1, gene name NFE2L1), also often called TCF11, together with the related Nrf2 (NFE2L2) protein, is responsible for the coordinated induction of proteasome genes. In normal situations, if the proteasome becomes rate limiting by dysfunction, as seen upon chemical inhibition, or because the metabolic needs increase, Nrf1 is induced by a “bounce-back” mechanism [27, 28]. In brief, Nrf1 is normally bound to the ER membrane where it is actively degraded by the ubiquitin-proteasome-

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dependent ERAD (ER-associated degradation) system. When proteasomes become limiting, Nrf1 accumulates, is retrotranslocated from the ER, and N-terminally cleaved by the aspartyl protease DDI2 in the cytosol to produce the mature, active protein that migrates into the nucleus to activate proteasomal genes. Nrf2 is also involved in the cell response to proteasome inhibition, but its activation occurs mostly under stressful conditions such as starvation or oxidative stress [26, 29]. Interestingly, while concerted induction of all proteasome genes is an efficient process to increase proteasome abundance, individual overexpression of the catalytic subunits β5 and β1 is enough to do the same [30, 31]. This suggests that incorporation of these subunits is somehow limiting for overall assembly of the whole complex, and that subtle regulation of their expression could also control proteasome levels. Additionally, the individual expression level of certain other subunits could impact on the composition of the assembled 20S proteasome. It has been shown for example that the stability and thus the level of the α4 subunit is tightly regulated by the antagonizing action of the c-Abl/Arg tyrosine kinase, which stabilizes the protein, and the BRCA1 Ub-ligase, which destabilizes it [32]. Alterations in this regulation might explain how α4 is overexpressed in certain cancers [33], and might favor the assembly of alternative proteasome forms in which α4 replaces α3 [20]. Following upstream adjustments of 20S proteasome subunits expression, assembly of the complex is an ordered process. It involves different dedicated assembly chaperones that are not present in the final complex but favor efficiency of the process through association to assembly intermediates (reviewed in [34]). Assembly chaperone-dependent formation of 20S proteasomes is a process that is now relatively well understood. However, it is important to keep in mind that (1) overexpression of the assembly chaperone Ump1/POMP, which assists the incorporation of β subunits into hemiproteasomes (intermediate assembly forms of the 20S proteasome), is also sufficient to

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promote proteasome accumulation [35]; (2) a proteasome assembly chaperone specific for early embryonic development has been described in mice [36]; (3) the sequence of incorporation of β subunits is different for the immunoproteasome and the thymoproteasome, compared to the constitutive proteasome [37]; (4) a functional crosstalk between 20S proteasome assembly and DNA damage response has been demonstrated in yeast [38]; (5) 20S proteasome assembly precursors have been localized on the ER membrane in mammalian cells [39], in line with the fact that the GET/TRC pathway, which delivers proteins to the ER membrane [40], impacts on proteasome assembly [41]. Therefore, proteasome assembly appears to be a process controlled by many regulatory events whose molecular details and consequences remain to be fully understood. Once assembled, the 20S proteasome is a stable protein, with a half-life that has been initially estimated to be
5 days in HeLa cells [42] and between about 8 [43] and 12–15 days in rat liver [44]. In line with earlier studies showing that in rat liver a significant proportion of proteasomes was degraded in lysosomes [43], recent studies demonstrated that proteasomes can be degraded by autophagy, in a mechanism now called proteaphagy, following ubiquitylation of assembled proteasomal subunits [45–47]. This process is clearly seen under certain starvation conditions or after proteasome inhibition. It seems reasonable to assume that it occurs at a lower rate under physiological conditions, but this has not been demonstrated yet. While it has been shown in yeast that the 20S proteasome and its 19S regulatory complex are targeted independently to the vacuole upon nitrogen starvation [46], it remains to be determined whether the 20S proteasome is always degraded in its free form. In this respect, it is interesting to note that in yeast, the proteasome regulator Blm10 (PA200 in mammals) protects the 20S proteasome from autophagy under carbon starvation by promoting its storage in reversible PSGs (Proteasome Storage Granules) [48]. In cells, free 20S proteasome represents a significant proportion of all proteasomal forms [16, 49]. This proportion, as well as the

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proportion of each of the other forms of the proteasome system, is not the same in different cell types [16], suggesting that binding of the 20S proteasome to each of its regulators is a dynamic process that can be fine-tuned in response to specific needs. In fact, there are two welldescribed conditions, in which these proportions can rapidly and significantly change. Upon oxidative stress, the 26S proteasome dissociates into its two subcomplexes, the 20S proteasome and the 19S complex [50–54]. Concomitantly, PA28αβ is recruited on the 20S proteasome [55]. These adaptations help to get rid of oxidized proteins, the accumulation of which is highly toxic for the cells [56]. The other situation is seen upon proteasome inhibition. In this case, the level of free 20S proteasome decreases, as its association with its regulators is stabilized [57, 58]. The molecular determinants as well as the function of this phenomenon are not understood.

3.2.3

Activation of the 20S Proteasome

The best know mechanism of 20S proteasome activation is the so-called gate-opening process. When purified in conditions that preserve its integrity, the 20S proteasome is usually in a latent form with very low peptidase activities. This is due to a lattice formed by the N-terminal extremities of the α subunits that closes the pore of the complex and thus gates the entrance to the catalytic chamber [59]. The 19S, PA200, and PA28s regulators open this gate upon binding and therefore activate 20S peptidase activities. There are controversies regarding whether PI31 does the same and whether PA200 and PI31 have positive or negative roles on intracellular protein degradation [60, 61]. Interestingly, whereas activation of 20S proteasome requires insertion of the C-terminal ends of the regulators subunits into pockets located at the interface of α-subunits, different mechanisms of activation are observed. Some of the ATPase subunits of the 19S regulatory complex possess a C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is a

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tyrosine and X can vary) which is necessary for inducing 20S proteasome gate opening upon binding [62]. Remarkably, peptides corresponding to the seven last C-terminal residues of the ATPases Rpt2 and Rpt5 are sufficient to induce gate opening, as long as their HbYX motif is present [62, 63]. The insertion in the same pockets C-termini of PA28 subunits, which do not bear a HbYX motif [62], is also required to anchor the regulator on the proteasome [64, 65]. However, the interaction with the proteasome via an additional loop of PA28 subunits, called the activation loop, is necessary to provoke gate opening [66, 67]. PA200 binds to the 20S proteasome in a related but distinct manner, as it uses two HbYX motifinteracting pockets of the proteasome (one at the α5–α6 interface, using an extended HbYX motif, the other at the α1–α2 interface, using a loop whose sequence differs from the HbYX motif) [68, 69]. Besides its regulators, the 20S proteasome can be activated in vitro by a variety of molecules [70], including hydrophobic peptides [71], basic proteins such as poly-lysine [72] or histone H3 [73], poly-ADP-ribose [74] as well as low concentration of SDS [75]. Of note, utilization of this latter compound has become a recognized trick to activate latent 20S proteasome in vitro. It is therefore likely that, when present in cells as a latent protease, free of its regulators, the 20S proteasome can nevertheless degrade certain proteins. This degradative capacity seems to be particularly useful under oxidative stress, as discussed below.

3.2.4

Posttranslational Modifications (PTMs)

Thanks to both directed analyses and large-scale proteomics approaches, it is now well established that proteasome subunits are collectively heavily modified by a variety of PTMs [76], including phosphorylation, acetylation, ubiquitylation, etc. (see Table 1 in [77] for a detailed list of PTMs found on subunits of the cardiac proteasome). Each of these modifications is potentially

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affecting assembly, activity, interaction with other proteins—including its own regulators–, or the localization of the 20S proteasome, and therefore its exact impact can only be understood after thorough characterization. Unfortunately, such detailed analyses have rarely been done. Among these modifications, phosphorylation of specific subunits has been initially linked to the control of nucleo-cytoplasmic distribution of the complex [78, 79] and of its association with its regulators [80]. In line with this later notion, several data suggested a role of different kinases or phosphatases in regulating association of the 20S proteasome to its 19S regulatory complex (reviews in [81, 82]). Oxidative modifications, such as carbonylation, HNE (4-hydroxy-2nonenal) modification or glycation, are believed to participate to the decline of proteasome activity observed upon aging [83]. Finally, more recently, the previously identified general ubiquitylation of proteasome subunits [84] has been linked to the process of proteasome degradation by proteaphagy [45–47], as mentioned above.

3.2.5

Localization

Proteasomes are abundant molecules that are present in variable amounts, depending on the cell types. Whereas 20S and 26S proteasomes are present in both the nucleus and the cytoplasm, in proportions that depend on the cell types, the other forms of the proteasome family are spatially restricted, since PA28γ and PA200 are mostly nuclear whereas PA28αβ and PI31 are mostly cytoplasmic [85]. The precise intracellular localization of the 20S proteasome has been reported to be dynamic, for example during development [86] or cell cycle [87, 88], or upon specific disease states [89]. LMP2 (β1i)-containing proteasomes gradually move from the cytoplasm to the nucleus during interphase, and redistribute to both compartments during mitosis, after nuclear membrane breakdown [90]. During quiescence in yeast, proteasomes are sequestered in cytoplasmic granules called Proteasome Storage Granules (PSGs) [91]. These granules are reversible, and dissolve upon exit from quiescence.

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Importantly, proteasome recruitment in PSGs depends on its Blm10 regulator and protects them from autophagy upon carbon starvation in yeast [48]. As PSGs have been also identified in Arabidopsis [48], it is likely that similar protective proteasome reservoirs also exist in mammalian cells. Even though most of them diffuse freely and have a rather dispersed distribution within the cytoplasm and the nucleus [90], proteasomes have been identified over the years at numerous specific structures or localizations, such as ER [92], chromatin [93], or cytoskeleton [94, 95] for example. Whether they are transiently recruited to these locations by the presence of substrates or tethered by specific adaptors, including possibly their own regulators, remains presently unclear. They can be actively transported within cells to answer specific needs, as shown in growing axons where their active retrograde transport, dependent on the ECM29 protein, has been demonstrated to play an important role in axon development [96]. Recently, the proteasome regulator PI31 has also been involved in retrograde axonal transport of the proteasome, where it couples proteasome to dynein [97]. After proteasome inhibition or in other proteotoxic situations, proteasomes can be found concentrated on centrosomes. This led to the hypothesis that proteasome-dependent proteolysis might be restricted or at least enriched in specific proteolytic centers where the substrates would be transported to be degraded (reviewed in [98]). However the current view posits that it is the formation of large protein aggregates called aggresomes that is responsible for these observations [99]. Proteasomes tightly associated to the neuronal plasma membrane were recently described [100]. These proteasomes were proposed to play a role in calcium signaling, underlying a possible direct implication of proteasomes in the transfer of information between cells. It has also been proposed that proteasomes and their regulators could participate in cell-to-cell communication after their secretion, via microvesicles, exosomes, or other processes [101, 102], or as active complexes circulating in the blood [103].

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3.3

Proteasome Regulators: Structure and Regulation

As mentioned in the introduction, the proteasome family comprises distinct proteolytic complexes that differ by the regulator(s) bound to the 20S proteolytic core. While the roles and mechanisms of action of the 26S proteasome, formed by association of the 19S complex and the 20S proteasome, are well understood, those of the alternative proteasome complexes are less clear [104]. Their regulatory complexes are usually much simpler than the 19S complex and are considered as functioning in ubiquitin-independent proteasomal degradation. Phylogenetic analyses allow to trace back all the 20S regulators to LECA, the Last Eukaryotic Common Ancestor [105]. Their conservation throughout evolution shows that they undergo strong selection pressure, which in turn indicates that they all perform specific and critical functions within the intracellular proteolytic machinery. Curiously, however, whereas the 20S proteasome and the 19S regulator are essential for life, all the other regulators have been lost in certain eukaryote supergroups, suggesting that in these eukaryotes the specific function(s) they fulfill either became dispensable or have been taken over by other factors. Despite exhibiting distinct functionalities, both 19S regulators and the alternative activators PA28αβ, PA28γ, and PA200 share the properties to bind to the α-ring of the 20S proteasome and to induce opening of its axial pore through mechanisms involving their C-terminal regions (see above). This gate opening is required to allow entry of substrates into the catalytic chamber of the 20S for proteolytic cleavage [59].

3.3.1

19S Regulator and Controlled Assembly of 26S Proteasomes

The 19S regulator, which forms the 26S proteasome together with the 20S core, is at the heart of the ubiquitin–proteasome system [106]. While balancing ubiquitylation and de

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ubiquitylation of individual proteasomal substrates is key to the control of the half-life of specific proteins, regulation of the 26S proteolytic activity allows fine-tuning of overall protein degradation according to the state of the cell and emerges as a novel concept in cell biology and disease pathogenesis [2, 107, 108]. Of interest, this concept is paralleled by an emerging finetuning of ribosome form and function [109, 110]. The 19S complex is built of two subcomplexes called base and lid [111]. The base contains a ring of six ATPases as well as three additional non-ATPase subunits, which have structural and ubiquitin-binding properties. The ATPases mediate binding to the 20S core and drive ATP-dependent unfolding of substrates and conformational shifts required for 26S activation [112–115]. The lid is composed of at least nine—mainly structural—subunits but also contains a deubiquitinating enzyme (Rpn11/ PSMD14). Further ubiquitin-binding and deubiquitinating proteins such as Rpn10/ PSMD4 and USP14 might be associated to the 19S lid [111]. Several recent studies have provided unprecedented insight into the structure of the 26S proteasome in its different substrate bound, inactive and active states as well into the kinetic of substrate degradation [116–119]. The reader is referred to these publications for details while we will here focus on the cellular regulation of the mammalian 26S proteasome complexes, which can operate at several levels [120]: (1) subcellular localization, (2) abundance, (3) assembly of the 19S with 20S, or (4) activity of the 26S complex.

3.3.1.1

Subcellular Localization of 26S Proteasomes 26S proteasomes are the most abundant proteasome complexes in the cell together with free 20S [16, 22]. They are specialized in the degradation of ubiquitylated proteins, but can also degrade certain proteins without their prior ubiquitylation. 26S proteasome complexes are critical for regulated and selective intracellular proteolysis, and participate in the degradation of abnormal proteins. Due to the high abundance of proteasome complexes in the cell [121], small

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changes in 26S abundance and activity can thus have a major impact on cellular protein turnover. 26S complexes are distributed within the nucleus and cytoplasm of mammalian cells [122]. Recent reports, however, also suggest defined subcellular functions for specialized 26S complexes such as at the primary cilium [123–125] and during synaptic signaling by controlled transport into dendritic spines [126]. These localized proteasome-enriched structures resemble yeast PSGs and may serve important functions for maintaining proteostasis also in mammalian cells [91, 127]. Of note, 26S proteasomes have been shown by in situ cryoelectron tomography to be enriched in the vicinity of nuclear pore complexes in the green alga Chlamydomonas reinhardtii [128]. It is not clear however whether this feature is conserved in metazoans. Finally, mammalian 26S proteasome complexes are specifically recruited to localized stress granules upon protein stress, where they are required for the degradation of defective ribosomal products to resolve protein stress [129, 130]. They may also be present in aggresomes which correspond to heaps of aggregated proteins observed in protein misfolding diseases or upon proteasome inhibition [99, 131]. In line with this, cryo-electron tomography in intact neurons has shown that 26S proteasomes are recruited to poly-GA aggregates seen in amyotrophic lateral sclerosis and frontotemporal dementia [132].

3.3.1.2

Regulating Abundance of 26S Proteasomes Besides house-keeping gene regulation, expression of both 19S and 20S subunits is concertedly regulated via the stress-related transcription factors Nrf1 and Nrf2, as outlined above. Transcriptional activation of 26S proteasomes is observed under conditions of growth stimulation such as EGF and mTOR signaling in vitro [133, 134] and conditions of immune cell activation [135]. Transcriptional repression of 19S proteasomal subunits has been recently demonstrated in proteasome inhibitor resistant multiple myeloma, as a mechanism participating to resistance [136–138]. The recently discovered

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process of proteasome removal via autophagy, i.e., proteaphagy, has also been observed for mammalian 26S proteasome complexes under conditions of amino acid starvation as detailed above for the 20S proteasomes [47, 120, 139] and represents an additional layer of regulating the abundance of 26S proteasome complexes in the cell. The details on the involved signaling pathways are, however, still enigmatic.

3.3.1.3

Assembly and Disassembly of 26S Proteasomes Assembly of 26S proteasome complexes depends on dedicated assembly chaperones [34, 140] but also on chaperones with broader specificity such as Bag6 [41]. Regulating the expression of these assembly factors provides a means to fine-tune 26S proteasome abundance and function according to cellular needs, e.g., upon activation of the mTOR pathway [107, 141]. Of note, the assembly chaperone p28/PSMD10 has independently been identified as the oncoprotein gankyrin which regulates cyclin-dependent kinase 4 and the tumor suppressors Rb and p53, thereby promoting oncogenesis [142]. Assembly of the 26S proteasome is also fostered by the 19S lid component Rpn6 which serves as a molecular clamp to stabilize the interaction of the 19S regulator with the 20S [143]. Rpn6/PSMD11 is required for assembly of 26S complexes and maintaining high 26S activity during pluripotency in human embryonic stem cells [144]. It has been recently shown that Rpn6 is upregulated by the profibrotic cytokine TGFβ to stimulate assembly of 26S proteasome complexes which is required for differentiation of fibroblasts into fibrogenic myofibroblasts [145]. In contrast, disassembly of the 26S proteasome has been shown to occur in vitro upon cellular stress such as oxidative stress [53, 146, 147] or cigarette smoke [148]. In disease, 26S proteasome complexes have been found to disassemble in end-stage heart failure [149], in neurons of Parkinson patients [150], and in lungs of patients with chronic obstructive pulmonary disease (COPD), a devastating lung disease whose main risk factor is cigarette smoke exposure [151] (see below).

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3.3.1.4

Fine-Tuning Activity of 26S Proteasomes by Posttranslational Modifications Assembly of 20S proteasome and 19S regulators into 26S proteasomes is also regulated via posttranslational modifications. Recent phosphoproteomic screens revealed that 26S proteasome components are dynamically phosphorylated under physiological and pathological conditions as reviewed recently in detail [82]. Phosphorylation and acetylation of proteasomal subunits as well as the S-glutathiolation of 20S subunit α5/PSMA5 were reported to enhance proteasome activity, whereas glycosylation and oxidative modifications such as occurring during aging and upon oxidative stress inhibit proteasome activity [2, 81, 82, 120, 152, 153]. Rpn6/ PSMD11 has been observed to be phosphorylated by protein kinase A thereby promoting assembly and activation of 26S proteasomes [154, 155]. Moreover, phosphorylation of 19S subunit Rpt3/PSMC4 was shown to induce 26S proteasome activity by enhancing substrate translocation during cell cycle progression [156]. Tyrosine nitrosylation of Rpt4/PSMC6 has been observed upon endothelial dysfunction contributing to increased 26S proteasome assembly [157]. Binding of NADH was observed to stabilize 26S complexes [158] while O-GlcNAc modifications inhibit 26S activity [159]. 26S proteasome activity was also demonstrated to be inhibited in vitro by mutant and aggregated proteins [160, 161] which may contribute to protein misfolding diseases such as neurodegenerative disorders, hereditary lung and heart diseases [162–164]. Chemical inhibition of the 26S proteasome is most likely the cause for the observed inhibition of 26S activity in blood and tissue upon exposure to diesel exhaust and cigarette smoke, respectively [165, 166]. Taken together, these numerous studies suggest a complex regulation of 26S proteasome function on multiple layers in the cell and also reveal dysregulation of these processes in disease. In most of the reports, the evidence for 26S dysfunction is mainly descriptive and we still do not

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know the causal contribution of single regulatory circuits in disease pathogenesis. This remains to be urgently explored.

3.3.1.5

Regulation of 26S Proteasome by (Transiently) Associated Proteins In addition to the subunits invariably found in the 26S proteasome, other proteins are observed in variable substoichiometric amounts in 26S proteasome samples. It is currently unclear whether these proteins should be considered as genuine subunits loosely bound to the complex and consequently easily lost during purification, or as accessory proteins recruited to the complex only for specific tasks. Such proteins include among others the protein ECM29 and various Ub-ligases or deubiquitylating enzymes. ECM29 ECM29 is a 200 kDa monomeric protein which contains multiple heat repeats similar to PA200 [167, 168]. Yeast ECM29 is able to bind both 20S and 26S proteasome complexes and thereby regulates assembly of 26S proteasome complexes by tethering the 19S particle to the 20S core, thus preserving 26S stability, particularly upon ATP depletion in vitro [169, 170]. Accordingly, ECM29 binds to both, the phosphorylated C-terminus of the α7 subunit of the 20S core and the 19S ATPase subunit Rpt5 [171, 172]. As such, ECM29 appears to be an important factor for quality control of 26S proteasome assembly in yeast by serving as a scaffold to promote assembly of 19S regulatory and 20S core particle assembly while blocking the substrate entry channel of immaturely assembled 20S complexes [173–175]. In mammalian cells, however, the function of ECM29 is less clear. Here, ECM29 was observed to be exclusively associated with 26S proteasomes [167, 176]. ECM29 knockout mice are viable and show no abnormalities [177]. ECM29deficient mouse embryonic fibroblasts (MEF), however, are more resistant to oxidative stress, which coincided with the stabilization of 26S proteasomes [177]. In addition, mammalian ECM29 appears to serve as an adapter that

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couples 26S proteasome complexes to defined cellular compartments: ECM29 was shown to localize 26S proteasomes to centrosomes and the ER/ERGIC (endoplasmic reticulum/endoplasmic reticulum-Golgi intermediate compartment) as well as endosome compartments [167, 178]. It might thereby regulate degradation and trafficking of proteins and may also mediate autophagy as shown for TLR3 [179]. In addition, ECM29 tethers 26S proteasomes to molecular motor proteins, including dynein which was crucial for active retrograde axonal transport of proteasome complexes in neurons [96, 178]. In disease, ECM29 was found to be mutated in a mammary carcinoma patient [180] and lower levels of ECM29 have been shown to correlate with improved survival of a subset of breast cancer patients [181]. Similarly, ECM29 was observed to be regulated by the microRNA-221/222 cluster whose downregulation correlated with increased levels of ECM29 and progression of prostate cancer patients [182]. Ub-Ligases, DUBs Associated to the 26S Proteasome Depending on the source and the purification procedure, numerous proteasome-interacting proteins (PIP) have been described over time. Among them, Ub-ligases and deubiquitylating enzymes (DUB) have been observed, suggesting that some Ub-ligases have a role in the targeting of their substrates to the proteasome and that, more generally, additional regulations of the ubiquitylation status of the substrates occur at the level of the proteasome itself [169, 183– 187]. Accordingly, dynamic remodeling of Ub-chains on the proteasome by the tightly associated Ub-ligase Hul5 and the DUB Ubp6 (USP14 in mammals) has been shown to regulate substrate degradation in Saccharomyces cerevisiae [188]. Drug targeting of the recruitment or the activity of these various PIPs, and particularly of the DUBs most tightly bound to the proteasome opens promising routes for therapeutic development [189]. For example, USP14 inhibition has been shown to enhance proteasome activity and resistance to oxidative stress by

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accelerating the proteins [190].

3.3.2

degradation

of

oxidized

PA28/REG

The PA28 (also called 11S or REG) proteasome regulators are formed in higher eukaryotes by three related proteins (PA28α, β, and γ) that associate in two distinct complexes [104]. PA28α and PA28β form a cytoplasmic heteroheptamer, PA28αβ, made of four PA28α and three PA28β subunits [191, 192], which participates in the immune response by favoring the generation of MHC class I antigens [193, 194]. By contrast, PA28γ forms a homoheptameric complex made of seven PA28γ subunits, which localizes in the nucleus and plays important roles in the control of cell proliferation [195]. Phylogenetic analyses show that PA28γ is the closest isoform from the common PA28 ancestor already present in LECA, and that PA28α and β appeared much more recently in evolution, through two sequential duplications contemporary to the apparition of the MHC peptide-based T cell recognition system [105]. Remarkably, some eukaryote supergroups have lost PA28 complexes [105]. The mechanisms of action of PA28 complexes remain elusive. When expressed, similarly to all proteasome regulators, PA28 complexes can bind to each ends of the 20S proteasome. This interaction opens the pores of the proteasome and activates its peptidase activities in vitro [196, 197]. Recent results suggest that their binding to the 20S proteasome α-rings might be asymmetrical [198]. Artificial peptide substrates commonly used to measure proteasome activities are believed to passively diffuse through the 20S pores, and therefore PA28s’ stimulation of their degradation can largely be explained by 20S-pore opening. However, the exact roles of these regulators in the degradation of protein substrates are less clear. Indeed, how PA28 complexes recruit protein substrates and deliver them to the proteasome is not understood, as these complexes are a priori inert molecules that, unlike the 19S complex, do not possess any ATPase activity that

could provide energy and movement to unfold the substrates and inject them into the 20S proteasome. A likely hypothesis is that PA28 substrates are essentially unstructured proteins, being either intrinsically disordered or unfolded by chaperones or by deleterious modifications. In line with this hypothesis, PA28αβ has been shown to collaborate with HSP90 and other chaperones [199, 200] and both PA28αβ and PA28γ enhance degradation of oxidized proteins by the 20S proteasome [55]. Recently, transposable short-charged sequences enriched in basic and flexible amino acids have been shown to efficiently promote proteasomal degradation mediated by both PA28αβ and PA28γ [201]. Of note, if PA28s can form so-called one- or two-capped proteasome complexes, in which the 20S proteasome is bound by one or two PA28 complex(es), they are also found in hybrid proteasomes in which the 20S proteasome is bound by one PA28 on one side and one 19S complex on the other side [49, 58, 202]. Although they are considered as functioning in ATP- and Ub-independent protein degradation by the proteasome, this suggests that they might nonetheless be also involved in the degradation of ubiquitylated substrates. Quantitative analyses have shown that a large proportion of PA28 complexes are not bound to the 20S proteasome [49, 58]. However, they can be largely recruited on 20S proteasome in certain conditions, as demonstrated upon proteasome inhibition [57, 58]. This suggests that PA28-20S proteasome interaction is probably quite dynamic, and that cells possess a reservoir of “free” PA28 complexes that can be mobilized and recruited on proteasomes to fulfill specific needs. Despite their similarity, PA28αβ and PA28γ seem to play very different roles in cells, as described below. These functional differences are most likely due in large part by the so-called homolog-specific inserts that characterize each PA28 isoform [203]. These inserts are disordered and not seen in the actual crystal structures of PA28, but most likely are located near the entrance pore of PA28s and therefore might act

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as gatekeepers that control specificity toward substrates and/or interactors [204].

3.3.2.1 PA28ab PA28αβ has been mainly and thoroughly analyzed for its functions in MHC class I antigen production [194, 205]. The fact that its expression is induced by IFNγ, similar to the inducible β-subunits of the immunoproteasome, indeed prompted many studies on its contribution to the processing and subsequent presentation of individual epitopes. The results led to the conclusion that PA28αβ is not a general “optimizer” of antigen presentation, since, while it favors the generation by the proteasome of many antigens, it impairs production of others [206]. Of note, whereas it is not induced by IFNγ, PA28γ also mediates the processing of specific antigens [207]. Despite numerous analyses, the exact role (s) of PA28αβ in antigen presentation still remains obscure, knowing that in any case its absence does not impair overall MHC class I response in vivo [208]. In vitro studies have shown that the effect of PA28αβ on the nature of the peptides produced by the proteasome is not mainly related to alterations in cleavage specificity of the protease, but rather that it influences the production of a subset of the peptides already produced by the proteasome alone [208]. In addition, while PA28αβ tends to reduce the overall size of the peptides, it increases the hydrophilicity of those that have a size compatible for antigen presentation [209]. One possible interpretation to rationalize the complex literature on that matter is that PA28αβ acts in fact as a molecular sieve controlling peptide efflux from the proteasome, globally favoring release of certain peptides while retaining other peptides and favoring their further degradation into smaller species [194]. Depending on the MHC haplotype [210], PA28αβ could therefore help the production of peptides having strong affinity for MHC class I molecules [194, 208]. The roles of PA28αβ and the proteasome in MHC class I antigen presentation certainly explain why many viruses have developed strategies to temper with their activities. As far

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as PA28αβ is concerned, it has been shown for example that HIV1 Tat [211] and the HBV HBX [212] can compete with PA28αβ for the binding to 20S proteasome, and that viruses such as Epstein-Barr virus can interfere with PA28αβ expression [213]. Aside of its functions in antigen presentation, PA28αβ, and to a lesser extent PA28γ, seem to be important effectors in the degradation of abnormal and particularly oxidized proteins [55] (see below). Of note, the protein 14-3-3ζ binds to PA28αβ and limits its interaction with the 20S proteasome, and favors myeloma cell growth and sensitivity to proteasome inhibitors [214].

3.3.2.2 PA28g To date, the functions and mechanisms of action of PA28γ remain largely elusive. As a regulator of the proteasome, and possibly independently of the proteasome in some cases, it is involved in the control of many nuclear processes, but the underlying mechanisms are often not understood. Furthermore, as mentioned above, although its ancestor was already present in the first eukaryotes [105], suggesting that it is an important actor of cell homeostasis, it has been lost in certain eukaryotic supergroups and is not essential for survival when expressed, as illustrated for instance by the fact that PA28γ/ mice are viable and fertile [215]. To explain the apparent contradiction between its very high conservation during evolution, which suggests strong selective pressure to maintain its functions, and its seemingly facultative character, a likely hypothesis is that the role of PA28γ is particularly critical under stress. In line with this hypothesis, PA28γ has been shown to be important (1) for the response to oxidative stress [216, 217], possibly in part by directly participating to the elimination of oxidized proteins [55], (2) for the response to proteasome stress, since it is massively recruited on proteasomes after nontoxic treatment of cells with proteasome inhibitors [58], suggesting that it might participate to a cellular rescue process when proteasome activity is impaired, and (3) for the response to genotoxic stress, as it has been shown to be an ATM target recruited at DNA double-strand break (DSB) sites, and to be

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required for the rapid accumulation of the 20S proteasome at these sites as well as for the coordination of DSB repair pathways [218]. This probable general role in stress response is consistent with the fact that PA28γ/ mice suffer from premature aging [219]. Nevertheless, PA28γ functions do not seem to be restricted to stress. Several observations show that PA28γ plays a central role in intranuclear dynamics through the regulation of (1) nuclear bodies (including nuclear speckles [220], Cajal [221] and PML [222] bodies), (2) nuclear trafficking of splicing factors [220], and (3) chromatin compaction [223] or integrity [218, 224]. PA28γ/ mice show growth retardation after birth, indicating that PA28γ functions as a regulator of cell proliferation [215]. Consistent with this phenotype, and although only a limited number of proteins whose degradation can be regulated by this complex have been described [104], it is noteworthy that many of them, including cell cycle inhibitors such as p21Cip1, p16INK4A, and p19Arf [225–227], the oncogene SRC-3 [228], the critical mitotic checkpoint protein securin/PTTG [229, 230], or the tumor suppressor p53 [231], are involved in the control of cell proliferation. Importantly, most of these proteins have been shown to be also degraded by other pathways, particularly Ub-dependent proteasomal proteolysis, illustrating the fact that PA28γ impacts on their turnover only under certain circumstances or cellular contexts. The effect of PA28γ on a particular substrate might be itself cell-context dependent, as indicated by apparently conflicting results describing a positive [232] and a negative [233] effect of PA28γ on c-Myc stability. Interestingly, the effect of PA28γ on p53 degradation is not direct, but through enhancing its ubiquitylation by the Ub-ligase MDM2 [231], suggesting that similar indirect effects on stability could be true for other of its substrates. PA28γ further regulates the Mdm2/ p53 loop through the degradation of Casein Kinase 1 delta (CK1δ) [219]. Overall, even if the mechanistic details are not understood, its various functions seem to converge toward conferring on PA28γ proliferation-promoting and antiapoptotic properties favoring tumorigenesis,

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as illustrated by the fact that PA28γ-/- mice show increased resistance to induced skin carcinogenesis [234], that PA28γ at high level can inhibit caspase activity [235]—even though it can be a caspase substrate at low level [235, 236]—and that PA28γ is found overexpressed in many cancers and often associated with poor prognosis [237–240]. PA28γ expression and functions are regulated at many levels. Transcription of its gene has been shown to be regulated by AP-1 [234], p53 [241, 242] (note that in this case contradictory results have been obtained, probably due to differential status of other signaling pathways, see below), and NF-κB [243], and several transcripts variants have been described [244]. Three microRNAs, miR-195-5p, miR-7 and miR-7-5p, all identified as tumor suppressors, target PA28γ [245–248]. PA28γ is also subjected to PTMs, including phosphorylation by ATM [218], Chk2 [249] and MEKK3 [250], SUMOylation and acetylation. SUMOylation is induced upon coxsackievirus infection [251] and provokes the cytoplasmic translocation of PA28γ to the cytoplasm [252]. Acetylation promotes assembly of PA28γ heptamers in mammalian cells [253]. Finally, PA28γ associates in cells with its tightly bound partner PIP30 (Fam192A) [254]. PIP30 favors association of PA28γ to the 20S proteasome, but inhibits its interaction with certain cellular proteins, as shown for the Cajal body marker protein coilin. In vitro, PIP30 alters PA28γ activating effects on peptide degradation by the 20S proteasome [254]. Over the years, PA28γ has been shown to be involved in the regulation of many signaling pathways. In inflammatory diseases such as bowel disorders [243] or testicular inflammation [255], PA28γ participates in NF-κB activation by mediating the degradation of the inhibitor IκBε. As NF-κB positively regulates both IκBε and PA28γ expression, these pathologies could be explained at least in part by the loss of control of the reciprocal positive-feedback regulation between PA28γ and NF-κB. Another reciprocal feed forward control between PA28γ and NF-κB, this time through PA28γ-mediated destabilization of the KLF2 negative transcriptional regulator of

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NF-κB, has been described upon bacterial infection [256]. However, upon Staphyloccus aureus infection, PA28γ rather inhibits NF-κB [257]. PA28γ has also been shown to control Hippo/Yap signaling through the degradation of Lats1 [258] and of the kinase CK1ε [259]. As mentioned above, PA28γ enhances p53 degradation by facilitating its interaction to its Ub-ligase Mdm2 [231]. It has been proposed that this effect is mediated by increased mono-ubiquitylation dependent nuclear export of p53 [260]. Since as mentioned above p53 modulates PA28γ expression, there is a reciprocal regulation between the two proteins that allows balancing their levels. However, this regulation is complex and probably cell-context dependent since the results suggest that when p53 is overexpressed [242] or mutated [241] it activates transcription of PA28γ gene, whereas upon TGF-β signaling p53 cooperates with SMAD3 to inhibit PA28γ expression [241]. Interestingly, this PA28γ/p53 regulatory loop has been demonstrated to be targeted in different ways by viruses for their own benefit [251, 261, 262], and different results suggest that its alteration could contribute to cancers [241, 263] or to aging [219]. Another important signaling pathway regulated by PA28γ is the Wnt/β-catenin pathway, which is critically controlled by the kinase GSK3. PA28γ mediates the degradation of GSK3-β [234, 264, 265], thereby blocking the phosphorylation- and ubiquitylationdependent degradation of β-catenin and allowing it to accumulate, translocate in the nucleus, and activate its target genes. Downstream of GSK3, PA28γ stimulates the degradation of MafA, a critical regulator of insulin expression [266]. In anaplastic thyroid cancer, PA28γ promotes cell dedifferentiation by degrading Smad7 and thereby activating TGF-β signaling [267]. Finally, PA28γ has been shown to control angiogenic VEGF signaling by mediating degradation of the catalytic α-subunit of PKA, and therefore preserving FoxO1 transcriptional activity [268]. The same PA28γ/PKA/FoxO1 axis controls expression of cyclophilin A in endothelial cells, an observation that strengthen the hypothesis of a possible role of PA28γ in atherogenesis [269]. Of note, PA28γ may also play a

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role in atherosclerosis, by mediating the CKIP-1dependent degradation of the transcription factor Oct-1 [270]. PA28γ has been shown to have inhibitory activity toward autophagy. Two mechanisms have been described for this. One is through the degradation of the deacetylase SirT1 [271], which plays a positive role in autophagy induction. Of note, SirT1 can de-acetylate PA28γ on lysine 195, a residue acetylated by CBP [253]. The other is through the degradation of LC3-I, a critical effector of autophagy, in which PA28γ cooperates with the antiapoptotic IAP Bruce/Apollon [272], a chimeric E2–E3 Ub-ligase [273]. To promote their infection cycle and/or camouflage themselves from cellular and organism defenses, including immune response, many viruses use the proteasome system and within it PA28γ. For example, the accessory protein p30 of HTLV-1 and HTLV-2 recruits both PA28γ and its cofactor PIP30 (NIP30, Fam192A) [274] to maintain latency [275]. PA28γ has been shown to bind to the multifunctional hepatitis C virus (HCV) core protein and to regulate its nuclear retention and stability [276]. This interaction promotes liver pathologies induced by HCV, including steatogenesis and hepatocarcinogenesis [277], as well as insulin resistance [278].

3.3.3

PA200

The proteasome activator 200 (PA200) was first described by Ustrell et al. [279] as a 200 kDa, monomeric, nuclear proteasome activator that promotes degradation of peptides in an ATPand ubiquitin-independent manner. These early in vitro experiments with PA200 purified from bovine testis indicated that PA200 activates all catalytic sites but mainly stimulates the caspaselike activity of the 20S proteasome [279]. A recent study that reconstituted human PA200 and 20S proteasomes in insect cells and resolved the cryo-EM structure of PA200-proteasome complexes at high resolution, however, demonstrated activation of the trypsin-like activity which might be explained by an increased

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accessibility of the β2 substrate-binding pocket upon PA200 binding [69]. These conflicting results need to be solved in future research. PA200 and its yeast homologue Blm10 bind both to 20S but can also be found as hybrid proteasomes together with the 19S regulator [68, 279, 280]. PA200 is highly conserved among mammals and also found in Saccharomyces cerevisiae, Caenorhabditis elegans, and Arabidopsis thaliana, but not in Archae or Drosophila melanogaster [105, 279]. Sequence homology of the human PA200 and its yeast homologue Blm10 is limited with only 17% amino acid similarity. Both proteins are composed of multiple and variable HEAT repeats providing structural preservation [105, 168, 281]. They also share a conserved motif at the C-terminal region, which conjointly with another domain anchors PA200 to the 20S core particle and induces its gate opening [69, 282]. Crystal structure and cryo-electron microscopy of Blm10 and PA200 show that the activator adopts a dome-like structure that caps the 20S core particle [69, 282, 283]. The recently resolved high-resolution structure of reconstituted human PA200-20S proteasome complexes shows that binding of PA200 to the α-subunits of the 20S induces opening of the 20S gate and allosteric regulation of proteasome catalytic sites [69]. However, the pore of the 20S proteasome is largely obstructed by PA200, indicating that only linear peptides but not fully folded proteins are able to enter the 20S proteasome upon PA200 binding [69]. The cryo-EM structure of human PA200 also shows two positively charged grooves/channels at the top of PA200, bound by negatively charged inositol phosphates, the cellular relevance or function of which remains to be determined [69]. Despite structural similarity of mammalian PA200 and yeast Blm10, they appear to have distinct cellular functions. Blm10 regulates proteasome maturation and assembly [284, 285] and mitochondrial and genomic integrity [286, 287]. In mammals, PA200 is ubiquitously expressed in different organs but it is most abundant in testis [279], where it participates to degradation of acetylated histones [19]. There, PA200 is present in proteasome complexes that

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contain the testis-specific α4s subunit. The functional relevance of this interaction, however, is unknown [19]. PA200 preferentially binds to standard 20S proteasomes and not to immunoproteasomes, which accords well with the observation that its expression is not inducible by INFγ [16, 280]. PA200/ mice are viable and do not exhibit developmental abnormalities except from a decrease in male fertility which might be due to defective degradation of acetylated histones by PA200/proteasome complexes [19, 288, 289]. The role of PA200 in DNA repair is controversial: PA200-containing hybrid proteasomes were reported to accumulate on chromatin upon ionizing radiation and PA200 silencing decreased cell survival upon the treatment [279, 290]. In contrast, PA200-deficient embryonic stem cells did not show an increased sensitivity to irradiation-induced DNA damage [288]. PA200 has been suggested to function in glutamine homeostasis due to its possible enhancing effect on the post-glutamyl peptidase activity of the proteasome [291]. Of note, PA200 was recently claimed to be a positive regulator of proteasome activity in a study that reported downregulation of PA200 by miR-29b [292]. Currently, this notion has also been put forward by others who proposed that PA200 might serve as a proteasomal adaptor protein recruiting proteasome complexes to defined cellular compartments [60, 66, 293]. The function of PA200 in mammalian cells thus remains still enigmatic and its dysregulation in disease has not been described so far.

3.3.4

PI31

PI31 (proteasome inhibitor of molecular weight of 31 kDa) is the least understood proteasomal regulator. While initial in vitro studies described PI31 as an inhibitor of proteasome activity, this has been controversially discussed in recent studies [61, 294–296]. PI31 is prominently expressed in mammalian cells on the cytosolic side of the nuclear envelope and ER membrane but interacts with the proteasome only to a minor extent [16, 61, 295]. The cellular function of PI31 is

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currently unknown. While homozygous PSMF1 (gene name for PI31) knockout mice are embryonically lethal (http://www.mousephenotype.org/ data/genes/MGI:1346072), silencing or overexpression of PI31 in mammalian cells did not affect cell growth or morphology [61, 295]. Recent studies in yeast suggest a role for PI31 in assembly of defective 20S proteasomes [297]. In accordance, Zaiss et al. reported that overexpression of PI31 impaired assembly of immunoproteasomes and interfered with the generation of immunoproteasomedependent MHC I epitopes upon induction by IFNγ [295]. However, as endogenously expressed PI31 preferentially complexes with standard proteasomes and only to a minor extent with immunoproteasomes, a regulatory role for PI31 in MHC I antigen presentation is questionable [22]. A recent study suggested that TLR2mediated phosphorylation of PI31 positively regulates MHC class I peptide loading and immunoproteasome stability at endoplasmic reticulum exit sites [298]. Other studies reported that protein PI31 forms a dimer and interacts with human Fbxo7, a component of the SCF-type E3 ubiquitin ligase, and with the E3 ubiquitin ligase Nutcracker in Drosophila, but its exact function remains unknown [296, 299]. Regulation of PI31 by ADP-ribosylation is still controversial [61, 300] but recent data suggest a role for PI31 as an adaptor for axonal transport of proteasomes in Drosophila [97], in which it participates to the regulation of longevity and locomotor ability [301]. Only circumstantial evidence links PI31 dysfunction to disease, e.g., SNP variations with onset of Alzheimer’s disease in an Arab-Israeli community [302], and hypermethylated promotor regions in children with high risk of B cell acute lymphoblastic leukemia [303].

3.4

Proteasomes in Diseased States

In this chapter, we will give an overview on the different forms of proteasomal dysfunction ranging from gene mutations to aberrant expression of proteasome complexes, proteasome regulators, or

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single subunits in disease. Much knowledge on proteasome regulation in specific diseases has already been summarized in excellent reviews. Therefore, we here mainly focus on the different levels of proteasome dysregulation such as mutations, expression, assembly, and disassembly of 20S complexes with their regulators under pathological conditions and in disease.

3.4.1

Mutations Affecting 20S Proteasome Function

As the 20S proteasome is an essential cellular component, mutations impairing its functions are rather scarce and usually do not completely shut down all its activities. Nevertheless, over time a certain number of such mutations have been identified, several of them leading to pathologies [304]. A specific group of such genetic diseases is called Proteasome-Associated Autoinflammatory Syndromes (PRAAS). These correspond to extremely rare pathologies, with only few tens of cases reported so far [305]. It is of importance to know that until recently PRAAS were described as separate conditions under distinct names, some of which are still in use: Nakajo– Nishimura syndrome [306]; joint contractures, muscle atrophy, microcytic anemia and panniculitis-induced lipodystrophy (JMP) syndrome [307]; Japanese autoinflammatory syndrome with lipodystrophy (JASL) [308]; chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome [309]. All those syndromes were originally described as autosomal recessive genetic disorders with mutations in PSMB8 gene encoding immunoproteasome subunit β5i/LMP7. However, new cases of CANDLE patients lacking PSMB8 mutations have been reported [305], suggesting that the cause of the disease may be more complex than originally anticipated. Indeed it has later been shown that CANDLE patients can carry mutations in genes encoding other proteasome subunits (such as PSMA3, PSMB4, PSMB9), or the Ump1/POMP chaperone [310]. As a result, the term PRAAS was

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forged, now referring collectively to autoinflammatory syndromes caused by genetic alterations that decrease proteasome levels and/or activity. Although there are differences in clinical manifestations and severity of the disease between PRAAS patients, which have been described in detail by clinicians over the past years [311, 312], there are symptoms that remain common. Initial signs of inflammation are observed in new-borns and infants, including recurring fever and pernio-like nodular rash on the skin infiltrated by immune cells—neutrophils and monocytes [313]. As the disease progresses with age the symptoms of severe inflammation become more apparent and burdensome, among which the most striking are progressive loss of muscular and fat tissues, arthritis and joint contractures, enlargement of the liver, and central nervous system inflammation (basal ganglia calcification). Patients are also reported to have increased levels of pro-inflammatory cytokines and acute-phase reactants, e.g., IL-6, TNF, and C-reactive protein. It has also been shown that inflammation is driven by increased expression of interferon-stimulated genes caused by activation of type I interferon response [305, 310], and consequently PRAAS are now classified as a new group of interferonopathies [314]. Given the severity of those symptoms, the prognosis for PRAAS patients is poor because in most cases chronic systemic inflammation leads to organ failure or cardiac arrest at young age, or causes strong developmental defects [312]. Unfortunately treatment of PRAAS still remains challenging since known anti-inflammatory therapies relieve only part of the symptoms or are not effective at all [315]. However, in a recent clinical trial the JAK1/2 inhibitor baricitinib, inhibiting the Janus kinases downstream of the type I interferon receptor, was used for CANDLE patients and has been shown to reduce disease symptoms and improve the quality of life of the patients [316, 317]. Over the past decade various mutations causing PRAAS were described, leading to the definition of four subclasses, named PRAAS1PRAAS4, respectively, to better reflect the

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heterogeneity of these syndromes. The first one, PRAAS1 (MIM#256040), involves patients carrying mutations in PSMB8 gene. It includes cases with homozygous mutations, among which the best characterized are G201V [306] and T75M [307], as well as compound heterozygous ones [318]. The list of PSMB8 mutations is continuously increasing and most probably the newest Nakajo–Nishimura syndrome cases will be included in this group as well [319, 320]. PRAAS1 patients can also have digenic heterozygous mutations, meaning that only one allele of PSMB8 is affected. So far, the known cases carry the second mutation in genes encoding standard 20S proteasome subunits— PSMA3 encoding α7 and PSMB4 encoding β7 [310]. PRAAS3 (MIM#617591) groups disorders caused by monogenic or digenic heterozygous mutations in other proteasome and immunoproteasome genes. Currently, the described cases include patients with double heterozygous mutations in immunoproteasome gene PSMB9 encoding β1i/LMP2 and PSMB4 encoding subunit β7, as well as a case of compound heterozygous mutations in PSMB4 gene [310]. In contrast to the syndromes described above, genes affected in PRAAS2 and PRAAS4 encode proteasome assembly chaperones. PRAAS2 (MIM#618048) patients carry dominant compound heterozygous frameshift mutations in POMP that lead to generation of unstable or truncated, nonfunctional proteins. Interestingly, this haploinsufficiency of POMP is sufficient to trigger strong systemic inflammation in those patients, highlighting how important this chaperone is for proteasome function and, by extension, human health [310, 321]. Finally, the most recently reported patient with compound heterozygous mutations in PSMG2 gene encoding PAC2 has been classified as the newest PRAAS4, which has not yet been assigned a MIM number [322]. The primary consequence of PRAAS mutations is decreased activity of proteasomes because majority are loss-of-function mutations that can either affect directly proteolytic activity of the catalytic subunits, maturation and stability

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of mutated proteins, or incorporation efficiency into new complexes. Unquestionably, mutations in proteasome chaperones directly decrease proteasome levels by impairing 20S assembly, which was also suggested for PSMB4 mutations. Moreover, mutations in PSMB4 and PSMA3 have been shown to impair interaction of 20S core with PA28αβ regulator [310]. Ultimately, the deficiency of proteasome activity also leads to accumulation of ubiquitylated and oxidized proteins [310, 321, 323]. Interestingly, carrier parents and siblings do not show symptoms of PRAAS and appear clinically healthy. To elucidate this contradiction it was proposed that due to digenic pattern of inheritance in PRAAS the patients not only have decreased probability of assembling non-mutated 20S complexes, but they can also assemble a complex with three or four impaired subunits making them more predisposed to have higher quantities of non-functional proteasomes [310]. It still remains unclear how proteasome deficiency and autoinflammation are linked together. Interestingly, decreased proteolytic activity in sterile conditions in vitro, achieved by silencing of proteasome genes or treatment with proteasome inhibitors, is sufficient to induce expression of type I interferon stimulated genes [310, 324]. However, this may not explain correctly the systemic, chronic inflammation observed in PRAAS and therefore a more probable model of pathogenesis has been proposed. Briefly, the decreased proteolytic activity of the proteasome impairs ability of patients’ cells to resolve immune response after common childhood infections because they are unable to remove the accumulated ubiquitylated, misfolded and foreign proteins. Consequently, the signaling pathways involved in innate immune responses remain activated and continue to induce expression of pro-inflammatory cytokines and interferons. In parallel, the increasing proteotoxic stress causes activation of unfolded protein response, leading to activation of inflammasome pathway, and increases reactive oxygen species (ROS) production, which altogether aggravate the inflammatory state in cells [325]. This model is supported by the fact that ER-stress and the

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unfolded protein response are strongly activated in patients with heterozygous truncation mutations in POMP [321]. An alternative model of PRAAS proposes a superior role of mitochondrial ROS in activation of innate immune responses, supported by experiments using pluripotent stem cells generated from Nakajo– Nishimura syndrome patients’ samples [326, 327]. However, the exact mechanisms by which all PRAAS mutations result in severe systemic inflammation require to be further elucidated using more appropriate cellular and animal models. Finally, it is important to mention a mutation of POMP that causes an autosomal recessive genetic disorder known as Keratosis Linearis with Ichthyosis Congenita and Sclerosing Keratoderma (KLICK) [328], a proteasomerelated syndrome not classified as PRAAS. The mutation in KLICK syndrome only results in formation of skin lesions, characterized by thickening and dryness of the epidermis with linear papules on flexural parts of the body. The onset of the disease has been also reported in early childhood like in PRAAS, but no systemic health issues have been described so far. The patients have a deletion in the 50 UTR of the POMP gene, which causes a switch in POMP transcription that favors synthesis of a negative regulatory transcript instead of the protein-coding one. Still, it is unclear how this mutation affects protein expression of POMP, but nevertheless, the malfunctioning of POMP transcription does affect keratinocyte differentiation and processing of filaggrin, a key component of epidermis, as shown by in vitro studies using reconstituted human epidermis [329]. In addition to these 20S proteasome diseaserelated mutations, a mutation of the 19S subunit Rpn5 was recently described in patients with hereditary neurodevelopmental disorder [330]. Several small nucleotide polymorphisms (SNPs) have been identified in the promoters, introns, and coding regions of different proteasomal genes. Some of the identified allelic variations result in codon and thus amino acid change. For a summary of the identified SNPs and their potential functional relevance, the

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reader is referred to an excellent overview given in table 2 of Gomes et al. [304]. Not much is known regarding allelic variations of proteasome regulators, only SNP variations of PI31 have been linked to the onset of Alzheimer’s disease in an Arab-Israeli community [302]. The evidence for a prominent association of proteasomal gene variation with human disease is generally rather scarce and dependent on distinct populations with no coherent picture emerging for a functionally relevant association of proteasomal SNPs with human diseases.

3.4.2

Proteasomes and Oxidative Stress

Conditions of oxidative stress pose a particular challenge to cell homeostasis and contribute to various acute and chronic diseases in all organs. Oxidative stress is mainly caused by ROS, which are constantly produced in the cell as by-products of respiratory chain function and are highly damaging for biological macromolecules. Despite robust cellular anti-oxidative systems, ROS cause different types of damage, some of them irreversible, to cellular proteins [331]. Beside the possible alteration of their function or regulation triggered by these modifications, oxidized proteins are often unfolded and represent an additional risk for the cell due to their tendency to aggregate. There is thus a need to eliminate irreversibly oxidized proteins, a cellular process in which the proteasome plays a key role when the insult is not too strong, even though autophagy is also involved, particularly when insoluble aggregates appear [332, 333]. Oxidative damage is a dose- and timedependent process with regard to protein structure, solubility, and degradation [331, 334]: mildly oxidized proteins tend to partially unfold and are usually good substrates for the 20S proteasome, while the most heavily oxidized proteins eventually end up in cross-linked aggregates of denatured, insoluble proteins that are not susceptible to proteasome degradation but are toxic for the cells. While the 26S proteasome

can also degrade oxidized proteins, the 20S proteasome, possibly associated to some of its ATP-independent regulators [55], seems to be the proteasome species best fitted to degrade slightly or mildly oxidized proteins, most likely because their partial unfolding allows the complex to efficiently capture them. Accordingly, the cellular antioxidant defense system involves the Nrf2-dependent transcriptional activation of 20S gene expression levels, as mentioned above, allowing the cells to eventually prevent accumulation of oxidized proteins [335]. However, oxidative stress by inhibition of the respiratory chain using rotenone was shown to be compensated by upregulation of 26S proteasomes via the Nrf1dependent pathway [324]. Oxidative stress has been shown to impact the assembly and activity of proteasome complexes [336]. Different processes leading to 20S activation after oxidative damage have been described which might act as an adaptive response to cope with the degradation of oxidatively modified proteins. One is the rapid activation of the nuclear 20S proteasome by a PARP-1-dependent process [74, 337]. In addition, binding of DJ1, a sensor of cellular redox homeostasis, fine-tunes 20S mediated protein degradation in conditions of oxidative stress [338]. Another mechanism is to liberate 20S proteasomes by dissociating the 26S proteasome [53, 146, 147]. It remains unclear whether these two mechanisms work synergistically or in parallel pathways. Disassembly of 26S complexes may involve the chaperone HSP70 [51] or oxidative stress-induced posttranslational modifications which destabilize the interaction between 20S and 19S regulators [147, 148]. In addition, ECM29 has been shown to compete with other proteasome regulators for binding to the 20S and thereby facilitate 26S disassembly, as outlined above. Association of ECM29 with the proteasome is regulated by stress such as toxins, ethanol, and oxidative stress [50, 167, 176] suggesting that mammalian ECM29 is indeed involved in the regulation of the stability of 26S complexes in response to oxidative stress [50, 54]. Both PA28αβ and PA28γ are important for the degradation of oxidatively modified proteins

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[55]. The impact of PA28αβ on this process is well documented [339, 340], and its induction is required for cellular adaptation to this stress [341]. Assembly of PA28αβ-containing complexes may thus provide an alternative or additional means to adjust proteasome function in order to cope with the removal of potentially proteotoxic proteins [342]. Accordingly, proofof-concept experiments based on overexpression of PA28α show that increasing PA28αβ activity is a valuable strategy to enhance degradation of oxidized proteins and thereby protect cells against oxidative stress in cardiomyopathies [342] as well as in inherited retinal degenerations [343]. There is less detailed information regarding the involvement of PA28γ. Nevertheless, PA28γ has been shown to target oxidized hemoglobin and hemoglobin δ subunit [344], and to be critical for the oxidative-stress induced degradation of p21 and of HCV core protein [216]. Supporting a role of PA28γ in oxidative stress response, male mice double-knockout for PA28γ and PA200 are completely infertile, most likely due to a defective response to oxidative damage [217]. In addition, oxidative stress may activate gene expression and assembly of immunoproteasomes to cope with the degradation of oxidatively modified proteins [345, 346]. As immunoproteasomal subunits are not activated by the antioxidant master regulator Nrf2 [347], the signaling pathway may involve inflammatory stimuli such as proposed for the activation of the immunoproteasome via IFNγ [345] or upon ischemia/reperfusion injuries [348]. As the proteasome is also a protein, it is subject of deleterious oxidation [336, 349]. Therefore, if oxidative stress is too strong or too long, there is an increasing imbalance between the cellular rate of degradation of damaged proteins, due to the oxidation-induced decline of proteasome activity, and the rate of formation of damaged proteins. If the cell is unable to adapt to this imbalance, e.g., through increase of proteasome levels as outlined above, a vicious circle takes hold as oxidized proteins start to unfold and form small aggregates (or oligomers) that are either degraded by the proteasome or by chaperone-mediated autophagy, or even

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condense into larger aggregates normally degraded by macroautophagy [332, 350, 351]. However, as some oligomers strongly impair proteasome function [352], there is a gradual decrease of proteasome activity contributing to the vicious cycle which further promotes aggregate formation and eventually overwhelms the autophagy system and deeply perturbs proteostasis. This may further exhaust proteasome degradative properties for reasons that are not entirely clarified [332, 353]. Additionally, the proteasome may also be directly modified and inhibited by environmental chemicals such as cigarette smoke components or diesel exhaust [165, 166], as mentioned above.

3.4.3

Cancer

In cancer, proteasome function is essential to ensure ongoing proliferation and survival. Ubiquitin-mediated turnover of key proteins involved in cell-cycle progression, such as the cyclins, p27 and p53, and in cell survival pathways, such as the NF-κB pathway, is essential for the hyperplastic growth of tumor cells and their resistance to apoptosis and senescence [354]. This observation initially led to the successful development of several types of catalytic proteasome inhibitors, as outlined below, which effectively block tumor cell growth [4, 355]. The important contribution of proteasome in tumorigenesis is also supported by the finding that inhibition of POMP expression, which is the major 20S assembly factor as outlined above, effectively suppressed cancer cell growth and in a tumor mouse model [356]. POMP-1 mRNA expression is negatively associated with breast cancer survival. Micro-RNA 101 was identified as a major regulator of POMP expression and might function as an endogenous inhibitor of proteasome function. Only recently, however, the oncogenic addiction of tumor cells to high 26S proteasome expression was demonstrated [357] and the molecular pathway that activates proteasomal gene expression in tumor cells was identified. Mutant p53 cooperates with Nrf2 to upregulate proteasomal gene

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transcription making p53 mutant tumor cells more resistant to proteasome inhibition [358]. Of note, cancer stem cells appear to have reduced proteasome expression and activity (reviewed in [359]) which is associated with a lower proliferative capacity of these cells. Thereby, cancer stem cells might escape therapeutic approaches that aim at the inhibition of tumor cell growth. In accordance with this notion, it was recently observed that proteasome inhibitor resistant multiple myeloma cells have reduced levels of 19S proteasome subunits [136, 137] suggesting that diminished proteasome expression and activity is an evolutionary beneficial strategy to develop resistance to chemotherapeutic proteasome inhibitors. As these proteasomeresistant cells have a unique metabolic profile, novel concepts of overcoming drug resistance are currently emerging [360]. Quite in contrast to standard proteasome expression, increased expression of immunoproteasomal genes correlates with improved survival of lung tumor patients [361]. This is most likely due to the beneficial role of immunoproteasomes in MHC I antigen presentation where high levels of immunoproteasomes might allow more effective processing of tumor antigens to raise tumorspecific CD8+ T cell responses [361]. With regard to proteasome activators in carcinogenesis, there is a strong correlation between PA28γ and cancer, thanks to its proliferationpromoting and antiapoptotic properties. These properties are resulting from its multiple roles (outlined above) in response to DNA damage [218, 249], in apoptosis inhibition [235, 362, 363], and in the control of critical signaling pathways such as NF-κB, β-catenin and p53 (see above). An additional function of PA28γ that is beneficial to tumor cells is to increase cell resistance to energy shortage through the degradation of SirT7 [364]. Regarding PA28αβ, aside of its roles in the processing of specific antigens relevant for the immune response against cancer cells [365], there is little information concerning its connections with cancer. However, its roles in oxidative stress response are most likely an important asset used by cancer cells. Accordingly, PA28αβ has been found overexpressed in

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different types of cancers [366–368]. For more details on the distinct proteasome subtypes and complexes and their differential regulation in cancer, the reader is referred to a recent comprehensive summary by Morozov and Karpov [369].

3.4.4

Muscle Atrophy and Hypertrophy

Pathological muscle atrophy occurs rapidly under conditions of denervation but also as a systemic condition in aging (sarcopenia), cancer (cancerassociated cachexia), organ failure, sepsis, burns and trauma, in chronic lung diseases and diabetes [370]. Loss of muscle mass is due to excessive protein degradation without compensatory protein synthesis. Concerted transcriptional activation of 20S and 19S proteasomal gene expression has been observed early on in various conditions of skeletal muscle breakdown such as acidosis, cachexia, and sepsis [371–374]. Proteasomal genes are thus part of a category of atrophy related genes—so called atrogenes—which also include several E3 Ub-ligases required for ubiquitin-mediated breakdown of contractile proteins, as well as autophagy-related genes. Activation of the UPS appears to be a universal feature of conditions of muscle protein breakdown and is also observed in diaphragm muscle atrophy in acute and chronic lung diseases [375]. For further details on the dysregulation of the UPS in muscle wasting diseases, the reader is referred to a recent review [370]. Of interest, activation of the proteasome system has also been observed under the opposite condition of muscle atrophy, i.e., hypertrophic muscle growth. While on first sight puzzling, this activation in proteasomal degradative capacity might be explained by the need for an increased protein turnover to compensate the increased rate of protein synthesis under conditions of hypertrophic growth [376]. Activation of mTOR signaling, a key signaling pathway for hypertrophic cell growth, may result in Nrf1mediated transcriptional activation of proteasome gene expression [377]. Accordingly, proteasome expression and activity are upregulated in cardiac

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hypertrophy (reviewed in [378]). Recent data also indicate activation of 26S proteasome assembly by protein kinase A mediated phosphorylation of the 19S lid-factor Rpn6 (see above) in skeletal muscles of exercising humans, and in electrically stimulated rat muscles [155]. Transcriptional upregulation of Rpn6 may also be responsible for the observed increased assembly of 26S proteasome complexes in experimental right heart hypertrophy [379]. Aging Aging is a complex biological process involving multiple cellular pathways including imbalanced proteostasis [380, 381]. There is a strong link between aging and oxidative stress, as aging is characterized by a life-long accumulation of abnormal (mostly oxidized) proteins that escaped degradation, and an age-dependent decline of proteasome activity [331, 332, 382]. Notably, a study comparing oocytes from young versus aged mice clearly showed that proteasome activity is lower in the older cells, and negatively correlates with the accumulation of oxidatively induced protein damage [349]. The protective action of proteasomes against aging is suggested by many observations, including that artificially decreasing proteasome activity in mice by constitutive expression of the β5t subunit of the proteasome, which has lower chymotrypsin-like activity than β5, results in the development of age-related phenotypes, accumulation of oxidized proteins, and shortened life span [383]. Indeed, it has been shown in diverse mammalian tissues and cells that proteasome expression is reduced in older cells or individuals resulting in diminished proteasome function (reviewed in [384]). In contrast, expression of the immunoproteasomal subunits has been shown to be elevated in aging mammalian tissues such as brain [385], muscle [386], and the lung [387]. Moreover, elevated expression of immunoproteasome in fibroblast was positively correlated with organismal aging across different species [388]. Dysregulation of proteasome activators in mammalian aging has been observed as well. It has been noted in rats that there is no change in PA28αβ content in aged muscle compared to young muscle. However,

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since there is more 20S proteasome in aged muscle, there is in fact a marked drop in the ratio PA28αβ/20S proteasome that may participate to the weakening of aged muscle [386]. PA28γ knockout mice show signs of premature aging, indicating that PA28γ functions to protect the organism against the deleterious effects of age [219]. Deficiency of PA200 does not seem to have an effect on mouse aging [288]. Vice versa, overexpression of 20S proteasome subunit β5 in whole organisms such as Caenorhabditis elegans [389] and Drosophila melanogaster [390] resulted in extended life span. In addition, it has been shown that increasing proteasome content and activity by overexpression of β5 [31], or assembly chaperone Ump1/POMP [35] enhances resistance to oxidative stress and delays senescence. In line with this observation, tissue from long lived animals such as the naked mole-rat have higher expression of immuno- and 26S proteasomes [391] and fibroblasts from human healthy centenarians have augmented proteasome activity compared to controls [392]. There is thus a solid array of presumptions that increasing proteasome activity could favor healthy aging [393], even though it is clear that many other pathways are at play in such a complex process.

3.4.5

Neurodegenerative Disorders

Neurodegeneration can be viewed as a pathologic form of brain aging [394]. Indeed, despite distinct and complex etiologies, neurodegenerative diseases such as Alzheimer (AD), Parkinson (PD) or Huntington (HD) diseases share proteinopathic phenotypes at later disease stages with large proteinaceous deposits in certain parts of the brain [395, 396]. These deposits are aggregates enriched in specific proteins, the nature of which permits diagnosis at autopsy (for example α-synuclein in PD, Aβ and tau in AD, and mutated (with polyQ expansion) huntingtin in HD). In most cases, these aggregates also contain protein chaperones and ubiquitin. There have been controversies

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regarding whether or not these large deposits are the cause of neurodegenerative pathologies. The present consensus is that it is rather the metastable forms and/or the small aggregates of the proteins that are toxic and not the large aggregates/ deposits, which could in fact represent a protective response by sequestration of toxic metastable proteins from the other cellular proteins [395]. In addition, aberrant function of the proteasome appears to contribute to defective clearance of proteotoxic proteins in various neurodegenerative diseases [397]. In mouse models with neuronal deletion of single 19S subunits, progressive neurodegeneration was observed, thus confirming the importance of balanced 26S proteasome function for maintaining neuronal health [398]. Of note, oligomeric proteins have been demonstrated to directly impair proteasome function thereby contributing to the gradual decrease of proteasome activity that drives the vicious cycle of protein aggregation and proteostasis collapse [332]. α-Synuclein, Aβ and mutated huntingtin apparently share a three-dimensional structure that is able to bind and inhibit the proteasome [352]. In addition, it was demonstrated that tau directly associates with and inhibits the activity of 26S proteasome in mice with tauopathy [399]. Activation of PKA signaling using the drug rollipram attenuated proteasome dysfunction, reduced aggregated tau levels and improved cognitive performance in these mice [399]. These effects might be due to the activation of 26S assembly via phosphorylation of Rpn6 [154]. A different level of proteasomal dysregulation in neurodegenerative disorders might involve the induction of immunoproteasomes possibly driven by elevated immune cell infiltration of the brain and/or glia cell activation. As inhibition of the immunoproteasome or deficiency of immunoproteasomal subunits, however, did not alter neurodegenerative defects in experimental mouse models, this might speak against the involvement of immunoproteasome function in the onset of neurodegenerative disorders (reviewed in [400]). 20S proteasome activators are also actively mobilized to degrade proteins aggregated or en route for aggregation. For example, PA28αβ is recruited into the protein aggregates made by a

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pathologic form of huntingtin [401]. However, the amount of PA28αβ in inclusion bodies depends on the cell type [402]. Overexpression of PA28γ improved survival in neuronal model cells [403] and the overall clinical tableau in a Huntington disease mouse model [404] while reduction of PA28γ levels had no beneficial effect in mice [405]. In Parkinson’s disease, neuronal cells with low levels of 19S complex and PA28αβ are the more susceptible to neurodegeneration [406].

3.4.6

Cardiovascular Disorders

Similar to neurodegenerative diseases, the proteasome has been found to be dysregulated in several heart diseases, namely cardiac hypertrophy, heart failure, and myocardial ischemia. Cardiac hypertrophy develops upon increased workload of the heart, e.g., upon arterial or pulmonary hypertension, and can be regarded as an adaptive response to meet the increased demand of cardiac function. The hypertrophic growth response of cardiomyocytes is characterized by an increased protein turnover involving both protein synthesis and degradation. Accordingly, proteasome expression and activity were found to be increased in experimental models of left and right cardiac ventricle hypertrophy and in hearts of patients with dilated cardiomyopathy ([379], reviewed in [407, 408]). In dilated cardiomyopathy, proteasome composition was investigated in detail and numerous studies revealed posttranslational modifications such as phosphorylations of 20S subunits which might add to altered 20S function or assembly with proteasome regulators [379, 409]. In contrast, heart failure and myocardial infarction are characterized by loss of proteasome function, which might be related to severe oxidative stress under these conditions, as outlined above. Experimental inhibition of proteasome activity by application of proteasome inhibitors attenuated both left and right heart cardiac hypertrophy but promoted cardiac failure [378]. Mild genetic inhibition of the proteasome promoted heart failure as well, demonstrating the crucial role of balanced

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proteasome activity for proper cardiac function [410]. The formation of amyloid-like abnormal protein aggregates in idiopathic or ischemic cardiomyopathies suggests that these proteotoxic oligomers, e.g., αB-crystallin, inhibit the proteasome and contribute to the development of heart diseases similar as described above for neurodegenerative disorders (reviewed in [378, 411]). Induction of immunoproteasome subunits and altered composition of 20S proteasomes have also been described for various heart diseases (reviewed in [407]). Only recently, the immunoproteasome subunit β5i/LMP7 was shown to regulate cardiac hypertrophy in experimentally induced hypertrophy of transgenic mice [412]: cardiomyocyte-specific deletion of β5i/ LMP7 attenuated, while its overexpression aggravated, experimental cardiac hypertrophy. In this model, β5i/LMP7 was found to interact and promote degradation of the autophagy-related gene ATG5. Similarly, β5i/LMP7 regulates activation of the angiotensin receptor, a major signaling molecule involved in cardiac arrhythmia, by promoting degradation of its negative regulator ATRAP [413]. These data identify the immunoproteasome as a putative therapeutic target for cardiac hypertrophy and cardiac arrhythmia. In contrast, immunoproteasome activity is required for effective virus clearance at conditions of virus-induced myocarditis (reviewed in [414]). In vascular diseases, proteasome activity and expression was also found to be dysregulated. Impaired activity of the proteasome has been mainly attributed to the increased levels of oxidative stress and inflammatory signaling, as outlined above. Therapeutic targeting of the proteasome has been proposed for these diseases but strongly depends on the stage and extent of atherosclerotic lesion formation (reviewed in [415]). The activity of the immunoproteasome seems to be unrelated to atherosclerotic plaque formation in experimental mouse models [416]. The specific role of proteasome regulators has not been investigated in detail in cardiovascular diseases. However, increased assembly of 26S proteasomes has been recently demonstrated in experimental right heart hypertrophy which is possibly mediated by

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transcriptional activation of the 19S subunit Rpn6. Rpn6 was also found to be overexpressed in human hearts of patients with right ventricle hypertrophy [379]. Regarding PA28s, it has been shown that PA28αβ expression is beneficial in cardiac proteinopathies in mice [417] and that PA28γ, by mediating CKIP-1-dependent degradation of the transcription factor Oct-1, plays an anti-atherosclerotic role [270].

3.4.7

Respiratory Diseases

The lung is particularly exposed to environmental challenges such as cigarette or wood smoke, particles and chemicals, which contribute to protein damage and misfolding as well as cause severe oxidative stress. Life-long or acute exposure to hazardous substances leads to chronic lung diseases such as lung cancer, chronic obstructive lung diseases (COPD), asbestosis, and idiopathic pulmonary fibrosis (IPF) (https:// www.erswhitebook.org/). There is accumulating although sparse evidence for dysregulation of the proteasome system in several of these diseases [2]. Some hereditary lung diseases, such as cystic fibrosis, hereditary surfactant protein C (SP-C) deficiency in IPF, and alpha1 anti-trypsin (α1AT) deficiency in familial forms of COPD, can be regarded as diseases of protein homeostasis where mutant or aberrantly folded proteins disturb the proteostasis network and causally contribute to disease [162]. For mutant SP-C it was shown that this protein inhibits proteasome activity similar to the abovementioned oligomeric proteins in neuronal and heart diseases [418]. In sporadic forms of COPD, proteasome activity is downregulated while expression of 20S and 19S proteasome subunits and also of proteasome regulators was not altered [151]. Inhibition of proteasome activity was observed both for standard and immunoproteasomes in the lung. Native gel analysis revealed impaired assembly of 26S proteasomes, which may reflect disassembly of 26S proteasome complexes upon severe oxidative stress as outlined above. These findings are supported by the observation that cigarette smoke inhibits proteasome activity both in vitro

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and in acutely cigarette smoke-exposed mice and destabilizes 26S proteasome complexes [148, 151, 166, 419]. The crucial role of the proteasome to cope with oxidatively modified proteins and to maintain proteostasis in response to cigarette smoke exposure, was also demonstrated in a transgenic mouse model with an overexpressed β5t subunit. These mice show slightly reduced chymotrypsin-like activities and are more susceptible to cigarette smoke-induced lung damage [420]. In contrast, proteasome activity is increased in experimental models of pulmonary fibrosis [145]. Augmented proteasome function was mainly due to increased assembly of the 26S proteasome, which was—at least in vitro—driven by TGFβ-mediated induction of the 19S subunit Rpn6. Rpn6-induced 26S proteasome assembly was required for differentiation of primary human fibroblasts into myofibroblasts which are key drivers of fibrotic tissue remodeling. Elevated levels of Rpn6 were also detected in lung tissue of patients with IPF supporting the idea that increased assembly of 26S proteasome is a pathogenic feature of fibrotic organ remodeling. The role of other proteasome activators and the immunoproteasome in fibrotic lung diseases still remains to be investigated. Of interest, conditions of acute lung injury result in the release of active 20S proteasome complexes into the extracellular environment, possibly via exosomal secretion [102, 421–423]. The function of these active extracellular 20S proteasome complexes, however, is not solved yet, but they might contribute to immune regulation as proposed recently for allograft rejection [424].

3.4.8

Involvement of Individual Proteasome Subunits in Disease

Over the years, many results have suggested the involvement of specific 20S and 19S proteasome subunits in pathological processes (see for example [33, 412, 425–427]). It is often unclear in these studies whether these roles are due to subunits integrated into the proteasome or not. Assuming that functional subunits are indeed integrated, such results suggest that within the

complex individual subunits might link the proteasome, possibly through direct interactions, to specific substrates or pathways. Alternatively, if they are not integrated into the complex, then these “orphan subunits” could compete with the proteasome by binding to potential substrates and therefore alter the regulation of certain pathways. Alternatively, they might sequester rate limiting proteasome subunits and thereby interfere with assembly of 20S proteasome complexes. Interestingly, some of the 19S regulator assembly chaperones (RACs, as introduced above) have been found dysregulated in several types of cancer suggesting an oncogenic role in tumor growth and metastasis. One example is gankyrin or p28, whose upregulation associates with poor prognosis in numerous tumors [428]. In any case, targeting these specific interactions could be of therapeutic interest for highly selective interventions in certain disorders.

3.5

Modulation of Proteasome Activity in Human Therapy

The many levels of regulation of proteasome activity highlighted above show that there are potentially numerous entry points to modulate proteasome activities in order to correct pathologies. Nevertheless, to date, the only approach adopted in the clinics has been direct chemical targeting of the 20S proteasome active sites. In the meantime, several research programs are pursued in view of identification of proteasome activators, which could be used in situations where proteasome activity is limiting.

3.5.1 3.5.1.1

Proteasome Inhibition

Development of Proteasome Inhibitors Since almost three decades, a large, still ongoing effort has been made to develop proteasome inhibitors (PI) targeting the active sites of the 20S proteasome [429–431]. Covalent inhibitors, which can bind reversibly (e.g., MG132), quasiirreversibly (e.g., bortezomib) or irreversibly

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(e.g., epoxomicin), generally consist of two pharmacophores: a peptide scaffold and an electrophilic anchor, which can be a boronate, an α,β0 -epoxyketone, an aldehyde or an β-lactone to only name a few examples [430, 432]. Reversible inhibitors were described to have less side- and off-target effects than irreversible inhibitors that induce sustained proteasome inhibition and exhibit a negative pharmacodynamic profile [430]. All proteolytic subunits bind to covalent inhibitors via a common mechanism involving the nucleophilic attack by their Thr1 hydroxyl group to the inhibitor, analogously to the nucleophilic attack of peptides for degradation. Of note, both the composition of side chains and the reactive group contribute to the substrate specificity of the inhibitor [433]. Besides covalent inhibitors, different classes of molecules interacting with the proteasome catalytic subunits in a non-covalently fashion have been generated, such as cyclic or noncyclic peptides [432]. Interestingly, covalent and irreversible PIs have also been modified with a reporter tag, usually a fluorophore, to design activity-based probes (ABPs) allowing the monitoring of active proteasome complexes in cells and tissues [434, 435]. The development of PIs has largely influenced proteasome research and also cancer therapy, as discussed below [436, 437]. In 2003, bortezomib (BZ) (Velcade®) was approved by the United States Food and Drug Administration (FDA) as the first 20S PI for third-line treatment of relapsed and refractory mantle cell lymphoma and later also as a front-line treatment of newly diagnosed multiple myeloma patients. In 2012, the secondgeneration inhibitor carfilzomib (Kyprolis®) was approved for the treatment of multiple myeloma exhibiting reduced side effects compared to the previously approved BZ [438]. The third PI currently used in the clinics is ixazomib (IXZ, Ninlaro®), approved in 2015 as the first oral PI drug. Three other PIs are presently in clinical trials: oprozomib (OPZ, ONX-0912, PR-047), delanzomib (CEP-18770) and marizomib (NPI-0052, salinosporamide A) [437]. Most of the first-generation PIs were targeting primarily the chymotrypsin-like activity of the proteasome, but were also hitting the other sites

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and, to a much lesser degree, other cellular proteases. In recent years, many efforts were aimed at developing inhibitors with stricter specificity toward unique sites of the proteasome, in order to increase clinical efficiency while reducing deleterious side effects [432]. Indeed, specific inhibition of distinct active sites of the proteasome may affect turnover of different substrates to different extents [439]. An additional step toward defined specificity is the development of inhibitors targeting preferentially the immunoproteasome, which has been identified as a potential target in autoimmune or neurodegenerative diseases, inflammation, and certain cancers [440]. Two compounds, the β1i selective IPSI-001 and the β5i selective ONX-0914 (formerly PR-957) are at an advanced stage of clinical drug development. In mouse models, ONX-0914 prevented experimental colitis and colitis-associated cancer, lupus- and rheumatoid arthritis-like disease, Hashimoto’s thyroiditis, acute myocarditis, microglial activation following central nervous system injury and allograft rejection, without apparent toxicity (reviewed in [441–443]). More recently, DPLG3, a non-covalent β5i-specific inhibitor, and LU-005i, a pan-immunoproteasome inhibitor that targets all three active subunits, have shown therapeutic efficacy in immune diseases in mice [444, 445]. Because the immunoproteasome is highly expressed in immune cells, immunoproteasome-specific inhibitors selectively affect the function of activated immune cells while sparing other cell types. Although these studies are at the preclinical stage, they hold great promise for the treatment of immune diseases [18]. KZR-616, an ONX-0914 derivative is presently being tested in a study in patients with Systemic Lupus Erythematosus (ClinicalTrials. gov). Finally, another exciting development is the design of species-specific PIs. Indeed, there is a huge interest in developing PIs able to target the proteasome of deadly pathogens with no or little impact on the activity of the human proteasome [446]. Despite the high conservation of the active sites, important progress has been made in this area with compounds showing high preference

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over human proteasome for the proteasome of Mycobacterium tuberculosis [447–449], and of the parasites Plasmodium falciparum [450, 451], Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei [452].

3.5.1.2

Proteasome Inhibitors in the Clinic In the clinic, the main application of proteasome inhibitors has been the exploitation of their anticancer properties [436], as they efficiently kill tumor cells that most of the time highly depend on proteasomal protein degradation [432]. For bioavailability and pharmacological reasons, the main application of bortezomib, the first-in-class clinically approved selective proteasome inhibitor, has been for the treatment of hematologic malignancies [453]. Alone or in combination with other drugs, it has proven to be highly efficient against multiple myeloma and mantle cell lymphoma, and is also evaluated against other pathologies. Despite its great success, both negative side effects and development of resistance limit its efficacy. To variable extent, the same is true for the other inhibitors that have entered the clinic. Side effects, which are manageable in most cases, include peripheral neuropathy, thrombocytopenia, gastrointestinal disturbances, fatigue, or cardiovascular complications [438, 454– 456]. But the major problem is that many patients become resistant to the treatment. Although the resistance mechanisms are multiple [457, 458], direct changes at the level of the proteasome, such as overexpression or mutation of the catalytic site of its β5 subunit (β5 is the main target of bortezomib) [459], or overexpression of POMP/ Ump1 and Nrf2 [460] have been described. Recent studies also unraveled defective expression of 19S subunits resulting in diminished formation of 26S proteasome complexes as an additional underlying mechanism of cancer cell resistance to PI [136]. Most recently, it was observed that resistance to PIs is associated with distinct mitochondrial metabolic states, which can be targeted to overcome PI resistance [360].

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3.5.2

Activation of the Proteasome in Human Therapy

There is presently a large effort to isolate proteasome-activating molecules that could be used in vivo to treat pathologies where proteasome activity is limiting, such as aging or many neurodegenerative diseases [5, 70]. Conceptually, such drugs could act at many levels, including expression and assembly of the 20S proteasome, direct activation of free, latent 20S proteasome, enhancement of its association to its regulators, modulation of the properties/activities of the regulators. There are already results showing that indeed many routes exist to enhance proteasome activities, and that depending on the exact target, each approach can impact differentially different sets of proteasome substrates. For example, inhibition of the 26S proteasome-associated DUB USP14 has been shown to increase the degradation rate of ubiquitylated substrates [190]; conversely, the small molecule TCH-165, which both activates the free 20S proteasome and promotes its accumulation in cells by disassembly of the 26S proteasome, enhances the degradation of intrinsically disordered proteins (IDPs) but not of folded proteins or protein domains [461]; similarly, different molecules have been identified through screenings that can stimulate α-synuclein or IDPs turnover in cells [462–464]; inhibitors of the p38 MAPK lead to a global activation of the catalytic sites of the proteasome in cells, through a mechanism that remains obscure, which does not destabilize cellular proteostasis but allows a more efficient degradation of genuine proteasome targets such as ubiquitylated proteins or aggregating, toxic α-synuclein [465]; finally, activation of protein kinase A attenuated tau-driven 26S proteasome impairment and cognitive dysfunction in an experimental model of degenerative neuropathy [399]. Interestingly, many natural products have been described to enhance intracellular proteolysis, sometimes acting positively on both the ubiquitin–proteasome and the autophagy–lysosome pathways [466]. In line with this notion,

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Mediterranean diet, which is rich in nutrients beneficial for proteasome functions [467], is known to favor healthy aging.

3.6

Conclusion

As a central actor of cell homeostasis, the proteasome system is involved in the control of most if not all cellular processes, and is therefore a nodal point at the crossroad between health and diseases. An obvious conclusion is that it represents a very promising target for therapies aiming at modulating the intracellular levels of specific proteins to treat diseases. This rationale justified a crucial and ongoing worldwide effort to develop proteasome inhibitors with increasing specificity and bioavailability. However, since most of these drugs target the proteolytic sites of the 20S catalytic core complex, there is no specificity with regard to the different proteasome complexes as all proteasomal forms are inhibited, leading to broad cellular effects and limiting the use of these molecules due to their toxicity. As far as proteasome inhibition is concerned, one direction of research is therefore to develop drugs targeting only subpopulations of the proteasome complexes, for example drugs hitting proteasomes bearing specific catalytic subunits or directed against a specific regulator of the 20S proteasome. Such strategies are currently being developed [190, 440, 468] and one can expect important progress in the future. However, this will require a deeper understanding of the exact roles and mechanisms of action of each subpopulation of the proteasome system. As illustrated in this review, our current knowledge of the specific impact of the 20S proteasome regulators on health and diseases is still limited, except perhaps for the 19S complex. It is therefore important to develop new research programs to better understand the biological roles of each proteasome regulator and to better apprehend the interest of their targeting in human pathology. Acknowledgments The authors wish to thank Prof. Elke Krüger (Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald,

83 Germany) for her critical reading of the manuscript and helpful comments. They also acknowledge the support of the COST program, Action Proteostasis BM1307.

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4

Proteostasis Dysregulation in Pancreatic Cancer Leena Arpalahti, Caj Haglund, and Carina I. Holmberg

Abstract

The most common form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), has a dismal 5-year survival rate of less than 5%. Radical surgical resection, in combination with adjuvant chemotherapy, provides the best option for long-term patient survival. However, only approximately 20% of patients are resectable at the time of diagnosis, due to locally advanced or metastatic disease. There is an urgent need for the identification of new, specific, and more sensitive biomarkers for diagnosis, prognosis, and prediction to improve the treatment options for pancreatic cancer patients. Dysregulation of proteostasis is linked to many pathophysiological conditions, including various types of cancer. In this review, we report on findings relating to the main cellular protein degradation systems, the ubiquitin–proteasome system (UPS) and autophagy, in pancreatic cancer. The L. Arpalahti · C. I. Holmberg (*) Medicum, Department of Biochemistry and Developmental Biology, University of Helsinki, Helsinki, Finland e-mail: leena.arpalahti@helsinki.fi; carina.holmberg@helsinki.fi C. Haglund Research Programs Unit, Translational Cancer Medicine Program, University of Helsinki, Helsinki, Finland Department of Surgery, Helsinki University Hospital, University of Helsinki, Helsinki, Finland e-mail: caj.haglund@hus.fi

expression of several components of the proteolytic network, including E3 ubiquitinligases and deubiquitinating enzymes, are dysregulated in PDAC, which accounts for approximately 90% of all pancreatic malignancies. In the future, a deeper understanding of the emerging role of proteostasis in pancreatic cancer has the potential to provide clinically relevant biomarkers and new strategies for combinatorial therapeutic options to better help treat the patients. Keywords

Proteostasis · Pancreatic cancer · Pancreatic ductal adenocarcinoma · Biomarkers · Ubiquitin-mediated proteolysis · Proteasome · E3 ubiquitin-ligase · Deubiquitinating enzyme · Autophagy

4.1

Introduction

Pancreatic cancer is globally the seventh leading cause of cancer-related mortality, with an increasing incidence in the Western world and an overall 5-year survival rate of 9% in the USA [1, 2]. For exocrine pancreatic ductal adenocarcinoma (PDAC), the prevalent form (90% of all cases) of pancreatic cancer, the survival rate is only 5% [3–5]. Regrettably, the underlying causes of this complex and lethal disease still remain to be fully resolved. One of the main paths to malignant neoplastic transformation involves progressively

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_4

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accumulating genetic mutations [6]. Additionally, major risk factors for developing pancreatic cancer include smoking, diabetes, obesity, and chronic pancreatitis [7–11]. Radical surgery is the standard operation of care and can improve 5-year survival to 20–25% [12–14]. Unfortunately, only 10–20% of all patients are electable for surgery [15, 16], as the lack of obvious initial symptoms and effective early screening methods contribute to a generally late diagnosis. Adjuvant chemotherapy is commonly used in combination with surgery, increasing 5-year survival to almost 30% [17]. However, taking into account the frequently high therapy resistance, successful treatment of pancreatic cancer is challenging [18]. Both the incidence and disease outcome vary widely, depending on the cell type of origin [6]. PDAC usually originates in the head of the pancreas and metastasizes to nearby lymph nodes, liver, lungs, spleen, and the peritoneal cavity [19]. PDAC tumors are considered to develop from microscopic precursor lesions, known as pancreatic intraepithelial neoplasias (PanIN) [20, 21]. The lesions progress in a stepwise and histologically defined manner (graded from I to III) toward malignant transformation, amassing increasing genetic alterations on the way [6]. The estimated timeframe for pancreatic cancer development is 15 years from initial mutation to a metastatic disease [22].

4.1.1

High Genetic Variability in PDAC

Pancreatic cancer is a variable disease, proven difficult to categorize beyond its morphology [3]. Histopathology alone is insufficient for making informed treatment decisions in patient care [3], as tumors with matching morphologies do not necessarily possess a similar molecular composition. Large stromal contribution, in comparison to the amount of actual cancer cells, interferes with primary tumor sequencing [23], and defining accurate molecular signatures corresponding with pancreatic cancer types is

Table 4.1 Common altered genes in PDAC Genes KRAS RB/CDKN2a/INK4A TP53/p19ARF TGFβ/SMAD4 BRCA1 BRCA2 PALB2 MLH1 MSH2 MSH6 PMS2

References [25–27] [25–27] [25–27] [25–27] [28] [28] [28] [29] [29] [29] [29]

problematic. Therefore, a clinically applicable molecular subtyping system for PDAC does not currently exist (as reviewed in [3]). High-grade PanINs often have many of the same genetic alterations as PDAC itself [24]. Extensive genetic instability, pervasive mutations, and chromosomal translocations are typical for PDAC tumors (commonly altered genes summarized in Table 4.1) [30], presenting as excessive inter- and intratumoral molecular heterogeneity [6, 31]. Interestingly, only 10% of patients have a family history of pancreatic cancer [32–34], yet germline mutations in oncogenes and tumor suppressors are present unexpectedly often also in the case of a sporadic PDAC [35, 36]. Familial mutations include BRCA1 or BRCA2, PALB2 [28] and alterations in the DNA mismatch repair genes (e.g., MLH1, MSH2, MSH6, and PMS2), which all increase the risk of developing pancreatic cancer [29]. Almost all PDAC tumors harbor mutations in KRAS and RB/CDKN2a/INK4A (over 90%), and a high mutation rate of TP53/p19ARF (75%) and TGFβ/SMAD4 (55%) is also common [25– 27]. Constitutively active oncogenic KRAS activates major signaling pathways, including the Raf/MEK/ERK, PI3K/PTEN/AKT, and Ral guanine nucleotide exchange factor cascades that regulate, e.g., cell survival and growth [23, 37–40]. Large-scale genomic studies report approximately 60 affected genes converging on 12 fundamental signaling pathways [27, 41], reflecting the high genetic variability of pancreatic tumors.

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Proteostasis Dysregulation in Pancreatic Cancer

4.1.2

PDAC Tumor Microenvironment

Pancreatic cancers have an unusually large (up to 80% of the tumor volume) stromal component, compared to most other cancer types [42]. The tumor microenvironment (TME) supports increased proliferation, immune evasion, and metastasis [43]. Stromal constituents include, but are not limited to, endothelial cells, infiltrating immune cells, and cancer-associated fibroblasts (CAFs) [44]. Pancreatic cancer stem cells (PCSCs) comprise a small subset of cells present in the TME [43]. PCSCs are particularly important for cancer initiation and progression [45], as well as in playing a role in metastasis, therapy resistance, and cancer recurrence [46, 47]. Desmoplasia, extensive fibrosis of the stroma, is a prevalent condition in PDAC. It induces hypoxia due to poor tumor vascularization [48], often combined with increased acidosis of the TME [49]. Stromal fibrosis also results, e.g., in increased hydrostatic pressure and decreased nutrient availability [49]. These events in turn promote cancer aggressiveness, cause changes in metabolic pathways, decrease apoptosis, and increase proliferation [50]. Rampant persisting pancreatic inflammation promotes immunosuppression, thereby stimulating PDAC development and tumor growth [51]. The high level of inflammation enhances metastasis via the induction of epithelial–mesenchymal transition (EMT), cell dissemination, and invasion [52]. Both cancer and stromal cells secrete immunosuppressive cytokines to induce desmoplasia and to repress tumor immune detection [53–56]. In turn, desmoplasia has been proposed to invite intense leukocytic infiltration of, e.g., inhibitory immune regulatory cells to the stroma, to further facilitate PDAC immune evasion [57, 58].

4.1.3

PDAC Biomarkers

Despite the large body of research studies, only a few clinically relevant biomarkers exist for PDAC. Carbohydrate antigen 19-9 (CA 19-9),

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which is a sialylated Lewis blood group antigen, is the only US Food and Drug Administration (FDA) approved biomarker currently in clinical use (reviewed in [25]). High levels of this biomarker are linked to poor prognosis [59]. The clinical use of CA 19-9 is limited, due to sensitivity and specificity problems [60], and because approximately 5–10% of the Caucasian population do not express CA 19-9 [59]. Of resectable PDAC patients, only 65% exhibit elevated CA 19-9 levels [61, 62]. Consequently, CA 19-9 is not applicable as an early screening marker for PDAC [63]. Combining CA 19-9 with other biomarkers derived from a variety of sources (including saliva, serum, urine, stool, or pancreatic juice) could improve its reliability remarkably [12]. A recent review by Ansari et al. [25] includes a comprehensive list of potential biomarkers for the clinic. As the therapies in current use, especially those for patients suffering from therapy-resistant and relapsed cancer, are often ineffective ([64] and references therein), development of innovative new therapeutic strategies is critical. The rest of this review focuses on the emerging roles of proteostasis pathways in PDAC, and their potential relevance in the detection and treatment of this fatal disease.

4.2

Intracellular Protein Degradation Systems

The maintenance of cellular protein homeostasis (proteostasis) is a balancing act between protein translation and degradation. In all cells, protein degradation is accomplished predominantly by two parallel and interconnecting systems: the ubiquitin–proteasome system (UPS) and the autophagy–lysosome system. In the UPS, three classes of enzymes, i.e., E1 ubiquitin-activators, E2 ubiquitin-conjugators and E3 ubiquitinligases, work sequentially to covalently attach ubiquitin (Ub) to substrate proteins and mark them for degradation [65]. In human cells, the substrate specificity of the UPS is conveyed by approximately 650 different E3 ubiquitin-ligases [66]. Around 100 deubiquitinating enzymes or

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deubiquitinases (DUBs) in turn remove ubiquitin from the substrates [67, 68]. Polyubiquitinated proteins are recognized and degraded by the 26S proteasome, an essential multisubunit enzymatic complex present in all eukaryotic cells [69]. The main form of the proteasome, 26S, is composed of a 20S core particle, containing the proteolytic activity, and one or two 19S regulatory particle (s), involved in, e.g., substrate recognition, unfolding, and translocation into the 20S proteolytic chamber [69]. UPS participates in virtually all cellular processes, including protein quality control, DNA repair, apoptosis, signal transduction, cell-cycle control, transcription and production of antigens for the major histocompatibility complex I (MHC-I) [70, 71]. UPS dysregulation is linked to multiple often aging-related disorders, such as neurodegenerative diseases, inflammation, and various types of cancer [70, 72]. Degradation by the autophagy–lysosome system targets defective organelles, such as damaged mitochondria (a process called mitophagy), and protein aggregates or individual proteins. The autophagy–lysosome system is further divided into macroautophagy (hereafter termed autophagy), microautophagy (pinocytosis), and chaperone-mediated autophagy. In autophagy, a double-membraned autophagosome forms to sequester the cytoplasmic component destined for degradation, in a selective or nonselective manner [73, 74]. The other autophagic systems use partially different mechanisms in substrate acquisition, but all converge on the lysosome for substrate degradation [75]. Basal levels of autophagy are detectable in almost all cells and play an important role in maintaining proteostasis and reducing cytotoxicity [23]. Under normal conditions, autophagy is also required for anticancer immunosurveillance and the inhibition of malignant neoplastic transformation [76].

4.3

UPS Dysregulation in PDAC

Due to the central role of UPS in degradation of key regulatory proteins involved in, e.g., cell proliferation and death, dysfunctions of the UPS are an integral part of many cancers, including

PDAC. Most studies on UPS report on changes in the expression levels or inactivation of UPS components, conceivably because measuring proteasome activity itself from PDAC patient samples is challenging, in part due to tumor heterogeneity. A transgenic pancreatic lesion mouse model expressing a fluorescent reporter for ubiquitin-independent proteasome activity revealed that an increase in proteasome activity is important for the initiation of precancerous lesions [77]. Proteasome inhibition can be used to reverse the cancerous phenotype in cells lacking oncogenic KRAS, but active mutant KRAS induces constitutively high proteasome activity and PanIN progression. These results suggest that proteasome activity may be involved in initiation, but not in the maintenance, of PanINs. Recently, PSMA6, one of the 20S core proteasome subunit genes, was identified in a genome-wide CRISPR screen using PANC-1 cells for gene-specific variation in response to the common PDAC chemotherapeutic, gemcitabine [78]. Knockout of PSMA6 induced apoptosis along with reduced spheroid formation, whereas high expression correlated with significantly shorter overall survival of PDAC patients [78]. However, further studies are warranted to clarify the roles of proteasome core subunits and activity in pancreatic cancer initiation and progression.

4.3.1

E2 Ubiquitin-Conjugating and E3 Ubiquitin-Ligase Enzymes in PDAC

Of the approximately 40 known E2 ubiquitinconjugating enzymes, the expression of UBCH6/UBE2E1 has been shown to be upregulated in PDAC [79, 80]. In contrast to the E2s, there is an accumulating number of reports on various E3 ubiquitin-ligases and DUBs involved in PDAC (summarized in Table 4.2). These UPS components contribute to the development of cancer via the deregulation of the ubiquitination/deubiquitination pattern of, e.g., multiple oncogenes and tumor suppressors and can in some cases also affect overall degradation

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Table 4.2 Examples of dysregulated proteasome subunits, E2s, E3s and DUBs in PDAC Gene/ protein PSMA6 UBCH6/ UBE21 RNF43

Type Proteasome subunit E2 E3

βTRCP1 NEDD4 SMURF1 SMURF2

E3 E3 E3 E3

MDM2

E3

CHIP CBL (two proteins) USP22

E3 E3

USPS11

DUB

USP9X

DUB

USP5

DUB

UCHL1 UCHL5

DUB DUB

DUB

Dysregulation and effects High expression, shorter overall patient survival

References [78]

Upregulation

[79, 80]

Recurring mutations and diminished expression, role in PDAC development Elevated expression, chemoresistance in vitro Elevated expression in vitro, PDAC development and progression Amplification/overexpression, promotion of invasiveness in vitro Activation of apoptosis, inhibition of cell invasion and migration connected to decreased level/inhibition Amplification/overexpression in pancreatic cancer, inhibition promotes PDAC apoptosis and cell-cycle arrest in vitro Lowered expression in serum, knockdown increases tumor growth in vitro Lowered expression connected to decreased PDAC survival and EGFR activation Overexpression induces autophagy, correlates with poor PDAC prognosis

[81–87]

Intermitted high expression in patients, inhibition decreases PDAC survival in vitro in a BRCA2-dependent manner Inactivation correlates with tumorigenesis in mice, low expression with reduced patient survival; expression supports survival in vitro Overexpression in PDAC tissue and cells, promoting tumorigenesis in vitro Increased expression in PDAC, correlates with poor patient survival High nuclear expression correlates with increased survival in PDAC

capacity by binding to the proteasome itself. The E3 ligases are divided into three families: homologous to E6AP carboxyl-terminus (HECT) E3 ubiquitin-ligases, really interesting new gene (RING) E3 ubiquitin-ligases and RING-BetweenRING (RBR) E3 ubiquitin-ligases [116]. In the following, we discuss examples of E3 ligases found to be dysregulated in pancreatic cancers, particularly PDAC. A recent report identified significant recurring mutations in the E3 ligase RNF43 gene by wholeexome sequencing from PDAC patient samples [81]. RNF43 is often mutated in pancreatic cancer cell lines [82], and analysis of patients with advanced PDAC has shown diminished RNF43 immunoexpression in comparison to low-grade in situ neoplasms or normal pancreatic tissue [83]. Further, PDAC precursor lesions, including intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, exhibit high

[88] [89–91] [92–97] [95–98] [95–97, 99–101] [102] [103, 104] [78, 105, 106] [107] [108–111] [112, 113] [114] [115]

frequency of RNF43 mutations [84, 85], indicating a potential role for RFN43 in PDAC initiation. In addition, RNF43 has been shown to regulate the Wnt/β-catenin pathway by promoting degradation of the Frizzled receptor through the autophagy–lysosome system, thereby functioning as a tumor suppressor [82, 86]. As RNF43 also inhibits the transcriptional activity of TP53 [87], RNF43 could affect PDAC tumorigenesis in various ways. βTRCP1 is a member of the F-box family of RING E3 ligases reported to function as an oncogene in PDAC [88]. Immunohistochemical staining of PDAC tumor sections showed significant βTRCP1 expression in approximately 65% of the studied patient samples, whereas normal tissue was βTRCP1 negative [88]. Increased βTRCP1 expression was also described in chemoresistant PDAC cell lines, possibly connected to the constitutive activation of

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NF-κB in these cells [88]. Another E3 ligase, NEDD4, has been linked to neoplastic transformation and tumor progression in multiple cancers, including PDAC [89, 90]. NEDD4 expression, as studied by Western blot, was not uniformly high in PDAC cell lines, but the expression was still elevated in comparison to normal immortalized epithelial pancreatic duct cells [91]. Further, NEDD4 knockdown decreased cell growth and migration, as well as suppressed AKT phosphorylation [91]. Thus, PI3K/Akt signaling might mediate the NEDD4induced effect in PDAC tumorigenesis, progression, and metastasis. The SMURFs (SMURF1 and SMURF2) take part, e.g., in the degradation of key members of TGFβ/BMP signaling, including the TGFβ receptor and different SMADs, which have been implicated to mediate PSC-induced carcinomacell migration [117–119]. Amplification of the SMURF1 gene has been documented both in pancreatic cancer cell lines and in PDAC tumor samples [92, 93], promoting invasiveness at least in vitro [94]. On the other hand, SMURF2 downregulation in TGFβ-treated PANC-1 cells increased TGFβ-mediated SMAD signaling and activated apoptosis [98]. Further, decreased SMURF2 expression inhibited invasion and migration in these cells [98]. In a mouse xenograft tumor model, downregulation of SMURF2 also decreased PANC-1-derived TGFβ-induced tumor growth [98]. The dysregulation of SMURFs has therefore wider implications in both PDAC development and maintenance. SMURFs also interact with MDM2, another E3 ligase and a crucial negative regulator of TP53 stability and activity [95–97]. MDM2 itself is amplified or overexpressed in pancreatic cancer and has been connected to metastasis and advanced disease [99–101]. Chemical MDM2 inhibition was discovered to promote PDAC apoptosis and cellcycle arrest, as well as inhibit survival and proliferation in pancreatic cancer cell lines, independent of their TP53 status [97]. In addition, treatment with a MDM2 inhibitor decreased tumor growth in xenograft and orthotopic mouse models [97].

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The E3 ligase/E4 conjugating factor CHIP, which also functions as a co-chaperone, was shown to have lower expression in pancreatic cancer patient tissue samples compared to corresponding normal-appearing adjacent tissue [102]. CHIP expression in the serum of PDAC patients was also lower compared to healthy controls [102]. Importantly, higher CHIP tissue expression in patients correlated with better prognosis [102]. Further, knockdown of CHIP increased tumor growth both in PDAC cell lines and in a mouse xenograft model, and active CHIP diminished invasion and migration of pancreatic cancer cells in vitro and in vivo [102]. Additionally, active CHIP was shown to regulate EGFR in PDAC cells, followed by inhibition of downstream pathways [102]. Taken together, CHIP appears to function as a tumor suppressor inhibiting, e.g., tumor formation, proliferation, and metastasis in pancreatic cancer. Low levels of CBL proteins with E3-ligase activity have also been observed to result in EGFR activation in PDAC tissue samples, cell lines, and tumor xenografts [103]. A multidimensional array analysis of PDAC tumors further linked low CBL mRNA levels with decreased patient survival [103, 104]. All aforementioned studies reflect on the importance of different E3 ligases in modulating activity and stability of key regulatory proteins involved in, e.g., tumorigenesis and cancer development.

4.3.2

Deubiquitinating Enzymes in PDAC

DUBs are divided into six families on the basis of their sequence homology and domain conservation [120]. These are the five cysteine peptidase families that consist of motif-interacting with ubiquitin-containing novel DUBs (MINDYs), Machado–Josephin domain-containing proteases (MJDs), ovarian tumor proteases (OTUs), ubiquitin carboxy-terminal hydrolases (UCHs) and ubiquitin-specific proteases (USPs), and a sixth additional zinc metalloprotease family of JAB1/MPN/MOV34 (JAMMs) [120]. More than 40 out of the 100 different DUBs have been

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reported to be involved in cancer [121, 122], and several of them show links to pancreatic cancers such as PDAC, as exemplified below. Dysregulation of USP22, a deubiquitinating subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, is involved in key epigenetic changes contributing to tumor development in PDAC and several other cancer types ([105], reviewed in [106]). In an immunohistochemistry study, overexpression of USP22 was especially high in the tissue samples of patients with advanced PDAC, whereas adjacent, normalappearing tissue was negative for this DUB [105]. In almost half of the tumor tissue samples, USP22 overexpression coincided with elevated levels of the autophagy marker LC3, and USP22 was shown to induce autophagy by affecting LC3 levels in PANC-1 cells [105]. Further, both USP22 and LC3 tumor immunoexpression correlated with poor patient prognosis [105]. Moreover, USP22 was another hit in the screen for gene-specific response variation to gemcitabine treatment [78]. A high-throughput chemical screen for USP11 inhibitors identified six small molecules that impede USP11 enzymatic activity, one of which, mitoxantrone, decreased cell survival in PDAC cell lines [107]. High USP11 mRNA levels correlated with inhibitor sensitivity, except in a BRCA2-mutant cell line, indicating a requirement for functional BRCA2 for this effect [107]. USP11 mRNA was highly expressed in almost half of PDAC patient tumor samples [107], though contamination by normal cells in the stroma can affect the readout [123]. Considering the high amount of PDAC tumors with intact BRCA2 function (90% of sporadic cases) [124, 125], targeting USP11 might be a future therapeutic option in PDAC. Several studies have reported important roles for the X-linked deubiquitinase USP9X in PDAC, either as a tumor suppressor or as an oncogene. In a genetic screen for KRAS interactors, based on transposon-mediated insertional mutagenesis, inactivation of the USP9X gene was detected in over 50% of the murine pancreatic tumors and correlated with accelerated tumorigenesis [108]. No mutations in the USP9X gene were

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detected, but low USP9X protein and mRNA expression correlated with poor survival of PDAC patients after surgery and associated inversely with metastatic burden in advanced PDAC [108]. In contrast, USP9X expression has also been shown to support cell survival and malignant phenotype in several human pancreatic tumor cell lines [109, 110]. It has been suggested that USP9X may have a context-dependent function involving tumor repression during development and growth promotion in advanced stages of PDAC [109]. USP9X may in part affect tumorigenesis by regulating the turnover of the E3 ligase ITCH [108], the substrates of which are linked to cell proliferation and survival. However, further complexity arises from results showing that USP9X inhibition repressed autophagy and thereby improved gemcitabine sensitivity in a pancreatic cancer mouse model [111]. USP5 was identified as one of the candidate genes in a short-hairpin (shRNA) library screen in pancreatic cancer cells for novel genes promoting cell survival and cancer progression [112]. USP5 mRNA is overexpressed in PDAC patient tissue and in several PDAC cell lines [112, 113]. Loss of USP5 resulted in accumulation of DNA damage, followed by an increase in p27, cell cycle perturbations and apoptosis [112, 113], whereas upregulation of USP5 promoted tumorigenesis by stabilizing the expression of the transcription factor FoxM1 [113]. Though normally exclusively a neuronal protein [126, 127], UCHL1 is a prominent oncogene linked to multiple cancers [128]. In pancreatic cancer, almost half of the patient samples showed UCHL1 tumor immunoexpression independent of neuronal status of the cell, whereas in healthy donors UCHL1 immunoexpression was neuron specific [114]. Further, positive UCHL1 tumor immunoexpression was shown to correlate with decreased patient survival [114]. Taken together, UCHL1 may play a role in pancreatic cancer tumorigenesis and could potentially be used as a prognostic marker in resected pancreatic cancer patients in the future [114]. Three DUBs, POH1/RPN11, USP14, and UCHL5, are known to bind to the proteasome, and they are also connected to cancer

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[121, 122]. These proteasome-associated DUBs are considered especially attractive therapeutic targets, due in part to their regulation of the ratelimiting deubiquitination step prior to proteasomal substrate degradation [121]. Interestingly, positive nuclear UCHL5 immunoexpression associates with increased PDAC patient survival in patients with regional disease (stages IIB-III), lymph-node positivity and age over 65 years [115]. Additionally, positive nuclear UCHL5 expression was reported to be an independent prognostic factor in PDAC in multivariate analysis [115]. Taken together, a growing number of studies emphasize the involvement of different UPS components in pancreatic cancer. However, as many of these reports rely on work with cell lines or murine models, validation of their clinical relevance with patient cohorts is of critical importance for, e.g., diagnostic, prognostic, and treatment purposes.

4.4

Autophagy Dysregulation in PDAC

A growing number of reports describe an important role for autophagy in pancreatic cancer. High levels of autophagy proteins, such as LC3 and BECLIN-1, correlate with poor prognosis of pancreatic cancer patients [129, 130]. In PDAC, autophagy is elemental in regulating tumor growth both in vitro and in vivo [131]. Autophagy inhibition in turn promotes tumor regression in PDAC xenografts and likewise induces death in PDAC cell lines [132]. Decreased autophagy also impairs mitochondrial function, causing a subsequent drop in cellular ATP levels in PDAC cells [132, 133]. Autophagy levels are often elevated in PDAC [23], and patient-derived activated pancreatic stellate cells (PSCs, type of cancer-associated fibroblasts) in particular exhibit higher autophagy in pancreatic cancer [134]. This enhancement is derived at least in part from tumor–stroma interactions and reduced nutrient availability [134], which activates autophagy generally through the mTOR pathway

[135]. However, the MiT/TFE family of transcription factors can also independently increase autophagy and they are often constitutively active in a nutrient-independent manner in PDAC [136]. Interestingly, also PCSCs show high levels of autophagy crucial for their survival [137]. Elevated levels of autophagy provide metabolic plasticity for pancreatic tumor cells struggling with nutrient deprivation, acidity, and hypoxia [23, 132, 138, 139]. Autophagy upregulation directly increases ATP levels by repurposing cellular macromolecules for metabolic use [76, 140]. In addition, PSCs secrete alanine in response to cancer cell signaling in an autophagy-dependent manner to supplement tumor cell metabolism and growth [141]. IL-6, one of the main activators of PSCs [142, 143], also promotes autophagy in cancer cells [144] and IL-6 secretion from the PSCs was reported to be regulated through autophagy in a positive feedback loop in vitro [134]. However, autophagy cannot create an increase in tumor biomass, because the cells are essentially devouring themselves [23]. To supplement their nutrient supply, pancreatic cancer cells therefore employ microautophagy to internalize large portions of extracellular fluid (containing, e.g., ECM materiel, such as proteins and lipids) [23, 145]. This additional nutrient uptake is essential for PDAC metabolism [23] and the process is dependent on the activation of oncogenic KRAS [146].

4.5

Developing New Therapies Related to Proteostasis

Combination of surgery with the development of adjuvant chemotherapies has improved the treatment outcome of PDAC patients during the last decade, but one of the main challenges is still the lack of reliable biomarkers in clinical use. These would be particularly important for the early diagnosis of the disease, but also for deciding the best course of treatment, and for determining a dependable prognosis. The current research landscape suggests that the use of a panel of biomarkers would be much more effective, than

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any single biomarker alone [147]. Finding a working treatment strategy for pancreatic cancer is demanding, due in part to the presence of multiple subclones within individual tumors, caused by the high genetic and epigenetic heterogeneity, in addition to the complexities of the tumor microenvironment [6, 64]. Another key challenge is the lack of effective therapeutic targets. Targeting protein degradation has emerged as an attractive strategy for treating hematological malignancies and solid tumors [148, 149]. Proteasome inhibitors such as bortezomib, the first FDA-approved proteasome inhibitor in clinical use, have been successful in the treatment of multiple myeloma and mantle cell lymphoma. Because of the essential role of the proteasome in maintaining proteostasis and its vast number of substrates, including key regulatory proteins, inhibition of proteasome activity triggers multiple cellular events, converging in cell death. These include the induction of endoplasmic reticulum stress leading to an unfolded protein response and apoptosis, the inhibition of the pro-survival NFκB pathway and induction of pro-apoptotic proteins [149]. Combinatorial treatment with proteasome inhibitors (e.g., bortezomib) with anticancer drugs such as gemcitabine has been shown to enhance cytotoxicity compared to any treatment alone in pancreatic cancer cell lines and xenograft models [150, 151]. However, Phase II studies have not reported any survival benefits of combined treatment with proteasome inhibitors and gemcitabine for patients with metastatic pancreatic cancer [152, 153]. One explanation could be that PDAC tumors with oncogenic KRAS mutations (90% of all) are potentially less sensitive to proteasome inhibitors [77, 154]. Proteasome inhibition can also result in ER stress-mediated activation of autophagy in pancreatic cancer cells [154, 155], which can in turn have either a proor anti-tumorigenic effect in a context-dependent manner. As concurrent autophagy inhibition increases proteasome inhibitor-mediated apoptosis, an advantage could be gained from combining proteasome and autophagy inhibitors in the treatment of patients [149].

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Modulating cellular metabolic pathways, including autophagy and microautophagy, is emerging as an attractive target in the treatment of PDAC [6]. Especially interesting are targeting mechanisms connected to KRAS-dependent metabolic changes [156, 157]. Acute suppression of KRAS resulted in increasingly elevated autophagy in human and murine PDAC cell lines [158], and in addition, knockdown of KRAS in murine PDAC cells created a subpopulation (approximately 10%) of cells that were dependent on autophagy and mitochondrial ATP production [159]. Inhibiting ERK was analogous to KRAS suppression, inducing autophagy in a similar manner [158]. Further, targeting autophagy suppressed tumor growth and enhanced anticancer agent effect in PDAC cells in vitro [129, 132, 138]. If possible, a two-pronged approach to autophagy seems therefore prudent: inhibition of autophagy in CAFs would decrease nutrient availability for the pancreatic cancer cells, and this coupled with simultaneous promotion of autophagy in the cancer cells would in turn further suppress their growth [19]. However, combinatorial treatments, disrupting also additional metabolic processes concurrently with autophagy, might be more successful, as the inhibition of autophagy alone has produced limited results [158]. Effectiveness of autophagy inhibition could additionally be increased in combination with chemotherapy, as it promotes cancer reliance on autophagy [138, 160, 161]. The dose-limiting toxicities and drug resistance of proteasome inhibitors in clinical use for cancer therapy, in particular of the first generation of inhibitors, have turned the attention into identifying alternative targets that modulate the UPS and/or the proteasome. Given the high numbers and diversity of E3 ligases and DUBs and their distinct association with particular genetic or biochemical pathways, specific targeting of these UPS components provides potential for minimizing off-target side effects, while developing drugs for cancer therapy [120]. This approach could potentially be used as a strategy to modulate expression of distinct proteins linked to better

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(or worse) patient survival: for example, high expression of the degradation-prone β-catenin has been shown to associate with lower tumor grade and lower risk of death in PDAC [162]. A growing number of E3 ligases and DUBs have been implicated in PDAC tumorigenesis, as discussed above. One promising example is the development of strategies to target the SIAH E3 ligase, which is the most downstream signaling molecule of the KRAS pathway (reviewed in [64]). Immunohistochemical staining revealed SIAH expression exclusively in proliferating PDAC tumor cells, while SIAH was absent in normal pancreas and nonmalignant TME cells [64], suggesting that SIAH could be a tumor-specific biomarker for cycling pancreatic tumor cells. Blocking SIAH function prevented tumorigenesis of PDAC cell lines in athymic nude mice [163], as well as in KRAS-mediated metastasis of human pancreatic cancer cells in xenograft models [64]. Further studies are required to validate whether inhibition of SIAH could countervail hyperactive KRAS signaling and reverse malignant transformation in PDAC patients. Similarly, detailed understanding of the function of other E3 ligases and DUBs implicated in PDAC is required to commence clinically relevant validation studies. Currently, new emerging technologies based on proteolysis-targeting chimeras (PROTACs) are attracting considerable attention as these first anticancer therapeutics are entering clinical trials [164, 165]. In this eventdriven approach, the target protein is tagged for elimination by exploiting the cell’s own protein quality control machinery. Potential advantages of PROTACs usage include reduced systemic drug exposure and the turning of undruggable targets druggable. PDAC is a harrowing disease, with an abysmal prognosis for the patients whose lives it affects. Molecular profiling of human pancreatic tumors and concurrently taking into account the tumor microenvironment will help to design better precision therapies to improve overall survival in pancreatic cancer. In conclusion, exploiting the proteolytic systems of the cell, in combination with more conventional treatment strategies, is emerging as a significant option for combatting this debilitating cancer.

L. Arpalahti et al. Acknowledgments We apologize to all the authors, whose original work could not be cited due to space limitations. This work was supported by grant to C.I.H. from the Academy of Finland (#297776), Sigrid Jusélius Foundation and Medicinska Understödsföreningen Liv och Hälsa r.f. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).

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Divergent Modulation of Proteostasis in Prostate Cancer Petek Ballar Kirmizibayrak, Burcu Erbaykent-Tepedelen, Oguz Gozen, and Yalcin Erzurumlu

Abstract

Proteostasis regulates key cellular processes such as cell proliferation, differentiation, transcription, and apoptosis. The mechanisms by which proteostasis is regulated are crucial and the deterioration of cellular proteostasis has been significantly associated with tumorigenesis since it specifically targets key oncoproteins and tumor suppressors. Prostate cancer (PCa) is the second most common cause of cancer death in men worldwide. Androgens mediate one of the most central signaling pathways in all stages of PCa via the androgen receptor (AR). In addition to their regulation by hormones, PCa cells are also known to be highly secretory and are particularly prone to ER stress as proper ER function is essential. Alterations in various complex signaling pathways and cellular processes including cell cycle control, transcription, DNA repair, apoptosis, cell adhesion, P. Ballar Kirmizibayrak (*) Faculty of Pharmacy, Department of Biochemistry, Ege University, Izmir, Turkey B. Erbaykent-Tepedelen Faculty of Science, Department of Molecular Biology and Genetic, Uludag University, Bursa, Turkey O. Gozen Faculty of Medicine, Department of Physiology, Ege University, Izmir, Turkey Y. Erzurumlu Faculty of Pharmacy, Department of Biochemistry, Suleyman Demirel University, Isparta, Turkey

epithelial–mesenchymal transition (EMT), and angiogenesis are critical factors influencing PCa development through key molecular changes mainly by posttranslational modifications in PCa-related proteins, including AR, NKX3.1, PTEN, p53, cyclin D1, and p27. Several ubiquitin ligases like MDM2, Siah2, RNF6, CHIP, and substrate-binding adaptor SPOP; deubiquitinases such as USP7, USP10, USP26, and USP12 are just some of the modifiers involved in the regulation of these key proteins via ubiquitin–proteasome system (UPS). Some ubiquitin-like modifiers, especially SUMOs, have been also closely associated with PCa. On the other hand, the proteotoxicity resulting from misfolded proteins and failure of ER adaptive capacity induce unfolded protein response (UPR) that is an indispensable signaling mechanism for PCa development. Lastly, ER-associated degradation (ERAD) also plays a crucial role in prostate tumorigenesis. In this section, the relationship between prostate cancer and proteostasis will be discussed in terms of UPS, UPR, SUMOylation, ERAD, and autophagy. Keywords

Prostate cancer · Ubiquitin · Ubiquitin-like · Deubiquitinase · Autophagy · Unfolded protein response

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_5

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P. Ballar Kirmizibayrak et al.

Introduction

Protein homeostasis, or proteostasis, is maintained by the concerted efforts from various cellular processes that control the synthesis, folding, posttranslational modification, assembly/disassembly, localization, and degradation of proteins in the cell [1]. Ubiquitination (also referred to as ubiquitylation) is one of the major posttranslational modifications regulating proteostasis. Ubiquitin (Ub), a highly conserved small polypeptide composed of 76 amino acids, is ubiquitously expressed in the eukaryotic cells [2]. Ubiquitination is a process where ubiquitin is covalently attached to a lysine of target proteins via an isopeptide bond and works in three sequential steps involving Ub-activating (E1), Ub-conjugating (E2), and Ub-ligase (E3) enzymes [3]. E1 initiates the process by forming a thioester bond between activesite Cys residue of the E1 and C-terminal carboxyl group of ubiquitin in an ATP dependent manner. Then, activated ubiquitin is transferred to a thiol group on the active site of E2 via a transacylation reaction. The final step of the ubiquitination, where in ubiquitin is transferred to the substrate, is catalyzed by E3 ligases. Depending on the E3, substrate ubiquitination occurs by direct transfer to the substrate from the E2 or after thioester formation of ubiquitin with the E3 [3]. The human genome encodes two E1 genes, 30 E2 genes, and over 600 E3 genes [4]. The E3 ubiquitin ligases determine the substrate specificity [5] and can be classified into three families: really interesting new gene (RING), homologous to E6AP carboxyl terminus (HECT), and UFD2 homology (U-box). The RING family is the largest family of E3 ubiquitin ligases [6]. The numerous combinations of E2 and E3 enzymes dictate the type of ubiquitin modification as well as the chain linkage type on protein substrates and allow selective tagging thereby affecting the fate of specific proteins within the cell [7]. Ubiquitin can be attached to substrates as single ubiquitin(s) via a process called as monoubiquitination or polyubiquitin chains can modify substrates via polyubiquitination. Seven

lysine residues of the ubiquitin polypeptide involved in the formation of the polyubiquitin chains are K6, K11, K27, K29, K33, K48, or K63. The type of polyubiquitin chain determines the mode of regulation of the conjugated proteins. For instance, K48-linked chains mainly lead to the intracellular degradation of substrates by 26S proteasome, while K63-linked chains can alter the protein’s activity, interaction, or localization [7]. Like other posttranslational modifications, ubiquitination is a highly dynamic reversible reaction. Deubiquitinating enzymes (DUBs), a member of proteases family, selectively trim ubiquitin from substrates and also disassemble polyubiquitin chain. Almost 100 DUBs play a role in this process [8]. Besides regulating the ubiquitin-mediated proteasomal degradation of different cellular proteins, DUBs also control the availability of free ubiquitin pool [8]. It is clear that the action of E3 ubiquitin ligases and DUBs control the balance of ubiquitination/deubiquitination in cellular levels [9]. Not surprisingly, DUBs have been associated with the development and progress of tumorigenesis by modifying key proteins that regulate the cell cycle, gene transcription, DNA repair, and apoptosis [10]. The ubiquitin–proteasome system (UPS) and autophagy are the two main protein quality control and intracellular degradation mechanisms crucial for proteostasis. UPS is essential for practically almost all cellular processes and maintenance of cellular homeostasis. It plays a crucial role in the regulation of cellular proteins implicated in the regulation of many biological processes via recognition and degradation of misfolded, damaged or tightly regulated, shortlived proteins functioning in cell cycle regulation, DNA damage response, cell growth, cell migration, transcription, apoptosis, cell adhesion, angiogenesis and tumor growth, antigen processing, and inflammatory responses [11]. While the UPS targets short-lived proteins and soluble misfolded proteins for degradation, autophagy is mainly responsible for the degradation of long-lived proteins as well as insoluble protein aggregates [12]. There are three types of autophagy—macroautophagy, microautophagy,

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Divergent Modulation of Proteostasis in Prostate Cancer

and chaperon-mediated autophagy, which utilize different mechanisms to target cargos for degradation by lysosomal enzymes [13]. Macroautophagy (hereafter referred to as autophagy) is initiated by phagophore formation, where ATG proteins play a critical role. Recent findings suggested that ubiquitin system has a role in the regulation of autophagy [14]. Approximately 30 ATG genes [15] have been identified to serve in the different steps of autophagy flux. Disruptions in the organization of subunits in these complexes lead to the deterioration of functional autophagy. Selective autophagy is the most well understood autophagy pathway involving ubiquitin [16], where selected cargos conjugated with ubiquitin chains are targeted for autophagydependent degradation. These ubiquitinated cargos, including protein aggregates are recruited to the phagophore via an autophagy receptor with an ubiquitin-binding domain and a LC3-interacting region. Ubiquitination of autophagy regulators with Lys48-linked ubiquitin chains often affects their stability. Lastly, ubiquitin can also be utilized as a UPS-independent tag via non-degradation-type ubiquitin chains to regulate autophagy [16]. Ubiquitin-like modifiers are important players of proteostasis and control several cellular events varying from protein stability to signal transduction [17, 18]. Small ubiquitin-like modifier (SUMO) is a highly conserved ubiquitin-like protein that covalently modifies a large number of proteins [17–19]. Like ubiquitination, SUMOylation is a dynamic process mediated by E1, E2, and E3 enzymes, which are distinct from ubiquitin E1, E2, and E3. Deconjugation of SUMO (deSUMOylation) is catalyzed by a family of proteases called SUMO-specific proteases or SUMO isopeptidases [20]. In contrast to ubiquitination cascade, Ubc9 is identified as the only known E2 conjugating enzyme for SUMOylation [21]. SUMOylation regulates many cellular events, including nuclear signaling, nuclear translocation, transcription activities, and DNA repair [22]. ISG15 is a small ubiquitin-like protein that is one of the most upregulated genes by type-I interferon and can be conjugated to target proteins by an enzymatic cascade using

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enzymes analogous to the ubiquitination [23]. ISG15 is conjugated more than 150 substrates including proteins having role in central immune signal pathways such NF-κB, JNK, and IRF-3 [24]. Although ISG15 conjugation (ISGylation) plays mainly an important role in innate immunity by regulating antiviral and antibacterial properties of type-I IFN, it has been suggested that ISG15 may be also involved in multiple cellular processes such as DNA repair, autophagy, and protein translation as well as in additional pathologies including cancer [25]. Protein NEDDylation is a posttranslational modification by a covalent conjugation with the neural precursor cell expressed, developmentally downregulated 8 (NEDD8) in a manner similar to that described for ubiquitination. As others NEDDylation is also a reversible process. While the family of cullin proteins is the most established target for NEDD8, additional targets including transcription factors and coregulators, signaling receptors, components of the protein synthesis and apoptotic machineries, E3-ligases, and histones have been identified [26]. Approximately one-third of cellular proteins is folded in and traffic through the endoplasmic reticulum (ER) before their transport to their proper subcellular or extracellular locations [27]. The ER contains chaperones and folding enzymes and provides a Ca+2 rich, oxidizing environment supporting the formation of disulfide bonds during protein folding. ER maintains a unique homeostasis via its Protein Quality Control mechanism to ensure that only properly folded proteins are transported exit the ER en route to functional destination. When the unfolded or misfolded protein load exceeds the capacity of the ER to fold protein, the cells are subject to a condition called “ER stress.” ER stress in turn activates a series of coordinated adaptive mechanisms like Unfolded Protein Response (UPR) and to alleviate the stress and restore ER proteostasis [28]. The accumulation of misfolded proteins in the ER lumen is the primary signal that activates the UPR [29] via three ER membrane-associated proteins, PERK (protein kinase RNA-like ER kinase), IRE1α (inositolrequiring transmembrane kinase/

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endoribonuclease 1), and ATF6α (the activating transcription factor-6). These three proteins act as ER stress sensors and they bind to the ER luminal chaperone BiP under basal conditions. However, when misfolded proteins are accumulated in the ER, BiP dissociates from ER stress sensors which in turn activates the sensors to transduce signals to the cytosol and nucleus to restore ER stress through various pathways [30]. IRE1α RNase activity cleaves XBP1 (the transcription factor X-box binding protein 1) mRNA to a spliced form of XBP1 that translates XBP1s to upregulate UPR genes encoding factors involved in protein folding, ER-associated protein degradation (ERAD), protein quality control, and organelle biogenesis [27, 28]. Activation of PERK attenuates general protein synthesis through phosphorylation of the eukaryotic translation initiator factor 2α (eIF2α) decreasing protein efflux into the ER. eIF2α phosphorylation selectively stimulates translation of the ATF4 mRNA, which encodes a transcription factor that induces the expression of some UPR genes involved in antioxidant responses, amino acid metabolism, autophagy, and apoptosis. ATF4 controls the expression of the proapoptotic components GADD34 (growth arrest and DNA damageinducible 34) and C/EBP-homologous protein (CHOP). GADD34 also binds protein phosphatase 1C (PP1C) to dephosphorylate eIF2α [27, 28]. ATF6 is localized at the ER in basal conditions and when UPR is initiated ATF6 translocates to the Golgi where it is processed by specific proteases releasing its active cytosolic domain which controls the upregulation of some UPR target genes [27, 28]. Chronic or severe ER stress activates the UPR leading to the cell death. It has been suggested that PERK–eIF2α–ATF4 signaling is a primary determinant for apoptosis [31]. The activation of IRE1α during severe ER stress has been also implicated in cell death mediated by apoptosis signaling kinase 1 (ASK1) through their interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2) [32]. IRE1α also degrades selected mRNAs through a process called regulated IRE1-dependent decay (RIDD), which can lead to cell death [33].

P. Ballar Kirmizibayrak et al.

In summary, proteostasis regulates key cellular processes such as cell proliferation, differentiation, transcription, and apoptosis. Several studies suggest that cancer cells are highly dependent on the mechanisms of proteostasis [34]. Since it also targets oncogenes and tumor suppressors; mechanisms by which proteostasis is regulated is crucial and the deterioration of cellular proteostasis has been significantly associated with tumorigenesis. Importantly, proteostasis is involved in prostate tumorigenesis in various ways by modulating prostate cancer-related genes/proteins such as AR, NKX3.1, cyclindependent kinase inhibitor p27, cyclin D1, and PTEN.

5.2

Prostate Cancer

The prostate is a secretion gland, which is a part of a man’s reproductive and urinary systems [35]. It produces seminal fluid that composed of wide range of proteins, antioxidants, minerals and protects the sperm [36]. One of the most critical contents of prostate is “Prostate Specific Antigen (PSA)” which is a serine protease with chymotrypsin-like substrate specificity produced by secretory epithelial cells. PSA exhibits its enzymatic activity against most important gel-forming proteins of semen, namely Seminogelin 1-2, and enhances sperm motility by preventing seminal fluid coagulation [37, 38]. The androgen signal plays a critical role in the normal development, proliferation, and differentiation of the prostate gland and is mediated by the androgen receptor (AR) [39]. The androgen signal is essential in almost every stage of prostatic growth and differentiation [40, 41]. Normal growth of the prostate continues throughout the life, but prostate is very prone to developing adenocarcinoma. Prostate cancer (PCa) is the second most common cancer diagnosed and the second most common cause of cancer death in men worldwide. In the USA, 164,690 new cases of PCa and 29,430 associated deaths were estimated in 2018 [42]. PCa is more frequently diagnosed in men over the age of 65.

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Screening for PCa is based on the PSA blood test; however, abnormal levels require prostate biopsies for definitive diagnosis as well as digital rectal examination. Combinations of tumor surveillance, surgery, radiation, hormonal, and chemotherapies are used in the management and treatment of PCa. Prostate tumors are derived from secretory luminal epithelial cells that are the major cell type in the prostate. Genetic changes such as AR, PTEN, p27, and RNaseL mutations, hypermethylation of the GSTP1 promoter, and TMPRSS2-ETS chromosomal translocation, transform the normal prostate epithelium into lesions called as Prostatic intraepithelial neoplasia (PIN) [43]. Several evidence suggest that PIN lesions are the precursors of human PCa [44–46]. PCa is a disease that generally develops in two phases. In the first phase, cancer cells need androgens for their survival and proliferation [47, 48]. Androgens have been shown to activate the proliferation of PCa cells along with peptide growth factors. Conversely, inactivation of AR inhibits DNA replication and transition to S phase in PCa cells and induces cell death. Thus, decreasing AR activity attenuates survival mechanisms while increasing AR activity leads to proliferation [47]. Androgen deprivation therapy (ADT) is usually used for the treatment of this stage of the disease and includes either reduction of androgen production by surgical or medical castration or inhibition of AR function by antiandrogens [49, 50]. Initially, ADT leads to decreased tumor cell proliferation and reduced tumor size. However, after long-term ADT, the disease usually progresses to second phase that represents highly aggressive castration-resistant metastatic prostate cancer, which does not respond to traditional treatment [47– 50]. Although androgen-independent, hormoneresistant, or hormone-refractory terms are used for the second phase of prostate cancer, in recent years this disease has been defined as castrationresistant prostate cancer (CRPC) due to evidences that indicate the prostate tumors still depend on androgen signaling and AR activity [41, 51, 52]. In these tumors, mutations of AR may increase the specificity of the AR against other

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steroids and even non-androgen molecules. Improper activation of the AR results in transactivation of androgen-regulated genes that significantly affect tumor progression [53]. For the development of CRPC, several models have been proposed in relation to the aberrant regulation of AR activity [48, 49, 54, 55]. These models include AR gene amplification, change of expression level, mutation of coregulators, kinase activation of AR via growth factor signaling, AR somatic mutations, overexpression of antiapoptotic Bcl-2 and other oncogenes that activate AR, and finally the selection of malignant stem cells that are endogenously androgen independent [35, 41, 48, 50, 54]. Consequently, each mechanism changes the ability of PCa cells to respond to androgens as independence or hypersensitivity [56]. Since all these mechanisms can alter the activation of the AR signaling pathway, ADT is not successful at this second phase [55]. Therefore, it is very important to determine the tumorigenesis potential of prostate and to understand the molecular basis of PCa for early diagnosis and treatment [48, 57]. Unfortunately, the molecular basis of PCa is quite complex and mutations in many genes have been implicated including AR, NKX3.1, TP53, RB1, EZH2, BMI, and PTEN [58–60]. On the other hand the frequency of reported genetic changes is highly variable in prostate cancer patients representing the heterogeneous and multifocal feature of prostate cancer [37]. In light of all of the identified mutations, PCa appears to be caused by dysregulation of many different cellular signaling pathways especially those involved in cell survival and apoptosis. Androgens that act through AR are essential for the growth and development of the prostate. Also NKX3.1, PTEN, and p27 regulate the growth and survival of prostate cells in normal prostate. Inadequate amounts of PTEN and NKX3.1 lead to a decrease in p27 level through various mechanisms and thus causes to an increase in proliferation and a decrease in apoptosis. All these molecular changes define specific targets for the diagnosis and treatment of prostate cancer (Table 5.1).

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Table 5.1 Key proteins involved in prostate tumorigenesis Protein AR

Type Transcription factor

NKX3.1

Homeobox gene/tumor suppressor

PTEN

Lipid phospatase/ tumor suppressor

Allelic loss, gene silencing by methylation, posttranslational modifications, decreased expression, stability

p27

CDK inhibitor/ tumor suppressor Fusion oncogene

Allelic loss, decreased expression, stability

Transcription factor/ oncogene Tumor suppressor

Chromosome 8q gain, gene amplification, increased expression Allelic loss, somatic mutations, posttranslational modifications stability

RB1

Tumor suppressor

Allelic loss, decreased expression stability

Endothelin 1/Bombesin/ Neurotensin

Neuropeptides

Increased expression

TMPRSS2 : ERG C-MYC

p53

5.3 5.3.1

Alterations in PCa Amplification, chromatin rearrangement, somatic mutations, change on expression level (itself and coregulators), stability, posttranslational modifications, intracellular localization Allelic loss, hypermethylation, decreased expression stability

Chromosomal translocation, gene fusion, overexpression

Key Proteins Involved in Prostate Tumorigenesis Androgen Receptor

Androgens are essential factors for the growth and development of prostate epithelial cells acting through the AR, a member of the nuclear receptor superfamily [41, 61, 78]. AR plays a crucial role via AR-regulated genes in the normal growth and development of the prostate gland, as well as in PCa and in the transition to more aggressive stage of disease [62].

Role in PCa Has a crucial role in the normal development of the prostate gland and PCa, regulates the expression of almost 100 genes including itself functions in the transition to CRPC.

References [53, 54, 61–67]

Regulates the growth and survival of prostate cells, negative regulator of AR-associated signaling pathways, promotes p53 half-life and activity Involved in cell survival, proliferation, and energy metabolism, abolishes PI3K-AKTmTOR pathway, increases p27 levels Inhibits pRB phosphorylation, blocks cell cycle progression

[46, 55, 68–70]

Functions as a key regulator of cell proliferation, contributes to the development of CRPC Triggers DNA replication and cell cycle progression, involved in the transition to CRPC Regulates of genomic instability, functions in G1/G2 and G2/M transitions, stimulates DNA repair genes, initiates apoptosis Regulates the cell cycle by inhibiting the progression to the S-phase Activates PI3K/AKT and NF-κB pathways, induces cell survival and migration

[43, 71– 74]

[43, 75]

[41, 48, 76] [50]

[37, 43, 77]

[43]

[48]

AR protein consists of four different structural and functional regions: N-terminal domain (NTD), DNA-binding domain (DBD), Ligandbinding domain (LBD), and Hinge Region. The Hinge Region separates LBD from DBD as well as contains the nuclear localization signal (NLS) required for nuclear translocation of AR [51, 61, 62, 79]. LBD of AR includes AF-2 (transcriptional activation function 2) region, which is important for forming coregulator binding site and mediating direct interactions between NTD and LBD. Additionally, the nuclear export signal (NES) that is responsible for exporting of AR to

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Divergent Modulation of Proteostasis in Prostate Cancer

the cytoplasm upon ligand withdrawal located in LBD [54, 62, 79, 80]. DBD is responsible for targeting the receptor to specific sequences ARE (androgen response elements) associated with the AR-regulated genes in the genome. NTD mediates the vast majority of AR transcriptional activity and has AF-1 region [52, 63, 80]. This AF-1 region consists of two distinct activation units termed as TAU-1 and TAU-5. While, TAU-1 is required for agonist stimulated AR activity, TAU-5 is provided constitutive AR activity in the absence of the LBD [54, 62, 63]. To date, approximately 200 AR coregulator proteins have been identified. These coregulators modulate a wide range of processes associated with AR activity such as intracellular localization, DNA binding ability, nuclear translocation, chromatin rearrangement, and AR stability [63– 65]. Coregulator proteins are divided into four main classes: (1) Molecular chaperones that control AR maturation and movement, (2) Histone modifiers, (3) Transcription coordinators, and (4) DNA structural modifiers [63, 65]. Several p160 class coactivators such as steroid receptor coactivator 1 (SRC1), transcriptional intermediary factor 2 (TIF2) and its mouse homolog glucocorticoid receptor interacting protein 1 (GRIP1) bind to the NTD-TAU-5 site and regulate the AR transactivation directly by their intrinsic histone acetyltransferase activity and also indirectly via acting as a platform for the collection of secondary coactivators. Examples of these secondary coactivators are CBP/p300 and pCAF, which have chromatin remodeling and acetyltransferase activity, and CARM1 or PRMT1 that have methyltransferase activity [63]. NCoR1 (the nuclear receptor corepressor) and SMRT/ NCoR2 (the silencing mediator of the retinoid and the thyroid hormone receptor) represent the most well-defined AR corepressors. These corepressors compete for the same AR binding surface with coactivators such as the p160 family, and therefore, they are important factors in the regulation of androgen-regulated genes and in the determination of the AR signaling level [54, 63]. There are several other coregulators with important roles in the regulation of AR transcriptional activity during the progression of PCa.

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In the absence of ligand, AR is localized in the cytoplasm in a conformation associated with heat shock proteins (HSP90, HSP70, HSP56, HSP40), cytoskeleton proteins, and cochaperones that prevent binding to DNA [48, 54, 62, 63, 81]. HSPs retain AR in the cytoplasm through cell skeletal proteins and regulate AR conformation for an effective ligand binding [54]. Androgen binding triggers a conformational change on the receptor that allows the dissociation of HSPs, interaction of coactivators such as SRC1 and SRC2, the entry of the complex to the nucleus by uncovering the NLS in the hinge region and interaction with coregulators such as importin-α that bind to the NLS [54, 81]. In addition, androgen binding leads to recruitment of kinases and serine phosphorylation that protects AR from proteolytic degradation [54]. The ligand-induced conformational change also facilitates the formation of the AR homodimer complex that binds to the ARE sequences in the promoter region of the target genes. Coactivators of AR facilitate the recruitment of histone modifying enzymes such as histone acetyltransferases and histone arginine methyltransferases [48] to relax nucleosomes allowing the access of the AR complex to ARE elements in target promoters. AR regulates the expression of almost 100 genes including itself, PSA, NKX3.1, probacin, kallikrein, keratinocyte growth factor, p21, the ornithine decarboxylase [53, 54, 61]. AR can up- or downregulate its target genes depending on the availability of the corepressors and coactivators [61]. Notably, AR activity is regulated by a variety of posttranslational modifications such as phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination [66, 67]. Phosphorylation of AR plays role in the protein stability, transcriptional activity, and nuclear localization [51]. AR has potential phosphorylation sites specific for several kinases such as casein kinase II, PKA, calcium calmodulin II kinase, protein kinase C, MAP kinases, AKT/PKB and is generally phosphorylated from the residues of serine, threonine, and tyrosine localized in the NTD region [51, 63]. Moreover, posttranslational modifications of coregulators play an important role in recruiting transcriptional

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complexes and increasing AR transactivation [63]. The regulation of AR by ubiquitination, deubiquitination, Ub-like modifications, and autophagy will be described in the following subsection.

5.3.2

Tumor Suppressors

The tumor suppressor PTEN (phosphatase and tensin homolog), which has lipid phosphatase activity, is involved in several cellular processes such as cell survival, proliferation, and energy metabolism by abolishing the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway which stimulates cell growth and survival [71]. It is one of the frequently lost or mutated tumor suppressors in PCa and its expression level has shown to be inversely correlated with the initiation, latency, and progression of PCa in a dose-dependent manner [82, 83]. Although the mechanisms such as gene deletion, mutation, and silencing by methylation are responsible for selective loss of PTEN protein expression in PCa, posttranslational ubiquitination also controls PTEN stability, activity, and localization [72–74]. The loss of the most potent tumor suppressor gene retinoblastoma (RB1) and RB1 mutations are observed in localized and invasive prostate tumors. The retinoblastoma gene product, pRB, regulates the cell cycle by inhibiting the progression to the S-phase. It is also thought that pRB regulates apoptosis in prostate cells, particularly in response to androgen removal [43]. A cyclin-dependent kinase (CDK) inhibitor p27 has role in controlling cell growth and division through inhibiting pRB phosphorylation and blocking cell cycle progression [75]. p27 inactivation may occur due to its loss of expression or its increased degradation which is triggered by its abnormal CDK-dependent phosphorylation and subsequent ubiquitination [43]. In addition, PTEN increases the p27 protein level by inhibiting the PI3K-AKT pathway. Therefore, loss of PTEN results in low p27 levels and

abolished p27 expression in PCa is associated with tumor grade [43, 84]. Allelic loss of the chromosome 17p13 harbors p53 locus is seen in 50% of advanced prostate cancer and metastasis [77]. p53 is the key regulator of G1/G2 and G2/M transitions and plays an important role in stimulating DNA repair genes. p53 may either stimulate cell cycle arrest and DNA repair or initiate apoptosis depending on the length of stress. As a result, inactivation of p53 results in decreased DNA repair mechanism and increased genomic instability [37, 43]. NKX3.1 expression is found to be lost or decreased in 50% of PIN lesions and 80% of metastatic tumors [46]. This loss is believed to be an early genetic change in the development of prostate tumors, so it is thought that NKX3.1 is one of the candidate prostate tumor suppressor genes [68, 69]. NKX3.1 expression is largely prostate specific and it is slightly expressed in the testis [68, 85, 86]. The homeodomain of NKX3.1 mediates the binding of DNA as a transcription factor as well as interaction with many proteins [40]. Although NKX3.1 expression is positively regulated by androgen, NKX3.1 inhibits AR expression [70]. On the other hand, AR modulates AKT activation by the PI3Kdependent mechanism, while NKX3.1 controls AKT phosphorylation via AR/PI3K-dependent mechanism. Thus, NKX3.1 and AR form an important feedback loop and NKX3.1 function as an important negative regulator in controlling AR expression and AR-associated signaling pathways in this cycle. Furthermore, NKX3.1 promotes p53 half-life by modulating nuclear MDM2 activity independent of AKT activation. NKX3.1 also binds to HDAC1 and stimulates p53 acetylation and activity by releasing p53 from the p53-MDM2-HDAC1 complex. In the absence of NKX3.1, the majority of p53 is located in the MDM2-HDAC1 complex, which leads to p53 degradation. Decreased p53 activity and activation of PI3K/AKT pathway due to increased AR level lead to increased cell proliferation, decreased cell death, and initiation of prostate cancer [45].

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5.3.3

Other Proteins Involved in Prostate Cancer

Protooncogene c-Myc is a transcription factor that is activated in many human cancers and triggers DNA replication and cell cycle progression. The two most commonly used prostate tumor cell lines LNCaP and PC-3 have significant c-Myc amplification and increased expression [50]. In addition, c-Myc overexpressing cells continue to proliferate during androgen deprivation therapy and c-Myc-specific amplifications is observed in approximately 72% of CRPC patients. Therefore, it is thought that ADT increases c-Myc amplifications and c-Myc activation is important in the development of CRPC [50]. One of the signal transduction pathways associated with the occurrence of castration resistance is neuropeptides and their membrane receptors. Loss of cell membrane-associated enzyme neutral endopeptidase increases the levels of neuropeptides such as endothelin-1, bombesin, and neurotensin. The interaction of these neuropeptides with their receptors induces cell survival and cell migration. The pro-survival signaling initiated by neuropeptides involves the activation of PI3K/AKT, which then activate the NF-κB transcription factor by phosphorylating its inhibitor IκB. Phosphorylated IκB is ubiquitinated and degraded by the proteasome, thus allowing NF-κB to translocate to the nucleus [48]. Activation of the NF-κB pathway results in increased AR levels and cell proliferation. In addition, NF-κB regulated cytokines such as IL-6 and IL-8 also activate AR [41]. PCa cells are reported to have constitutive NF-κB activity, partly due to increased activity of the I-κB kinase complex which is consistent with increased proteasome activity in prostate tumors [87]. Wnt-1 is another signaling pathway whose regulation is impaired in the progression of prostate carcinogenesis and resulted in activation of the β-catenin transcription factor. In the absence of Wnt-1 signals, β-catenin is either retained in the cytoplasm via binding to E-cadherin or phosphorylated, ubiquitinated and degraded with the proteasome. Conversely, the inhibition of

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phosphorylation and ubiquitination of β-catenin allows its translocation to the nucleus and initiates its transcriptional activity in the presence of the Wnt-1 signal. β-catenin/TCF4 transcription plays an important role in PCa, especially when the AR signal is inactive [41, 48, 50]. E-twenty-six transformation-specific sequence (ETS) family transcription factors such as Etwenty-six related gene (ERG) also play a critical role in CRPC. ERG is an oncogene that functions as a key regulator of cell proliferation, differentiation, angiogenesis, inflammation, and apoptosis. ERG gene is fused to the androgen-responsive gene transmembrane protease serine 2 (TMPRSS2) in almost 50% of PCa, resulting in its overexpression [88]. This fusion event causes the ETS transcription factors to respond to androgen and ERG overexpression contributes to the development of CRPC [41, 48, 76]. In conclusion, alterations in various complex signaling pathways and cellular processes including cell cycle control, transcription, DNA repair, apoptosis, cell adhesion, epithelial–mesenchymal transition (EMT), and angiogenesis are known to be involved in prostate carcinogenesis through key molecular changes on the PCa-related genes/proteins, including AR, NKX3.1, PTEN, p53, cyclin D1, and p27.

5.4

Ubiquitin-Related Processes in Prostate Cancer

The E3 ubiquitin ligase enzymes have important roles on the regulation of the comprehensive signaling network of prostate tumorigenesis by controlling stability and/or activity of many critical proteins, including AR, cofactors of AR, several other main tumor suppressors, and oncogenes (Table 5.2).

5.4.1

The Ubiquitin-Mediated Regulation of Androgen Receptor

Several E3 ubiquitin ligases have been identified to interact with and ubiquitinate AR. These E3s

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Table 5.2 Ubiquitin ligases and DUBs implicated in prostate tumorigenesis MDM2 SKP2

Function RING E3 ligase SCF E3 ligase-RING

E6AP

HECT E3 ligase

Substrates involved in PCa AR/AR-V7, p53 AR, p27, p21, BRCA2, p57, p53, p130, FOXO1, DAB2IP AR, NCoR1, p27, PML

SIAH2 SPOP-CUL3RBX1 STUB1/CHIP NEDD4 SAG/RBX2/ROC2/ RNF7 MYLIP TOPORS β-TrCP

RING E3 ligase Cullin3/BTB-RING E3 ligase U-BOX E3 ligase HECT E3 ligase SCF E3 ligase-RING

AR, NCoR1, ATM, HIF1α AR, SRC3, ERG, DDIT3, TRIM24, SENP7, EgIN2, ATF2, c-MYC, FASN AR/AR-V7, PRMT5, p53, HIF1α AR, PTEN, IRS2 PHLPP1, DEPTOR, p21, p27, N0XA

RING E3 ligase RING E3 ligase SCF E3 ligase-RING

AR NKX3.1 IκBα, IκBβ, β-Catenin, δ-Catenin, Gli2, REST, HIF1α

RNF6 RNF126 HUWE-1

RING E3 ligase RING E3 ligase HECT E3 ligase

AR p21 p53, BRCA1, c-MYC

USP2

Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase Ubiquitin-specific peptidase

FAS, Cyclin D1, ASAH1, Aurora-A, MDM2/MDMX

[127] [128, 129] [121, 130– 133] [134] [135] [102, 136– 139] [140–144]

AR, p53, MDM2, PTEN, FOXO

[145–148]

PBX1, ERG, IGF-IR, IRS-2

[149–151]

AR, H2A.Z, G3BP2, p53

[152–157]

AR, MDM2

[158–160]

AR

[161, 162]

IRS-2

[124, 163]

AR

[164]

AR, MDM2

[160]

USP7 (HAUSP) USP9x USP10 USP12 USP14 USP15 USP26 USP46

consequently regulate the AR transcriptional program either positively or negatively by using distinct mechanisms to modulate the expression of AR target genes, including local turnover of AR chromatin complex, recruitment of AR coactivators, and the stability of global AR. Proteostasis of AR is a complex process that could occur both in the absence or presence of androgen. Androgen-independent AR degradation occurs mainly in response to stimulation of cytokines such as IL-6 and growth factors

References [48, 61, 66] [89–102] [48, 61, 103] [104–107] [106, 108– 117] [118–121] [122–124] [125, 126]

including insulin-like growth factor 1 (IGF-1) [61]. These growth factors and cytokines activate the PI3K/AKT kinase pathway, which promotes AR phosphorylation at Ser210/213 and Ser790/ 791. Phosphorylation of these residues has been shown to suppress AR transactivation via inhibiting the interaction of AR with one of its coactivators namely ARA70 [61]. ARA70 functions in AR translocation to the nucleus. Therefore, inhibition of AR-ARA70 interaction may lead to AR accumulation in the cytoplasm,

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where it is susceptible to 26S proteasomemediated degradation. On the other hand, overexpression of proteasome alpha subunit 7 (PSMA7) of the 20S core results in an increase in AR transactivation by influencing the androgen-induced translocation of AR to the nucleus [61]. 20S proteasome inhibitor MG132 is able to block the interaction of AR with ARA70 [61]. Moreover, AKT-mediated phosphorylation of AR induces ubiquitin E3 ligase MDM2mediated AR ubiquitination and its proteasomal degradation [61]. Recently it was reported that AR-negative signature of PCa stem cells is due to constant degradation of AR by MDM2 conserving PCa stem cell integrity [165]. AR amino acid sequence contains a KLKK motif, the lysine residues of which are modified with acetylation/deacetylation by p300/CBP and HDAC1, respectively [61]. Notably, this KLKK motif is very similar with a KSKK motif of p53, a well-known MDM2 substrate and deacetylation of p53 by HDAC1 is required for its MDM2mediated degradation [61]. It is noteworthy that the NLS (628–669), KLKK sequence (630–633) and PEST sequence (638–658) overlap in AR protein sequence, yet, it has been suggested that initial ubiquitination of this motif may provide a signal for the binding of the 20S proteasome and coregulators which regulate AR translocation. Furthermore, mutation of lysine residues in the KLKK motif delays ligand-dependent translocation and results in colocalization of mutated AR with proteasome, implying that proteasome is also involved in nuclear translocation [166]. The MDM2 E3 ligase-mediated AR ubiquitination and degradation is regulated via several distinct mechanisms in PCa cells. The balance between protein phosphatase-1 and AKT kinase controls the phosphorylation status of Ser213 in both full-length AR and truncated AR-V7 variant, which is important for MDM2 interaction [66]. Similarly, PIM-1S mediated phosphorylation of AR at Ser213 destabilizes AR by recruitment of MDM2 [167]. AR phosphorylation at Ser578 and MDM2 at Thr158 and Ser186 by p21-activated kinase 6 (PAK6) was also reported to facilitate MDM2-AR interaction,

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enhance degradation of AR, and inhibit prostate cancer growth [168]. Nuclear receptors like glucocorticoid receptor, progesterone receptor, and estrogen receptor undergo rapid turnover in the presence of their ligands [61]. However, AR is not degraded after ligand binding, yet, it is upregulated in the presence of the androgen. Moreover, it has been revealed that mRNA level of AR was decreased by androgen treatment, while its protein level was augmented probably due to the stabilization of the ligand–receptor complex after androgen binding [61]. On the other hand, it was suggested that AR is ubiquitinated in response to androgen treatment. Treatment of androgen-dependent PCa cells with synthetic androgen resulted in the presence of mono- and tri-ubiquitinated AR species that are stable for 2 h after androgen treatment. Afterwards, there was a decline in AR stability, which was correlated with the accumulation of polyubiquitinated AR forms [61]. UPS is also involved in the regulation of transcription and AR transactivation. It has been revealed that a RING domain containing E3 ligase hPIRH2 (human p53-induced ringcontaining H2) interacts with AR, and also reduces HDAC1 levels, which in turn stabilizes histones in the acetylated state [48] and increases AR-mediated transcription. Meanwhile, ubiquitination of corepressors CtBP1/2 and NCoR/SMRT facilitates their proteasomal degradation releasing transcriptional repression allowing the interaction of the transcription complex with DNA [48]. It has been further suggested that UPS components may be recruited into AR transcription complexes by transducin-β-like (TBL) proteins. Indeed, TBL1 is recruited to the promoter of AR target genes after androgen treatment, and is required for transcriptional activation by AR [61]. Functionally, TBL1 recruits the ubiquitin-conjugating enzyme UbcH5 and E3 SNURF (small nuclear RING finger protein) [61]. SNURF binds to the hinge region of AR and enhances AR-dependent transcription by enhancing recruitment of general transcription factors and facilitating AR import to the nucleus. TBL1 and TBLR1 also serve as bridging factors

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for E6-AP, a steroid hormone receptor coactivator to the transcriptional complex [61]. E6-AP is a HECT domain containing ubiquitin E3 ligase and in conjunction with its E2 enzyme UbcH7 enhances the androgen-dependent transcriptional activity of AR and the degradation of NCoR [61]. Besides inducing AR transactivation, E6-AP has been shown to promote PCa by mediating the degradation of several tumor suppressors like p27 and the promyelocytic leukemia (PML) [103]. Both ubiquitination and proteasome itself have critical roles in promoter clearance, which is necessary for the transcription continuation by new androgen-bound AR molecules [48]. It is well evidenced that several AR coactivators interacts with the promoter of target genes in a transient and cyclic manner [48]. Moreover, E6-AP and MDM2 are recruited to the complex with or after DNA binding and promote ubiquitination of AR [48, 61]. A transient monoubiquitinated form of AR is stabilized by TSG101 (tumor susceptibility gene 101) [48]. Therefore, binding of TSG101 to monoubiquitinated AR prevents its polyubiquitination in order to promote transcription [48]. After a few rounds of transcription, ubiquitin ligases attach four ubiquitin molecules to each of the AR molecules for proteasomal degradation. Meanwhile, proteasome is actively involved in the transcription of AR target genes. After the induction of transcription, the ubiquitin chain arises giving a signal for the 19S proteasome to recruit the 20S proteolytic core [48, 61]. Consistently, it has been revealed that inhibition of the 26S proteasome demolishes AR transcriptional activity without affecting AR nuclear translocation [61]. Aside from MDM2 and E6-AP, there are other E3 ligases that interact with AR and regulate its stability and/or interaction patterns. Siah is a RING finger E3 ubiquitin ligase that mediates ubiquitination and degradation of several substrates involved in stress-induced signaling pathways such as cell cycle control, apoptosis, and DNA repair [169]. In humans, Siah has two isoforms, Siah1 and Siah2. Siah2 has been suggested to have tumor-promoting role since its inhibition blocks the development of several

P. Ballar Kirmizibayrak et al.

types of cancers [170]. Biochemical studies showed that Siah2 interacts with AR, and overexpression of Siah2 results in the K48-linked polyubiquitination and proteasomedependent degradation of AR [104]. Furthermore, NCoR1 corepressor is also a Siah2 substrate [105]. Surprisingly, knockdown of Siah2 has no effect on global levels of AR and NCoR1. Instead Siah2 targets the degradation of transcriptionally inactive AR/NCoR1 complexes associated with a small number of AR target genes. Removal of this inactive complex under androgen deprivation promotes recruitment of AR/coactivator complexes to enhance the transcription of only selective AR target genes, including those involved in lipid metabolism, cell motility, proliferation, and steroid biosynthesis [105, 106]. Furthermore, Siah2 protein level is tightly regulated in PCa cells. An RNA helicase named DHX15 was suggested to be a novel AR coactivator and was found to be upregulated in PCa tissues. DHX15 and Siah2 interact with AR, where DHX15 stabilizes Siah2 and enhances its E3 activity causing AR activation [171]. Similarly, an androgenic steroidogenic enzyme AKR1C3 implicated in CRPC development was also shown to inhibit Siah2’s auto-ubiquitination, which in turn increases Siah2 protein levels [172]. Consistently, nuclear Siah2 levels are first decreased upon androgen deprivation therapy but then increased upon disease recurrence [104]. Siah2 expression is also markedly increased in human CRPCs and Siah2 was shown to be required for the expression of selective AR target genes in prostate tumors in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model and in castrate resistant tumor xenografts. Therefore, it has been suggested that Siah2 is crucial for CRPC development [107]. In addition to AR, Siah2 regulates the levels of several proteins involved in cancer biology including DNA repair factors in PCa cells. In particular, Siah E3 enzymes target ATM whose expression is controlled by AR [169]. It has been also well demonstrated that Siah2 contributes the transcription, activity, and stability of HIF1α, a key regulator of hypoxia signaling [107]. Consistently, studies in TRAMP mouse model with Siah

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deficiency or Siah inhibition showed a correlation between reduced HIF1α levels and decreased cell proliferation [107]. This Siah-HIF1α axis has been suggested to be especially important in the development of more aggressive prostate tumors [107]. RNF6 is another RING finger E3 ubiquitin ligase that is upregulated in androgen-insensitive prostate cancer cells and in CRPC tissues. Silencing of RNF6 expression has shown to inhibit the growth of PCa cells under androgen deprivation and growth of xenograft tumors in castrated mice [134]. PIM-1L kinase induces Thr-850 phosphorylation of AR, which facilitates the binding of RNF6 to AR. RNF6 induces polyubiquitination of AR via non-canonical K6- and K27-linked chains. Moreover, the RNF6-AR interaction stabilizes AR and induces AR-mediated transcription under low androgen conditions [134]. The cullin–RING ubiquitin ligases (CRLs) are multi-subunit complexes that include a cullin scaffold protein, a RING domain protein (Rbx1 or Rbx2) that interacts with E2, and a substrate adaptor protein that determines substrate specificity. Speckle-type POZ protein (SPOP) is the substrate adaptor protein for the cullin3/Rbx1 CRL, and it selectively recruits substrates for their ubiquitination. Notably, SPOP is one of the most frequently affected genes by somatic mutations in PCa and it is reported to be mutated in up to 15% of prostate cancers [173, 174]. While overexpression of wild-type (wt) SPOP induces the ubiquitination and degradation of AR [106], knockdown of SPOP in PCa cells or hemizygous knockout of SPOP in mouse prostate tumor xenografts increases AR protein levels [106, 108]. PCa-related mutant SPOPs cannot bind with and degrade AR. Thus, the expression of mutant SPOPs increases AR stability and transcriptional activity, which in turn promotes cell proliferation in vitro in PCa cells and tumor growth in mice [106]. SPOP also induces the ubiquitination and degradation of SRC-3 [109], another coactivator of AR that is necessary for cancer cell proliferation, survival, metabolism, and metastasis. It has been shown that SPOP interacts with SRC-3 and promotes its Cullin3dependent ubiquitin-mediated degradation, which

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results in suppressed AR transcriptional activity. PCa-related SPOP mutations cannot interact with SRC-3 [106, 109]. Hence, PCa-related mutant SPOP-mediated increase of AR transcriptional activation may also be due to the elevation of SRC-3 levels. One another critical substrate of SPOP is wt ERG protein. Wt SPOP ubiquitinates and facilitates degradation of wt ERG, and mutant SPOPs related with PCa abrogate the SPOPmediated ERG degradation. Notably, the majority of TMPRSS2-ERG fusions encode truncated proteins resistant to SPOP-mediated degradation [88, 108]. Several studies revealed that there are other substrates of SPOP involved in PCa tumorigenesis. DDIT3 is an ER stress-responsive transcription factor functioning on the apoptotic execution pathways triggered by ER stress. DDIT3 is a SPOP-CUL3-RBX1 E3 substrate and SPOP mutations seen in PCa are defective in promoting its degradation [110]. TRIM24 is another substrate of SPOP and stabilized in PCa with SPOP mutations. On the other hand, TRIM28 interacts with TRIM24 and protects it from SPOP-mediated degradation. Consistently, TRIM28 promotes PCa cell proliferation and upregulated in aggressive PCa [111]. DEK, an oncogenic effector, is stabilized due to PCa-related SPOP mutations and promotes prostate epithelial cell invasion [112]. Likewise, SPOP induces senescence through targeting SENP7 deSUMOylase for degradation. SENP7 degradation increases senescence via increasing the SUMOylation of epigenetic remodeler heterochromatin protein 1 α (HP1α) and related epigenetic silencing. SENP7 was found to be upregulated in SPOP mutated PCa specimens [113]. EgIN2, a prolyl hydroxylase, hydroxylates proteins and its dysfunction facilitates PCa growth. AR transcriptionally upregulates EgIN2. Meanwhile, SPOP interacts with and mediates degradation of EgIN2 [114]. ATF2 (Activating Transcription Factor 2), a transcription factor that heterodimerizes with members of the JUN and FOS transcription factor families, is another bona fide substrate of SPOP-CUL3-RBX1 E3 ligase complex. It has been suggested that ATF2 might be an important mediator of SPOP mutation-induced cell proliferation, invasion,

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and migration [115]. Wt SPOP has been also reported to interact with c-Myc and promote its ubiquitination. Therefore, it has been suggested that in addition to amplification of c-myc locus in PCa, mutant SPOPs may increase c-Myc protein levels [116]. Very recently, fatty acid synthase enzyme (FASN), which is vital for lipid homeostasis via de novo fatty acid synthesis in PCa cells, was suggested to be a novel substrate of wt SPOP. Once again SPOP mutants are not capable of binding FASN and downregulation of SPOP levels increases the FASN expression and triggers lipid accumulation in PCa cells [117]. STUB1/CHIP (C-terminus of Hsp70interacting protein) is a U-box E3 ubiquitin ligase enzyme functioning in the proteostasis of AR and truncated AR-V7 variant proteins [118, 119]. Functionally, CHIP disassociates AR/AR-V7 from HSP70, which in turn facilitates AR/AR-V7 ubiquitination and degradation [118, 119]. CHIP also mediates ubiquitination and proteasomal degradation of PRMT5, a protein arginine methyltransferase, which regulates many cellular processes via epigenetic control. PRMT5 expression is upregulated in several human cancers and has role in cell proliferation, transformation, cell cycle progression in PCa cells suggesting that PRMT5 may serve as an oncoprotein [120]. Another E3 that targets AR for proteasomal degradation is MYLIP (myosin regulatory light chain interacting protein). MYLIP in conjunction with its E2 enzyme UBE2D1 ubiquitinates and targets AR for degradation. Inhibition of the interaction of MYLIP with UBE2D1 by expression of CNPY2 (canopy FGF signaling regulator 2) has shown to inhibit MYLIP-mediated AR ubiquitination and degradation. Consistently, the expression levels of CNYP2 and AR target genes are positively correlated in PCa samples [127]. The E3 NEDD4 (neural precursor cell expressed developmentally downregulated protein 4) is suggested to involve in AR degradation via PMEPA1, a NEDD4-binding protein. While PMEPA1 is a direct transcriptional target of the AR [175], its overexpression downregulates AR protein levels and AR target genes in PCa cells probably via recruitment of NEDD4 [176]. This

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was further supported by a study showing that AR downregulation by NEDD4 is PMEPA1 dependent [122].

5.4.2

Regulation of p27, p21, PTEN, and NKX3.1 Tumor Suppressors via E3 Ubiquitin Ligase Enzymes

SCF (Skp1-Cullin 1-F-box) type of E3 ligase complex is a member of RING-type E3 ligases and consists of four components: the linker component Skp1 (S phase kinase associated protein 1), RING finger protein Rbx, the scaffold protein Cullin1, and the interchangeable F-box protein that functions as a receptor for target proteins [3, 23]. It has been shown that there are 69 F-box proteins in human genome [6, 7], whereas there are only two RING family members of RING proteins that are in SCF E3 ligase complexes, RBX1/ROC1, and SAG/RBX2/ROC2/RNF7 [5, 8–10]. Three major subfamilies have been characterized based on the substrate binding domains: the FBXW (F-box with the WD40 motif), FBXL (F-box with the LRR motif), and the FBXO (F-box only) subfamily [7]. These F-box proteins target a wide range of substrates, including several tumor suppressor proteins for ubiquitin-mediated degradation and subsequently regulate cellular processes such as cell cycle, cell proliferation, apoptosis, angiogenesis, and metastasis [5]. Skp2 (FBXL) and β-TrCP are wellestablished F-box proteins involved in PCa tumorigenesis. It has been suggested that ubiquitination of cyclin-dependent kinase inhibitor, p27 is another main mechanism involved in PCa. p27 is downregulated in PCa mainly due to aberrant ubiquitin-mediated degradation and its low level correlates with tumor aggressiveness and poor prognosis in PCa [177, 178]. The F-box protein Skp2, a major regulator of p27 levels, is overexpressed in human prostate tumors [89, 90]. The inverse correlation between the low levels of p27 and increased expression of Skp2 might be explained by the findings

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suggesting that Skp2 acts as an oncoprotein probably through promoting the degradation of p27. Hence, the overexpression of Skp2 causes significant downregulation of p27 both in vivo in prostate glands of transgenic mice and in PCa cells [91, 92]. The Skp2/SCF complex also targets numerous other substrates for degradation, many of which are negative cell cycle regulators including p21, BRCA2, p57, and, p130, and FOXO1 (Forkhead box O1) [93, 94]. As p27, BRCA2 is inversely correlated with Skp2 expression in PCa and Skp2 overexpression has shown to reduce BRCA2 protein level [95]. Tsai et al. showed that Skp2 also targets DAB2IP (DOC-2/DAB2 interacting protein), which is a GTPase-activating protein, acting as a novel tumor suppressor gene [96]. Loss of DAB2IP is associated with the high risk of PCa [97]. While DAB2IP is regulated by Skp2-mediated proteasomal degradation in the prostate cell lines, it conversely suppresses Skp2 protein expression through AKT signaling [96]. The regulation of Skp2 expression level is very critical for prostate tumorigenesis since it targets several tumor suppressor proteins for degradation. Blocking PI3K/AKT signaling in PCa cells resulted in decreased Skp2 protein expression, suggesting that PI3K/AKT signaling regulates Skp2 expression [90]. PTEN is another regulator of the oncogenic functions of Skp2 and there is an inverse correlation between PTEN and Skp2 expression levels in PCa [90]. Downregulation of PTEN in prostate cancer DU145 cells has showed the presence of PTEN/AKT-dependent regulation of Skp2 and p27 [98]. Furthermore, androgen and AR are also involved in the regulation of Skp2. It has been demonstrated that Skp2 expression is inhibited by androgen in an AR-dependent manner [99] and androgen depletion was found to decrease PCa cell proliferation partly through downregulation of Skp2 [100]. Interestingly, Skp2, in turn, serves as an essential downstream effector of the AR in promoting proliferation [101]. p21 is another cyclin-dependent kinase inhibitor that serves as tumor suppressor. It can arrest cell cycle progression in G1/S and G2/M transitions [179]. Besides Skp2, a novel oncogenic E3 ligase, RNF126 has been linked with p21 degradation. RNF126 silencing was reported to increase p21

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levels by enhancing its stability, which in turn resulted in significant decrease on cell viability and delay on cell cycle G1-S progression in PC3 cell line. Therefore, it has been suggested that RNF126 promotes PCa cell proliferation by targeting the tumor suppressor p21 by ubiquitinmediated proteasomal degradation [135]. SAG/RBX2/ROC2/RNF7, the RING component of SCF E3 ubiquitin ligase was found to be overexpressed progressively from early-to-late stages of PCa. SAG/RBX2/ROC2/RNF7 knockdown suppresses the growth and survival of PCa cells due to accumulation of PHLPP1 and DEPTOR. PHLPP1 is a phosphatase that directly dephosphorylates AKT and DEPTOR, an endogenous inhibitor of both mTORC1 and mTORC2 functions as a tumor suppressor. Induced proteasomal degradation of PHLPP1 and DEPTOR by SAG/RBX2 leads to activation of PI3K/AKT/mTOR axis and thus SAG/RBX2 suggested to act as an oncogenic gene that promotes prostate tumorigenesis by activating the PI3K/AKT/mTOR signaling [125]. Consistently, silencing SAG/RBX2 in CRPC cells attenuates proliferation and enhances sensitivity of PCa cells to cisplatin treatment possibly due to accumulation of tumor suppressive proteins p21, p27 and NOXA [126]. NEDD4 family ubiquitin ligases, specifically NEDD4-1 and WWP2, are described as the E3 ubiquitin ligases for the tumor suppressor PTEN [123]. It has been shown that upregulation of NEDD4 can posttranslationally suppress PTEN in cancers [123]. While AR downregulation by NEDD4 is PMEPA1 dependent, PTEN downregulation by NEDD4 is independent [122]. Furthermore, it has been shown that monoubiquitination of PTEN at specific lysine residues promotes its shuttling into the nucleus and PTEN is excluded from the nucleus in many cancer cells including PCa [180]. While proteostasis of PTEN is critical in PCa, PTEN itself plays important roles on proteostasis of other proteins. It has been shown that PTEN competes with the F-box protein FBXL2 for IP3R3 (inositol 1,4,5-trisphosphate receptor) binding and PTEN loss promotes IP3R3 degradation by FBXL2 in PCa cells. Of the note, IP3R3 is

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the major player in Ca2+-dependent apoptosis and thus PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumor growth [181]. Additionally, nuclear PTEN directly enhances the tumor suppressive activity of the APC/C (Anaphase Promoting Complex/Cyclosome) complex, which controls cell cycle progression [182]. Interestingly, Skp2 is one of the APC/CDH1 substrates. As described before, PTEN has a regulatory role on Skp2 function, which might be partly attributed to the ability of PTEN to activate the E3 ligase activity of APC/CDH1 [182]. NKX3.1, which is downregulated in PCa is a transcription factor that acts as haploinsufficient tumor suppressor. The low correlation between the mRNA and protein levels of NKX3.1 in carcinoma indicates that posttranscriptional modifications of NKX3.1 play a critical role in prostate tumorigenesis [183]. It has been shown that the upregulation of NKX3.1 decreases cell proliferation in vitro and cancer growth in vivo [184, 185]. Phosphorylation status of certain amino acids on NKX3.1 directly regulates its stability. It has been revealed that protein kinase CK2 phosphorylates the N-terminal Thr89-93 residues of NKX3.1 and delays its ubiquitination and degradation [186]. Conversely, the level of NKX3.1 is affected by inflammatory cytokines such as TNF-α and IL-1β, which induce phosphorylation C terminal serine residues and ubiquitin-mediated degradation of NKX3.1 protein [128]. A RING-type E3-ubiquitin ligase, namely TOPORS (TOP1 binding arginine/serine rich protein) has been reported as the E3 enzyme responsible for the ubiquitination and proteasomal degradation of NKX3.1 [129]. Additionally, ubiquitination of NKX3.1 is also reported to alter its subcellular localization and protein–protein interaction [187].

5.4.3

Other Players of Prostate Cancer and Their Ubiquitin-Mediated Regulations

Besides Skp2, several other F-box proteins, as components of Cullin RING ligases, are

implicated in PCa tumorigenesis. F-box protein, beta-transducin repeat-containing protein (β-TrCP) is overexpressed in many cancers and possesses mainly oncogenic characteristics [121]. β-TrCP mediates the degradation of a variety of targets, including IκBα, IκBβ, and β-catenin [130]. In the absence of Wnt-1 signals, β-catenin is either retained in the cytoplasm via binding to E-cadherin or phosphorylated, ubiquitinated, and degraded through a process that is regulated by protein kinase glycogen synthase kinase-3 beta (GSK-3β) and β-TrCP [130]. β-TrCP also interacts with δ-catenin and regulates δ-catenin levels by ubiquitination and degradation. δ-catenin has been shown to promote E-cadherin processing involved in the epithelial cell–cell adhesion, which is also implicated in PCa progression. Hence, β-TrCP might be involved in angiogenesis by modulating δ-catenin levels in the PCa cell line [131]. One another substrate of β-TrCP is Gli2, a transcription factor functioning in the transduction of Hedgehog signals. Gli2 protein level, but not mRNA expression, is upregulated in PCa cell lines and primary tumors [132]. Intriguingly, β-TrCP is also implicated in the regulation of the AR transcriptional activity. AR inhibition therapies depletes RE-1 silencing transcription factor (REST), which is a mediator of AR actions on gene repression, and expression of REST is negatively correlated with disease recurrence after prostatectomy [133]. β-TrCP facilitates REST protein degradation and thereby affects the AR-induced gene repression [133]. Very recently, it has been revealed that β-TrCP regulates HIF-1α stability through abolishing the interaction between HIF-1α and the CHIP E3 ligase complex. Thus, β-TrCP stabilizes HIF-1α protein by antagonizing another E3 ligase and increases HIF-1α transcriptional activation in PCa cells by decreasing its ubiquitination [121]. The suppressor of cytokine signaling 2 (SOCS2), a substrate recognition module of Cullin5/Rbx2 ubiquitin ligases, acts as a tumor suppressor which is downregulated in advanced CRPC [188]. It has been shown that SOCS2/ Cullin5/Rbx2 ubiquitin ligase complex interacts with NDR1, a serine threonine kinase, and regulates its turnover via K48-linked

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ubiquitination in PCa [189]. NDR1 induces TNFα-stimulated NF-κB activity and suggested to function as an oncogene in PCa. Therefore, SOCS2 overexpression antagonizes NDR1induced TNFα-stimulated NF-κB activity [189]. Furthermore, a correlation has been reported between SOCS2 expression and AR expression in malignant tissue of PCa patients [190]. It has been further suggested that SOCS2 mediates the cross talk between androgen and growth hormone signaling in PCa [188]. This notion was supported with findings indicating that AR and STAT5 (signal transducer and activator of transcription 5 protein) cooperate to induce SOCS2 expression and consequentially, SOCS2 inhibits growth hormone activation of Janus kinase 2, SRC, and STAT5 in vitro in PCa cells [188]. Oncogenic transcription factor c-Myc is one of the frequently altered genes in human cancers, including PCa [191] and regulates cell proliferation and transformation. Overexpression of c-Myc found to lead the transformation of primary human prostate epithelial cells in vitro [192]. Similarly, prostate-specific overexpression of c-Myc promotes tumor development in mouse prostate [193]. Furthermore, upregulation of c-Myc is associated with PCa recurrence and poor prognosis [193]. The degradation of c-Myc by several E3 ubiquitin ligases, including Fbxw7, Skp2, Pihr2, and HUWE1 is one of the mechanisms to regulate c-Myc levels in cells [102]. The HUWE-1 E3 ubiquitin ligase enzyme (HECT, UBA and WWE Domain Containing 1, E3 Ubiquitin Protein Ligase) catalyzes both monoubiquitination and K6-, K48-, and K63-linked polyubiquitination of its substrates [192]. It regulates ubiquitin-mediated degradation of several substrates including p53 [102], BRCA1 [136], and Myc [137, 138]. HUWE-1 overexpression significantly reduces PCa cell proliferation and migration [139]. One another mechanism regulating c-Myc expressions is via the histone demethylase JMJD1A. JMJD1A promotes recruitment of AR to the c-Myc gene enhancer to enhance AR-dependent expression of c-Myc mRNA. Interestingly, JMJD1A also interacts with HUWE-1 E3 ligase and suppress

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HUWE-1-mediated c-Myc degradation. Concomitantly, JMJD1A knockdown antagonizes the proliferation and survival of PCa cells [194]. TRIM (tripartite motif-containing protein) ubiquitin ligase family is also associated with PCa. As described above, TRIM28 protects TRIM24 from SPOP-mediated degradation and promotes PCa progression [111]. TRIM25 is defined as one of the ubiquitin ligase enzyme responsible for the proteasomal destruction of ERG [195]. A putative E3 ligase TRIM68 is preferentially upregulated in PCa cells and enhances the transcriptional activity of AR in the presence of androgens via its interaction with TIP60 and p300, coactivators of AR [196]. In summary, indispensable roles of many E3 ubiquitin ligases have been reported in prostate tumorigenesis. Several other ubiquitin ligase enzyme families, including WW-HECT domain E3 family like WWP1, SMURF1, and SMURF2 proteins and TRAF E3s such as TRAF4 and TRAF6, have shown to be upregulated in several types of PCa [197, 198]. They are involved in prostate tumorigenesis by enhancing either proteolytic or nonproteolytic ubiquitination of several modulators including receptor tyrosine kinases, components of TGF-β tumor suppressor pathway, and membrane cytoskeletal linkers [199–202]. However, due to space limitations we were unable to include all of the PCa-related E3 ubiquitin ligases and their substrates in this chapter.

5.5

The Role of Deubiquitinating Enzymes in Prostate Cancer

As described earlier, AR is ubiquitinated by several E3 ubiquitin ligases resulting in either its proteasomal degradation or enhanced transcriptional activity. Thus, AR protein levels as well as AR transactivation are tightly regulated by the balance between E3s and DUBs in PCa (Table 5.2). Ubiquitin-specific protease 12 (USP12) is one of the most commonly overexpressed cancer-associated genes and has been identified as a novel positive regulator of AR. USP12, in conjunction with its cofactors,

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Uaf-1 and WDR20 deubiquitinates AR to enhance receptor stability and transcriptional activity [158]. USP12 also deubiquitinates MDM2, which in turn controls the levels of the p53 and AR in PCa. Therefore, USP12 levels are predictive of PCa development and patient’s therapy resistance, relapse, and survival [158– 160]. Functional analysis showed that silencing of USP12 significantly reduces PCa cell proliferation and induces apoptotic cell death [158, 203]. Similar to USP12, USP46 is responsible for increased AR stability and also regulates MDM2-p53 signaling pathway. Notably, USP12 and USP46 selectively target only the full length AR, but not the AR variants [160]. USP26 is another DUB functioning as a regulator of AR signaling. It binds to AR directly and deubiquitinates the receptor. As a nuclear protein, it interacts with AR in subnuclear foci and counteracts androgen-induced AR ubiquitination and regulates AR transcriptional activity [164]. DUBs associated with the 19S proteasome regulatory particle are emphasized as critical therapeutic targets in numerous cancer types. USP14, a novel regulator of AR, inhibits the degradation of AR via deubiquitinating in the androgenresponsive PCa cells. Hence, downregulation of USP14 suppresses cell proliferation and colony formation, and induces G0/G1 phase arrest in PCa cells [161]. Recently, inhibitors of USP14/ UCHL5 (DUBs) of the 19S proteasome have been shown to downregulate the expression levels of AR in androgen-dependent PCa cells [162, 204]. Similarly, inhibition of USP14 accelerates the ubiquitin-mediated degradation of AR also in estrogen receptor-negative/AR-positive breast cancer cells and significantly suppresses cell proliferation in these cells by blocking G0/G1 to S phase transition and inducing apoptosis [205]. USP7 has been described initially as a herpes simplex protein function in lytic infection and termed as Herpes-associated USP (HAUSP). The expression of USP7 is directly correlated to aggressiveness of PCa [145]. USP7 interacts with AR in an androgen-dependent manner and mediates AR deubiquitination [146]. The suppression of the USP7 expression has been

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suggested to attenuate cell proliferation in PCa cell lines [146]. Intriguingly, USP7 is necessary to facilitate androgen-activated AR binding to chromatin. A transcriptome profile analysis of USP7-knockdown LNCaP cells has further revealed the essential role of USP7 in the expression of a subset of androgen-responsive genes [146]. USP7 has been also associated in the regulation of tumor suppressors p53, PTEN, and FOXO [147]. Interestingly, USP7 is able to deubiquitinate both p53 and MDM2. However, it preferentially binds to and deubiquitinates MDM2 which leads to an increase in p53 degradation and consequent antiapoptotic function. However, the balance is reversed and USP7 stabilizes p53 especially under DNA damage, where ATM is activated [147]. USP7 is also implicated in PTEN nuclear exclusion [145]. It is well known that monoubiquitination of PTEN facilitates its shuttling into the nucleus and PTEN is excluded from the nucleus in many cancer cells including PCa [180]. Hence, USP7 catalyzes the deubiquitination of PTEN in the nucleus leading to its export from the nucleus [145]. Similarly, monoubiquitination of FOXO3 and FOXO4 tumor suppressor proteins facilitates their nuclear retention and induces their transcriptional activity. As for PTEN, the ubiquitination of FOXO is reversed by USP7 [148]. Ubiquitin-specific peptidase 9x (USP9x) is another DUB with several PCa-related substrates. Pre-B-cell leukemia homeobox-1 (PBX1) is an oncogenic transcription factor that induces PCa cell proliferation and confers to resistance against several common anticancer drugs. USP9x interacts with and stabilizes PBX1 protein via its deubiquitinase activity. Inhibition of USP9x markedly induces PBX1 degradation and promotes PCa cell apoptosis [149]. USP9x also binds to and deubiquitinates ERG. USP9x silencing results in increased levels of ubiquitinated ERG and is coupled with depletion of ERG [150, 195]. USP9x is also implicated in Insulinlike growth factor (IGF) signaling. IGFs induce proliferation of cancer cells and activate IGF-I receptor (IGF-IR) tyrosine kinase, which targets Insulin receptor substrates (IRSs). IGF/IGF-IR/ IRS axis is very important in PCa since IRSs in

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turn function in the activation of downstream signaling pathways, including the Erk1/2 pathway. Furuta et al. showed that silencing of USP9x decreases both IGF-IR and IRS-2 proteins by causing increased ubiquitination, and also suppresses activation of Erk1/2 pathway [151]. In addition to USP9x, ubiquitin-specific protease 15 (USP15) was also reported to interact with preferentially ubiquitin-conjugated IRS. IRS2 is monoubiquitinated by NEDD4 and the NEDD4–IRS2 interaction is maximizing the activation of IGF-I signaling and mitogenic activity in PC3 cells [124]. On the other hand, USP15 antagonizes the effect of Nedd4 on IRS-2. Therefore, USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination [163]. Ubiquitin-specific protease 2 (USP2) is another DUB with oncogenic feature and overexpressed in almost half of human PCa patients [140]. It is upregulated in response to androgen in PCa cells [141] and leads to an increase in the levels of deubiquitinated substrates such as fatty acid synthase, cyclin D1, acid ceramidase, and Aurora-A [140–144]. MDM2 and its homolog MDMX are also substrates of USP2 and unlike USP7; USP2 does not deubiquitinate p53. Moreover, the c-Myc oncogene found to be upregulated in a subset of USP2 overexpressing PCa cells. Overexpression of USP2 downregulates a set of microRNAs that collectively increase Myc levels by MDM2 deubiquitination and subsequent p53 inactivation [206]. Therefore, it is well evidenced that upregulation of USP2 protects cells from apoptosis [140]. USP10 is another androgen-regulated DUB, which is transcriptionally induced with the recruitment of AR to an intronic region [152]. High expression of USP10 has been significantly associated with poor prognosis of patients with prostate cancer. Furthermore, it interacts with AR and modulates its functions via deubiquitination [153]. USP10 subsequently regulates androgen-mediated signaling and cell growth. Indeed, silencing of USP10 has been

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shown to repress the androgen-dependent induction of several AR target gene, which suggests the presence of a positive feedback loop of AR signaling in PCa [154]. Additionally, USP10 was found to deubiquitinate monoubiquitinated H2A. Z, a variant of the core histone H2A [152]. Monoubiquitination of H2A.Z is associated with transcriptional silencing of AR [152, 155], yet USP10 positively regulates AR-mediated transcription of AR target genes [156]. Another important substrate of USP10 is p53 and USP10 regulates both p53 localization and stability [157]. Strikingly, USP10 mainly localizes in the cytoplasm and interacts with and stabilizes p53 in unstressed cell. Following DNA damage, USP10 is upregulated and also translocates to the nucleus and contributes to p53 activation [157]. USP10 has also been reported to have a repressive effect on p53 signaling by increasing the stability of GTPaseactivating protein-binding protein 2 (G3BP2) expression [154]. G3BP2 exports p53 to the cytoplasm, thereby reducing p53 activity. USP10 silencing inhibits G3BP2-induced cell proliferation and p53 nuclear export [154]. In summary, high level of USP10 expression was found to be strongly correlated with high levels of AR, G3BP2, and p53 in the cytoplasm [154]. There are several other DUBs, some of which are upregulated and some are downregulated in PCa. For example, ubiquitin-specific peptidase 39 (USP39) is significantly upregulated in PCa and knockdown of USP39 in PCa cells inhibits colony formation and tumor cell growth, and induces G2/M arrest and cell apoptosis probably via decreasing the transcriptional elongation and maturation of EGFR mRNA [207]. On the other hand, ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is downregulated in PCa via hypermethylation of its promoter. Its overexpression suppresses LNCaP cell growth probably via p53-mediated inhibition of AKT/PKB phosphorylation and also via accumulation of p27 [208]. It is obvious that DUBs have emerging potential as therapeutic targets in PCa therapy.

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5.6

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Ubiquitin-Like Modifiers Involved in Prostate Tumorigenesis

Although AR has been reported as one of the SUMOylated proteins [209], the consequence of AR SUMOylation is not yet clear. It has been suggested that SUMO modification might influence AR transcriptional activity positively or negatively in a target gene- and pathway-selective manner [22]. Furthermore, SUMOylation also affects the half-life of AR in the nucleus, given that a SUMOylation-deficient AR mutant exhibited a shorter half-life compared with wildtype AR [67, 210]. Both SUMO E2 conjugating enzyme UBC9 and SUMO-E3 ligase PIAS (protein inhibitors of activated STAT) expression levels are elevated in PCa [211, 212]. PIAS1 has been revealed to serve as oncogenic factor through regulation of tumor suppressor p21 and the antiapoptotic protein Mcl-1 [211]. Additionally, both UBC9 and PIAS1 have been shown to interact with AR [209, 212, 213]. PIAS1 may function as a coregulator of the AR since it binds to and stabilizes AR. The protection of AR from proteasomal degradation by PIAS enhances AR transcriptional activity and expression of AR targets [214]. Genome-wide analyses further showed that PIAS1 functions as a coregulator of chromatin-bound AR and that it interacts with FOXA1, a transcription factor facilitating the DNA binding of AR [213]. Intriguingly, FOXA1 has shown to be also modified by SUMO-2/3 in LNCaP prostate cancer cells. Mutation of the FOXA1 SUMOylation sites was shown to abolish its mobility and enhance its chromatin occupancy as well as its activity on AR-regulated gene locus in LNCaP cells [215]. Although the interaction of ectopic SUMO E2 conjugating enzyme, UBC9 has been shown to interact with AR and enhances the AR-mediated transcription, this effect of UbBC9 appears to be independent of its ability to catalyze SUMOylation of AR [209]. Hence, it has been further suggested that UBC9 modulates the transcriptional activity of AR target genes through

SUMOylation of AR cofactors [216]. In line with this, several AR coregulators have been reported to be SUMOylated. For instance, SUMOylation of coactivator GRIP1 regulates its ability to function as an AR coactivator since mutations on SUMOylation sites on GRIP1 resulted in attenuated ability to enhance AR-dependent transcription [217]. Interestingly, HDAC4 was shown to interact with and inhibit the activity of the AR in a SUMOylationdependent manner. Silencing of HDAC4 increases the activity of AR and PCa cell growth, which is correlated with decreased AR SUMOylation. Thus, HDAC4 suggested being a positive regulator for AR SUMOylation, revealing a deacetylase-independent mechanism of HDAC action in PCa cells [218]. Pontin, a component of chromatin-remodeling complexes, is another PCa-related protein that is SUMOmodified and interacts with UBC9. Intriguingly, androgen treatment significantly increases the SUMOylation of pontin, which is required for transcriptional activation of AR target genes, and associated with an increase in proliferation and growth of PCa cells [216]. There are several identified UBC9 substrates such as Flotillin-1 (Flot-1) that are not directly related with AR signaling. Flot-1, a protein that regulates cancer progression has been shown to be SUMOylated via UBC9 activity in metastatic PCa. It has been shown that mitogen induces the SUMOylation and nuclear translocation of Flot-1, which interacts with Snail, and inhibits Snail degradation promoting EMT. Furthermore, SUMOylation of Flot-1 by UBC9 in PCa with high metastatic potential positively correlated with the stabilization of Snail and the induction of Snail-mediated EMT genes in the metastatic PCa [219]. DeSUMOylating enzyme, SENP1 (SUMOspecific cysteine protease 1) has been reported to be upregulated in PCa tissues and cell lines. Furthermore, high levels of SENP1 have been linked to advanced pathological stages, higher Gleason grade, positive lymph node status, and PSA recurrence [220, 221]. While androgen induces rapid and dynamic conjugation of SUMO-1 to AR,

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SENP1 and SENP2 are efficient in cleaving AR-SUMO-1 conjugates. Their expression promotes AR-dependent transcription in a promoter-selective fashion. Silencing of SENP1 attenuates the expression of several AR target genes and blunted androgen-stimulated growth [222, 223]. It has been also revealed that androgen-induced AR binds to specific ARE region of the SENP1 promoter and enhances its transcription directly [224]. Thus, a positive feedback mechanism exists between AR and SENP1 [224]. Intriguingly, mutation on SUMOylation residues on AR did not decrease SENP1’s effect of AR-dependent transcription suggesting this effect is not mediated by AR SUMOylation similar as it was described before for UBC9. Indeed, it was shown that SENP1 modulates the transcriptional activity of AR via deSUMOylation of the coregulator protein HDAC1 by diminishing HDAC1’s deacetylase activity and ability to suppress transcription [225]. Besides AR signaling components, SENP1 directly modulates several important transcriptional mediators in prostate cells, including c-Jun, SMAD4, HIF-1α, and cyclin D1 [223, 226]. Interestingly, SENP1 increases c-Jun’s transcription activity in PCa and its effect is critically dependent on its deSUMOylation activity but independent of the SUMOylation and phosphorylation status of c-Jun [223]. Huang et al. showed that SENP1 deSUMOylates the CRD1 domain of p300, a well-known c-jun coactivator, and releases the cis repression of CRD1 on p300 [223]. It has been also revealed that overexpression of SENP1 enhances the cyclin D1 expression and cell proliferation in LNCaP cells [223]. SENP1 is also implicated in the regulation of SMAD4, which is an important tumor suppressor involved in EMT. Zhang et al. has been reported that SENP1 promotes EMT of PCa cells via regulating SMAD4 deSUMOylation [227]. There are several studies implicating the role of other ubiquitin-like modifiers like ISG15 and NEDD8 in PCa. ISG15 found to be expressed exclusively in high-grade PCa with high Gleason scores. Furthermore, most enzymes involved in ISG15 conjugation are upregulated in tumor

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samples of PCa patients and regulated in specific androgen-mediated, AR-dependent manner [228]. While silencing ISG15 expression was shown to result in marked attenuation of PC cell survival, its overexpression promotes PCa cell growth, indicating its oncogenic property [229]. Additionally, the ISGylation system controls AR mRNA and protein expressions, as overexpression of Ube1L as a limiting ISGylation factor in the androgen-sensitive PCa cell line leads to a significant AR upregulation, accompanied by an increased proliferation even under androgen deprivation [228]. NEDD8, another Ub-like modifier has been also linked with prostate tumorigenesis. Specific inhibition of NEDDylation pathway with MLN4924 (also known as Pevonedistat) was found to suppress the proliferation and clonogenic survival of PCa cells. Mechanistically, MLN4924 treatment inhibits cullin NEDDylation, by inactivating Cullin-RING E3 ligases (CRLs), which in turn results in accumulation of tumor-suppressive CRLs substrates, including cell cycle inhibitors (p21, p27, and WEE1) and NF-κB signaling inhibitor IκBα. In conjunction with this, MLN4924 triggers DNA damage, G2 phase cell cycle arrest, and apoptosis [230]. Although inhibition of NEDDylation is an emerging strategy for cancer therapy, a recent report claimed that NEDD8 might facilitate cell migration by augmenting the Src-mediated phosphorylation of caveolin-1, which consequently increases the migration of PC3 prostate cancer cells [231].

5.7

Autophagy and Prostate Cancer

Autophagy is involved in both cell survival and cell death, depending on cell type, genetic context, stage of tumor development, and nature of the stress inducer [232]. It serves as prosurvival mechanism in cancer cells to adapt to the hypoxic, nutrient-deprived, and metabolically stressful tumor microenvironment [233]. Contradictorily, many human cancers have defective autophagy mechanisms and several Atg genes like Beclin1, Atg4, Atg5, Atg7, as well as autophagy regulators,

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such as p53 and PTEN are considered tumor suppressors implicated in tumorigenesis [234]. Furthermore, the PI3K/AKT/mTOR pathway, which is a negative regulator of autophagy, is constitutively active in cancer cells [235]. When autophagy is defective protein aggregates and damaged organelles are accumulated, which in turn increases production of reactive oxygen species promoting genomic instability [235]. Several proteins having central role in the autophagic process or in the autophagy regulation have also been implicated in prostate tumorigenesis. The gene encoding the pro-autophagic Beclin1 protein is monoallelically deleted in many prostate tumors, and the expression levels of Beclin1 and LC3 proteins are lower in prostate adenocarcinoma than in prostate benign hyperplasia suggesting that autophagy might serve as a tumor suppressor mechanism in the prostate [236–238]. On the other hand, about 35% of PCa has upregulation of key autophagy proteins like LC3, which have been shown to be correlated with a high Gleason score, indicating that autophagy signaling may be important for cell survival in high-grade PCa [239]. A study using genetically engineered mouse model with prostate tumor-specific Atg7 deficiency and PTEN deletion has revealed that Atg7, an essential autophagy gene, cooperates with PTEN loss to drive PCa tumor progression [240]. Santanam et al. further suggested that autophagy deficiency generates smaller prostate tumors, delays tumor progression, and results in decreased AR signaling accompanied with induced ER stress [240]. p62/SQSTM1, which is accumulated by the inhibition of autophagy serves as a selective autophagy receptor during autophagy process that modulates the direction of ubiquitinated proteins for degradation [241]. Several studies implicated that p62 levels in PCa tissues are significantly higher than those in benign prostate hyperplasia and strong p62/SQSTM1 levels found to be linked with high tumor grade and high intensity of metastasis [239, 242]. p62/ SQSTM1 has been suggested to have a role in increased PCa cell survival and tumorigenesis [243]. Hence, Mitani et al. reported that hypoxia induces the interaction of p62 and AR in the

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cytosol, enhances AR sequestration from the nucleus, and decreases AR at protein level but not at mRNA level. Furthermore, autophagic degradation of AR is mediated by enhanced phosphorylation of p62, which in turn suppresses apoptosis of PCa cells in hypoxia [244]. Additionally, p62/SQSTM1 interacts with and increases the levels of HDAC6 in PCa. Consecutively, increased deacetylase activity of HDAC6 stabilizes α-tubulin and microtubules promoting EMT of PCa cells [245]. Autophagy is especially important for prostate tumorigenesis since it was found to be associated with androgen pathway as well as androgen deprivation responses. Very recently, Ye et al. suggested that testosterone inhibits autophagy by inducing phosphorylation and activation of 4E-BP1 and ULK1, which are effectors of mTORC1 signaling [246]. Concordantly, several core autophagy-related genes including ATG4B, ATG4D, ULK1, and ULK2 are transcriptional targets of the AR [247]. In parallel with this notion it has been found that reduced androgen levels lead to an increase on autophagy [248]. Moreover, androgen depletion also induces hypoxia in the tumor microenvironment of mouse models [249]. Hypoxia, in turn, induces autophagy in human tumor cells mainly via HIF1α-mediated gene expression and mTOR kinase inhibition. Similarly blocking AR in a long-term androgen deprivation therapy causes energy deficiency and activates AMP-dependent protein kinase (AMPK) [250]. AMPK increases AMP/ATP ratio by directly suppressing mTOR activities, which promotes fatty acid oxidation, glycolysis, and autophagy to match energy imbalance [249, 250]. GABA(A) receptor-associated protein like 1 (Gabarapl1) has been reported to mediate androgen deprivation-induced autophagy in PCa. AR transcriptionally regulates the level of Gabarapl1, which has a repressive role in autophagy. Xie et al. suggested that androgen deprivation downregulates Gabarapl1, and consequently increases the autophagy flux in PCa cells [251]. Furthermore, elevated expression level of Gabarapl1 was found to repress the PCa cell proliferation [251]. Intriguingly, silencing AR expression in AR positive PCa cell lines led to

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increased autophagy, and the ectopic expression of AR in AR negative cell lines resulted in decreased autophagy. Thus, the induction of autophagic response after androgen deprivation may facilitate the transition of the PCa cells into androgen-independence phase. Therefore, autophagy may be associated with resistance to chemotherapy in malignant PCa tumors and serves an important role in the development of CRPC [250]. Due to the prosurvival function of autophagy in cancer cells, autophagy inhibition might be a possible strategy to sensitize cancer cells to different therapies and to defeat therapeutic resistance in PCa [249]. Indeed, it has been shown that inhibition of autophagy sensitizes cells against a Src tyrosine kinase inhibitor that inhibits androgen-independent growth of PCa cells [243]. Moreover, inhibition of autophagy during androgen deprivation autophagy synergistically induces cell death in PCa cells [249]. On the other hand, several studies suggested that autophagy induction may sensitize cells to apoptotic stimuli and radiation in androgenindependent PCa cells [249]. In conclusion, autophagy might have contradictory effect in prostate tumorigenesis depending on the cellular features.

5.8

The Role of Unfolded Protein Response and ERAD in Prostate Cancer

Cancer cells are highly dependent on protein quality-control (PQC) mechanisms. Firstly, cancer genomes with several point mutations in protein coding sequences may cause expression of mutated proteins with folding problems. Secondly, most human solid tumor cells have more than two copies of one or more chromosomes [252]. The transcription of these extra chromosomes results in synthesis of several proteins in excess, increasing the level of unoligomerised proteins [252]. Thirdly, cancer cells often experience impaired ATP generation, hypoxia, and hypoglycemia, which may perturb

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ER function and trigger UPR [252]. UPR activation can promote survival of cancer cells by increasing the protein folding capacity of the ER and enhancing ERAD to alleviate ER stress and maintain ER homeostasis [253]. On the other hand, chronic ER stress often activates cell death mechanisms; however, tumor cells may hijack the PQC machineries to provide survival signals to escape cell death [254]. Both ER stress and UPR activation are involved in prostate tumorigenesis [255]. It has been shown that the three UPR branches are downregulated during prostate tumorigenesis in both the NKX3.1:PTEN mutant and Myc-overexpression PCa mouse models [256]. On the other hand, there are several studies indicating positive correlation between UPR and prostate tumorigenesis, where androgens regulate the expression of different ER stress-associated genes in PCa [255]. In a brilliant study, Saatcioglu group showed that androgen activates the IRE1α-XBP1 arm via AR transactivation [257]. Furthermore, chemical or biological inhibition of IRE1α significantly decreases PCa cell proliferation and PCa growth in multiple preclinical models in vitro and in vivo tumor formation [257, 258]. In contrast to the IRE1α branch, limited knowledge is present about the functions of PERK-eIF2α and ATF6 in PCa tumorigenesis. It has been suggested that androgens simultaneously downregulate PERK-eIF2α pathway in LNCaP cells. However, while androgen has shown to downregulate PERK activation and eIF2α phosphorylation, expression of downstream targets ATF4 and CHOP were found to be increased at the protein level [257]. Contradictory to this findings, in the CRPC model 22Rv1 cells androgens activate eIF2α phosphorylation [259]. Therefore, more studies are required to better understand the mechanisms of androgenmediated regulation of the PERK pathway and its involvement in prostate tumorigenesis. The proper functions of molecular chaperones are vital for cellular homeostasis. The cytoprotective molecular chaperones are upregulated in PCa [260]. The most frequently studied chaperone in prostate tumorigenesis is BiP/GRP78 and its increased expression is

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correlated with PCa recurrence and advanced pathological stages [261]. BiP also has been suggested as a cancer-specific cell surface marker, since it has been detected on the cell surface in several cancers, including PCa, but not on normal cells [260]. Furthermore, its translocation from ER to the cell surface is implicated in the hormonal resistance of PCa [255]. Moreover, prostate apoptosis response 4 (PAR-4), a pro-apoptotic protein secreted by cancer cells, was shown to bind to cell surface BiP and induce apoptosis of PCa cells [262]. Intriguingly, acutely stimulated ER stress has been shown to induce expression PAR-4, which switches protective autophagy to apoptosis in androgen-independent PCa cells [263]. Besides BiP, expression of several other molecular chaperones, such as HSP27, HSP70, and HSP90 have been associated with PCa. HSPs are expressed at low levels under normal conditions, but upregulated by cellular stress [260]. Several HSPs has an integral role in AR function as described earlier in this chapter. Briefly, AR resides in the cytoplasm via its interaction with a complex of chaperone (HSP90, HSP70 and HSP40) and cochaperone proteins [260]. HSP90 protects AR from degradation and maintains a conformational structure of AR with a high affinity for its androgen [260, 264]. Binding of androgen causes a conformational change in the AR-HSP90/HSP70 complex in the cytoplasm. Meanwhile, HSP27 undergoes a rapid phosphorylation and displaces HSP90 from the complex with AR. This promotes AR nuclear trafficking and facilitates regulation of AR transcriptional activity [260, 264]. The complex disassembles in the nucleus releasing AR, which then facilitates transcription of AR target genes. Importantly, HSP27 is highly upregulated in CRPC cells and its overexpression is associated with resistance to chemotherapy by inhibiting apoptotic pathways (X). Moreover, HSP27 facilitates progression to androgen-independent PCa, and regulates EMT in PCa [265]. Similarly, HSP70 and HSP90 are highly expressed in PCa and possess cytoprotective effects by suppressing proapoptotic proteins and upregulating the antiapoptotic ones [266, 267]. Furthermore, inhibition of HSP70 decreases both AR and AR-V7 expression

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[268]. Thus, HSP inhibitors might have potential anticancer activity against CRPC. UPR activation also enhances ERAD pathway. ERAD is a multistep process, including the recognition and targeting of substrates, ubiquitination, retrotranslocation, and proteasomal degradation [269]. Recently, ERAD has been also implicated in PCA tumorigenesis. First evidence came from a study that aimed to identify novel androgenresponsive genes by sequencing of LongSAGE libraries [270]. In this study Romanuik et al. showed that SVIP (Small VCP interacting protein), the first identified ERAD inhibitor is one the downregulated genes by androgen in PCa cells [270]. Furthermore, gp78, an ER membrane resident E3 ubiquitin ligase was found to be specifically expressed in PCa rather than normal prostate tissue [271]. Similarly, elevated expression of p97/VCP, the key protein involved in retrotranslocation step of ERAD has been implicated in poor prognosis of PCa [272]. Consistently, overexpression of p97/VCP in PCa cells increases cell proliferation, migration, and invasion [273]. In line with all these observations, ERAD pathway was recently shown to be regulated by androgen [274]. Androgen induces the mRNA levels of ERAD ubiquitin ligases gp78 and Hrd1, as well as p97/VCP and its cofactors Ufd1-Npl4; however it abolishes ERAD inhibitor SVIP expression via AR transactivation. Furthermore, androgen treatment increases the degradation rate of ERAD substrates, which is positively related prostate tumorigenesis especially in terms of migration and malignant transformation [274]. In summary, ER chaperones, UPR and ERAD appear to be among the key players in prostate tumorigenesis and therapy resistance, making them of considerable interest as novel valuable therapeutic targets.

5.9

Conclusion Remarks

In this chapter we have described proteostasis and its roles in prostate tumorigenesis. The data that have accumulated indicate that proteostasis plays an important role in PCa by modulating multiple signaling pathways such as AR, AKT, PTEN, p27, Myc, mTOR, Wnt, and NF-κB. Given the

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pivotal role of proteostasis in regulating various steps of prostate tumorigenesis and metastasis as well as tumor cell apoptosis, targeting all aspects of proteostasis including proteolytic or nonproteolytic ubiquitination, proteasome function, ubiquitin-like modifiers, autophagy, PQC, heat shock chaperones and UPR could be considered as potential therapeutic targets for PCa. However, an increased number of clinical trials are warranted in order to validate them as viable therapeutic targets for PCa treatment. Acknowledgments Work by PBK is supported by the Scientific and Technological Research Council of Turkey (TUBITAK, SBAG-108S056/114S062), Ege University internal funds, BAGEP Award of the Science Academy with funding supplied by Pfizer-Turkey, COST Action (PROTEOSTASIS BM1307), and by COST (European Cooperation in Science and Technology).

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6

Resistance to the Proteasome Inhibitors: Lessons from Multiple Myeloma and Mantle Cell Lymphoma Maria Gonzalez-Santamarta, Grégoire Quinet, Diana ReyesGarau, Brigitte Sola, Gaël Roué, and Manuel S. Rodriguez

Abstract

Since its introduction in the clinics in early 2000s, the proteasome inhibitor bortezomib (BTZ) significantly improved the prognosis of patients with multiple myeloma (MM) and mantle cell lymphoma (MCL), two of the most challenging B cell malignancies in western countries. However, relapses following BTZ therapy are frequent, while primary resistance to this agent remains a major limitation for further development of its therapeutic potential. In the present chapter, we recapitulate the molecular mechanisms associated with intrinsic and acquired resistance to BTZ learning from MM and MCL experience, including mutations of crucial genes and activation of prosurvival signalling pathways inherent to malignant B cells. We also outline the preclinical and clinical evaluations of some potential druggable targets associated to BTZ resistance, considering the most meaningful findings of the past 10 years. Although our M. Gonzalez-Santamarta · G. Quinet · M. S. Rodriguez (*) ITAV-IPBS, CNRS USR3505, Toulouse, France e-mail: [email protected] D. Reyes-Garau · G. Roué Lymphoma Translational Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona (Barcelona), Spain B. Sola Normandie University, INSERM UMR1245, UNICAEN, Caen, France

understanding of BTZ resistance is far from being completed, recent discoveries are contributing to develop new approaches to treat relapsed MM and MCL patients. Keywords

BTZ resistance · Proteasome · Ubiquitin · Mantle cell lymphoma · Multiple myeloma

6.1

Introduction

Proteolysis is tightly regulated in eukaryotes through the superposition of sophisticated molecular mechanisms to ensure protein homeostasis. One of the major proteolytic activities is driven by the 26S proteasome that holds a catalytic core particle (CP) or 20S [1]. The proteolytic activity of the 26S proteasome requires the previous ubiquitylation of protein targets mediated by a cascade of thiol-ester reactions implicating at least 3 enzymes named activating (E1), conjugating (E2) and ubiquitin ligases (E3). Removal or remodelling of ubiquitin chains condition the stability, localisation and function of the modified target proteins. The ubiquitin tagging step and the 26S-mediated proteolysis constitute the Ubiquitin–Proteasome System (UPS). Some proteins directly degraded by the CP do not require ubiquitin tagging and are therefore destroyed by an ubiquitin-independent process. The CP can also include proteasome subunits that are specifically involved in the immune response, constituting the immunoproteasome. Furthermore,

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_6

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the CP can be associated to other regulatory subunits such as 11S, which have specialised cellular functions [1]. In sum, the proteasome acts as a central hub of cellular proteolysis, having an impact on multiple processes such as cell cycle, DNA repair, cell differentiation, immune response, amino acid recycling or apoptosis. For this reason, the proteasome became a privileged target for drug development to treat diverse disorders including cancers, infections and inflammation-related diseases, among others [2]. The proteasome inhibitor (PI) bortezomib (BTZ), also known as Velcade, was, in 2003 and 2006, the first Food and Drug Administration (FDA)-approved PI for the treatment of two haematological malignancies: multiple myeloma (MM) and chemotherapy-resistant mantle cell lymphoma (MCL), respectively. Despite the success of BTZ therapy, inherent and acquired resistance in patients were observed, encouraging the development of a new generation of PIs, as well as small molecules targeting enzymes of the UPS. Full understanding of the mechanisms underlying BTZ resistance in cancer is a prerequisite to design new strategies to recover sensitivity to these agents, or to use alternative treatments to lower apoptosis threshold in BTZ-resistant cells. To elucidate these mechanisms several laboratories have characterised a number of patient-derived MM and MCL cell lines with natural or acquired resistance to BTZ. In this chapter we summarise mechanisms of PI resistance reported in the last decade. Even if some of these acquired resistance mechanisms have not yet been confirmed in patients, their discovery may have an impact in upcoming clinical studies. We also review potential strategies to overcome PIs resistance mechanisms, including the use of new signalling pathways inhibitors regulating protein homeostasis.

6.2

Cancers Treated with Proteasome Inhibitors

Resistance to proteasome inhibitors has been observed in various cancer types including haematological, pancreatic or breast cancer [3]. Two of the best responding cancers are MM and MCL and for this reason, more knowledge

has been accumulated on BTZ resistance for these haematologic disorders [4].

6.2.1

Multiple Myeloma

MM is a plasma cell malignancy with bone marrow (BM) infiltration of clonal cells and monoclonal immunoglobulin protein in the serum and/or urine of patients. Genomic techniques have allowed a better understanding of the genetic abnormalities of MM by providing a better landscape of this collection of diseases with a common clinical phenotype [5]. Several genetic alterations including chromosomal translocations of the immunoglobulin heavy chain (IGH) gene leading to the overexpression of D-type cyclins, have been considered as primary events. Not less important are the secondary mutations and clonal evolution. The most frequent mutations occur in KRAS, NRAS, FAM46C, DIS3 and TP53, among others. These mutations affect multiple signalling pathways by altering the mRNA levels but also protein expression and stability. In the past decade, this knowledge has contributed to remarkable changes in the clinical practices, such as the implementation of more effective therapies including new classes of drugs like PIs. The combination of BTZ with immunomodulatory drugs (IMiDs) such as lenalidomide or dexamethasone are currently among the most effective treatments in MM (see Sect. 6.4). The success of BTZ as a MM treatment underlies its broad impact on the stability and activity of vital cellular factors.

6.2.2

Mantle Cell Lymphoma

MCL is an aggressive non-Hodgkin lymphoma (NHL) arising from pre-germinal centre of mature B cells and is typically incurable due to the inevitable development of drug resistance, leading to the worst prognosis among NHL subtypes [6]. Classical MCL cells show minimal mutations in the IGH variable region gene (IGHV) and express the transcription factor SOX11. Patients present tumours in lymph nodes or extra-nodal

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sites and cells overexpressing cyclin D1 are prone to acquisition of additional abnormalities in cell cycle, DNA damage response, or cell survival pathways, leading to a more aggressive disease behaviour. Less typical are leukemic non-nodal MCL developed through the germinal centre with IGHV somatic hypermutation and minimal SOX11 expression. These patients present MCL cells in peripheral blood, BM and spleen. Leukemic non-nodal MCL behaves in a more indolent way with genetic stability over time. Secondary genetic abnormalities, such as TP53 mutations, result in a more aggressive disease associated with poor outcome. Since BTZ was approved by the FDA in 2006 for the treatment of relapsed/ refractory (R/R) MCL, numerous phenomena have been described to explain innate or acquired resistance observed in more than half of patients [7]. It is known that the development of resistance to BTZ in MCL is an adaptive process, which takes place gradually and includes metabolic changes and/or deregulated (re)activation of adaptive processes like plasmacytic differentiation, autophagy, or improper activation of intracellular signalling pathways such as PI3K/AKT/ mTOR axis or NF-κB, among others.

6.2.3

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also drive proteolysis in other cellular compartments such as the nucleus [1]. Given the role of the proteasome in the degradation of many critical cellular factors, its potential as therapeutic target attracted the interest of many pharmaceutical companies. Approved in the 2000s by the FDA, BTZ has been used for decades as one pivotal treatment in MM and MCL. However, its association with neuropathy and the acquisition of resistance in the clinics highlighted the need to develop new PIs that would be more effective, less toxic and would reduce the occurrence of resistance. Each of these aspects was considered for the development of second generation PIs such as marizomib (MRZ), carfilzomib (CFZ), ixazomib (IXZ) and oprozomib (OPZ) [3] (Table 6.1). Unlike BTZ, some of them target all the catalytic sites of the proteasome, like MRZ. They carry a different administration way and reversibility than BTZ, hence reducing off-target effects and toxicities in patients. MRZ and OPZ are in early clinical development, and CFZ and IXZ have been already approved in combined treatment for R/R MM. However, preclinical adaptation to these new agents has already been reported, strengthening the need for alternative strategies to face PI resistance [8].

Proteasomes and Chemical Inhibitors

6.3 Proteasomes are macromolecular proteolytic complexes with distinct roles under multiple physiologic or pathologic situations. The 26S proteasome is composed by a 19S regulatory particle that recognises ubiquitin chain as degradation signals [1]. The catalytic core or 20S subunit contains 7 α and 7 β subunits of which β5, β2 and β1 hold the chymotrypsin-like, trypsinlike and peptidyl-glutamyl peptide-hydrolysing activity. Alternative β subunits named β5i, β2i and β1i are expressed in haematopoietic cells in response to pro-inflammatory signals such as cytokines or γ interferon and integrate the immunoproteasome. The 20S core can also associate with 11S, another regulatory particle also known as PA28, REG or PA26 which contributes to the action of the immunoproteasome but can

Molecular Origin of the Resistance to Bortezomib

In the last decade, several molecular mechanisms involved in BTZ resistance have been proposed. During the progression of the disease, complex genomic alterations promote the activation of different signalling cascades that contribute to the development of the resistant phenotype. These include defects in the initiation/regulation of cellular stress, cell differentiation, apoptosis and autophagy, in combination with mutations and alterations in the expression of the drug target. On the other hand, microenvironmental factors and epigenetics can be another source of inherent resistance mechanism, as these events can modulate the expression of critical genes, including

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Table 6.1 Proteasome inhibitors PI Bortezomib

Action Reversible

Family Boronate

Carfilzomib

Irreversible

Epoxyketone

Oprozomib CFZ oral analogue Ixazomib

Irreversible

Epoxyketone

Reversible

Boronate

Marizomib

Irreversible

β-Lactone

ONX0914

Irreversible

Epoxyketone

Target β5c β5i β1c β5c β5i β5c β5i β5c β5i β5c β2c β1c β5c β5i

IC50 (nM) 7 4 74 5 33 36 82 3 31 2.5 26 330 28 280

Data presented in the table have been compiled from [4]

tumour suppressors [9]. The acquired resistance to BTZ is also multifactorial including, among others alterations, the levels of expression of proteasome subunits, crosstalk with other proteolytic pathways or overexpression of efflux pumps (Fig. 6.1).

6.3.1

Inherent Resistance

In this part of the chapter, we will review available data about molecular mechanisms that have been proposed so far to be at the origin of the inherent BTZ-resistant phenotype in MM and MCL.

6.3.1.1

Mutations in PSMB5 Proteasome Subunit PSMB5 mutations are known to lower PI binding capacity and to impair the chymotrypsin-like catalytic activity of the 20S proteasome [10, 11] giving a benefit under PI stress. However, both in in vitro and in vivo settings, mutations were detected only in tumour cells that received heavy PI-based therapies, suggesting that the selected mutations emerge lately during the process of clonal selection besides the apparition of the resistant phenotype. Moreover, in vivo, at the time of relapse, cells exhibiting PSMB5 mutations could partially or totally disappear, questioning the role of such mutations at late stages of the disease [12]. Finally, the relevance of PSMB5 gene mutation in BTZ resistance has

recently been challenged. Soriano and colleagues have shown that proteasome activity is dispensable in BTZ- and CFZ-resistant MM cell lines suggesting that PSMB5 mutations are likewise not required or involved in the development of BTZ resistance [10] (Fig. 6.1). In support, no mutations of PSMB5 were found for years in MM primary cells, even using targeted or high-throughput sequencing techniques on large cohorts of patients including refractory patients or in relapse. The relevance of PSMB5 mutations and their functional impact were suggested recently. Four PSMB5 mutations were detected in a single MM patient that received prolonged BTZ-based treatments (Table 6.2) [12]. According to the Darwinian model of myeloma evolution, mutations evolved independently in different tumour subclones. For instance, C63Y and A27P are lost during the course of the disease, whereas A20T and M45I are maintained longer. When tested in vitro, all mutations were functionally relevant and provided PI resistance but at different degree according to both the mutation itself (A20 and M45 having a higher impact than C63 and A27), and the tested PI (BTZ, CFZ or IXZ). The response pattern was similar for BTZ and IXZ, but not for CFZ. CFZ response was less affected by PSMB5 mutations, likely due to its unique structure and binding [12]. No mutations of PSMB5, PSMB6 and PSMB7 were ever described in CFZ-adapted MM cell lines [10].

mutations in the β5 subunit of the proteasome. Arrows indicates up- or downregulation of the different molecular mechanisms when compared with non-pathological conditions. See main text for further details

Resistance to the Proteasome Inhibitors: Lessons from Multiple Myeloma and. . .

Fig. 6.1 Molecular mechanisms associated to BTZ resistance in acquired and resistant MCL and MM cell models. The pathways involved are: UPS and ALS degradation systems, UPR response, apoptosis, B cell differentiation, cell cycle regulation and

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Table 6.2 PSMB5 mutations associated to BTZ resistance in MCL and MM Gene PSMB5

Mutation c.322G>A

Protein p.A49T

c.247A>G c.322G>A c.310A>G c.310A>G c.235G>A c.256G>C c.312G>C c.365G>A

p.T21A p.A49T p.M45V p.M45V p.A20T p.A27P p.M45I p.C63Y

Tumour cells KMS-11 OPM-2 8226

Pathology MM

References [13]

MM

[11]

8226a AMO Primary cells

MM MM MM

[4] [10] [12]

The indicated cell lines have been exposed to a continuous pressure of BTZ; except those marked with a, exposed to PR-924, a selective inhibitor of the immunoproteasome

6.3.1.2 Apoptosis Failure Defective apoptosis signalling is a key oncogenic mechanism of drug resistance in haematological malignancies, mainly attributed to the deregulation of B cell lymphoma-2 (BCL-2) family members. This family of proteins is composed by prosurvival proteins such as BCL-2, BCL-XL, MCL-1, BCLW and BFL1/A1, as well as proapoptotic factors, represented by multidomain (BAX, BAK and BOK) and BH3-only (BIM, PUMA, NOXA, BAD, BID, BMF, BIK and HRK) proteins. Once activated upon cytotoxic or stress signals, the BH3-only proteins interact with their prosurvival counterparts, leading to the release and oligomerisation of BAX and BAK, permeabilisation of the mitochondrial outer membrane, and the cytosolic release of apoptogenic factors, culminating in the activation of the caspase family of proteases and ultimately, cell death [14]. In MCL cells, BTZ has been described to evoke intracellular accumulation of MCL-1, which harbours a PEST sequence at the origin of its targeting to the proteasome for its degradation. As MCL-1 can physiologically interact with and block the proapoptotic signalling of NOXA, which is transcriptionally activated upon cell exposure to BTZ, the increase in MCL-1 levels can counteract NOXAmediated activation of BAK, thus delaying the onset of cell death. Therefore, blocking NOXA expression or inhibiting MCL-1 was used to modulate the response to BTZ in MCL [15] (See Sect. 6.4). Despite concomitant overexpression of several antiapoptotic proteins of the BCL-2 family, MM cells depend primarily on MCL-1 for survival as

demonstrated by the use of small-molecule MCL-1 inhibitor and the knockdown of MCL-1 [16, 17]. MM cells are tightly dependent on their microenvironment known to promote MCL-1 expression in plasma cells. For example, bone marrow stromal cells (BMSCs) provide survival signals such as interleukin-6 (IL-6), vascular endothelial growth factor and insulin-like growth factor. IL-6 upregulates MCL-1 transcription and induces MCL-1 dependence [18]. Recently, it has been shown that the long non-coding RNA (lncRNA) H19 is present in the serum of MM patients and that an H19/miR-29b-3p axis promotes MCL-1 translation and BTZ resistance [19]. Thus, MCL-1 is certainly an important target for coping with MM drug resistance.

6.3.1.3 Signalling Cascades The NF-κB pathway is activated via canonical and non-canonical signalling mechanisms. The canonical pathway regulates inflammatory responses, immune regulation, and cell proliferation, whereas the non-canonical signalling cascade leads to B cell maturation and lymphoid organogenesis. These pathways regulate the expression of genes involved in cell survival and tumour-promoting cytokines. Therefore, its activation has a profound impact in tumorigenesis. The NF-κB pathway can be potentially targeted and is expected to have a high impact on the viability of malignant B cells, due to its interplay with other crucial pathways activated during B cell differentiation, such as B cell receptor (BCR), PI3K/AKT/mTOR, and toll-like receptor (TLR) signalling axes. Constitutive NF-κB activity is often present in MCL and MM. The inhibition of

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NF-κB is a primary mechanism to induce cell apoptosis after BTZ treatment and plays a role in evading the effect of this treatment in BTZ-resistant phenotypes [20]. A high NF-κB activity was found in tumour cells of BTZ refractory MM patients and in in vitro models of cell adhesion-mediated drug resistance (CAM-DR), reinforcing the notion that the NF-κB pathway signals BTZ resistance [21, 22]. In MCL, this constitutive NF-κB signalling and consequent lack of response to BTZ has been linked to a proteasome-independent degradation of the intrinsic NF-κB inhibitor, IκBα [20]. However, a number of studies have pointed out a lack of correlation between NF-κB activity and BTZ resistance status [23]. NF-κB pathway is also regulated by casein kinase 2 (CK2). CK2 is a multifaceted serine/ threonine kinase involved in several cellular processes, and is overexpressed and overactive in many blood tumours. CK2 regulates signalling cascades and molecules that are targeted by BTZ. For instance, it modulates IκBα protein turnover, p53 function, AKT activation, and has a role in the control of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) (see Sect. 6.3.2.3). Inhibition of CK2 enhances BTZ cytotoxic effect in MCL cell lines by down modulating NF-κB and signal transducer and activator of transcription 3 (STAT3) signalling cascades and by potentiating the proteotoxic effects of proteasome blockade. Altogether these results suggest that levels of CK2 are involved in MCL resistance to BTZ [24]. The BCR includes a heterodimer of CD79A/B molecules, and CD19, a key co-receptor. The upregulation of those molecules have been proposed to promote BTZ resistance in MCL cells. While BCR regulates cell survival and proliferation of MCL cells, in MM it has been only linked to monoclonal gammopathy of undetermined significance (MGUS), a premalignant phase of MM [25]. A human phospho-kinase array further pointed out an overexpression of phosphorylated BCR kinases LYN, LCK, and YES as well as a sustained downstream activation of PI3K/AKT/mTOR axis in BTZ-resistant cells. Among these kinases, LYN was functionally associated with the resistance

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phenotype, rending cells more sensitive to the SRC kinase inhibitor dasatinib and allowing to synergistic activity of the dasatinib/BTZ combination in vitro [26]. In MCL, the redox status has also been pointed out as a crucial mediator of BTZ efficacy, as PIs lead to the generation of large amounts of reactive oxygen species (ROS), modulating at least in part the transcription of NOXA and thus contributing to the cytotoxic activity of proteasome inhibition [27]. The nuclear factor NF-E2 p45-related factor 2 (NRF2) was identified as a key regulator of this response. Indeed, while under physiological conditions it is sequestrated by Kelch-like ECH-Associated Protein 1 (KEAP1) in the cytosol, when KEAP1 is oxidised by ROS, NRF2 is released to the nucleus where it initiates the transcription of genes involved in the adaptive oxidative stress response. Upon BTZ exposure, BTZ-sensitive MCL cells display a sharp increase in the expression of NRF2 target genes, as well as genes related to protein ubiquitylation or proteasome components, while resistant tumours show minimal changes. Accordingly, an elevated expression of NRF2 target genes at the basal level, predicts a poor sensitivity to proteasome inhibition [28]. In line with this, a recent study has highlighted the capacity of ROS to modulate some cancer stem cells (CSCs)-like subpopulations in MCL cell lines and primary cultures and to regulate cell sensitivity to BTZ. Authors showed that O2- was involved in the inhibition of CSC-like cells and in the sensitisation of MCL to BTZ, while H2O2 favoured a CSC-like phenotype, impairing BTZ-induced cell death [29]. This process was associated with transcriptional regulation of two O2- and H2O2 targets, namely MCL-1, and ZEB-1, a WNT-regulated transcription factor that interfered with MCL response to chemotherapeutics. This resulted in the activation of proliferation-associated genes including MYC and CCND1 and the induction of an antiapoptotic gene signature [30].

6.3.2

Acquired Resistance

PI-acquired resistance has multifactorial and interconnected causes and PI-resistant cells show cross-resistant profiles. Among the main

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mechanisms recognised in MM and/or MCL are upregulation of 20S proteasome subunits including β5c, downregulation of 19S proteasome subunits and overexpression of efflux pumps. Adaptive metabolic changes, modulation of the unfolded protein response, and alteration of autophagy signalling contribute also to PI resistance in MM cells.

6.3.2.1

Overexpression of Proteasome Subunits Beside PMBS5 mutations, overexpression of PMBS5 and (to a lesser extent) PMBS6 are frequent alterations found in MM cell lines adapted to increased concentrations of BTZ [11, 31, 32]. PSMB5 is overexpressed in one MM patient with clinical resistance to BTZ, compared to three BTZ-sensitive patients [33]. Franke and colleagues demonstrated a tight relationship between impaired proteasome activity carried on by a mutated β5c subunit and the β5c subunit overexpression. In cells harbouring homozygous PSMB5 mutations, the upregulation of β5c subunit was even more important when compared to cells harbouring heterozygous mutations. The authors proposed a model in which, the prolonged exposure of MM cells to BTZ leads first to the appearance of PSMB5 mutations, resulting in decreased BTZ binding. In turn, mutant cells compensate this reduced proteasome activity by upregulation of the β5c subunit. Moreover, in those BTZ-resistant MM cells the upregulation of β5c is associated with the downregulation of β5i to balance the total proteasome units and impairs any possible remaining BTZ-inhibition [11]. In two recent studies, the involvement of the 19S subunits of the proteasome has been highlighted. To identify genes controlling the sensitivity and adaptation of MM cells to CFZ, Acosta-Alvear and colleagues used a new-generation shRNA library screening [34]. They found that the knockdown of several subunits of the 20S proteasome core (including β5c) provides a strong sensitisation to proteasome inhibition. Paradoxically, the genetic depletion of most of the 19S regulatory components confers a marked resistance. They further confirmed that shRNAs-mediated knockdown of PMSC1, PMSC6, PMSD1, PMSD2, PMSD6 and PMSD12

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leads to resistance towards BTZ and CFZ in MM cell lines. Importantly, the authors showed that PSMC2 levels in MM patients seem predictive for the response towards CFZ-based therapy. In the second report, Shi and colleagues used a genome-scale CRIPSR-Cas9 library to identify genes associated with BTZ resistance. They validated PSMC6 depletion as the strongest hit conferring BTZ resistance in MM cells [35]. PSMC6 deficiency resulted in reduced BTZ ability to regulate chymotrypsin-like activity of β5c through changes in the proteasome structure. Mutations in other members of the PMSC group also individually impart BTZ resistance albeit less potently. No mutation of PSMC6 has been reported so far but the analysis was performed on a cohort of untreated MM patients [35]. Recent results from our laboratories comparing BTZ-adapted MCL cell lines and their parental counterparts revealed a reduced expression of 19S proteasome subunits in BTZ-resistant cells. Strikingly, when autophagy was blocked with inhibitors such as bafilomycin A or chloroquine, the level of those proteasome subunits increased in resistant cells only, suggesting an autophagymediated degradation. In MCL cell lines that naturally resist to BTZ, the accumulation of proteasome subunits after the chemical inhibition of autophagy was proportional to the level of BTZ resistance observed. The proteasome degradation by autophagy was named proteaphagy and has been observed in response to starvation or proteasome inhibition in several biologic models including human cells [36, 37]. Quinet et al. showed that proteaphagy can contribute to develop resistance to BTZ in MCL cells since inhibited proteasomes are degraded and BTZ reduces its impact on proteasomes and cell death. In other words, BTZ-resistant cells bypass proteasome inhibition relying on autophagy through degradation of proteasomes and perhaps other cellular proteins [38].

6.3.2.2 Metabolic Adaptation Metabolic reprogramming is a hallmark of cancer that has emerged as an attractive target for novel therapeutic strategies for cancer treatment. O-GlcNAcylation is an abundant, dynamic and

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nutrient-sensitive post-translational modification that corresponds to the addition of an O-linked β-N-acetylglucosamine (O-GlcNAc) moiety to the serine or threonine residues in proteins in response to changes in the hexosamine biosynthetic pathway. As this latest depends on various essential nutrients and metabolic intermediates like glucose, glutamine, acetyl-coA, and UTP, it provides an ideal machinery for cells to sense and respond to a variety of microenvironmental conditions [39]. Little was known about its role in MCL, until the recent study of Luanpitpong and collaborators who demonstrated that O-GlcNAcylation of tBID promoted apoptosis in MCL cells exposed to BTZ, and that this process could be amplified by co-treatment with the antifungal drug kenoconazole, an O-GlcNAcase inhibitor that blocks tBID ubiquitylation and subsequent proteasomal degradation [40]. ABC (ATP-binding cassette) transporters such as ABCB1 (multidrug resistance, MDR1 or P-glycoprotein, P-gp) mediate drug resistance by alterations of the absorption and elimination of xenobiotics and drugs. ABCB1 expression correlates with poor prognosis, treatment resistance and aggressiveness of the MM disease [41]. ABCB1 protein is overexpressed in CFZ-resistant compared to sensitive MM cell lines [10]. ABCB1 was expressed by circulating malignant plasma cells of MM patient at diagnosis and its expression increases along the course of CFZ treatment [42]. Overexpressed ABCB1 protein limits the proteasome-inhibiting activity and clearance of poly-ubiquitinated proteins by CFZ and reduces its cytotoxicity. Importantly, ABCB1 overexpression affects the cytotoxic activity of epoxyketone-type PIs (CFZ) significantly stronger than non-epoxyketone PIs (BTZ). Drugs targeting ABCB1 may resensitise MM cells to PI, in particular CFZ. Soriano and colleagues analysed by a combined quantitative and functional proteomics approach CFZ- and BTZ-adapted MM cell lines [10]. They found that resistance to BTZ/CFZ was independent of proteasome activity but relied on energy metabolism, redox homeostasis, protein folding and degradation. PI-resistant cells adapted themselves to a very low proteasome activity

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while continuing to synthesise immunoglobulins. In turn, the level of metabolic intermediates involved in oxidative glycolysis (pyruvate kinase), redox state (superoxide dismutase, glutathione peroxidase, peroxiredoxin), mitochondrial respiration (cytochrome c) were increased thus maintaining high stringent redox conditions. In agreement with previous studies, authors also confirmed that the most upregulated proteins in CFZ-resistant cells were ABCB1 [41, 42] and the heat-shock proteins HSP70 and HSP90, whose transcriptional regulator, HIF1, is involved in BTZ resistance in MM cells [43]. Conversely, the positive apoptosis regulators BAX, CASP and DIABLO were downregulated [10]. Similarly to Soriano et al., Dytfeld and colleagues conducted a comparative proteomic profiling of R/R patients vs. naïve MM patients [44]. In the proteomic signature associated with BTZ resistance, four sets of proteins were characterised including proteasomal proteins, some factors regulating the redox status, proteins signalling apoptosis, and proteins involved in the inflammation response. In particular, regulatory and catalytic components of the proteasome, including part of the 11S complex, were upregulated. The antioxidant thioredoxin, peroxiredoxin, and thioredoxin reductase were upregulated whereas annexins A1 and A2, that regulate the apoptotic process, were downregulated. PI resistance may thus be alleviated by manipulating the redox status and the energy metabolism. Although not revealed by the above proteomic studies, NRF2 seems to be a node for BTZ and CFZ resistance in MM. As it maintains redox homeostasis by inducing antioxidant and detoxification genes and by modulating energy metabolism [45], NRF2 indirectly regulates: (a) chaperoning activity [46]; (b) redox, metabolic and translational reprogramming [47]; and (c) activation of prosurvival autophagy. These major functions are supported by clinical data showing NRF2 upregulation in a subgroup of relapsed patients [47]. Other studies showed that high glutathione (GSH), whose levels are controlled by NRF2, dampens BTZ toxicity in MM cells [48].

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In the last decade, numerous studies have indicated that components of the BM stroma, extracellular matrix (ECM), cytokines, chemokines and growth factors are involved in BTZ resistance in MM cells [49]. The membrane protein myristoylated alanine-rich C-kinase substrate (MARCKS) is a protein that plays an important role in cell adhesion, spreading and invasion, and is crucial for metastasis [50]. MARCKS is overexpressed in MM cell lines and is involved in the cross-resistance to the farnesyltransferase inhibitor R115777 and BTZ, as well as in MM patients that do not reach a sustained response to BTZ therapy. In addition, the inhibition of MARCKS phosphorylation increases cytotoxicity in BTZ-resistant cells [51]. The insulin-like growth factor IGF-1, known as a growth factor for MM cells [52], is produced by plasma cells, and is present in the BM microenvironment. IGF-1 has been proposed to promote proliferation and drug resistance in MM cells through the activation of MAPK and PI3K/ AKT-signalling pathways [49]. According to these data, the IGF-1/IGF-1R signalling axis was detected to be upregulated in three BTZ-resistant MM cell lines, compared to parental cells. Kuhn et al. proved that a small molecule responsible for the inhibition of IGF-1R has the capacity to sensitise BTZ-resistant MM cells to the proteasome inhibitor [53].

6.3.2.3 Protein Homeostasis Because of their high capacity to synthesise and secrete immunoglobulins, MM cells exhibit an expanded ER network and an increased ability to cope with unfolded or misfolded proteins that accumulate in the ER. These conditions are referred to as ER stress. As a consequence, MM cells activate the UPR pathway as an adaptive strategy and are rendered dependent on this mechanism for their survival [54]. ER stress upregulates three UPR signalling branches: activating transcription factor 6 (ATF6), protein kinase R (PKR)-like ER kinase (PERK)-ATF4 and inositol-requiring enzyme 1 (IRE1)-X box binding protein 1 (XBP1) which suppress global translation and promote protein folding and degradation. During ER stress, ATF6 translocates into the

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nucleus and activates the XBP1 promoter allowing an upregulation of the protein. At the same time, IRE1 oligomerizes and autophosphorylates, resulting in the activation of its endonuclease activity that cleaves XBP1 mRNA. This results in a frameshift that modifies the unspliced inactive XBP1 form (XBP1u) into an active XBP1s form. XBP1s acts as transcription factor and activates genes encoding protein folding and chaperones (see Sect. 6.3.2.5). Previous studies done on cohorts of MM patients and confirmed in vitro on BTZ-adapted cell lines, defined a ‘low IRE1XBP1’ phenotype that predicts a poor response to BTZ [55, 56]. Moreover, XBP1 knockdown experiments in MM cell lines showed correlation with BTZ resistance, as the suppression of XBP1 lowers both the basal ER stress and the ER stress due to proteasome inhibition [56]. High expression of deubiquitinating enzymes (DUBs) and autophagy-related proteins have been detected in MM patients resistant to BTZ. These alterations in enzymes that are involved in deubiquitinating misfolded/unfolded proteins and in the turnover of proteins by the autophagy– lysosome system (ALS) suggest an important role of ubiquitin signalling pathways in BTZ resistance. Niewerth et al. showed that inhibition of USP14 and UCHL5 promotes apoptosis and helps to overcome BTZ resistance in MM patients [4]. Another enzyme that can be regulated to recover sensitivity to BTZ is the ubiquitinconjugating enzyme H10 (UbcH10). Wang et al. proved that through the expression of hsa-miR631, the negative regulation of UbcH10 transcription prevents MM cells to develop resistance against proteasome inhibitors [57].

6.3.2.4 UPS–ALS Crosstalk Eukaryotic cells have two interconnected mechanisms for protein degradation and removal of misfolded proteins and aggregates, the UPS and the macroautophagy (here referred to as ALS). Autophagy functions by double-membrane vesicles known as autophagosomes which sequester cytosolic proteins, followed by fusion with lysosomes for degradation. Autophagy is involved in several human diseases, such as neurodegenerative diseases and cancer [58]. While it

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appears to be tumour suppressive in normal cellular homeostasis, autophagy can mediate tumour cell survival under stress conditions [59]. For many years, UPS and ALS pathways were thought to function independently, but the recent observation that impairment of either pathway impacts the other has suggested that these two proteolytic systems do collaborate [58]. It is thought that upon proteasome inhibition, autophagy is initiated as a survival mechanism to eliminate UPS substrates [60], and thus, upregulated autophagy could play a role in BTZ resistance [61]. The ubiquitin-binding cargo autophagy receptor sequestrosome 1 (SQSTM1) or p62 is a critical link between UPS and ALS [62]. In CFZ-adapted MM cells, SQSTM1/p62 is elevated triggering a prosurvival autophagy however through two different mechanisms according to the settings. In the first model, the pluripotencyassociated transcription factor Kruppel-like factor 4 (KLF4), is overexpressed and contributes to CFZ resistance by activating the SQSTM1 gene [47]. In the second model, elevated levels of SQSTM1/p62 conduct CFZ resistance through both a prosurvival autophagy involving GABARAPL1 upregulation and the activation of the NRF2 pathway [47]. However, since KLF4 is also a target of NRF2, both factors could cooperate for maintaining a high level of SQSTM1 transcription. The activation of NRF2 occurs through the activation of the PERKeukaryotic translation initiation factor2α (eIF2α) axis of the UPR [47]. As stated before (see paragraph 3.2.2), NRF2 is a major actor for PI resistance through the reprogramming of metabolism and the control of redox status [10, 47]. In addition to eIF2α, another NRF2 target and translation initiation factor, eIF4E3 is overexpressed in CFZ-resistant cells, and increased EIF4E3 expression was found in a subgroup of patients with chemoresistant minimal residual disease and in R/R patients [47]. Interestingly, deficiency in BIM has been shown to contribute to adaptive resistance to BTZ in MM cells, mediated by increased autophagy, and that autophagy disruption by means of chloroquine could sensitise these cells

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to BIM-mediated cell death [63]. Of special interest, upon exposure to pharmacological inhibitors of autophagy like chloroquine or bafilomycin A, BTZ-resistant MCL cells can undergo a blockade of proteaphagy, leading to the stabilisation of proteasome subunits, and the recovery of BTZ sensitivity [38]. Importantly, sensitivity to autophagy inhibitors requires a significant degree of BTZ resistance, thus suggesting that modulating proteaphagy with specific inhibitors may be considered as a strategy to resensitise resistant cells to PI.

6.3.2.5 Stress Signals Subsequent studies have been focused to determine the interplay between the deregulation of the intracellular stress machinery and MCL loss of sensitivity to BTZ. A key defect in BTZ-dependent cell death was first identified within the ER stress pathway, because its activation in MCL cells exposed to BTZ is required to elicit NOXA transcription [64]. ER homeostasis is controlled by the immunoglobulin heavy chain binding protein (BiP), also referred as 78-kDa glucose-regulated protein (Grp78). BiP/Grp78 forms a large multiprotein complex with a set of other ER molecular chaperones, including the Hsp90 ER homologue, Grp94, protein disulphide isomerase, calcium binding proteins, and cyclophilin B [65]. Under non-stressed conditions, BiP/Grp78 binds to and maintains in an inactive monomeric state the ER transmembrane PKR-like ER kinase, IRE1, and ATF6 [66]. After proteasome inhibition, the accumulation of polyubiquitylated and misfolded proteins within the ER lumen leads to BiP/Grp78 dissociation from the luminal domains of these sensor proteins and the initiation of UPR (see Sect. 6.3.2.3) [67]. This coordinated cellular response initially promotes cell survival, but ultimately triggers apoptosis if cytoprotective mechanisms are overwhelmed. Supporting the observation that accumulation of some HSP proteins can promote cellular resistance to PIs, a correlation has been made between acquired and primary resistance to BTZ in MCL, and intracellular accumulation of BiP/Grp78 in proteasome-compromised MCL cells [68]. For this reason inhibitors of HSP90

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have been used to improve the BTZ-mediated cell death induction in BZT-resistant cells (see Sect. 6.4) [68]. In line with this study, deregulated expression of several cytosolic HSP70 family members has also been associated with BTZ resistance in MCL [69] and MM [70]. Supporting a role of HSP70 protein in MCL cell resistance to chemotherapeutic agents, B cells modified to overexpress cyclin D1, the genomic hallmark of MCL, presented strong alterations in their response to growth factor withdrawal [71]. Acquired BTZ resistance is also attributed to the upregulation of other HSPs such as HSP90 and HSP27, that promotes NF-κB activity [21].

6.3.2.6

B Cell Differentiation Programme It has been proposed that MM cells also achieve BTZ resistance via the dedifferentiation of plasma cells. A pool of XBP1low/ tumour progenitors or CSCs pre-exists drug treatment, contributing to tumour diversity [55]. CSCs recapitulate maturation stages between B cells and plasma cells. Tumour B cells and pre-plasmablasts survive PI treatment preventing cure, while maturation arrest of MM before the plasmablast stage enables progressive disease on PI treatment. These tumour progenitors should be targeted to allow a complete cure for MM patients. Although MCL was originally considered a neoplasm of naive lymphocytes that have not passed through the germinal centre (GC), a significant number of cases present somatic mutations in the immunoglobulin genes, suggesting that they have been in contact with the antigen in the GC. Some cases were also described that presented evidences of plasmacytic differentiation in patients harbouring the characteristic t(11;14) translocation [72]. A couple of studies have related the resistance to BTZ with the plasmacytic differentiation programme in MCL cells. Plasma cells are the final effectors of humoral immunity, which are devoted to the synthesis and secretion of immunoglobulins. BTZ-resistant MCL cells display some of the characteristics of the plasma cells, such as the overexpression of interferon regulatory factor

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4 (IRF4) and elevated membrane levels of CD38 and CD138 cell surface markers, but they do not present splicing of XBP1 or increase in immunoglobulin production [6]. It is postulated that during the acquisition of resistance to BTZ, the balance between the protein load and the proteasomal activity is key. When plasmacytic differentiation is induced through stimulation of TLR9 receptor, the sensitivity to BTZ changed throughout the process of B cell differentiation to final plasma B cell phenotype with the capacity to manage the future increase in protein loads. Since the cells do not acquire full secretory capacities, this mechanism granted them with an advantage against the antitumour activity of BTZ. Once MCL have completed this process of differentiation, they return to a BTZ-sensitive state [6]. Mouse xenograft models of MCL using different cell lines with different sensitivity to BTZ, including cells with acquired or primary resistance to the PI, demonstrated a tight correlation between increased tumorigenicity of BTZ-resistant tumours with a plasmacytic differentiation phenotype including upregulation of IRF4, PR domain zinc finger protein 1 (PRDM1/BLIMP-1) and CD38, and loss of the B cell markers PAX5 and CD19 [73]. Of note, beside its role as a transcription factor that represses the expression of proteins needed for B cell identity and proliferation, and that helps to drive B cells through their final differentiation stage to become antibody-secreting cells, BLIMP-1 is also a mediator of NOXA-induced apoptosis in MCL and is required for BTZ-induced apoptosis in MCL cell lines and primary tumour samples [74]. This finding further strengthens the interplay between NOXA and plasmacytic differentiation in BTZ-mediated anticancer activity in MCL.

6.4

Potential Targets to Recover Proteasome Inhibitors Sensitivity

Given the resistance to PIs observed in some patients, alternative drugs have been tested to overcome resistance as single or combinatorial

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treatments. Many agents have shown promising preclinical results in terms of safety, specificity and efficacy to treat PI-resistant MM and MCL cells. Already existent or new therapeutic drugs are used to tackle the adaptation of the cells to PIs (Table 6.3). Cellular mechanisms involved in PI resistance are hence targeted by a wide range of drugs, some of the most relevant are described below.

this effect was transient due to the compensatory upregulation of USP24 who sustains MM cell survival. By contrast, a novel compound EOAI3402143 with a dual USP9X/USP24 inhibiting activity displayed promising antimyeloma activity [80]. Associated with the inhibition of USP14 activity, VLX1570 led to an extended survival of xenografts models of myeloma including BTZ-resistant cells [81].

6.4.1

6.4.2

Deubiquitinases

Because proteasome degradation and UPR implicate ubiquitin signal, targeting factors regulating ubiquitin signal could potentially contribute to increase the sensitivity to BTZ and therefore be an option to overcome PI resistance. In this context, DUBs have been considered as therapeutic targets to overcome BTZ resistance. P5091 is a selective inhibitor of USP7, a DUB that targets the E3 ligase HDM2. Treatment of MM cell lines and primary cells from MM patient with P5091 inhibited growth and induced apoptosis in tumour cells including those resistant to BTZ, without affecting the viability of normal PBMCs [75]. The 19S regulatory particle inhibitor b-AP15 selectively blocked deubiquitinating activity of USP14 and UCHL5 without inhibiting proteasome activity. This led to an activated UPR and an inhibited tumour growth, in MM xenografts resistant to BTZ [76]. The antitumoral effects of b-AP15 were also demonstrated in MCL [77]. Another USP inhibitor, SJB3-019A tackling USP1 showed synergic toxicity in MM when combined with BTZ [78]. Song et al. found contribution of RPN11, a proteasomal deubiquitinase, in MM pathogenesis using gene expression analysis. Pharmacological inhibition of RPN11 with O-phenanthroline (OPA) or capzimin blocked proteasome function, induced apoptosis in MM cells and overcame resistance to BTZ [79]. USP9X is also highly expressed in MM patients and in particular in those with as short progression-free survival. The partially selective USP9X inhibitor WP1130 induced apoptosis through the downregulation of MCL-1. However,

Transport Modulators

PI cellular intake is regulated by transport modulators, and treatment efficiency is directly linked to intracellular concentration of drug. Specific inhibitors of ABCB1 such as verapamil and reserpine showed increased proteotoxic stress in CFZ-resistant MM cells [10]. The two HIV inhibitors nelfinavir and lopinavir counteract ABCB1 overexpression in CFZ-resistant MM, via the modulation of the mitochondria transition pore. This promising preclinical results encourage the clinical evaluation of both treatments [42]. Thanks to its capacity to modulate the UPR pathway, nelfinavir has also attested a safe and promising activity in combination with BTZ and/or dexamethasone, in a phase I clinical trial involving advanced BTZ-refractory MM patients [82].

6.4.3

Autophagy Signalling

Ubiquitin and ubiquitin-like molecules play an important role on the regulation of autophagy. In BTZ-resistant MCL cells, the use of autophagy inhibitors such as bafilomycin A and chloroquine, and the p62 inhibitor verteporfin revealed increased cytotoxicity and synergistic activity with BTZ, mediated by the reversion of the proteaphagic process [38]. Blocking autophagy also leads to multiple changes in the cell such as the accumulation of IκBα, which prevents BTZ-induced NF-κB activation. The combination of bafilomycin A with BTZ might therefore contribute to increase cytotoxicity in MM cells in this manner [83]. Finally, orlistat, a fatty acid

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Table 6.3 Drugs currently used to recover sensitivity to BTZ Drug name(s) P5091 b-AP15 SJB3-019A O-phenanthroline Capzimin WP1130 EOAI3402143 VLX1570 Verapamil Reserpine Nelfinavir Lopinavir Bafilomycin A Chloroquine Verteporfin Orlistat Perifosine Dactolisib NVP-BEZ235 Temsirolimus (Torisel) Deforolimus (Ridaforolimus) Perillyl alcohol Selinexor Ibrutinib (Imbruvica) Degrasyn Obatoclax cAMP Lenalidomide (Revlimid) Pomalidomide Dexamethasone JQ1 CPI203 PROTACs (ARV-825) Birabresib (OTX015) Ricolinostat (ACY-1215) Decitabine Vorinostat

Mechanism Ubiquitin signal Ubiquitin signal Ubiquitin signal Ubiquitin signal Ubiquitin signal Ubiquitin signal Ubiquitin signal Ubiquitin signal Drug transport Drug transport Drug transport Drug transport Autophagy Autophagy Autophagy Autophagy mTOR/Akt mTOR/Akt mTOR/Akt mTOR/Akt

Molecular target USP7 USP14 and UCHL5 USP1 RPN11 RPN11 USP9X, USP5 USP9X and USP24 USP14 ABCB1 ABCB1 ABCB1 ABCB1 Vacuolar ATPase ATP6V1A Lysosome p62 Fatty acid synthase Akt, PI3K PI3K, mTOR mTOR mTOR

Pathology MM MM MCL MM MM MM MM MM MM MM MM MM MM MM, MCL MCL MCL MCL MM MCL MCL MCL

References [75] [76, 77] [78] [79] [79] [80] [80] [81] [9] [9] [42, 82] [42] [38, 83] [38] [38] [84] [85] [86] [87] [88]

NF-κB NF-κB NF-κB NF-κB Bcl-2 proteins Bcl-2 proteins IMiD

TG2 signalling XPO1 BTK STAT, DUBs NOXA Mcl-1 CRBN, TNFSF11, CDH5, PTGS2 CRBN, TNF, PTGS2 NR3C1, NR0B1, ANXA1, NOS2 BRD4

MM MCL MM MM MCL MCL MCL MM MCL

[89] [90] [91–93] [94] [15, 95] [96] [97, 98]

MM MM MM

[99–101] [99, 100, 102] [103, 104]

BRD4

MM MCL

[73, 104]

BRD4 and other BET BRD2,3,4

MM MM

[105] [101]

HDAC6 DNA methyl transferase HDACI

MM MCL MCL MM MCL

[102, 106] [107] [108]

IMiD Glucocorticoid Immunosuppressant BET bromodomain inhibitors BET bromodomain inhibitors BET-specific target BET bromodomain inhibitors Epigenetic regulators Epigenetic regulators Epigenetic regulators

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synthase inhibitor that affects autophagy, sensitises MCL cells to BTZ through the inhibition of the autophagic degradation of NOXA [84].

6.4.4

Oncogenes and Signalling Pathways

Proteasome inhibition has pleiotropic effect within the cells. It affects a wide range of cellular factors, including important signalling cascades, oncogenes or epigenetic regulators. This modulation of critical factors during BTZ treatment can hamper the apoptotic effect of PI, and leads to resistance. As described above, adaptation of signalling pathways including mTOR and NF-κB, or modulation of important oncogenes such as BCL-2 or MYC proteins, have been directly linked to BTZ resistance in MM and MCL. Therapeutics agents targeting these factors have been proposed to overcome BTZ resistance.

6.4.4.1 mTOR/AKT Pathway Modulating mTOR/AKT, key proteins of a complex signalling cascade, has successfully reverted malignant cell adaptation to BTZ in preclinical studies. The dual PI3K and mTOR inhibitor dactoslisib (NVP-BEZ235) showed great results in MCL BTZ-resistant cells lines [86]. A new generation of mTOR inhibitors, such as temsirolimus and deforolimus have been also proposed for MCL treatment but limited clinical impact was obtained [87, 88]. The AKT inhibitor perifosine combined with BTZ revealed increase cytotoxicity in R/R MM patients previously treated with BTZ [85], warranting its use in BTZ-resistant cancers. 6.4.4.2 NF-kB Pathway Since NF-κB pathway is overactivated under BTZ treatment, its modulation has been used to treat PI-resistant MM and MCL. Selinexor is a reversible inhibitor of exportin 1 (XPO1) that blocks the nuclear export of NF-κB/IκBα complexes leading to NF-κB pathway inactivation [109]. Selinexor associated with BTZ or CFZ overcomes acquired PI resistance in MM models

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and patients. MM drug resistance to CFZ and BTZ is enhanced in hypoxic conditions [90]. In such conditions, selinexor is capable to overcome PI resistance [90]. Because Transglutaminase 2 (TG2) is a calcium-dependent enzyme, calcium blockers have been proposed to hamper high NF-κB expression in BTZ-resistant cells. The combination of such molecules with BTZ indeed improves cytotoxicity in MCL [89]. Degrasyn has been also proposed to target constitutive NF-κB and STAT3, and combined treatment with BTZ showed a synergic apoptosis in MCL [94]. Within the BCR pathway, Bruton’s tyrosine kinase (BTK) inhibitors lead to NF-κB inactivation and downregulation of MYC. Ibrutinib (Imbruvica), a first-in-class BTK inhibitor, was approved by the FDA in 2013 as second line treatment for MCL patients [91]. This promising drug leads to the best complete remission rate as a single agents when compared to the other three drugs licensed at that time for use in MCL (BTZ, temsirolimus (Torisel) and the IMiD drug, thalidomide-derivative lenalidomide (Revlimid)) [110]. Promising preclinical results were obtained combining ibrutinib and BTZ in MCL and MM BTZ-resistant cells [92]. Ibrutinib alone or in combination with dexamethasone went very recently through a phase II trial with R/R MM patients, with some positive results [93].

6.4.4.3 NOXA/BCL-2 Proteins BH3 mimetic compounds like obatoclax showed great results alone or in combination with BTZ in relapsed MCL. By neutralising BTZ-induced MCL-1 accumulation, obatoclax sensitises MCL cells to low doses of the PI [15]. A phase I/II study substantiated the tolerance of a combined treatment BTZ/obatoclax in patient with R/R MCL. However the synergism supported by the preclinical studies was not confirmed in patients [95]. Treatment of MM cell lines including BTZ-resistant cells and primary cells with cyclic adenosine monophosphate (cAMP) induces downregulation of MCL-1 and degradation of cyclin D1. Moreover, a synergy between BTZ and cAMP showed promising results in a murine xenograft model, warranting this strategy to overcome BTZ resistance [96].

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6.4.4.4 IRF4/MYC Signalling As described before, exacerbated de novo IRF4 signalling has been associated with MCL resistance to BTZ in vitro and in vivo, thus supporting the preclinical/clinical evaluation of IRF4targeting drugs. Following first observations in MM preclinical models where it efficiently suppressed IRF4-expressing cells [111], lenalidomide was found to be effective in vitro and in vivo in BTZ-resistant MCL tumours harbouring high IRF4 levels, while sparing IRF4 negative cases. Lenalidomide activity relied on a functional interaction with the component of the E3 ligase complex, cereblon (CRBN), and CRBNdependent lowering of IRF4 expression, leading to the blocking of B cell differentiation programme, as shown by increase in PAX5 and loss of CD38 and BLIMP-1. Consequently, BTZ-lenalidomide combination could overcome BTZ resistance [73]. Lenalidomide single agent was further validated in a phase II trial involving R/R MCL patients, including cases refractory to BTZ [97]. In contrast, in MM patients, therapy combining lenalidomide with BTZ failed in phase II trial [98]. Among the IMiD family, pomalidomide has been approved by the FDA for the treatment of R/R MM with at least two prior treatments, including BTZ. When added to BTZ or MRZ in combination with low dose dexamethasone, this agent went through successful phase I trial on heavily pretreated, high risks relapsing MM [99, 100]. Beside these approaches, the inhibition of the IRF4 target gene, MYC, in BTZ-resistant MCL cultures has been studied either with siRNAmediated gene knockdown or with treatment with an inhibitor of BRD4, a bromodomain and extraterminal domain (BET) protein. BET proteins mainly regulate epigenetics marks. They impact gene expression and in turn, participate in cancer pathogenesis. BET inhibitors (BETis) are very promising novel anticancer agents, and combinatory therapy with these inhibitors has been suggested. Because BETis target the NF-κB pathway, their impact on BCL-2 and c-MYC proteins among others, has been tested preclinically in MCL and MM. As a proof-of-concept, inhibition of BRD4 synergistically induced cell death in vitro and in vivo when combined with lenalidomide.

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This confirmed that exacerbated IRF4/MYC signalling is associated with MCL resistance to BTZ and warranted the clinical evaluation of the IMiDBETi combination in MCL cases refractory to the inhibition of proteasome [73]. Also, the BETi birabresib (OTX015) significantly synergises with BTZ, CFZ, IXZ and IMiD to improve MM response and overcomes resistance to PIs. It triggers the suppression of NF-κB pathway and decrease in c-MYC signalling. The birabresib/pomalidomide combination demonstrated great results to overcome adaptive resistance in MCL [101]. Moreover, JQ1, a thieno-triazolo-1,4diazepine has been characterised as the first indirect inhibitor c-MYC transcriptional network in MM cells [103]. Thereafter, the combination of BTZ with the JQ1 derivative, CPI203, was found to be synergistic in BTZ-resistant MM cell lines and in a primary culture from a MM patient refractory to BTZ therapy [104]. These studies supported the clinical evaluation of the IMiDBETis combination in MM and in MCL cases refractory to PIs. An alternative approach to directly inhibit BRD4 was to promote its degradation. This was recently achieved by the use of the protein-targeting chimeric molecule (PROTAC), ARV-825, which specifically induces BRD4 ubiquitination and degradation, granting activity and overcoming PI resistance in MM [105].

6.4.5

Epigenetic Modulators

Histone deacetylase 6 (HDAC6) mechanistically links the UPS and autophagy by facilitating the transport of protein aggregates along tubulin to juxtanuclear microtubule organising centres [112]. Aggregated ubiquitinated proteins are transferred to lysosomes via autophagy, and BTZ treatment contributes to aggresome formation. Cells that lack HDAC6 were found to be defective in the removal of protein aggregates and are not able to form large aggresomes. The combination HDAC inhibitors with BTZ sensitises MM-resistant cells to proteasome inhibition [113]. Ricolinostat (ACY-1215) is a specific

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inhibitor of HDAC6. When ricolinostat is combined with BTZ and dexamethasone, the response rate among BTZ-refractory MM patients raised up to 20%. This combined treatment appeared well tolerated, supporting the use of HDAC6 inhibitors to overcome PI resistance in patient [102]. In MCL, combined PI and ricolinostat treatments are still under preclinical investigations [106]. Vorinostat is an inhibitor of class I and II HDAC. In combination with BTZ, vorinostat revealed limited results in clinical phase II and I, respectively, for relapsing MCL and MM [108]. Others combinations involving this HDAC inhibitor are under investigation, and epigenetic regulation mechanisms such as DNA methylation have been subject of preclinical studies. Because NOXA and BCL-2 are demethylated during BTZ treatment, the DNA methytransferase inhibitor decitabine showed synergic effect with BTZ in PI-resistant MCL [107].

6.5

Concluding Remarks

Molecular mechanisms that explain inherent or acquired BTZ resistance have been essentially explored in MM and MCL. The identified alterations include mutations in proteasomal subunits and activation of prosurvival signalling pathways that have impact in cell cycle, cell differentiation, apoptosis, and stabilisation of critical cellular factors. Inside the cell, UPS and ALS are major regulators of protein homeostasis and contribute to maintain the balance required to accomplish all protein functions, including the capacity of the cells to respond to BTZ. Beside the malignant cell itself, microenvironment is a crucial factor for impairing PI activity, especially in MM cells, which are heavily dependent on external factors for their growth and response to drugs. New molecular mechanisms are regularly discovered that have been associated with drug resistance. Some of these mechanisms could be implicated in PI resistance, enhancing the complexity of this process. In the hypothesis of a multifactorial origin, the conception of new and more efficient approaches tackling PI resistance

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should include combinatorial approaches simultaneously targeting multiple cellular mechanism. In the case of MCL and MM, the development of new types of drug such as IMiDs and BETis used in combination with PIs led to promising results in vivo. Nevertheless, combinatorial treatment could also increase off-target effects and for this reason a better assessment of these treatments has to be performed before been used in patients. The perfect strategy to overcome drug resistance in MM, MCL and other cancer types is far from being identified. Improving our knowledge on the molecular mechanisms implicated in resistance would also open the possibility to elaborate more efficient treatments while reducing the undesired side effects on healthy cells. Acknowledgments MGS and MSR are part of the UbiCODE project and received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 765445. GQ is a fellow from the French Ministry of Education. MSR is also funded by the Institut National du Cancer, France (PLBIO16-251), LASSERLABEUROPE grant number 654148 and CONACyT-SRE (Mexico) grant 0280365. BS acknowledges support from Ligue contre le Cancer and Fondation Française pour la Recherche contre le Myélome et les Gammapathies. GR was financially supported by Fondo de Investigación Sanitaria PI15/00102 and PI18/ 01383, European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’.

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M. Gonzalez-Santamarta et al. 65. Ni M, Lee AS (2007) ER chaperones in mammalian development and human diseases. FEBS Lett 581:3641–3651. https://doi.org/10.1016/j.febslet. 2007.04.045 66. Kaufman RJ (2002) Orchestrating the unfolded protein response in health and disease. J Clin Invest 110:1389–1398. https://doi.org/10.1172/JCI16886 67. Szegezdi E, Logue SE, Gorman AM, Samali A (2006) Mediators of endoplasmic reticulum stressinduced apoptosis. EMBO Rep 7:880–885. https:// doi.org/10.1038/sj.embor.7400779 68. Roué G, Pérez-Galán P, Mozos A et al (2011) The Hsp90 inhibitor IPI-504 overcomes bortezomib resistance in mantle cell lymphoma in vitro and in vivo by down-regulation of the prosurvival ER chaperone BiP/Grp78. Blood 117:1270–1279. https://doi.org/ 10.1182/blood-2010-04-278853 69. Weinkauf M, Zimmermann Y, Hartmann E et al (2009) 2-D PAGE-based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma. Electrophoresis 30:974–986. https:// doi.org/10.1002/elps.200800508 70. Davenport EL, Moore HE, Dunlop AS et al (2007) Heat shock protein inhibition is associated with activation of the unfolded protein response pathway in myeloma plasma cells. Blood 110:2641–2649. https://doi.org/10.1182/blood-2006-11-053728 71. Roué G, Pichereau V, Lincet H et al (2008) Cyclin D1 mediates resistance to apoptosis through upregulation of molecular chaperones and consequent redistribution of cell death regulators. Oncogene 27. https://doi.org/10.1038/onc.2008.126 72. Orchard J, Garand R, Davis Z et al (2003) A subset of t(11;14) lymphoma with mantle cell features displays mutated IgVH genes and includes patients with good prognosis, nonnodal disease. Blood 101:4975–4981. https://doi.org/10.1182/blood-2002-06-1864 73. Moros A, Rodríguez V, Saborit-Villarroya I et al (2014) Synergistic antitumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma. Leukemia 28:2049–2059. https://doi.org/10.1038/leu.2014. 106 74. Desai S, Maurin M, Smith MA et al (2010) PRDM1 is required for mantle cell lymphoma response to bortezomib. Mol Cancer Res MCR 8:907–918. https://doi.org/10.1158/1541-7786.MCR-10-0131 75. Chauhan D, Tian Z, Nicholson B et al (2012) A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell 22:345–358. https://doi.org/10.1016/j.ccr.2012.08. 007 76. Tian Z, D’Arcy P, Wang X et al (2014) A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance. Blood 123:706–716. https://doi.org/10.1182/blood2013-05-500033

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Resistance to the Proteasome Inhibitors: Lessons from Multiple Myeloma and. . .

77. Kropp KN, Maurer S, Rothfelder K et al (2018) The novel deubiquitinase inhibitor b-AP15 induces direct and NK cell-mediated antitumor effects in human mantle cell lymphoma. Cancer Immunol Immunother 67:935–947. https://doi.org/10.1007/s00262-018-2151-y 78. Das DS, Das A, Ray A et al (2017) Blockade of deubiquitylating enzyme USP1 inhibits DNA repair and triggers apoptosis in multiple myeloma cells. Clin Cancer Res 23:4280–4289. https://doi.org/10. 1158/1078-0432.CCR-16-2692 79. Song Y, Li S, Ray A et al (2017) Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Oncogene 36:5631–5638. https://doi.org/ 10.1038/onc.2017.172 80. Peterson LF, Sun H, Liu Y et al (2015) Targeting deubiquitinase activity with a novel small-molecule inhibitor as therapy for B-cell malignancies. Blood 125:3588–3597. https://doi.org/10.1182/blood-201410-605584 81. Wang X, Mazurkiewicz M, Hillert EK et al (2016) The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells. Sci Rep 6:26979. https://doi.org/10.1038/srep26979 82. Driessen C, Kraus M, Joerger M et al (2016) Treatment with the HIV protease inhibitor nelfinavir triggers the unfolded protein response and may overcome proteasome inhibitor resistance of multiple myeloma in combination with bortezomib: a phase I trial (SAKK 65/08). Haematologica 101:346–355. https://doi.org/10.3324/haematol.2015.135780 83. Kawaguchi T, Miyazawa K, Moriya S et al (2011) Combined treatment with bortezomib plus bafilomycin A1 enhances the cytocidal effect and induces endoplasmic reticulum stress in U266 myeloma cells: crosstalk among proteasome, autophagylysosome and ER stress. Int J Oncol 38:643–654. https://doi.org/10.3892/ijo.2010.882 84. Heine S, Kleih M, Giménez N et al (2018) Cyclin D1-CDK4 activity drives sensitivity to bortezomib in mantle cell lymphoma by blocking autophagymediated proteolysis of NOXA. J Hematol Oncol 11:112. https://doi.org/10.1186/s13045-018-0657-6 85. Richardson PG, Eng C, Kolesar J et al (2012) Perifosine, an oral, anti-cancer agent and inhibitor of the Akt pathway: mechanistic actions, pharmacodynamics, pharmacokinetics, and clinical activity. Expert Opin Drug Metab Toxicol 8:623–633. https://doi.org/10.1517/17425255.2012.681376 86. Kim A, Park S, Lee J-E et al (2012) The dual PI3K and mTOR inhibitor NVP-BEZ235 exhibits antiproliferative activity and overcomes bortezomib resistance in mantle cell lymphoma cells. Leuk Res 36:912–920. https://doi.org/10.1016/j.leukres.2012. 02.010 87. Witzig TE, Geyer SM, Ghobrial I et al (2005) Phase II trial of single-agent temsirolimus (CCI-779) for relapsed mantle cell lymphoma. J Clin Oncol 23:5347–5356. https://doi.org/10.1200/JCO.2005.13.466

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Part II Neurodegeneration

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Altered Proteostasis in Neurodegenerative Tauopathies Katerina Papanikolopoulou and Efthimios M. C. Skoulakis

Abstract

Tauopathies are a heterogeneous group of neurodegenerative dementias involving perturbations in the levels, phosphorylation or mutations of the neuronal microtubulebinding protein Tau. Tauopathies are characterized by accumulation of hyperphosphorylated Tau leading to formation of a range of aggregates including macromolecular ensembles such as Paired Helical filaments and Neurofibrilary Tangles whose morphology characterizes and differentiates these disease states. Why nonphysiological Tau proteins elude the surveillance normal proteostatic mechanisms and eventually form these macromolecular assemblies is a central mostly unresolved question of cardinal importance for diagnoses and potential therapeutic interventions. We discuss the response of the Ubiquitin–Proteasome system, autophagy and the Endoplasmic Reticulum-Unfolded Protein response in Tauopathy models and patients, revealing interactions of components of these systems with Tau, but also of the effects of pathological Tau on these systems which eventually lead to Tau aggregation and accumulation. These interactions point to potential

K. Papanikolopoulou · E. M. C. Skoulakis (*) Institute for Fundamental Biomedical Research, Biomedical Sciences Research Centre “Alexander Fleming”, Vari, Greece e-mail: skoulakis@fleming.gr

disease biomarkers and future potential therapeutic targets. Keywords

Tauopathies · Proteostasis · Tau toxicity · Neurodegeneration · Alzheimer’s disease

7.1 7.1.1

Introduction The Microtubule-Associated Protein Tau

Tau is a microtubule-associated protein (MAP) found largely in axons and to a lesser extend in the dendrites of Central Nervous System (CNS) neurons. In general, Tau promotes the polymerization of tubulin, regulates the stability of microtubules, and determines microtubule spacing [1–3]. Recently however, Tau interactions with the neuronal plasma membrane and the actin cytoskeleton, as well as novel localizations including sub-synaptic sites and the nucleus have been identified, collectively suggesting varied additional functions for Tau beyond its microtubule regulating role [4]. In the adult human brain, alternative splicing of a single-copy gene generates six Tau isoforms that differ by the absence or presence of one or two inserts in the amino-terminal part (0N, 1N or 2N), in combination with either three or four imperfect repeats (3R or 4R) that possess the microtubule-binding activity of the protein, near

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_7

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its carboxy-terminal part. During embryogenesis, the smallest isoform 0N3R, predominates, while in the adult brain all six Tau isoforms are present with roughly equal amounts of the 3R and 4R isoforms, but the 1N, 0N, and 2N isoform comprise about 54%, 37%, and 9%, respectively, of total Tau [5, 6]. In addition, Tau functions and subcellular localization are highly regulated at the posttranslational level by various modifications, such as phosphorylation, acetylation, glycosylation, truncation and others [7], giving rise to an enormous heterogeneity of individual Tau molecules. Tau, in its longest 2N4R isoform contains 80 serine and threonine residues, mainly clustered in the proline rich domain (PRD) and a few phosphorylatable tyrosines. Numerous kinases and phosphatases target these sites, apparently with overlapping specificity in vitro [8] and this suggests that Tau may be targeted by different kinases as available in the various neuronal types expressing it in vivo. Phosphorylation, which is the most abundant posttranslational modification on Tau, is low during embryogenesis, but then it varies in other life stages probably in neuronal-type specific fashion. Biophysical studies suggested that Tau contains little stable secondary structure and is often referred to as an “intrinsically disordered protein” (IDP), probably because its high hydrophilicity is not conducive to stable hydrophobic region-driven secondary structure. Nevertheless, a “paper-clip” conformation has been proposed for Tau in which the C terminus folds over the microtubule-binding domain and the N terminus folds back over the C terminus [9]. However, transient or more stable higher order structures may arise and be resolved consequent of phosphorylation and dephosphorylation cycles or other posttranslational modifications and they in principle could alter Tau interactions or subcellular location. In pathological conditions, Tau isoforms self-assemble into disease-specific filamentous structures and adopt a C-shaped crossβ/β-helix structure in Neurofibrillary Tangles (NFTs) and a long J-shaped fold in the spherical Pick bodies [10, 11].

K. Papanikolopoulou and E. M. C. Skoulakis

7.1.2

Tauopathies and Tau Toxicity

Interest in Tau increased and has not waned since about 30 years ago when the protein was demonstrated to be the major component of NFTs, a neuropathological hallmark of Alzheimer’s disease (AD) [12, 13]. Since then, Tau misregulation has been linked to the pathogenesis of several dementing conditions now grouped under the term Tauopathies. In addition to AD, Tauopathies encompass a range of neurodegenerative dementias, including Pick’s disease (PiD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), and Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Only FTDP-17 is consequent of mutations in the tau gene, while all other Tauopathies involve altered abundance or posttranslational modifications of otherwise wild-type Tau isoforms [14–16]. To date, the mechanism of Tau aggregation remains elusive and several contributing factors may be envisaged including pro-aggregation aberrant posttranslational modifications. The most common posttranslational modification associated with NFTs is excessive Tau phosphorylation. Hyperphosphorylated Tau is thought to lose its biological activity, cause destabilization of the microtubule network, disrupt axonal transport, and redistribute from the axons to the somatodendritic compartment of neurons, where it aggregates in filamentous form [7, 17]. Similarly, FTDP-17-associated mutations appear to increase Tau phosphorylation via largely unknown mechanisms and impair its ability to bind microtubules [18, 19], underscoring the importance of hyperphosphorylation for Tau toxicity. However, it is possible that hyperphosphorylation of Tau may result in a “gain of function” situation endowing the protein with novel activities and functions including its subcellular re-localization and this may underlie its toxicity and aggregation [20]. Independently, or in addition to hyperphosphorylation, residues within the microtubule-binding repeat region are important not only for Tau function, but also

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Altered Proteostasis in Neurodegenerative Tauopathies

appear instrumental in the initiation of Tau aggregation. More specifically, hexapeptide motifs in that region have been shown to possess a high β-sheet-forming propensity and may function as a core to induce Tau fibrillization [10, 21]. Moreover, recent evidence suggests that there are different and distinct types of Tau filaments likely characteristic of different Tauopathies and this may underlie the distinct appearance of aggregates that characterizes and differentiates them [22]. In pathological conditions Tau isoforms selfassemble into diverse filamentous structures with the microtubule-binding repeats and adjoining sequences forming the filament core, whereas the amino-terminal half and the carboxyl terminus give rise to the so called “fuzzy coat” that surrounds the core and apparently offers potential binding sites for diverse cellular proteins [23– 25]. In AD, Chronic Traumatic Encephalopathy (CTE) and other Tauopathies, all six Tau isoforms are present in the filaments. In other diseases, such as PSP, CBD, Argyrophilic Grain Disease, and Globular Glial Tauopathy, filaments are made of 4R Tau isoforms, while, the spherical bodies characteristic of PiD (Pick bodies) are made only of 3R Tau [14, 16]. Moreover, even when they are made of the same isoforms, the morphologies of Tau filaments vary in accord with the distinct Tauopathies [14]. Ultrastructurally, NFTs from AD are composed of Paired Helical filaments (PHFs) and Straight filaments (SFs). PHFs and SFs are made of identical protofilaments, but differ in interprotofilament packing, hence they are ultrastructural polymorphs. Each C-shaped protofilament contains eight β-strands, five of which give rise to two regions of antiparallel β-sheets, with the other three forming a β-helix [11]. PHF protofilaments are arranged base to base and SF protofilaments back to base. Filament structure in CTE is distinct from AD in that the six isoforms assemble into C-shaped protofilaments with different β-strand packing than in the typical AD fold [26]. Cryo-EM studies yielded highresolution structures of PiD-derived Tau filamentsF and showed that they contain two types of filaments, a majority of narrow Pick

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filaments (NPFs) and a minority of wide Pick filaments (WPFs). The core of NPFs is made of a single J-shaped protofilament that consists of 3R Tau, which has adopted an elongated cross-β structure. WPFs are formed by the association of two NPF protofilaments at their distal tips [10]. Protein misfolding and aggregation into any of these filamentous forms can activate the cellular proteostasis network, which consists of the ubiquitin proteasomal pathway (26S pathway or UPP), autophagy and the endoplasmic reticulumunfolded protein response (ER-UPR) [27– 29]. However, the presence of aggregates and NFTs indicates that these mechanisms are either impaired or dysfunctional in Tauopathies. It is unclear at the moment whether pathological Tau formation attenuates these mechanisms resulting in aggregate accumulation, or that expedient accumulation of aggregates overwhelms and impairs these mechanisms. This is exaggerated by the uncertainty regarding possible mechanisms of Tau degradation during the aggregation process and it should be noted that these proteins often undergo a stepwise fragmentation to generate cleaved forms, some of which have enhanced aggregation propensities [30]. Growing evidence reveals a relationship between abnormal degradation of misfolded Tau proteins and the pathogenesis of AD [31–33], but their relative contributions to normal and pathological Tau clearance are still poorly understood. It is also unclear whether there is differential impairment of some or all proteostatic mechanisms in different Tauopathies. Given that reducing Tau levels attenuates neuronal dysfunction in animal models, there is growing interest in defining the degradative pathways that remove excess Tau [34, 35].

7.1.3

Proteostatic Failure or Inhibited Proteostasis?

Accumulation of pathological, hyperphosphorylated Tau in an age-dependent manner characterizes all Tauopathies and underlies NFT formation [14]. But does nonphysiological Tau evade detection by the three main proteostasis

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systems and therefore accumulates in affected neurons? Or does pathological Tau actually inhibit directly or indirectly these physiological mechanisms typically clearing misfolded or aberrant proteins? Does the age dependence for the presentation of Tauopathies reflect the known age dependent change in the efficiency of proteostatic mechanisms [36] that finally fail to clear the pathological species? Answers to these questions are essential to understand Tauopathies and other neurodegenerative aggregopathies such as Parkinson’s disease or ALS, but also to devise potential therapeutic interventions. With these overarching questions in mind, we review the involvement of the main proteostatic systems in Tauopathies below.

7.2 7.2.1

Proteostatic Mechanisms in Tauopathies Ubiquitin–Proteasome System and Tau Degradation

The ubiquitin–proteasome system (UPS) is a highly conserved intracellular pathway contributing to the maintenance of protein homeostasis and elimination of damaged, misfolded, and mutant proteins in both, the cytoplasm and the nucleus [37, 38]. Proteasomal degradation by the UPS requires the conjugation of multiple 8.5 kD Ubiquitin (Ub) peptides onto the substrate protein through an enzymatic cascade. This involves the ubiquitin activating enzyme E1, the ubiquitinconjugating enzyme E2, and ubiquitin ligase E3, which tags a protein for recognition by the proteasome. The abundance and specificity of the E3 ligases identified to date suggest that they determine the substrate selectivity of the UPS [39, 40]. In addition to ubiquitination, similar modifications such as SUMO-ylation and NEDDylation contribute to protein degradation [31]. The initial link between Tau and the UPS was revealed when ubiquitin was found to be a component of NFTs [41–43]. Additionally, Tau isolated from human AD brains appears to be physically associated with various proteasome subunits [44]. This suggests that Tau is a substrate

of the proteasome, but may also indicate impaired degradation resulting in prolonged association with it, thereby facilitating co-purification. This putative shift in degradation kinetics could inhibit proteasome activity, likely underlying the significant impairment in overall degradative activity of UPS in postmortem human AD brains [45, 46]. Consistent with this notion, independent studies reported that proteasome activity, but not the levels of their protein constituents, was strikingly impaired in AD-affected compared to unaffected brain regions [44, 45, 47], which might be related to the presence of inclusions [48]. Moreover, the trypsin-like catalytic activity was significantly decreased in postmortem AD brains [46] and appeared most critical for the degradation of full-length Tau and its truncated forms [49]. Related to the data above, reversible and irreversible proteasome inhibitors including lactacystin, leupeptin, and epoxomicin delayed the degradation of endogenous and/or transiently overexpressed Tau [49–51]. Therefore, affected neurons appear to attempt pathological Tau degradation via the ubiquitin–proteasome system. However, this likely impairs its function and contributes at least in part, to the intraneuronal elevation of pathological species and aggregate formation. Proteasome inhibition has also been linked to Amyotrophic Lateral Sclerosis (ALS) and its impairment in primary neuronal cultures yielded aggregate accumulation [52]. Therefore, proteasome impairment characterizes neurodegenerative brains, but it is not observed in healthy, age-matched subjects [53] and is the likely reason for the significantly increased levels of Ub in AD brains [54]. The physiological roles of E3 Ub-ligases in AD are still poorly understood, but several of them, such as CHIP, Parkin, and RNF182 have been suggested to play a role in the progression of AD and other neurodegenerative diseases [55, 56]. In fact, Parkin mutations are common familial causes for another aggregopathy, Parkinson’s disease [57]. Paradoxically, E1 and E2 enzymes have been reported downregulated in AD brains [58]. Ubiquitinated Tau proteins occur in both the mono-ubiquitinated and polyubiquitinated forms although the latter is less abundant. Initial studies

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Altered Proteostasis in Neurodegenerative Tauopathies

identified that ubiquitinated Tau at residues Lys254, Lys257, Lys311, and Lys317 was mainly in the Lys48-linked form [59], a typical signal for proteasomal degradation [60]. Further mass spectrometric and immunological studies added evidence that Tau pulled down from AD brain was ubiquitinated at Lys254, Lys311, and Lys353, not only through Lys48-, but also through Lys6-, Lys11-, and Lys63-linked polyubiquitin chains [61–63], strongly suggesting attempted UPS-mediated Tau clearance. The fate of these Ub–Tau species is still rather unclear. However, evidence from co-transfection with Tau and Ubiquitin in HEK cells indicated Tau accumulation in the insoluble fraction, an effect enhanced further by ALLN or MG-132-mediated proteasome inhibition. Therefore, Tau is at least in part degraded by the ubiquitin–proteasome system and attenuation of this inhibition by still poorly understood factors, possibly including Tau hyperphosphorylation-mediated local structure stabilization, results in elevated aggregate formation. Remarkably, all the identified ubiquitination sites of Tau in PHFs identified via LC–MS/MS are located in the microtubule-binding region [61]. Similar to phosphorylation, other modifications in this domain, such as acetylation at Lys280, impair Tau–microtubule interactions and facilitate aggregation [64]. By analogy, Tau ubiquitination is thought to weaken microtubulebinding affinity as documented for polyubiquitinated Tau in HEK293 cells [65]. Quantitative comparison of Tau modifications revealed that most lysines targeted by acetylation were also ubiquitination targets suggesting competition between these processes [66, 67]. Notably, ubiquitination modifies a critical lysine (Lys311) in one of the hexapeptide motifs (306VQIVYK311) that regulate the aggregation propensity of Tau [21]. The influence of ubiquitination on the solubility of Tau is still unclear, but there is evidence supporting the view that ubiquitination promotes the formation of Tau aggregates as shown in cell-culture models [63]. Hence although ubiquitination is meant to

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target it to the proteasome for degradation, it may in fact favor the competing action of aggregation leading to Tau accumulation and inclusion formation. Further evidence of UPS-mediated Tau degradation came from the identification of Tau-targeting E3 ligases. These are the carboxyl terminus of the Hsc70-interacting protein (CHIP)-Hsc70 complex with UbcH5B as the E2 enzyme and the Tumor Necrosis Factor Receptorassociated Factor 6 (TRAF6) [56, 68] and they co-localize with NFTs in AD brains. In vitro, CHIP ubiquitinates Tau through both ubiquitin Lys48 and Lys63 linkage [68], whereas TRAF6 only through ubiquitin Lys63 [65]. Even though, the actual lysines on Tau modified by these two ligases have not been determined in vivo, in cultured cells, CHIP seems to selectively ubiquitinate Tau phosphorylated at proline directed serine/threonine sites [56, 69]. In contrast, phosphorylation in the microtubule-binding domain at KXGS motifs prevents CHIP binding and ubiquitination [69, 70]. This suggests that the phosphorylation state at particular Tau sites precedes and promotes or occludes Tau ubiquitination. We hypothesize that the outcomes of such interactions will be dictated by the spatiotemporal availability of the relevant enzymes and may underlie the neuronal type specificity of different Tauopathies, or their progression. In accord with this hypothesis, overexpression of CHIP in cultured cells promoted the formation of polyubiquitinated Tau aggregates [56, 68], while deletion of CHIP in mice caused accumulation of hyperphosphorylated Tau without aggregate formation [71]. This strongly suggests that ubiquitination contributes to and may even be necessary for Tau aggregation. Additional work is necessary to establish and characterize this further, but a pathology promoting cycle appears likely, whereby ubiquitination necessary for excess Tau degradation may in fact promote aggregation, which may impede proteasome function resulting in further Tau elevation and aggregate formation.

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7.2.2

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Autophagy-Dependent Tau Degradation

Although the UPS–proteasome system accounts for the degradation of up to 80–90% of proteins, the majority of aggregates and proteins too large to be processed through the proteasome barrel, as well as long-lived proteins typically undergo autophagic degradation [27, 72, 73]. Macroautophagy (heretofore called autophagy) is a major protein degradation pathway whereby cytoplasmic contents are sequestered into a double-membrane vesicle called the autophagosome, which by fusing with the lysosome enables degradation of its contents [74]. Autophagy is regulated by signaling cascades mediated through the liver kinase B1 (LKB1)/ AMP-activated protein kinase (AMPK), or the Class I phosphatidylinositol 3-kinase (PI3K)/Akt pathways which converge on the mammalian target of rapamycin (mTOR) kinase through the tuberous sclerosis complex/Ras homolog enriched in brain (TSC/Rheb) signaling [75]. Inhibition of mTOR sets in motion a sequence of events coordinated by complexes of autophagyrelated (Atg) proteins that initiate the formation of the autophagosome [76]. Conversely, chaperonemediated autophagy (CMA) is selective autophagy of proteins carrying a KFERQ-like motif. This motif is recognized by chaperones like Hsc70, which targets client proteins via the lysosome-associated membrane protein2 (LAMP-2) to the lysomal lumen. Notably, Tau contains two KFERQ motifs in the microtubule-binding repeat domain and hence is expected to be degraded, at least in part in lysosomes [77], as long as the motif is not occluded by posttranslational modification-driven conformations. Using an N2a neuroblastoma cell line that expresses the repeat domain of Tau with an FTD-17 mutation (TauRDΔK280), Wang et al. demonstrated that Tau aggregates can be degraded by autophagy [77]. In mouse cortical neurons, autophagy seems to be the main pathway of degradation of a proteolytically cleaved Tau at Asp421 (tauDeltaC), a species found in the brains of AD patients [78]. In patients, Nixon and colleagues first reported evidence of defective

autophagy in EM images of biopsied neocortex with clear AD pathology. They describe various types of autophagic vacuoles (AVs) representing “intermediate” stages in autophagy, suggesting that the process is likely stalled in AD neurons [79]. Ample further evidence corroborates the view that autophagy is defective in Tauopathies/AD. For example, levels of Beclin 1, a key component of the pre-autophagosomal structure, appear reduced in AD brains [80, 81]. This appears to be consequent of caspase-3-mediated cleavage of Beclin 1 and co-localization of the cleaved product with NFTs [81], suggesting its tight association with pathological Tau clearance. Although impaired autophagy appears to be critical in AD, the exact impairment(s) and the stage it occurs in the process remain elusive. Recently, gene expression analysis of the CA1 hippocampal region of AD patients revealed that autophagosome formation and lysosomal biogenesis genes were upregulated during early stages of the disease [82]. This suggests upregulation of autophagic activation early in disease onset and progression. Upregulated autophagy may be cytoprotective, reducing, or masking symptoms, but it may become compromised in later AD stages, possibly as a consequence of the lengthy continuous upregulation [83], or of aging. Interestingly, the Alz-50 antibody recognizing a pathological folded conformation of phosphorylated Tau containing amino acids 2–10 and 312–342 [84, 85], co-localizes with lysosomes in AD brains [86, 87]. This is a clear indication that aberrantly folded Tau proteins are targeted for autophagy. In accord with this notion, hyperphosphorylated Tau co-localizes with the Atg8 family autophagy markers and the autophagy cargo-receptor SQSTM1/p62 in CBD and PSP patients [88]. Conversely, Tau in NFTs from AD patients associates with the neighbor of BRCA1 gene 1 (NBR1), another selective autophagy receptor [89] and the p62 autophagosome cargo protein [90]. This differential interaction with autophagy cargo receptors likely reflects recognition of distinct modifications or conformations of Tau that

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characterize the different Tauopathies. It is possible then that these distinct Tau–cargo protein interactions can serve as Tauopathy-type-specific distinguishing biomarkers. Strong evidence that pathological Tau is degraded by autophagy is provided by studies targeting its negative regulator, mTOR. mTOR is a 289-kDa serine/threonine kinase, which consists of two multiprotein complexes, mTORC1 and mTORC2 [91]. mTORC1 controls cellular homeostasis by activation of p70S6 kinase (S6K), and is inactivated by rapamycin. In contrast mTORC2 is insensitive to rapamycin and controls cell survival via PI3K and phosphoinositide-dependent kinase (PDK-2) signaling [92]. mTOR activation inhibits autophagy and results in accumulation of pathological Tau species and exaggerated pathology [93]. Studies on human cells have shown that mTOR mediates the intra-, but also the extracellular distribution of Tau [94], the latter of which mediates pathological Tau spreading from affected to healthy neurons. In addition, mTOR regulates directly or indirectly Tau phosphorylation, and accumulation [90]. Independent evidence of the importance of autophagy in pathological Tau proteostasis is provided by studies utilizing autophagy inhibitors such as NH4Cl, chloroquine, 3-methyladenine (3-MA) and cathepsin inhibitors. All these treatments delayed Tau degradation and enhanced formation of high molecular weight species [95– 97]. In contrast, the autophagy inducer rapamycin facilitated the degradation of insoluble forms of Tau and also protected against its toxicity in Drosophila [98]. Consistent with this result, rapamycin reduced Tau phosphorylation at Ser214 in human neuroblastoma SH-SY5Y cells and decreased the levels of insoluble Tau in P301S Tau transgenic mice [99, 100]. Furthermore, the mTOR-independent activator of autophagy, trehalose [101], prevented Tau accumulation in primary neurons and reduced the level of aggregates in the brains of Tauopathy model mice [102–104]. Methylene blue, which has been shown to directly inhibit Tau aggregation, can also induce autophagy and reduce total and phospho-Tau levels and improve cognitive

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performance in Tau transgenic mice [105]. These results suggest that the autophagylysosomal pathway contributes to Tau degradation and with neurons profoundly relying on autophagy for clearance of toxic protein aggregates, impairment in this proteolytic system could contribute to AD pathogenesis [106]. However, Tau hyperphosphorylation seems to be a key factor in autophagy perturbation and defects as it results in neuronal microtubule destabilization, hence affecting the placement and function of lysosomes [107]. In support of this finding, evidence from a mouse and a Drosophila model of FTD indicates that pathological Tau disrupts axonal vesicle transport by impairing the dynein–dynactin complex, increasing the number of autophagosomes and contributing to Tau-induced toxicity [108, 109]. This appears to constitute a vicious cycle, as autophagic dysfunction induces Tau hyperphosphorylation as shown in the mouse. Silencing of Atg7 expression increased the levels of phosphorylated Tau without aggregate formation [97, 110] and knocking out of p62 led to accumulation of hyperphosphorylated Tau and impairment of spatial memory [111]. In contrast, induced expression of the autophagy adaptor protein NDP52 facilitated the clearance of phosphorylated Tau in neurons and reduced aggregate levels [112]. A different type of interaction is afforded by the ability of Tau to bind lysosomal membranes and perturb the permeability of the organelle in vitro and in a mouse AD model [77, 113]. Defective lysosomal membrane integrity was also found in AD patients [114], and increased levels of LAMP-1 and the lysosomal protease cathepsin D were reported in CBD and PSP patients [88]. Moreover, Tau may also affect autophagy [115] via inhibition of HDAC6 activity, a noncanonical function of which involves binding of polyubiquitinated misfolded proteins [116, 117]. Collectively then, autophagy can be considered as a target for controlling the progression of Tauopathies, but therapeutic strategies require careful consideration and additional investigation since studies in animal models suggest that enhancing autophagy early in disease has

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beneficial effects but is ineffective in late disease states [118, 119].

7.2.3

Endoplasmic Reticulum-Unfolded Protein Response in Tauopathies

The ER-UPR is a major proteostatic protein quality control system activated by the accumulation of misfolded proteins in the ER [29] and closely connected to the proteolytic machinery of the cell. Proteins that misfold in the ER are exported to the cytosol and degraded by the proteasome [120]. However, once the UPR is activated, autophagy is increased and this becomes the major proteolytic system [121–123]. UPR activation is also sensitive to different cytoplasmic alterations including changes of calcium homeostasis, defective or failed autophagy, oxidative stress, proteasome inhibition, and mitochondrial dysfunction [124]. A successful outcome of the ER-UPR response is the correction of protein folding and the restoration of ER’s capacity to control protein synthesis, processing, and secretion. The ER-UPR relies on three major signaling pathways, each represented by a sensor protein. These ER transmembrane proteins are the activation transcription factor 6 (ATF6), doublestranded RNA-dependent protein kinase (PKR)like ER-resident kinase (PERK), and inositol requiring enzyme 1 (IRE1) [125, 126]. Under basal conditions, ATF6, PERK, and IRE1 are inactive with their luminal domains bound to the Binding immunoglobulin Protein/78-kDa glucose-regulated protein (BiP). ER stress leads to enhanced production and binding of BiP to misfolded proteins and its concomitant dissociation from the three sensor proteins. This promotes homo-dimerization and autophosphorylation of IRE1 and PERK and the translocation of ATF6 to the Golgi apparatus where it is processed. In addition, there is activation of eukaryotic initiation factor 2 alpha (eIF2α) upon phosphorylation by activated PERK, which results in reduced protein translation for newly transcribed genes [127]. Concurrently, the

phospho-PERK branch of the UPR selectively increases the translation of activating transcription factor 4 (ATF4), which induces UPR-related gene expression [128] and upregulation of ER-associated degradation (ERAD) to promote clearance of misfolded proteins [129]. The cleavage product of ATF6, termed ATF6f, translocates to the nucleus and drives the expression of different chaperone and ERAD component-encoding genes [130]. Hence, the UPR is a multilevel protein quality control system with a broad proteostatic protective role. However, prolonged ER stress with its consequent hyper-activation of ER-UPR can induce protracted broad apoptotic responses that can finally result in cell death [131]. Therefore, prolonged production of misfolded pathological Tau may result in a significantly prolonged ER-UPR, which may underlie degeneration and death at least in some types of CNS neurons. Experimental data from postmortem AD brains support the involvement of ER stress in AD pathogenesis as the levels of markers specific for UPR activation such as BiP and phosphoPERK were found elevated in the cortex and hippocampus [132, 133]. Interestingly, immunostaining for phospho-PERK in neurons bearing NFTs was scarce as compared to neurons with more diffuse hyperphosphorylated Tau reactivity. This suggests that tangle formation follows initial pathological Tau-dependent dysregulation of ER-UPR in the early stages of AD. In accord, the levels of BiP and the occurrence of phosphoPERK immunoreactivity in AD neurons correlated well with the NFT-abundance-based Braak staging [134] of pathology classification and localization in autopsied patient brains. This further supports the notion that ER stress spreads with disease progression. Congruently, in cellular models of Tauopathies using induced pluripotent stem cells from fibroblasts of patients harboring the FTD-associated Tau mutations N279K and V337M, ER-UPR was shown to be activated and BiP and phospho-PERK were significantly elevated [135]. Experimental support for the role of Tau in activating the ER-UPR was provided by a Drosophila Tauopathy model expressing

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pan-neuronally human wild-type Tau, the FTDP17-associated R406W mutant, and a phosphomimic construct. Activation of the UPR and the IRE pathway was shown to be age depended and more pronounced upon pseudophosphorylation at 14 S/P and T/P sites, suggesting that ER-UPR is downstream of Tau hyperphosphorylation [136]. Congruently, in mice expressing the human 2N4R tau bearing the FTD causing mutation P301L, phosphoPERK immunoreactivity was barely detected at 8,5 months of age, but 18,5 month-old mice displayed a large number of phospho-PERKimmunoreactive granulovacuolar lesions [137] and similar results were obtained in the hippocampi of 21-month-old mice expressing a 0N4R isoform bearing the P301L mutation. Interestingly, hyperphosphorylation, conformational changes, and Tau aggregation resulting in tangle-like pathology in these mice become apparent at 8 months of age [138]. This provides independent support of the model that ER-UPR follows pathological Tau generation. Moreover, increased levels of phospho-PERK and phosphoeIF2α were detected in the hippocampus of 21-month-old mice expressing human 0N4R Tau with the P301L mutation compared to age-matched controls [139] and this phosphoPERK immunoreactivity co-localized with hyperphosphorylated Tau. Therefore, the phospho-PERK ER-UPR marker and pathological hyperphosphorylated Tau co-localize in these hippocampal neurons. Further demonstration of the dynamic relationship of pathological Tau and ER-UPR came from the doxycycline repressible Tauopathy model of the rTg4510 0N4R-P301L Tau mice. These animals develop severe tangle pathology at 3 months of age [35], but when pathology is advanced, around 9 months-old, levels of phospho-PERK and BiP in the hippocampus and cortex were highly elevated [140]. However, after doxycycline treatment for 35 days to suppress Tau expression, both phospho-PERK, and Tau levels were significantly reduced compared to untreated animals. Finally, subsequent studies in the same animal model revealed PERK activation at 6 months of age and increased levels of

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phospho-eIF2α and ATF4 consistent with decreased rates of protein synthesis [141]. Collectively, these results suggest that ER-UPR sensor proteins and other components of the system, may in fact serve as early, potentially diagnostic biomarkers for AD and possibly other Tauopathies. In support of this hypothesis, recent studies showed increased levels of phospho-PERK and phospho-eIF2 with no differences in BiP levels between AD and controls [142]. Significantly, there appears to be a linear correlation between the degree of neuropathology and the levels of phosphorylated IRE1 [143] in these AD brains. Furthermore, chaperones, such as Hsp72, Grp94 and PDI [144, 145] and the pro-apoptotic UPR transcription factor CHOP [146] were found elevated in such AD brains. Analysis of other Tauopathies including CBD, PSP, PiD and the hereditary FTDP-17, also showed selectively increased phospho-PERK, phospho-eIF2α, and phospho-IRE1 in affected brain areas [147] and for ATF6 in PiD samples as well [148]. Finally, brain stem neurons, a region typically affected in PSP showed enhanced immunoreactivity for phospho-PERK and phospho-eIF2α, whereas a genome-wide association study for PSP and AD revealed single-nucleotide polymorphisms within EIF2AK3, the gene that encodes PERK [149– 151]. Importantly, pharmacological inhibition of PERK for 2 months significantly reduced the levels of phospho-PERK, phospho-eIF2α, and ATF4 compared to vehicle-treated animals and restored protein synthesis rates to normal. Significantly, this treatment also reduced Tau phosphorylation at disease-associated epitopes in 8-monthold mice. Therefore, the data from human postmortem Tauopathy brains and animal models presenting features of an activated UPR provide multiple converging lines of evidence that the progressive accumulation of Tau can induce ER stress. Pharmacological modulators of PERK activity are currently under development [152, 153], but further studies are required to fully understand the potential of the ER proteostasis network as an ameliorative target for Tauopathies.

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7.3 7.3.1

K. Papanikolopoulou and E. M. C. Skoulakis

Synthesis and Perspectives Failure to Clear Pathogenic Tau or Inhibition of Proteostasis?

Despite significant evidence that Tau is targeted by both autophagy and the proteasome, it is still not understood how Tau clearance in the cell is actually regulated. It is also unclear whether and how these proteostatic systems differentiate pathological from physiological Tau. One possibility is that the state of Tau, such as the number and type of posttranslational modifications, or its polymerization are the determinants according to which Tau molecules are targeted and which proteostatic system is engaged. Although Tau is thought of as a “natively unfolded” protein, particular posttranslational modifications or interactions may in fact stabilize particular conformations within certain Tau domains that brand these molecules as aberrant and targeted for degradation. In fact, certain pathogenic phosphorylations have been named “gatekeeper” because occupation of these Serines and/or Threonines mediates further pathogenic phosphorylations, presumably because they stabilize certain conformations making normally occluded sites accessible [154, 155]. On the other hand, pathogenic Tau may in fact suppress the proteostatic clearance systems and this could account for the multiply demonstrated increase in its levels and accumulation in affected neurons. For example, recent evidence has emerged that FTDP-17-linked Tau mutations suppress the physiological age-dependent elevation in expression of the autophagy activator Rhes [156]. Given that despite the multiple clearance systems pathological Tau accumulates in oligomers and high molecular weight aggregates, it is possible that such suppression of the proteostatic response may be more common that considered to date. Perhaps neuronal-type specific suppression of proteostatic clearance systems may be reflected in the differential brain distribution and characteristic differentiating pathology of Tauopathies.

In addition, evidence from Tau animal models indicates that NFT formation alone is insufficient for neurodegeneration and suggests that pre-filamentous Tau aggregates, e.g., Tau oligomeric intermediates may be the most toxic Tau species [35, 157, 158]. However, if NFTs are not the pathology-causing species, approaches targeted at eliminating them could in fact be pathogenic, if soluble Tau species are not also identified by the clearance systems and eliminated.

7.3.2

Multiplicity of Potential Tau Clearance Mechanisms

Coordination of activities between the proteasome and autophagy has been extensively demonstrated [159, 160], where inhibition of proteasome activity induced autophagy. However, impaired autophagy led to decreased UPS flux, rather than upregulation [161], whereas more recent studies suggest negative-feedback coordination between proteasomal activity and autophagic flux. With respect to Tau, the UPS has been proposed to be more dominant when Tau is monomeric, whereas Tau oligomers and higher order aggregates might be more efficiently degraded by autophagy [162]. Very recently however, a fibril-fragmenting function of the proteasome holoenzyme 26S has been identified that targets fibrils assembled from full-length Tau and reduces them into smaller aggregates [163]. This suggests that all three systems may in fact be differentially activated by the various states, conformations, or even isoforms of pathogenic Tau. In agreement with this notion, proteasomal activation achieved by Ubiquitin Specific Peptidase 14 (USP14) delayed the fusion of autophagosomes with the lysosome and Tau oligomerization. Speaking for the differential outcomes depending on the mode of activation, if cellular autophagy was induced via nutrient deprivation, proteasomal activity was reduced and oligomerization elevated [164]. Therefore, identification of the pathogenic Tau species in different neuronal populations is essential to

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understand the type of dominant proteostatic system engaged and to attempt outcome prediction. This may in fact lead to Tauopathy-specific, or even personalized ameliorative approaches.

7.3.3

Potentially Therapeutic Proteostatic Interventions

Inhibitors of specific proteasome proteins or regulators have been the subject of many studies, in contrast to the discovery of activators that has proved to be a difficult task [165]. A selective inhibitor of USP14, known as IU1 has been shown to increase proteasomal activity in vivo [64, 166]. However, it is not clear whether USP14 inhibition will be a successful approach, since its deletion in mice did not affect Tau levels and actually increased its phosphorylation [167]. Thus far, only resveratrol has reached Phase III clinical trials as a therapeutic candidate for AD [168], but its effect on proteasomal activity is unclear [169]. Since autophagy fails in Tau-associated neurodegenerative disorders, it may be the primary defect responsible for loss of proteostasis, or the target of toxicity caused by pathogenic Tau. To that end, a number of autophagy inducers, mainly mTOR inhibitors, have been shown to be effective in facilitating Tau degradation and may be beneficial in early stages of pathogenesis [170]. However, it remains to be determined whether activation of autophagy is safe, or under certain conditions detrimental because different cell types present distinct autophagy responses to stressors [171] and growing evidence suggests variable vulnerability to autophagy malfunction in different brain regions [172, 173]. Thus, diseasemodifying compounds capable of targeting autophagy in specific brain regions and cell types seem to be required for effective treatment. Although the list of small molecules that target the IRE1 and PERK branches of UPR is also growing [174], the consequences of its manipulation as therapeutic target are unpredictable. Crosstalk between the three signaling pathways was demonstrated by PERK deletion which led to increased activity of the parallel stress pathway

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mediated by IRE1α [175]. Therefore, inhibition of one pathway may in fact increase signaling through one, or both of the other two. Another limitation arises from the use of animal models characterized by fast rates of Tau misfolding and accumulation for practical purposes, which do not reproduce the slow time course of human Tauopathies. Therefore, the neuroprotective effects of PERK inhibitors in the aggressive Tauopathy rTg4510 mouse may be unique for this animal model and should not be generalized [141]. Significantly, the Rhes-targeting farnesyltransferase inhibitor Lonafarnib already approved for use in human cancers activates the lysosome response and reduces Tau pathology in the aggressive rTg4510 mouse model [156]. Rhes is a GTPase of the Ras superfamily and an mTORindependent autophagy activator [176], whose levels are suppressed by FTDP-17-linked Tau mutations [156]. However, as this condition affects mostly the frontotemporal lobe, it remains to be seen whether this mechanism and the inhibitor will prove effective in cases of Alzheimer’s disease and other Tauopathies. A very promising emerging strategy for ablating specific proteins takes advantage of the proteasomal degradation machinery coupled with the extraordinary specificity of Positron Emission Tomography (PET) tracer probes. Such a chimeric PROTAC (PROteolysis Targeting Chimera) probe [177] contains the clinically advanced tau PET tracer 18F-T807, which binds to phosphorylated Tau in a conformationdependent manner, linked via a short linker to the CRL4 E3 ligase-recruiting moiety Cereblon. The Chimera binds with high efficiency to FTDP-17 mutation carrying Tau proteins that produce AD-like PHF pathology [178] and induces their specific ubiquitination and subsequent proteosomal degradation in patient-derived neurons in culture [179]. Although it is unknown whether this will work and with what efficiency in patients, this targeted degradation of aberrant Tau proteins holds great proteosome-dependent therapeutic promise and may lead to similar strategies co-opting the ER/UPR and autophagy systems in the near future.

188 Acknowledgments The authors acknowledge support for their research by: KP: A grant from the Stavros Niarchos Foundation to the Biomedical Sciences Research Center “Alexander Fleming,” as part of the Foundation’s initiative to support the Greek research center ecosystem, Fondation Sante and ELIDEK. EMCS: Fondation Sante and the project “Strategic Development of the Biomedical Research Institute ‘Alexander Fleming’” (MIS 5002562) which is implemented under the “Action for the Strategic Development on the Research and Technological Sector,” funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

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8

The Ubiquitin System in Alzheimer’s Disease Lee D. Harris, Sarah Jasem, and Julien D. F. Licchesi

Abstract

Alzheimer’s disease (AD) is the most common form of dementia, most prevalent in the elderly population and has a significant impact on individuals and their family as well as the health care system and the economy. While the number of patients affected by various forms of dementia including AD is on the increase, there is currently no cure. Although genome-wide association studies have identified genetic markers for familial AD, the molecular mechanisms underlying the initiation and development of both familial and sporadic AD remain poorly understood. Most neurodegenerative diseases and in particular those associated with dementia have been defined as proteinopathies due to the presence of intra- and/or extracellular protein aggregates in the brain of affected individuals. Although loss of proteostasis in AD has been known for decades, it is only in recent years that we have come to appreciate the role of ubiquitin-dependent mechanisms in brain homeostasis and in brain diseases. Ubiquitin is a highly versatile post-translational modification which regulates many aspects of protein

fate and function, including protein degradation by the Ubiquitin–Proteasome System (UPS), autophagy-mediated removal of damaged organelles and proteins, lysosomal turnover of membrane proteins and of extracellular molecules brought inside the cell through endocytosis. Amyloid-β (Aβ) fragments as well as hyperphosphorylation of Tau are hallmarks of AD, and these are found in extracellular plaques and intracellular fibrils in the brain of individuals with AD, respectively. Yet, whether it is the oligomeric or the soluble species of Aβ and Tau that mediate toxicity is still unclear. These proteins impact on mitochondrial energy metabolism, inflammation, as well as a number of housekeeping processes including protein degradation through the UPS and autophagy. In this chapter, we will discuss the role of ubiquitin in neuronal homeostasis as well as in AD; summarise crosstalks between the enzymes that regulate protein ubiquitination and the toxic proteins Tau and Aβ; highlight emerging molecular mechanisms in AD as well as future strategies which aim to exploit the ubiquitin system as a source for next-generation therapeutics. Keywords

L. D. Harris · S. Jasem · J. D. F. Licchesi (*) Department of Biology & Biochemistry, University of Bath, Bath, UK e-mail: [email protected]

Alzheimer’s disease · Ubiquitin-proteasome system · E3 ubiquitin ligases · Deubiquitinases · Tau · Amyloid beta

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_8

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Abbreviations Aβ AD DLB DUB E1 E2 E3 E-NFTs FTD FTD-U HD HECT LTP MAM MLKL mTOR NEDD4 NFTs PHFs PM PROTACs PSD PTM RBR RING RIPK SCF USP

8.1 8.1.1

Amyloid beta Alzheimer’s disease Dementia with Lewy Bodies Deubiquitinase E1-activating enzyme E2 conjugating enzyme E3 ubiquitin ligase Extracellular neurofibrillary tangles Frontotemporal dementia Frontotemporal dementia with ubiquitin pathology Huntington’s disease Homologous to E6AP carboxyl terminus Long-term potentiation Mitochondria-associated membranes Mixed lineage kinase domain-like Mammalian target of rapamycin Neuronal precursor cell-expressed developmentally downregulated 4 Neurofibrillary tangles Paired-helical filaments Plasma membrane Proteolysis targeting chimeric molecules Postsynaptic densities Post-translational modification RING-between-RING Really-Interesting new gene Receptor-interacting serine/threonine protein kinase Skp, Cullin, F-box containing E3 ligase complex Ubiquitin-specific protease

Alzheimer’s Disease Dementia

Neurodegeneration is an umbrella term used to describe the degeneration and eventual death of

neurons which usually occurs late in life or in individuals with an associated neurodevelopmental defect. Amongst neurological disorders, dementia is caused by the progressive loss of cognitive functions which leads to symptoms such as memory loss and disorientation. The main differentiating factor that assists in characterising the various forms of dementia is whether the pathology primarily affects the cortical regions (outer layer of the cerebrum) or non-cortical regions (involving the thalamus, basal ganglia and vasculature). Cortical dementias include Alzheimer’s Disease (AD) which is the most common form of dementia with around two-thirds of cases, Vascular Dementia (VD) which accounts for 20% of dementia cases, dementia with Lewy bodies (DLB) which represents 10–15% of cases, and Frontotemporal Dementia (FTD) with less than 5% of dementia cases [1]. AD is defined by a progressive decline in mental, behavioural and cognitive functions, with a median survival time from diagnosis of less than 5 years [2]. FTD, commonly termed Pick’s Disease, can be easily confused with AD at the time of diagnosis. Both dementias are characterised by progressive cognitive decline, the build-up of aggregated proteins and cortical atrophy. However, FTD can occur in younger individuals (above 35 years age) and is detected by more rapid, extreme changes in personality. FTD-U is distinct from FTD in the fact that that it contains inclusions which stain positive for the small protein modifier ubiquitin but not for Tau, α-synuclein or polyglutamine antibodies [3, 4]. In contrast, Parkinson’s Disease (PD), Huntington’s Disease (HD) and progressive supranuclear palsy disorders affect subcortical structures. Both DLB and PD are characterised by α-synuclein inclusions although other studies have suggested that these aggregates can also be found in other neurodegenerative diseases including familial and sporadic AD [5–7]. PD dementia (PDD) is characterised by a slow progressing dementia with impairment in memory and executive functions, and it is usually observed after 10 years of diagnosis [8]. Individuals exhibiting signs of cognitive decline within 1 year of PD diagnosis are likely exhibiting DLB rather than PDD.

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The Ubiquitin System in Alzheimer’s Disease

8.1.2

Genome-Wide Association Studies (GWAS)

Genome-Wide Association Studies (GWAS) have identified genetic variants, or single nucleotide polymorphism (SNPs), which may be associated with familial AD mostly. The original genetic studies identified mutations in amyloid precursor protein (APP) [9] and Presenilin (PSEN1/PSEN2) [10] as genetic risk factors for familial early-onset AD. In contrast, genetic loci for late-onset AD include TREM2 [11–13], ABCA7 [14], TP53INP1 and IGHV1 [15], phospholipase D3 [16]. Recent comprehensive metaanalysis identified 29 risk loci and 215 potential causative genes, suggesting roles for the immune system, lipid-related processes, APP and protein degradation in AD [17, 18]. GWAS have also identified specific genetic variants in different types of dementia including in FTD [19] and DLB [20], and have also revealed potential overlap between the genetic make-up of dementia and Parkinson’s Disease [21, 22]. GWAS have been less consistent for sporadic AD, for which ageing remains the greatest risk factor. Nevertheless, Apolipoprotein Epsilon 4 (APOE ε4) has emerged as the most prevalent genetic risk factor for both familial and sporadic late-onset AD [23, 24]. ApoE regulates lipid homeostasis by mediating lipid, fat-soluble vitamin and cholesterol transport between cells and tissues. In the brain, it is produced and secreted by astrocytes and recognised by the ApoE receptor on the surface of neurons [25]. The APOE ε4 variant is less efficient than other alleles during neuronal repair, and this may increase susceptibility to AD. Current estimates suggest that by 2050 the number of AD cases will have reached 115 million worldwide, which represent a threefold increase compared to 2010 [26]. Yet, the development of AD drugs showed a 99.6% failure rate between 2002 and 2012 [27]. The impending dementia epidemic is being closely monitored at the global level, and research priorities have now been identified, beyond the amyloid hypothesis. Amongst these, fundamental research into the

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basic mechanisms of dementia has been highlighted as holding the greatest potential for identifying novel drug targets for AD [28, 29].

8.1.3

Molecular Mechanisms in Alzheimer’s Disease

8.1.3.1 Amyloid-Beta (Ab) The amyloid precursor protein (APP) is a transmembrane glycoprotein found in human, C. elegans (APL-1) and D. melanogaster (APPL) but not in prokaryotes, plants or yeast [30]. A current hypothesis suggests that APP has been acquired during evolution at the same time as the development of a central nervous system with functioning synapses [31]. The human APP gene contains 18 exons and is prone to splicing. In fact, tissue-specific APP variants have been identified in smooth, cardiac and skeletal muscle, as well as organs such as the kidney and pancreas. The brain-specific APP variant excludes exon 7 and 8, which suggests that the function of APP proteins is tissue specific. Of the three major splice isoforms of APP (APP695, APP751, APP770), APP695 is the predominant neuronal form. Studies in the invertebrate models C. elegans and D. melanogaster have revealed that APP orthologues play a role in axonal transport and neuronal signalling. The conditional knockout of APP in mouse brains results in long-term potentiation (LTP) phenotypes strongly suggesting that APP plays a role in maintaining neuronal plasticity and brain homeostasis [32]. APP proteolytic processing is key to its function in regulating neuronal activity [33], and can occur through the amyloidogenic and non-amyloidogenic pathways [34–36]. The non-amyloidogenic pathway results in the initial cleavage of APP via α-secretase [37], resulting in a truncated APP protein that lacks the Aβ N-terminus. Subsequent cleavage via γ-secretase results in the non-pathogenic truncated Aβ peptide p3 and the amyloid precursor protein intracellular domain (AICD) [38]. In the amyloidogenic pathway, APP is cleaved by the

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β-secretase enzyme BACE which releases sAPPβ. It is the second cleavage by γ-secretase which leads to the intracellular release of AICD in the nucleus and of extracellular Aβ peptides (40 and 42). Aβ peptides can aggregate into oligomers to form fibrils and ultimately plaques [39]. Interestingly, the release of Aβ peptides into the brain interstitial fluid is activity dependent and requires APP endocytosis and its processing by the endocytic recycling machinery, which emphasises the role of APP processing and Aβ in normal neuronal activity [40, 41]. In contrast to Aβ peptides, sAPPα has been shown to bind BACE and decrease soluble Aβ and Aβ plaques [42]. Since plaques are also found in the brain of elderly AD-free individuals, the toxic potential of these protein aggregates remains unclear. Indeed, soluble oligomeric pools of Aβ have also been implicated in driving the neurotoxic effects of Aβ [43]. Interestingly, recent studies have shed light on the dynamic nature of Aβ conformations, by showing that plaques can be readily removed from the brain during sleep, and that sleep disturbance may affect metabolite clearance and can lead to Aβ accumulation [44, 45].

8.1.3.2 Tau In contrast to APP which is an integral membrane protein, Tau is a cytosolic protein found to bind to and stabilise microtubules [46]. Human Tau consists of six isoforms with varying amino acid chain lengths. These isoforms have a microtubule binding domain which consist of C-terminal repeats (R1–R4) and an N-terminal projection domain composed of N1 and N2 motifs. The projection domain mediates interaction with the neural plasma membrane as well as cytoskeletal proteins through the microtubule binding protein MAP1A [47, 48]. Tau isoforms are named according to the presence of these motifs and include 2N4R, 1N4R, 0N4R, 2N3R, 1N3R and 0N3R [49]. The balance between 3R and 4R isoforms is lost in Tauopathies and Tau mouse models and this leads to Tau hyperphosphorylation, insolubility and in turn cognitive impairment [50]. In the Tau hypothesis, hyperphosphorylated Tau accumulates into pathological inclusions and tangles which are

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observed in AD [51]. Protein phosphorylation leads to Tau dissociation from microtubules and the formation of oligomeric species that further aggregate into paired-helical filaments (PHFs) and neurofibrillary tangles (NFTs) [52, 53]. Although the pathological trigger leading to hyperphosphorylated Tau is still poorly understood, the main kinases responsible for its phosphorylation have been identified and include Cyclin Dependent Kinase 5 (CDK5), Mitogen Activated Protein Kinase (MAPK) [54] and Glycogen synthase kinase 3 (GSK3) [55]. Hyperphosphorylated Tau, which exists in a soluble oligomeric form, mediates neuronal toxicity by breaking down microtubules and impairing axonal transport [56–58].

8.1.3.3

Loss of Proteostasis in Alzheimer’s Disease Seminal studies in C. elegans and D. melanogaster have established that Insulin Growth Factor (IGF) and mTOR pathways regulate lifespan [59]. In C. elegans for example, daf-2 (IGF1R in human) mutants are long lived and stress resistant. Given that ageing is the most reliable risk factor for sporadic AD, it is perhaps not surprising that these pathways, through their control of protein synthesis, play important roles in AD. In support of this, daf-2 RNAi reduces Aβ42-induced proteotoxicity [60]. Protein synthesis is inherently error prone and an estimated 30–90% of newly synthesised proteins are defective and improperly folded [61]. In eukaryotic cells, the Ubiquitin–Proteasome System (UPS), the endosomal–lysosomal pathway and autophagy participate in protein quality control to prevent the accumulation of non-functional and misfolded proteins [62]. The endosomal– lysosomal pathway for instance handles monomeric Aβ42, while autophagy and the UPS clear Aβ42 oligomeric species [63]. The first link between proteasomal dysfunction and AD came from histopathological analysis of protein aggregates from AD brains, which revealed the presence of the small protein modifier ubiquitin [64–66]. GWAS studies have also added to mounting evidence in support for a role of altered ubiquitin signalling, with the discovery

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The Ubiquitin System in Alzheimer’s Disease

of E3 ubiquitin ligases TRIM15 [67] and UBR5 as potential genetic markers [68]. In addition to this, ubiquitin (Sect. 8.3.3), as well as a growing number of E3 ubiquitin ligases and deubiquitinases, have also been implicated in AD (Table 8.1). Proteomics has also been particularly useful in order to identify changes that occur at the proteome level during AD progression. This include for example the remodelling of UCHL5 interactome [123], alterations in overall protein ubiquitination [124] and overall protein levels [125].

8.2 8.2.1

The Ubiquitin–Proteasome System Ubiquitin

Ubiquitin is a highly conserved 76 kDa protein modifier found in all eukaryotes. The yeast Saccharomyces cerevisiae ubiquitin sequence varies by only two amino acids compared to the mammalian one [126]. In contrast, ubiquitin is not found in prokaryotes and instead prokaryotic ubiquitin-like protein (Pup) and Small Archaeal Modifying Protein (SAMP) drive protein degradation [127, 128]. Ubiquitin is a versatile posttranslational modification (PTM) which mediates a plethora of functions including protein degradation as part of the UPS but also proteasomeindependent functions [129, 130]. These include the sorting of receptors at endosomes [131], the recognition of protein complexes and organelles by autophagic receptors [132], as well as the recruitment of the DNA repair machinery at sites of DNA double-strand brakes [133]. In addition to ubiquitin, mammalian cells also encode a number of ubiquitin-like modifiers (UBLs) such as SUMO [134], NEDD8 [135] and ISG15 [136]. The structure of ubiquitin is characterised by a β-grasp fold and a flexible six-residue C-terminus tail which is important for its conjugation onto lysines of protein substrates [137]. The hydrophobic patch formed by Ile 44, Leu 8, Val 70 and His 68 is key for its recognition by most ubiquitinbinding domains (UBDs) [138]. The hydrophobic surface which centres around Ile 36 (Ile 36, Leu

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71 and Leu 73), seems to be particularly important for the transfer of ubiquitin between E2-conjugating enzymes and E3 ubiquitin ligases [139] as well as for its interaction with UBDs [140] and DUBs (Deubiquitinating enzymes) [141, 142]. Protein ubiquitination can result in the addition of one molecule of ubiquitin onto a single lysine (i.e. monoubiquitin) or on multiple lysines residues (multi-monoubiquitination). In addition to this, multiple ubiquitin molecules can be assembled together to form a ubiquitin chain. This is mediated between the C-terminal glycine residue (G76) on a donor ubiquitin and the free amino group of the N-terminal methionine (M1) or of any of the seven lysines from an acceptor ubiquitin. Therefore, as many as eight distinct linkages can be formed between two ubiquitin molecules and these include Met-1, K6, K11, K27, K29, K33, K48 and K63 [143, 144]. Although the canonical ubiquitin signal that mediates protein degradation via the UPS was originally found to be K48-linked polyubiquitin chains, additional ubiquitin chain types including the more complex mixed and branched chains have since now been reported [145, 146]. This, together with the fact that ubiquitin itself can be modified with other PTM, further expands the complexity and functional diversity of ubiquitin.

8.2.2

Components of the Ubiquitin System

Ubiquitin is covalently tagged onto target proteins via an enzymatic cascade which include E1-activating, E2-conjugating and E3 ubiquitin ligases. The C-terminal glycine residue of ubiquitin is first adenylated in the presence of ATP, followed by its transfer onto the catalytic cysteine (Cys) residue of an E1-activating enzyme via a thioester bond [147, 148]. The activated ubiquitin is then transferred in a transthioesterification reaction onto the catalytic residue of an E2-conjugating enzyme, prior to its loading onto lysines residues of protein substrates [149]. E3 ubiquitin ligases mediate this final step, enabling the formation of an isopeptide bond

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Table 8.1 E3 ubiquitin ligases and DUBs in Alzheimer’s Disease Protein Protein name family E3 ubiquitin ligases Parkin RBR

Evidence/mechanisms

References Nemes et al. [69] Ye et al. [70]

Baker et al. [79]; Cruts et al. [80]; Gass et al. [81]; Galimberti et al. [82] Hou et al. [83]

BRCA1

RING

CHIP/ STUB1

RING

Dactylidin/ RNF146 GRN/ Progranulin

RING

Parkin is found in neurofibrillary tangles. Parkin depletion during the course of AD in hAPP neurons and AD patient brains. Mitophagy inhibits Aβ and Tau pathology. Loss of function leads to learning and memory impairment phenotypes in mice. Increase activity through elevated expression of the activator Cdh1 prevents cell cycle re-entry of neurons via suppression of cyclin B. Reduced BRCA1 levels found in the brain of AD patients. Ubiquitin-dependent degradation of phosphorylated Tau. Binds to and functions in a complex with CRL4CRBN to ubiquitinate APP via the APP cytosolic region. RNF146 is upregulated in AD brains.

RING

GRN mutations are associated with FTD-U.

RING

HRD1 is expressed in neurons and reactive astrocytes and might be associated with the ubiquitin-dependent degradation of hyperphosphorylated Tau. HRD1 is inhibited by soluble Tau accumulation which leads to activation of the unfolded protein response. Loss of HRD1 leads to APP and Aβ accumulation. Loss of function leads to Aβ generation.

APC/c

HRD1/ synoviolin

RING

MARCH7

RING

MGRN1 MYLIP/ IDOL TRAF6

RING RING

ZNRF1

RING

FBXO2 CCNF

SCF SCF

SLIMB

SCF

TRIM11

TRIM

TRIM21

TRIM

TRIM28

TRIM

RING

MARCH7 ubiquitinates 4-repeat (4R) tau and impairs microtubule binding. Involved in the maturation and trafficking of APP. Targets LDLR-dependent APOE and Aβ for clearance. Candidate therapeutic target for AD. Prevents Aβ-induced neuronal death through ubiquitindependent degradation of p75 neurotrophin receptor. ZNRF1 promotes neurodegeneration through the degradation of Akt via the UPS. Regulates APP levels and processing in the brain. Abnormal ubiquitination and accumulation of TDP-43 and SCF substrates in amyotrophic lateral sclerosis and FTD. SLIMB mediates the ubiquitin-dependent UPS degradation of active and phosphorylated PAR-1. This impacts on tau-mediated postsynaptic toxicity of Aβ. TRIM11 targets humanin, a neuroprotective peptide that suppresses AD-related neurotoxicity, to the UPS. TRIM21 mediates the UPS-mediated degradation of tau and this inhibits seeding aggregation. Stabilises tau and α-synuclein nuclear accumulation.

Fang et al. [71] Kuczera et al. [72] Aulia and Tang [73]

Suberbielle et al. [74] Shimura et al. [75]; Petrucelli et al. [76] Del Prete et al. [77] Rotz et al. [78]

Abisambra et al. [84] Kaneko et al. [85] Maeda et al. [86]; Kaneko et al. [85]; Tanabe et al. [87] Flach et al. [88] Benvegnù et al. [89] Choi et al. [90] Geetha et al. [91] Wakatsuki et al. [92] Atkin et al. [93] Williams et al. [94]

Lee et al. [95]

Niikura et al. [96] McEwan et al. [97] Rousseaux et al. [98]; Rousseaux et al. [99] (continued)

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Table 8.1 (continued) Protein name TRIM32/ 37 NEDD4

Protein family TRIM HECT

NDFIP1

NEDD4 adaptor

HUWE1A

HECT

UBE3A

HECT

UBR5

HECT

Deubiquitinases OTUB1 OTU USP46

USP

USP14

USP

USP8

USP

UCHL1

UCH

Evidence/mechanisms Altered expression in AD brains.

References Yokota et al. [100]

Regulates AMPR ubiquitination, turnover and trafficking. Aβ increases turnover of AMP receptor through NEDD4-dependent ubiquitination and lysosomal degradation Aβ-induced synaptic alterations correlate with AMPAR ubiquitination by NEDD4. Ubiquitination of GluA1 by NEDD4–1 is activated by neuronal signalling and leads to GluA1 endocytosis and lysosomal trafficking. NDFIP is implicated in the degradation of divalent metal transporter 1 (DMT1). Loss of NDFIP leads to increased iron dyshomeostasis and Aβ production. Proteome-wide study identifies lower HUWE1A protein levels in AD brain. UBE3A-deficient AD mice have reduced Aβ levels and plaque formation and show accelerated cognitive and motor deficits compare with AD mice. Novel loci associated with disease progression in subjects with mild cognitive impairment in AD.

Lin et al. [101]; Hou et al. [102]

OTUB1 deubiquitinates tau in vivo and in vitro and regulates tau oligomeric forms. Aβ induces internalisation and subsequent ubiquitination and lysosomal degradation through NEDD4 upregulation and downregulation of USP46. USP14 inhibition accelerates degradation of WT tau as well as pathological tau mutants P301L and P301S, and the variant A152T. Regulates the ubiquitination, trafficking and lysosomal degradation of BACE1. Regulates α-synuclein clearance in Lewy bodies. Elevated protein levels in AD patients, decreased mRNA and protein level in DLB. Controversial genetic association between UCHL1 polymorphism and sporadic AD. DUB activity required for normal synaptic and cognitive function. Overexpression accelerates APP lysosomal degradation and reduces Aβ production. Aβ affects BDNF retrograde trafficking through inhibition of UCHL1 activity. Inhibition of UCHL1 DUB activity decreases tau binding to microtubules and increases its phosphorylation.

Zhang et al. [103]

Rodrigues et al. [104] Schwarz et al. [105]

Tian et al. [106]

Ho Kim et al. [107] Singh et al. [108]

Hu et al. [68]

Wang et al. [109] Zhang et al. [103]; Huo et al. [110] Boselli et al. [111]

Yeates and Tesco [112] Alexopoulou et al. [113] Ohrfelt et al. [114]; Barrachina et al. [115] Xue and Jia [116]; Forero et al. [117]; Shibata et al. [118] Gong et al. [119] Zhang et al. [120] Poon et al. [121] Xie et al. [122]

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between the carboxyl group of G76 of ubiquitin and the free amino group on lysine(s) of a protein substrate [150]. Depending on the mechanism of ubiquitin transfer onto the substrate, E3 ligases have been divided into three main families. Really interesting new gene (RING), characterised by their RING finger zinc domain or U-box domain, are E3 ligases that act as scaffolds to facilitate the transfer of ubiquitin from the E2, directly to the target substrate [151]. RING finger domains have been shown to adopt a ‘cross-brace’ arrangement through binding two zinc ions [152]. RING fingers have also been suggested to activate E2s allosterically [153]. There are over 600 RING E3s encoded in the mammalian genome, making this family of E3s the most diverse [154]. The fact that BRCA1/BARD1 assembles different ubiquitin chains depending on the E2 it partners with, has led to the recognition that, in the case of RING E3s, polyubiquitin chain specificity is primarily determined by E2s [155]. In contrast, homologous to E6AP carboxyl terminus (HECT) E3s show intrinsic catalytic activity by accepting ubiquitin from the E2, before it is transferred to the substrate [156]. The C-terminal HECT domain is characterised by an N- and a C-lobe, connected by a flexible glycine hinge region linking the lobes, and with the catalytic cysteine residue located on the C-lobe [157]. In contrast to RINGs E3s, polyubiquitin chain specificity in HECT ligases is determined by the HECT domain itself, as changing the E2 that interacts with the HECT domain does not affect ubiquitin linkage type [158]. RING-in-between-RING (RBR) E3 ligases represent a hybrid family between RINGs and HECTs. They have a RING domain for E2 recruitment which subsequently transfers ubiquitin via transthioesterification onto a cysteine residue of a RING-like domain [159]. PARK2 is perhaps the most relevant example for neurodegeneration, given that loss of Parkin ubiquitin ligase activity in PD and AD is associated with mitophagy inhibition and the accumulation of damaged mitochondria [70, 160, 161]. One of the main functions of ubiquitin is to label proteins for degradation by the 26S

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proteasome [162]. The 26S proteasome is a multi-protein complex organised into the 19S regulatory cap and the 20S catalytic core which is responsible for the proteolytic cleavage of most intracellular and soluble proteins. The 20S core particle has two outer (α) and two inner (β) rings, with each ring composed of seven subunits. The catalytically active β-rings which make up the proteolytic region, consists of N-terminal nucleophile hydrolases, with threonine functioning as the active nucleophile [163]. These hydrolases include trypsin, chymotrypsin and peptidylglutamyl-peptide [164]. The 19S regulatory lid on the other hand has multiple subunits serving as UBDs for the docking and processing of ubiquitinated substrates by the 20S. For example, Rpn10 is a 19S regulatory subunit which recognises polyubiquitin chains through its Ubiquitin-Interacting Motif (UIM) [165]. At least 99 DUBs are encoded in the human genome and these have been grouped into the JAMM/MPN, OTU, MJD, UCH, MINDY and ZUP1 subfamilies based on their catalytic domain, with new families still being discovered [166]. Some DUBs, including those from the OTU family, have shown some remarkable specificity towards particular chain types [166, 167] In addition to their role in editing and modulating cell signalling through reversal of protein ubiquitination, DUBs also have important roles at the surface of endosomes and on the 19S lid of the proteasome where they ensure the recycling of ubiquitin thereby maintaining the pool of ubiquitin in cells. Rpn11, Ubp6/USP14 and UCH37 are the resident proteasomal DUBs in charge with substrate deubiquitination prior to unfolding and degradation [168–170].

8.2.3

Ubiquitin Signalling and Neuronal Homeostasis

Neuronal cells communicate through synapses, formed between the axon of a sending neuron and the dendrite of the receiving neuron, with the pre- and postsynaptic membranes separated by the synaptic cleft. Each of these compartments have a number of cell adhesion molecules and

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scaffold proteins which enable the release of neurotransmitter from the presynaptic density into the synaptic cleft, in response to an action potential [171]. Postsynaptic membranes contain the receptors that bind the released neurotransmitters, leading to the activation of downstream signalling pathways which regulate synaptic plasticity, learning and memory [172]. Glutamate is a major excitatory neurotransmitter in mammalian brains and the regulation of its receptors is crucial for neuronal plasticity. The binding of glutamate onto NMDA (N-methyl D-aspartate) receptors (NMDAR) triggers the opening of this ion conductance channel protein, and the influx of calcium. This increase in intracellular calcium levels is a key signalling event in neurotransmission and therefore it needs to be tightly regulated. Astrocytes play an important role in this process by metabolising excess extracellular glutamate at synapses [173]. In AD, it has been shown that Aβ can impede the function of neuronal receptors including NMDAR, AMPAR (α-amino-3-hyroxy-5-methyl-4-isoxazolepropionate acid receptor), the Prion Protein cell surface (PrPC), as well as drive inflammation in astrocytes which further contributes to neurotoxicity [174–177]. Loss of glutamate homeostasis at synapses leads to the activation of extrasynaptic NMDA receptors. The ensuing excitotoxicity is mediated by the stabilisation and activation of calpain-dependent cleavage of the striatalenriched protein tyrosine phosphatase (STEP), the activation of p38 mitogen-activated protein kinases and neuronal apoptosis [178, 179]. Calcium signalling is intimately linked with ubiquitin ligase function. For instance, the HECT E3 NEDD4 (neuronal precursor cellexpressed developmentally downregulated 4) binds calcium through its N-terminal calciumbinding module (C2 domain). In the absence of calcium, the C2 domain binds to the C-terminal HECT domain of NEDD4, inhibiting its ubiquitin ligase activity. In contrast, the increase in intracellular calcium concentration outcompetes this interaction and releases the HECT domain. NEDD4 then translocates to the plasma membrane where it ubiquitinates synaptic proteins and contributes to neuronal activity [180, 181].

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Normal brain function requires a precise and dynamic control over the remodelling of synaptic signalling processes. Ubiquitin plays key roles in regulating synaptic receptors through proteasome-dependent and proteasomeindependent mechanisms [182, 183]. Both NMDA and AMPA receptors are regulated by protein ubiquitination [184]. For example, AMPA receptor surface expression is regulated by the opposing activities of the E3 ligase NEDD4 and the DUB USP8 (Ubiquitin-Specific Protease 8) [101, 102, 180, 185]. The NMDA receptor is a substrate of FBXO2 and MIB2 E3 ligases, and loss of ligase activity disrupts synaptic function [93, 186]. In addition to synaptic receptors, the UPS also regulates the turnover of postsynaptic scaffolding proteins such as PSD-95 [184]. Another interesting study showed that proteasomes are recruited to synapses, further implicating the UPS as a key regulator of synaptic proteins [187]. Together, these and other studies implicate ubiquitin signalling as an important regulator of the trafficking and turnover of ionotropic glutamate receptors during neuronal activity [188].

8.3 8.3.1

Ubiquitin Signalling in Alzheimer’s Disease Ubiquitin and Protein Aggregation

Some of the earliest studies linking UPS dysfunction and AD pathology reported the accumulation of ubiquitin-bound proteins in NFTs [65, 66]. Ubiquitin was also found in neuritic plaques and NFTs in the cortex of AD brains, in Lewy bodies and filaments associated with PD and Pick’s disease as well as HD [64, 65, 189, 190]. Furthermore, ubiquitin is detected in both intracellular inclusions as well as extracellular NFTs (E-NFTs) in AD brains [191, 192]. Components of the proteasome were also found in NFTs in AD and DLB [193]. Whether these represent active or functional proteasomes particles is still unclear. Importantly, the role and function of protein ubiquitination with regards to aggregate-prone

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proteins is starting to emerge. NEDD4 was recently shown to ubiquitinate α-synuclein filaments, suggesting that other protein inclusions might also be targeted by ubiquitin-mediated clearance mechanisms [194]. However, some types of protein aggregates might exert protective functions. This is the case for inclusion bodies found in HD which are thought to be important to ‘soak up’ mutant huntingtin elsewhere in neurons, thereby decreasing neuronal toxicity [195]. Similarly, higher order protein conformations seem to sequester the toxic effect of soluble Aβ and Tau [196]. Cryo-EM has emerged as a powerful technique to further our understanding of protein aggregation in neurodegeneration, by providing detailed structural information of the fibril structures of Aβ42 [197] and Tau filaments [198]. These studies revealed that Tau oligomerisation and aggregation into NTFs is likely to be disease specific [199]. It will be interesting to examine the composition of these aggregates in conjuction with quantitative proteomics methods to generate more complete structural models. Interestingly, there seems to be some preferential enrichment of particular ubiquitin chain types in some protein aggregates. For instance, Lewy bodies in PD brains are enriched with K29- and K63-linked ubiquitin chains [200]. Linear (Met-1) and K48-linked ubiquitin were detected in NTFs and PHFs [201]. However, while K29 chains were found in NFTs [69], a more sensitive quantitative approach revealed that K11, K48 and K63 but not K29 are found in AD or DLB [202]. It will be interesting to determine whether more complex ubiquitin chain types such as mixed and branched chains can also be found in some of these proteins inclusions. Proteomics studies have been useful for the non-biased determination of the protein composition of these aggregates. For example, Lewy bodies were shown to contain multiple components of the ubiquitin system including E1-conjugating enzyme, the DUBs Otubain 1 and UCLH1, proteasome subunits, SCF E3 ligases and the E4 ubiquitin ligase UBE4B [203].

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8.3.2

Crosstalk Between Tau, Ab and Ubiquitin Signalling

The UPS also plays a role in synaptic plasticity, a vital element of synaptic function, through the ubiquitination of Protein Kinase A (PKA) subunits as well as multiple other synaptic proteins [182, 204]. Reduction in NMDA and AMPA receptor expression is associated with AD and elevated Aβ decreases their surface expression [205, 206]. In fact, a recent study showed that Aβ overexpression mediates the ubiquitination of AMPA receptor and its turnover at the plasma membrane, which leads to synaptic weakening [104]. The underlying mechanisms linking Aβ abnormalities and UPS dysfunction in AD are also becoming clearer. AD patients exhibit decreased proteasomal activity in the hippocampus, a particularly vulnerable area of the brain during the early stages of the disease [207]. The presence of aggregated proteins has been shown to reduce proteasome function and this is likely exacerbated by Tau and Aβ aggregation [208]. Scanning transmission electron microscopy revealed that Aβ40 binds to the interior of the proteasome and inhibits the chymotrypsin activity of the 20S catalytic core [209, 210]. In vitro proteasomal assays further confirmed that oligomeric Aβ40-Aβ42 indeed inhibits chymotrypsin but also petidylglutamyl activity [211]. Expression of the ubiquitin conjugating enzyme E2K (E2-25K/HIP-2) is upregulated in neurons exposed to Aβ, and has also been shown to inhibit proteasomal function through its association with mutant ubiquitin UBB+1 [212]. In the case of Tau, aggregated species found within PHFs co-immunoprecipitate with proteasome subunits [213]. In addition, proteasomes found in PHFs also appeared to be less active, and in line with this, aggregated Tau inhibited proteasomal activity in AD brain samples. The E3 ligase CHIP (C-terminus of heat shock protein 70-interacting protein) might contribute to this given that ubiquitination of microtubule-associated Tau increases its aggregation propensity [75, 76].

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Mechanisms leading to loss of function of components of the proteasome have also been proposed in AD. For example, the proteasome ubiquitin receptor Rpn10 is cleaved by calpainmediated cleavage following inhibition of the electron transport chain in neurons [214]. Furthermore, the DNA-damage-inducible 1 protein Ddi1, a ubiquitin receptor that aids recruitment of ubiquitinated substrates to the proteasome, is mutated in early-onset AD [215]. Protein deubiquitination on the proteasome lid is a key step which needs to be tightly coupled to substrate processing and degradation. However, the proteasomal DUB USP14 can release partially ubiquitinated substrates from the proteasome prematurely and prior to their degradation and this reduces proteasome activity [216]. Excitingly, inhibition of USP14 DUB activity was shown to enhance proteasome activity and improve Tau degradation [111, 217]. In Table 8.1 we provide a summary of some of the E3 ubiquitin ligases and deubiquitinases which have been linked with AD.

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does not affect the proteolytic activity of the 20S directly, it inhibits the activity of proteasomal DUBs [224]. UBB+1 cannot be used like wild-type ubiquitin to covalently modify proteins due to the absence of the G76 residue. However, it can still be ubiquitinated, primarily with mixed K29/K48-linked polyubiquitin chains although other linkages might also be implicated [225, 226]. Interestingly, the toxic potential of the ubiquitinated pools of UBB+1 appears to be different between organisms. UBB+1 ubiquitination with K29- and K48-linked chains is required for full inhibition of proteolysis in mammalian cells but not in yeast [226, 227]. The finding that Aβ42 increases the expression of the E2-conjugating enzyme E2-25K/Hip2 is particularly interesting given that E2-25K/Hip2 interacts with UBB+1 and drives apoptosis [212]. This study therefore highlights functional links between AD hallmarks and the UPS.

8.4 8.3.3

Mutant Ubiquitin UBB+1

APP and the ubiquitin gene UBB have been found mutated by a process called molecular misreading. The resulting mutant protein APP+1 and UBB+1 were found to accumulate in NFTs of frontal and temporal cortices of the hippocampus in AD and Down’s Syndrome individuals [218]. Molecular misreading is caused by dinucleotide deletion at GAGAGA sequences in mRNA transcripts, resulting in frame shift and the expression of +1 proteins with an aberrant C-terminus. In the case of ubiquitin, this results in an uncleavable 19 amino acid extension at the C-terminus of ubiquitin (UBB+1). Although molecular misreading was first reported in prokaryotic cells, neurons seem particularly prone to such transcriptional error [219]. While low levels of UBB+1 are turned over by the proteasome, its accumulation drives mitochondrial stress and neuronal cell death [220–223]. UBB+1 accumulation seems disease specific since it is not found in synucleinopathies [221]. Although UBB+1

8.4.1

Emerging Topics in Proteostasis and Alzheimer’s Disease Mitochondrial Dysfunction

Mitochondria are the energy generating organelles of all eukaryotic cells by producing ATP molecules through the oxidative phosphorylation pathway. Mitochondrial dysfunction in AD was first suspected through microscopy studies [228]. Since then, most if not all hallmarks of AD, including Aβ, Tau and UBB+1, have been associated with mitochondrial dysfunction [222, 229–231]. Amyloid-β for instance was shown to accumulate in the mitochondrial matrix of neuronal cells from AD transgenic mice [232]. Mitochondrial homeostasis is maintained by multiple pathways including fission and fusion as well as through the removal of damage mitochondria via mitophagy [233]. Increased mitochondrial fragmentation for instance is observed in AD brains and it is associated with Aβ expression. In fact, Aβ physically interacts with mitochondrial dynamin-related protein

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1 (Drp1), a protein involved in mitochondrial fission [234]. This results in increased mitochondrial fragmentation which affects mitochondrial dynamics and causes synaptic damage. Ubiquitin signalling is tighly connected to mitochondrial homeostasis. For example, the E3 ubiquitin ligase MITOL/MARCH5 regulates Mitochondrial fission 1 protein (Fis1) and Drp1 levels, which in turn impact on mitochondrial dynamics [235]. Loss of MITOL was shown to preserve mitochondrial function during neuronal stress, including during Aβ-induced stress. The RING-Between-RING (RBR) ligase Parkin is perhaps the best studied mitochondrial E3 ligase identified to date. Parkin and PTENinduced putative protein kinase 1 (PINK1) form the core components of mitophagy, including in neuronal cells [161, 236, 237]. Mutations in the Parkin gene associated with PD were first identified in 1998 [70, 160, 238, 239]. Since then, Parkin mutations have been linked to the pathogenesis of a number of additional brain diseases including AD and multiple sclerosis [70, 240]. PINK1 also appears to be important in AD, since restoring its expression lowers Aβ levels and also reduces mitochondrial and synaptic dysfunction [241]. The accumulation of damaged mitochondria seen in AD brains is a result of inadequate Parkin-mediated mitophagy [70]. Recent structure–function studies have revealed how Parkin mutations found in PD affect its ligase activity and function during mitophagy [242]. This is also informative for AD, given that PARK2 mutations have also been found in some individuals with sporadic early-onset AD [243]. Calcium is another major regulator of mitochondrial function which is highly relevant to AD. The atypical Rho GTPases Miro for example anchors mitochondria to motor proteins and regulates mitochondrial trafficking in a calciumdependent manner [244]. Under conditions that lead to excess intracellular calcium concentration, just as those observed during excitotoxicity, Miro recruits and is ubiquitinated by Parkin, which drives the removal of damaged mitochondria. Under these same conditions, Miro depletion prevents Parkin mitochondrial translocation and this protects neurons from glutamate-induced

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mitophagy [245]. These studies highlight novel crosstalk between calcium signalling, the UPS and mitochondrial function and these are also likely to be relevant in the context of AD.

8.4.2

ER-Mitochondria-Associated Membranes

The endoplasmic reticulum (ER) has emerged as an important network which is tightly connected to most, if not all, membrane-bound organelles including the plasma membrane (PM), peroxisomes, mitochondria, golgi, lipid droplets and endosomes [246]. Contact sites between the ER and mitochondria, known as ER-MitochondriaAssociated Membranes (ER-MAMs), were first visualised under electron microscopy over 60 years ago [247]. ER-MAMs regulate organelle dynamics [248], autophagy [249], lipid metabolism and trafficking [250], calcium dynamic [251, 252], metabolism [253], and apoptosis [254]. At ER-MAMs, ER resident IP3R (inositol 1,4,5triphosphate receptor) interacts with the outer mitochondrial membrane (OMM) protein VDAC (voltage-dependent anion-selective channel). Both IP3R and VDAC function as calcium channels, while Grp75 mediates and regulates IP3R-VDAC interactions, thus increasing mitochondrial calcium uptake efficiency [255]. IP3R and VDAC are both substrates of protein ubiquitination, which suggests interesting crosstalks between the ubiquitin system and inter-organelle communication [256, 257]. For example, the E3 ligase MITOL regulates ER-tethering to the mitochondria, through Mitofusin 2 ubiquitination and degradation [258]. ER-MAMs are also highly relevant in AD, with Aβ shown to associate with VDAC1, which enhances its conductance and leads to apoptotic cell death [259]. Recent data also indicate that C99, the APP fragment produced by β-secretase-mediated cleavage, increases at ER-MAMs and interferes with mitochondrial function in a cellular model of AD [260]. The other fragment produced from that cleavage event, Aβ, increases the number of ER-MAMs contact sites which raises mitochondrial calcium levels [261]. The hyperphosphorylated Tau P301L mutant, a hallmark on FTD, is enriched at

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ER-MAMs which might further explain the mechanisms underlying Tau-mediated mitochondrial toxicity [262]. Together, these and other studies strongly implicate ER-MAMs dysregulation in AD and related dementias [263].

8.4.3

Necroptosis and Alzheimer’s Disease

Apoptotic cell death is the best studied mode of progammed-cell-death in eukaryotes. It can be triggered by the extrinsic (e.g. Tumour Necrosis Factor-α, Fas Ligand) or intrinsic (e.g. translocation of BAX to the mitochondrial matrix) pathways, which lead to the activation of caspases. In AD, glutamate-induced excitotoxicity mediates neuronal apoptosis. More recently, necroptosis which originally was not believed to be regulated at the molecular level, has emerged as a second mode of programmed-cell-death relevant in AD [264, 265]. Both apoptosis and necroptosis are activated by ligand binding of TNFα onto its cognate receptor TNFR1. Necroptosis likely acts as a fail-safe mechanism for cell death since inactivation of apoptosis is a prerequisite for necroptosis induction. While apoptotic cells release their content through the form of apoptotic bodies, necroptotic cells directly leak theirs into the extracellular space. In addition to this phenotypic difference, both cell death pathways are regulated by distinct mechanisms. In contrast to apoptosis, necroptosis does not involve the sequential activation of caspases but rather, it relies on the activation of the ripoptosome, a multi-protein complex which contains serinethreonine kinase receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) [266]. RIPK1/ K3 converge to induce the phosphorylation of MLKL (mixed lineage kinase domain-like). Necroptosis culminates in pore formation which is mediated by MLKL, directly and/or indirectly. In the direct model, MLKL phosphorylation leads to exposure of a 4-helical bundle domain which results in its relocalisation and oligomerisation at the PM where pore formation occurs [267]. In contrast, the indirect model suggests that MLKL

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recruits Ca2+ channels which increases calcium influx and leads to PM rupture [268]. Recent evidence have reported the upregulation of MLKL and RIPK1 in post-mortem AD brains suggesting that necroptosis is activated in sporadic AD [269]. In support of this, viral-vector mediated expression of MLKL led to more severe cognitive deficits in an AD mouse model, and these effects could be blocked by necrostatin-1, a RIPK1 inhibitor. Cellular inhibitors of apoptosis protein 1 and 2 (cIAP1 and 2) are E3 ligases mediating apoptosis. In contrast, much less is known regarding the role of ubiquitin in necroptosis. A recent study reported that expression of the HECT E3 ubiquitin ligase SMURF1 is increased upon lipopolysaccharide (LPS)-induced inflammation, and this is accompanied by an increase in the necroptosis marker RIPK1 [270]. Examining the therapeutic potential of RIPK1 and of the ubiquitin system could lead to new and exciting approaches for AD. Encouraging data have shown that the proteasomal inhibitor Carfilzomib prevents ripoptosome complex formation and reduces necroptosis in multiple myeoloma cells, although the exact molecular mechanisms remain to be determined [271].

8.4.4

Targeting the Ubiquitin System in Alzheimer’s Disease

The prevailing dogma suggests that maintaining proteostasis throughout life might protect or at least delay age-related disease including AD. Although the protective effect of caloric restriction on health and survival in rhesus monkeys has been controversial, it nevertheless seems to show some cognitive benefits [272]. Studies in rodents have provided strong evidence to suggest that a decrease in protein synthesis through mTOR inhibition can decrease the toxic effect associated with protein aggregation, while loss of autophagy can cause neurodegeneration in mice [273–275]. Enhancing autophagy and UPS function have been put forward as promising strategies to maintain neuronal health [276, 277]. In the context of the UPS, this

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could be achieved through increasing peptidase activity within the proteasomal core. Alternatively, USP14 inhibition could reduce the premature release of partially deubiquitinated substrates from the proteasome and therefore prevent the accumulation of non-functional proteins [216]. Another strategy could be to increase the recruitment of proteasomal particles to dendritic spines, which are key synaptic contact points where neuronal processes occur [187]. The stimulation of 26S proteasomal activity has also been achieved through the cAMP-PKA-mediated phosphorylation of proteasomal subunits, such as Rpn6/PSMD11, which enhances the degradation of misfolded proteins [278, 279]. Cyclic nucleotide phosphodiesterases (PDEs) act as part of a negative feedback loop to dampen the effect of second messengers. PDE inhibition, which prolongs the activation of proteasomal function induced by cAMP, has already shown encouraging results in AD mouse models [280, 281]. Another emerging approach is the targeted degradation of specific molecules by proteolysis targeting chimeric molecules (PROTACs) [282]. These molecules can recruit the ubiquitin machinery to target specific proteins for degradation [283]. PROTACs are hetero-bifunctional molecules, composed of a linker attached to two ligands. One ligand recruits the E3 ligase while the other recruits the target protein. The recruitment of the target protein to the E3 ligase results in its ubiquitination and subsequent degradation. In theory, this technology has the potential to utilise the endogenous activity of a whole spectrum of E3 ligases in the mammalian genome to degrade a vast array of target proteins. Initially, in vivo applications of PROTACs had been problematic due to their poor cellular permeability, but the development of specialised in vivo PROTACs containing poly-arginine tags for improved permeability seem to facilitate the degradation of target proteins [284]. Small molecule-based PROTACs have also improved cell permeability over their peptide-based counterparts. The first small molecule-based PROTAC was composed of the E3 ubiquitin ligase MDM2 ligand Nutlin and a non-steroidal androgen receptor ligand,

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which efficiently degradaded the androgen receptor [285]. Additional PROTACs have since been developed to degrade Cellular Retinoic Acid-Binding Protein 1 (CRABP1) in neuroblastoma cell lines [286], bromodomain and extra-terminal (BET) proteins in leukaemia [287] and prostate cancer cells [288]. Excitingly, Arvinas’ Androgen Receptor (AR)-PROTAC™ entered the first phase 1 clinical trials for patients with metastatic castration-resistant prostate cancer in 2019. In the context of AD, PROTACs could in theory be developed for the targeted degradation of mutant Tau or of proteases involved in Aβ production. The latter option is perhaps not straightforward given that Aβ also has a normal physiological role in neurons. In addition, Tau, Aβ and UBB+1 can impede UPS activity, and this might affect the efficacy of PROTACs in AD [207]. Therefore, an approach that combines enhancing proteasomal activity with PROTACs might be necessary to overcome this limitation. Another mode of targeted protein degradation which could prove useful in AD employs the cytosolic Fc receptor TRIM21 which was recently shown to neutralise misfolded Tau and its associated seeding propensity [97]. These new technologies, and the ability for the UPS to target some protein aggregates, have the potential to change the therapeutic landscape in AD over the next few years. Finally, the recent development of USP7 inhibitors as novel cancer therapies might also be promising in AD [289–293]. USP7 deubiquitinates and inactivates MDM2 ligase activity which in turns stabilises p53 and enables cancer cells to survive stress conditions. It will be interesting to see how USP7 inhibitors perform in clinical trials and also to explore other DUBbased therapies in AD (Table 8.1). For instance, USP14 inhibition accelerates the degradation of mutant Tau [111], while loss of USP8 DUB activity leads to the lysosomal degradation of BACE1 which in turn reduces Aβ production [112]. Importantly, DUBs and E3s likely have multiple substrates and the specificity of the mechanisms targeted will need to be closely monitored [294].

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8.5

Concluding Remarks

Despite the discovery of protein inclusions in the brain of patients with AD over a century ago, there is still no cure available. GWAS studies have been useful to identify biomarkers in familial cases, but the molecular mechanisms that initiate and drive AD remain elusive. Determining the properties and function of Aβ and Tau in normal brain homeostasis and AD has been a focus for many years and it now appears that soluble intermediate oligomers, rather than protein aggregates may well be the toxic species. AD and other brain proteinopathies have been associated with loss of proteostasis and the decrease in proteasomal function and autophagy contribute to this process. The brain seems to be particularly reliant on these pathways to maintain homeostasis, which likely reflects the inability of neurons to divide or replenish following injury. In this chapter, we have summarised some of the evidence linking dysregulation in ubiquitin signalling and AD, including crosstalk between Tau, Aβ and the UPS. We have also highlighted new ubiquitin-dependent mechanisms and in particular how changes in deubiquitinases and E3 ubiquitin ligases activity impact on synaptic and cognitive function. Neuronal necroptosis, mitochondrial homeostasis and calcium signalling at ER-MAMs have all emerged as exciting new fields of research relevant for AD. It will be interesting to determine how the writers (i.e. E1, E2, E3s), readers (i.e. UBDs) and erasers (i.e. DUBs) of the ubiquitin code impact on these mechanisms during healthy and pathological brain ageing. Technological advances including cryo-EM, quantitative proteomics, single molecule microscopy as well as improved in vitro and in vivo AD models will be instrumental in further defining how such proteinopathies initiate and develop. This fundamental knowledge should then feed into drug development with the aim to open new therapeutic avenues for AD. Acknowledgements We thank Dr. Robert Williams for feedback on the chapter. The authors acknowledge funding from Alzheimer’s Research UK (pilot grant

209 ARUK-PPG2015A-16) as well as Bath/Bristol ARUK network pilot grants. Lee Harris is funded by an ARUK PhD studentship (ARUK-PhD2017-28), Sarah Jasem was funded through a Kuwait Science PhD Scholarship. We also thank COST ACTION BM1307 – COST Proteostasis – as well as the Bath/Bristol ARUK Network for conference travel awards.

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The Interplay Between Proteostasis Systems and Parkinson’s Disease Diana F. Lázaro and Tiago F. Outeiro

Abstract

The proteostasis network controls the balance between protein synthesis, folding, function, and degradation, and ensures proteins are recycled when they are no longer needed or become damaged, avoiding unwanted aggregation and accumulation. In various neurological disorders, such as Parkinson’s disease (PD) and other synucleinopathies, the accumulation of misfolded and aggregated alphasynuclein (aSyn) is considered a central event in the onset and progression of disease. During aging, there is a decline in the activity of

D. F. Lázaro Department of Experimental Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany T. F. Outeiro (*) Department of Experimental Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK e-mail: [email protected]

various degradation machineries, and the overall buffering capacity of the proteostasis network starts to decrease. Such decline is thought to play a pivotal role in PD, causing aSyn to build-up due to compromised clearance, which in turn contributes to further disease progression. In this chapter, we summarize central findings related to aSyn accumulation and degradation, as well as to the consequences of the toxic effects caused by aSyn on proteostasis. We also highlight some of the factors and pathways that may be used as potential targets for therapeutic interventions in PD. Keywords

Proteostasis · Parkinson’s disease · Alphasynuclein · Protein aggregation · Neurodegeneration

9.1

Introduction

Protein homeostasis (proteostasis) is the process through which the concentration, conformation, interactions, and localization of proteins are regulated, and involves a series of transcriptional and translational changes in the cell. Heat, oxidative, osmotic, or pH are examples of different stresses that can affect proteostasis [1]. Aging and disease-associated mutations may also directly affect proteostasis due to the progressive decline in the activity of cellular quality

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_9

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control systems, leading to the buildup of harmful, aggregation-prone protein species. Thus, to maintain proteostasis, cells have evolved a wide variety of protein quality control systems, such as molecular chaperones and different degradation systems that constitute the proteostasis network [1, 2]. Deficiencies in this complex network can lead to a range of diseases known as protein conformational disorders [3], of which neurodegenerative diseases are chief examples, having as a common denominator the accumulation of misfolded/aggregated proteins.

9.2

Parkinson’s Disease

Parkinson’s disease (PD) is the most common progressive motor disorder, and the second most prevalent neurodegenerative disorder, after Alzheimer’s disease (AD), affecting around 1% of the worldwide population at the age of 60, and 4–5% of people over 85 [4]. PD is known for the progressive loss of dopaminergic neurons in substantia nigra pars compacta (SN), leading to dopamine (DA) deficiency in the striatum. This explains the cardinal features of the disease [5]. Alpha-synuclein (aSyn) is a natively unfolded protein considered as a major culprit in PD due to its accumulation in insoluble protein deposits known as Lewy bodies (LBs) or Lewy neurites (LNs) [6, 7]. In addition, missense mutations and multiplications of the SNCA gene (encoding for aSyn) [8–13] are associated with familial forms of the disease. The etiology of PD is highly complex, and involves a combination of genetic and environmental factors, together with aging. Together, these factors explain the majority of cases. Protein aggregation, as in other neurodegenerative disorders, is thought to play a central role in PD. Several studies demonstrated an association between aging and the gradual decline in cellular proteostasis capacity [1, 14]. Therefore, cells evolved several mechanisms to preserve proteostasis and to mitigate life-threatening effects of toxic protein aggregation. Below, we review how some of these proteostasis mechanisms have been linked to PD.

9.3

Regulation of Protein Folding and Aggregation in Mammalian Cells

Cells need to maintain the balance between protein synthesis, folding, trafficking, assembly, and degradation. Proteins known as molecular chaperones mediate protein folding, by stabilizing non-native regions, by assisting proteins to acquire their native state, or by preventing improper protein associations [15– 18]. Several of these chaperones can be induced under stress conditions, such as heat shock, but are also involved in trafficking, protein refolding, disaggregation, and degradation [19]. Some chaperones are often referred to as heat shock proteins (Hsps) because they are upregulated during heat stress. Hsp70 plays an important role in stabilizing the folding of newly synthesized polypeptides [15]. The mechanism of action is dependent on ATP hydrolysis to promote the folding of the target protein. In contrast, holdases (as small Hsps) are ATP independent since they do not participate in protein folding, and the association with their target is due to hydrophobic interactions. If energy is available, holdases can also deliver target proteins to foldases for folding, or to degradation systems such as the proteasome [20]. The association between molecular chaperones and PD was provided by the identification of several Hsp (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90, Hsp110) as components of LBs [21]. Several subsequent studies have investigated the impact of chaperones interaction on aSyn aggregation in vitro and in vivo [22, 23].

9.3.1

The Hsp70/Hsp40 System

Hsp70 and its constitutive relative heat shock cognate 70 (Hsc70) belong to conserved family of proteins that are the first to respond to cellular stress and misfolded protein accumulation [24]. Under physiological conditions, Hsp70

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The Interplay Between Proteostasis Systems and Parkinson’s Disease

primarily localizes in the cytoplasm but, upon stress, it can relocate into the nucleus and nucleoli, to influence gene expression [25]. Hsp70 binds and protects the hydrophobic region of client protein, preventing its aggregation in an ATP-dependent manner [26]. The ATPase cycle is normally modulated by several co-chaperones, like “J-domain” proteins [27]. So, Hsp70 relies on regulatory chaperone cofactors of the Hsp40 class (also known as DNAJ or J proteins), which will first bind to exposed hydrophobic portion on client proteins, and then recruits Hsp70 [28, 29]. Much of the function of Hsp70s is driven by Hsp40s—there are 11 Hsp70s and 41 J-proteins [28]. This allows Hsp70 to have more versatility multifunctionality. In proteinopathies such as PD, the Hsp70 system plays an important role. The inhibitory effect of Hsp70 on aSyn aggregation depends on the ratio between Hsp70/aSyn, and the relative levels of aSyn and ATP, or ADP [30]. In vitro, Hsp70 significantly increases the lag phase of aSyn aggregation [30]. Overexpression of Hsp70 in cell and animal models reduces aSyn toxicity [31, 32]. In mice, for example, Hsp70 reduces both high molecular and detergent-insoluble aSyn species [32]. The reduction of aSyn toxicity by Hsp70 is thought to be due to a modulatory effect on aSyn oligomeric [22], and fibrillar species [33] (Fig. 9.1). However, aSyn oligomers, but not aSyn monomers, strongly inhibit Hsp70/ Hsp40 activity [34] (Fig. 9.1). This is due to the competitive inhibition of the oligomers on J-domain co-chaperones. Furthermore, Hsp70/ Hsp40 activity can repress IkB and, therefore, repress NF-κB-mediated apoptosis [35]. Thus, the inhibition of Hsp70/Hsp40 by aSyn oligomeric species could potentiate the proteostasis imbalance in DA neurons, leading to neuroinflammation and, ultimately, to cell death.

9.3.2

Hsp90 Chaperone

Hsp90 is a highly conserved chaperone that acts downstream of Hsp70. Hsp90 is active as a homodimer, and is not normally a general

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chaperone for newly synthesized proteins [36]. Co-chaperones regulate Hsp90 function in different ways, such as by coordinating the interaction between Hsp90 and other chaperone systems, stimulating/inhibiting ATPase Hsp90 activity, recruiting specific clients or participating in different enzymatic activities through the chaperone cycle [36, 37]. Hsp90 can also act independently of ATP and prevent protein aggregation alone or with other chaperones, such as Hsp70. Hsp90 conserves client proteins in a foldingcompetent conformation and prevents their denaturation under stress and dependent on ATP binding [38]. Furthermore, when ATP is bound, Hsp90 can also promote the ubiquitination and degradation of a client protein [38, 39]. Hsp90 can co-localize with ubiquitinated aSyn both in human synucleinopathies and in animal models [39] (Fig. 9.1). A putative explanation for this co-localization relies on the fact that, under stress, aSyn may become denatured or aggregated, and Hsp90 may rescue aSyn from further denaturation/aggregation. However, if stress persists, and Hsp90 is no longer able to rescue aSyn, it may redirect aSyn to the proteasome, by facilitating aSyn ubiquitination [39]. An aspect that argues in favor of this theory is that proteasomal inhibitors (MG132 or lactacystin) lead to upregulation of Hsp90 and accumulation of ubiquitinated proteins [39]. Furthermore, NMR data unveiled that the interaction with Hsp90 occurs thought the aSyn NAC domain, which is highly hydrophobic [40]. This region is well known to be important for aSyn aggregation, so this might be another clue implicating Hsp90 on the aSyn aggregation cascade.

9.3.3

Small Heat Shock Proteins

LBs, LNs, and glial cytoplasmic inclusions (in multiple system atrophy) contain various proteins in addition to aSyn. Among these, small heat shock proteins (sHsps) have been identified in the different inclusions [21, 22, 41]. sHsps are ubiquitous proteins that assemble and operate in a conserved oligomeric structure that is capable of preventing inappropriate

Fig. 9.1 Proteostasis mechanisms. During the process of aSyn aggregation, several mechanisms are in place to maintain proteostasis. Misfolded aSyn can be recognized and refolded by molecular chaperones. When misfolding is not possible, the UPS and

ALP systems are called to action in order to attempt to degrade and recycle aSyn. When these systems fail and the aggregation process continues, aSyn can accumulate in the typical pathological inclusions—Lewy bodies and Lewy neurites

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associations between proteins, reversing misfolded proteins to their normal state, and promoting the clearance of misfolded proteins [42]. In humans, 11 different sHsps have been described but only a few can be considered as true heat shock proteins [43]. The most studied sHsps in mammals are αB-crystallin (aBc) and Hsp27, and these will be the subject of the following sections.

9.3.3.1 aB-Crystallin aBc is an oligomeric molecular chaperone that acts against abnormal protein aggregation. It is involved in pathologies like cataracts [44, 45], myopathies [46], and neurodegenerative diseases [47, 48], and it is also involved in cell cycle, apoptosis, and cancer [49]. aBc is organized in three domains: the central domain, with an alphacrystallin domain that is flanked by an N-terminal domain (NTD), and a short C-terminal extension [50]. Phosphorylation is the major regulator of aBc, and occurs in three different sites (Ser19, Ser45, and Ser59) all located within the NTD [51, 52]. In pathological situations, aBc accumulates in neurons and glia in the central nervous system [53]. In PD, aBc is detected in microglia in the SN and in the hippocampus [54]. aBc can interact with aSyn along the length of aSyn mature amyloid fibrils [55], via a conserved β4/β8 surface of the central alpha-crystallin domain. This transient interaction [43, 55, 56] is able to inhibit aSyn fibrilization both at early stages, and during the growth phase of fibril formation [57, 58]. Furthermore, aBc reduces aSyn toxicity in cellular models [22]. A recent study challenged the protective role of aBc by showing that it suppresses astrocytic autophagy by binding to BAG3, which negatively affects the degradation of aSyn aggregates. In this study, knockdown of aBc improved the clearance of aSyn pre-formed fibrils (PFFs) [59], suggesting that targeting aBc my hold potential as a therapeutic target in synucleinopathies. Nevertheless, additional studies will be necessary to clarify how

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manipulating the levels of aBc in different cells may affect the accumulation and toxicity of aSyn.

9.3.3.2 Hsp27 Hsp27 is a molecular chaperone expressed in several cell types and tissues at specific stages of development and differentiation [60]. Hsp27 modulates apoptosis [61], and plays an important role against oxidative stress [62]. Interestingly, Hsp27 has been found in the brains of AD patients, together with amyloid plaques [63], and elevated levels of Hsp27 mRNA are present in dementia with LBs [64]. Similarly to aBc, Hsp27 is able to transiently interact with aSyn fibrils [43], inhibiting the elongation of aSyn fibrils [55], and reducing aSyn toxicity in primary dopaminergic neurons [22, 65]. The effect of Hsp27-mediated inhibition of aSyn aggregation is dependent on the rate of aSyn, and on the relative concentration of monomeric aSyn to Hsp27 [43, 66]. This suggests that increasing aSyn concentration may allow aSyn to escape sHsps, and become more prone to aggregate. These findings are particularly relevant in the context of familial forms of PD with duplications and triplications of the SNCA gene, and suggest that, in such cases, upregulation of certain sHsps may be beneficial.

9.4

Proteolytic Recycling of Proteins

We still do not fully understand how misfolded proteins are distinguished from the correct folded counterparts. However, this is a crucial step in cellular homeostasis. Whenever the proteins are unable to fold or refold, despite intervention of chaperones, they must be removed by proteolytic degradation to avoid the accumulation of toxic species, and to enable the recycling of the amino acids. In eukaryotic cells there are two major pathways involved in the recycling of proteins: the ubiquitin proteasome system (UPS) and the autophagy–lysosomal pathway (ALP).

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9.4.1

D. F. Lázaro and T. F. Outeiro

The Ubiquitin Proteasome System (UPS)

The UPS is the primary degradation system in eukaryotic cells. This large multi-catalytic proteinase complex is present in the nucleus and in the cytoplasm of cells, and is responsible for the degradation of short-lived regulatory proteins and for the elimination of damaged, misfolded/ unfolded proteins [67, 68]. When proteins are targeted for degradation they are ubiquitinated at the lysine residues, by a sequential action of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligase (E3) enzymes. The formation of ubiquitin chains linked through K48 or K11 residues targets the substrate for degradation by the 26S proteasome, and K63-linked chains target proteins for lysosomal degradation [69, 70]. A common feature among neurodegenerative disorders is the deposition of polyubiquitinated protein inclusions in neurons [33]. In physiological conditions, aSyn is mostly degraded by the UPS system [71]. However, under chronic proteasome inhibition, aSyn can accumulate in non-ubiquitylated forms [72]. Dysfunction on 26S proteasomal via Psmc1 is sufficient to induce neurodegenerative disease. PSMC1 is a crucial subunit of the 26S proteasome and deleting this protein in mice causes the accumulation of intraneuronal LBs-like inclusions and neurodegeneration in the nigrostriatal pathway and forebrain regions [73]. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a multifunctional protein with high prevalence in the brain [74]. In vitro, UCH-L1 was found to possess a dimerization-dependent ligase activity responsible for K63-linked polyubiquitination of aSyn [75], which potentially blocks its degradation and leads to its accumulation and aggregation within neurons. Yet, the link to PD remains controversial [76]. Subsequently, a polymorphism in the UCH-L1 gene (S18Y) was found to be associated with lower risk for developing PD, by conferring a protective effect through an antioxidant action

[75, 77]. However, more recent studies have failed to find significant association between S18Y polymorphism and reduced PD risk [78, 79], and this gene has not received much attention in the field. The neuronal precursor cell-expressed, developmentally downregulated gene 4 (Nedd4) is another protein involved in aSyn ubiquitination. Nedd4 is an ubiquitin ligase, which acts in the endosomal–lysosomal pathway [80]. Nedd4 ubiquitinates aSyn, and promotes its clearance via the endosomal–lysosomal pathway in cultured cells [70]. Deletion of the ortholog of Nedd4 in yeast, Rsp5p, leads to a decrease in aSyn degradation and leads to the formation of aSyn inclusions, and to toxicity [70]. Interestingly, N-aryl benzimidazole was found to protect cells from aSyn toxicity, because it mediates endosomal transport dependent on Rsp5/Nedd4 ligase activity [81]. In Drosophila, Nedd4 expression prevents locomotor deficits associated with aSyn [82]. In vitro, aSyn filaments are a better substrate for ubiquitination than monomeric protein [83], which could imply that the formation of beta-sheet-containing filaments generates a surface for ubiquitin ligase recognition [83]. Thus, perhaps Nedd4 may act as modifier of aSyn accumulation and may be used as a target for neuroprotective therapies. The carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a co-chaperone of Hsp70 and Hsp90. CHIP is an E3 ubiquitin ligase to regulate proteasomal degradation of chaperone client proteins [84, 85]. CHIP has two specialized protein domains: a tetratricopeptide/Hsp70 binding domain, and a U-box/ubiquitin ligase domain that plays different roles in the fate of misfolded proteins [86, 87]. CHIP can mediate the degradation of misfolded proteins associated with PD [87, 88], AD [89, 90], Huntington’s disease [91], Spinocerebellar ataxia [92], and Spinal and bulbar muscular atrophy [93]. CHIP can rescue the cytotoxicity promoted by aSyn oligomers, and reduce the formation of higher molecular weight oligomeric species, in an Hsp70-dependent manner [87]. Furthermore, CHIP and aSyn form a protein complex mediated by BAG5

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The Interplay Between Proteostasis Systems and Parkinson’s Disease

(co-chaperone bcl-2-associated athanogene 5) [94]. BAG5 inhibits CHIP-mediated ubiquitination of aSyn, and regulates the ability of CHIP to decrease the levels of aSyn oligomeric species. Therefore, enhancing the CHIP E3 ubiquitin ligase activity or inhibiting BAG5 function can prevent aSyn toxicity, both of which may hold value as strategies to mediate aSyn toxicity. However, it is important to note that CHIP can regulate the degradation of tyrosine hydroxylase [95], which may compromise its usefulness as a therapeutic approach. SUMOylation, a posttranslational modification (PTM) that consists of the addition of a small ubiquitin-related modifier, is a similar process to ubiquitination, involving a series of enzymatic steps [96]. SUMO is conjugated to lysine residues in a wide range of proteins, regulating various biological processes [97]. In aSyn there are two SUMO acceptor residues (at position 96 and 102), and mutations in these residues significantly impair aSyn SUMOylation. Most importantly, a double mutation on K96/K102 residues increases the propensity of aSyn aggregate and to cause toxic effects in cell models and in dopaminergic neurons [98]. Recently, it was described that SUMOylation by PIAS2, an E3 SUMO ligase [97, 99], leads to the formation of aSyn inclusions in cells, impairs aSyn ubiquitination, prevents its degradation, and increases the release of aSyn to the medium [100]. Interestingly, we have recently shown that glycation, an age-associated PTM that consists of the covalent bonding between a sugar and a protein (or nucleic acid), can block lysine residues in aSyn, leading to its accumulation and aggregation [101, 102]. Therefore, glycation or other PTMs that block residues, which may normally be used to target proteins for degradation, may tip the proteostasis balance and cause cellular pathologies that may, ultimately, contribute to diseases such as PD.

9.4.2

The Autophagy–Lysosome Pathway (ALP)

The ALP acts on the degradation of long-lived proteins and protein assemblies, like misfolded

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and aggregated proteins, as well as of dysfunctional organelles, in lysosomes [103, 104]. The target components of the ALP can be delivered to the lysosomes by three main paths: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). CMA and macroautophagy (herewith designated as autophagy for simplicity) have been found altered in PD and in various models of the disease [105–107]. CMA is a selective process in which chaperones and co-chaperones first recognize a pentapeptide motif (KFERQ) in proteins, docking them to a CMA receptor at the lysosomal membrane, known as the lysosome-associated membrane protein type 2A (LAMP-2A). Afterwards, this complex is internalized into the lysosome for subsequent degradation [108]. In autophagy, the cytoplasmic components are engulfed by a double membrane structure, the autophagosome, and then fused with the lysosome to form the autophagolysosome, where the actual degradation will take place [109]. Autophagy can be activated via the mTOR (mammalian target of rapamycin) pathway [110], or the PI3kinase/beclin/vsp34 pathway (also known as mTOR-independent pathway) [111], both of which are extremely important for normal cellular function. Inhibition of the autophagy increases the levels of extracellular aSyn [112], and upregulates the association of aSyn with extracellular vesicles [113]. Certain PTMs on aSyn can influence its degradation by the CMA pathway in idiopathic forms of PD. For example, oxidation and nitration slightly inhibit aSyn degradation, and phosphorylation or dopamine modification of aSyn almost prevents CMA-dependent degradation [114]. In yeast, autophagy is the main mechanism for the clearance of aSyn, and a substitution of S129 in aSyn by an alanine residue (S129A), which blocks phosphorylation, impairs the autophagy-mediated degradation of aSyn [115]. It was recently shown that aSyn-induced toxicity could be prevented, in cultured cells, through autophagy stimulation [116]. The idea is to upregulate autophagy to provide protection when the degradation systems may already be

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impaired. Consistently, previous studies reported that increasing the expression of autophagic markers improves the survival of dopaminergic neurons [117]. When the levels of aSyn are increased, the transcription factor EB (TFEB), which is a master regulator of ALP [118, 119], cannot be translocated into the nucleus [117, 120]. This retention was observed both in rat and in human tissue, and TFEB was found to co-localize with aSyn in LBs [117, 120]. If the levels of TFEB are restored, nigral DA neurons are rescued from aSyn toxicity. aSyn is known to be, predominantly, a cytosolic protein. However, it can be present in the nucleus [121], and outside cells, as in cerebrospinal fluid (CSF) and plasma [122]. The precise mechanisms by which aSyn is released from cells, potentially leading to the spreading of pathology in PD, are unclear, but one possible route is via exosomes [123]. These small vesicles are abundant in various body fluids, and naturally carry mRNA, miRNA, and proteins [124, 125]. In exosomes, aSyn may exist in oligomeric or in other aggregated forms [126–128]. Inhibition of lysosomal function by ammonium chloride or bafilomycin increases aSyn release and its transfer to neighboring cells [129]. This suggests that impairment or blockade of ALP [130, 131] may trigger, or at least contribute to the spreading of aSyn pathology. External environmental factors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can have a profound impact on autophagic flux. MPTP can induce abnormal permeabilization of lysosomal membranes due to the increase of reactive oxygen species (ROS) derived from complex I inhibition in mitochondria. In turn, ROS will lead to defective protein clearance due to the accumulation of autophagosomes [132], and also can cross the blood–brain barrier [133]. Perhaps the strongest evidence supporting the connection of lysosomal dysfunction and PD comes from the finding that mutations in the GBA gene, encoding for glucocerebrosidase (GCase), strongly increase PD risk [134– 142]. GBA mutations are associated with Gaucher’s disease, a lysosomal storage disorder

D. F. Lázaro and T. F. Outeiro

characterized by the impaired lysosomal function [143, 144]. This strongly implicates lysosomal dysfunction in the etiology of PD, but the precise molecular mechanisms involved are still being investigated [145–148].

9.5

Conclusions and Final Remarks

Eukaryotic cells have evolved a variety of mechanisms to preserve a functional and balanced proteome. Several levels of organization are in place to protect the health status of cell and, in multicellular organisms, of the whole organism. We still not fully understand how these signaling processes are organized and regulated during development, aging, and disease. However, it is clear that, over time, the activity of these systems starts to decline, and they are no longer able to fulfill their tasks. This proteome imbalance facilitates the development of degenerative pathologies such as those characterized by the accumulation of misfolded and aggregated proteins. In neurodegenerative diseases such as PD, the characteristic protein deposits are enriched in aSyn, a protein whose function is still obscure. However, the molecular events that trigger the deposition of aSyn, and that may explain its apparent toxicity are unclear. Why, when, and how aSyn misfolds and aggregates are still open questions we need to tackle in order to be able to develop novel and effective therapeutic strategies capable of preventing disease or modifying disease progression. Thus, understanding the involvement of various proteostasis components in the disease process emerges as essential, not only as a window into future therapeutics, but also into possible biomarkers that may help diagnose and follow disease progression. In conclusion, as our knowledge of the molecular underpinnings of proteostasis evolves, there is justified optimism for the development of novel approaches to tackle PD and other protein misfolding disorders. Acknowledgments TFO is supported by an EU Joint Programme—Neurodegenerative Disease Research

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The Interplay Between Proteostasis Systems and Parkinson’s Disease

(JPND) project (aSynProtec) and by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 721802. TFO and DFL are supported by a grant from ParkinsonFonds Deutschland (PD).

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D. F. Lázaro and T. F. Outeiro 139. Sidransky E et al (2009a) Multicenter analysis of glucocerebrosidase mutations in Parkinson’s disease. N Engl J Med 361:1651–1661. https://doi.org/10. 1056/NEJMoa0901281 140. Sidransky E, Samaddar T, Tayebi N (2009b) Mutations in GBA are associated with familial Parkinson disease susceptibility and age at onset. Neurology 73:1424–1425., author reply 1425-1426. https://doi.org/10.1212/WNL.0b013e3181b28601 141. Tsunemi T, Krainc D (2014) Zn(2)(+) dyshomeostasis caused by loss of ATP13A2/ PARK9 leads to lysosomal dysfunction and alphasynuclein accumulation. Hum Mol Genet 23:2791–2801. https://doi.org/10.1093/hmg/ddt572 142. Wang R et al (2019) ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosomelysosome fusion. J Cell Biol 218:267–284. https:// doi.org/10.1083/jcb.201804165 143. Klein AD, Mazzulli JR (2018) Is Parkinson’s disease a lysosomal disorder? Brain J Neurol 141:2255–2262. https://doi.org/10.1093/brain/awy147 144. Lwin A, Orvisky E, Goker-Alpan O, LaMarca ME, Sidransky E (2004) Glucocerebrosidase mutations in subjects with parkinsonism. Mol Genet Metab 81:70–73 145. Beavan MS, Schapira AH (2013) Glucocerebrosidase mutations and the pathogenesis of Parkinson disease. Ann Med 45:511–521. https://doi.org/10.3109/ 07853890.2013.849003 146. Guedes LC et al (2017) Serum lipid alterations in GBA-associated Parkinson’s disease. Parkinsonism Relat Disord 44:58–65. https://doi.org/10.1016/j. parkreldis.2017.08.026 147. Yap TL et al (2011) Alpha-synuclein interacts with Glucocerebrosidase providing a molecular link between Parkinson and Gaucher diseases. J Biol Chem 286:28080–28088. https://doi.org/10.1074/ jbc.M111.237859 148. Ziegler SG et al (2007) Glucocerebrosidase mutations in Chinese subjects from Taiwan with sporadic Parkinson disease. Mol Genet Metab 91:195–200. https://doi.org/10.1016/j.ymgme.2007. 03.004

Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

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Nico P. Dantuma and Laura K. Herzog

Abstract

Machado–Joseph disease (MJD), also known as Spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant inheritable neurodegenerative disorder. After a long pre-symptomatic period, this late-onset disease progressively disables patients and typically leads to premature death. Neuronal loss in specific regions of the cerebellum, brainstem and basal ganglia as well as the spinal cord explains the spectra of debilitating neurological symptoms, most strikingly progressive limb, and gait ataxia. The genetic cause of MJD is a polyglutamine (polyQ) repeat expansion in the gene that encodes ataxin-3. This polyQ-containing protein displays a well-defined catalytic activity as ataxin-3 is a deubiquitylating enzyme that removes and disassembles ubiquitin chains from specific substrates. While mutant ataxin-3 with an expanded polyQ repeat induces cellular stress due to its propensity to aggregate, the native functions of wild-type ataxin-3 are linked to the cellular countermeasures against the very same stress conditions inflicted by polyQcontaining and other aggregation-prone proteins. Hence, a mixture of gain-of-function and loss-of-function mechanisms are likely to contribute to the neuronal demise observed in N. P. Dantuma (*) · L. K. Herzog Department of Cell and Molecular Biology (CMB), Karolinska Institutet, Stockholm, Sweden e-mail: [email protected]

MJD. In this review, we discuss the intimate link between ataxin-3 and cellular stress and its relevance for therapeutic intervention in MJD. Keywords

Ataxin-3 · Ubiquitin · Polyglutamine · Neurodegeneration · Spinocerebellar ataxia type 3 · Machado–Joseph disease

Abbreviations ALS AR-JP ASO Chk CUL DDR DRPLA DSB DUB ER ERAD HD HDAC6 JAMM KPNA3 MJD MTOC NER

Amyotrophic lateral sclerosis Autosomal recessive juvenile Parkinson’s disease Antisense oligonucleotides Checkpoint kinase Cullin DNA damage response Dentatorubral and pallidoluysian atrophy DNA double-strand break Deubiquitylating enzyme Endoplasmic reticulum ER-associated degradation Huntington’s disease Histone deacetylase 6 Jab1/Mov34/Mpn Karyopherin α-3 Machado–Joseph disease Microtubule-organizing center Nucleotide excision repair

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_10

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Nuclear export signal Nonhomologous end joining Nuclear localization signal Ovarian tumor Parkinson’s disease Polyglutamine Peptide nucleic acid Polynucleotide kinase/phosphatase RING-between-RING RING finger protein Spinal and bulbar muscular atrophy Spinocerebellar ataxia type 3 Small hairpin RNA SUMO-interacting motif Stable nucleic acid lipid particle Single nucleotide polymorphism DNA single-strand break Small ubiquitin-like modifier Ubiquitin-like Ubiquitin C-terminal hydrolase Ubiquitin-interacting motif Ubiquitin/proteasome system Ubiquitin-specific protease Valosin-containing protein

Machado–Joseph Disease

Four decades ago, clinicians reported the occurrence of a variety of neurological symptoms in Azorean patients from Portuguese descent [1]. Not long after the identification of these patients, it was recognized that a single genetic alteration was the underlying cause for the spectrum of neurological defects [2]. This was somewhat surprising, as the clinicians initially believed that, given the diversity in clinical manifestations, they were dealing with at least three distinct diseases, going under different names such as Machado or Azorean disease [1, 3, 4]. Presently, this disease is best known as Machado–Joseph disease (MJD)—after the first families in which the disease was identified—as well as under the more descriptive classification of Spinocerebellar ataxia type 3 (SCA3). MJD is 1 of at least 12 autosomal dominant inheritable forms of ataxias [5]. Despite it being a rare disease, MJD is the most common form of

dominant inheritable ataxia in many countries [6]. Throughout the years, it has gained considerable attention from the research community: from the early identification of the mutant gene responsible for MJD to the present attempts to develop therapeutic interventions for this incurable disease. Insights into the etiology may not only increase our understanding of MJD but also aid the identification of unifying concepts in the molecular mechanisms responsible for other neurodegenerative diseases, such as other dominant spinocerebellar ataxias as well as related progressive neurodegenerative diseases, including Huntington’s disease (HD) and spinal and bulbar muscular atrophy (SBMA) [7]. MJD patients are usually presymptomatic during the first part of their lives with the first symptoms typically appearing around middle age [8]. The presymptomatic stage is followed by a period of progressive decline, leading in severe cases to premature death. The disease manifests itself with several neurological symptoms, among which gradually worsening gait and limb ataxia (postural instability), severe spasticity (muscle weakness), fasciculation (muscle twitches), dysarthria (speech dysfunction), and ophthalmoplegia (visual dysfunction). Loss of neuronal function in selective brain regions is the primary cause for these symptoms with the most profound pathology occurring in the basal ganglia, brain stem, and spinal cord [8]. The pathology in the cerebellum of MJD patients is most prevalent in the dentate nucleus, while the Purkinje cells in the cerebellar cortex are relatively well preserved, unlike the situation in other related ataxias [5]. There is a wide phenotypic variability in MJD pathology and the disease manifestations range from a clear ataxic syndrome to a disease state that is difficult to distinguish from Parkinson’s disease (PD). The fact that PD-like symptoms are not uncommon in SCA3 may not be surprising given that one of the primary affected sites in both diseases is the substantia nigra. However, these MJD symptoms are, unlike PD, typically accompanied by signs of cerebellar dysfunction. In the early 1990s, the genetic cause of several late-onset, progressive neurodegenerative

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

diseases, some of which shared features with MJD, had been traced to the presences of triplet repeat expansions in largely unrelated genes [9– 13]. Screening of a human brain cDNA library for additional genes with CAG repeats led to the identification of a novel repeat-containing gene that was originally coined MJD1a to reflect it being a candidate gene for MJD [14]. The latter was based on the observation that this particular repeat was expanded in all affected members of a Japanese family with MJD. This link was further strengthened when repeat expansions in the same candidate genes were also found in patients in German pedigrees that had been diagnosed with SCA3, demonstrating that patients originally diagnosed with MJD or SCA3 suffer from the same inheritable neurodegenerative disease [6]. With this finding came also the realization that the disease was much more prevalent than originally anticipated as it was found to be the most common cause for dominant spinocerebellar ataxia in the German population [6]. The fact that the repeat was present in the open reading frame of the MJD1a transcript and predicted to encode a polyglutamine (polyQ) repeat raised the suspicion that MJD might share, at the molecular level, features with four earlier identified polyQ disorders [14]. Indeed, SBMA [11], HD [9], SCA1 [13], and dentatorubral and pallidoluysian atrophy (DRPLA) [10, 12] had been found to be caused by polyQ repeat expansions in otherwise unrelated proteins. Affirmatively, it was found that expression of the mutant protein responsible for MJD, named ataxin-3, gave rise to intranuclear inclusions that contained fragments of ataxin-3 and stained positive for ubiquitin [15]. Similar structures were also found in the affected regions of the brain in deceased MJD patients, recapitulating what had been observed for other polyQ diseases [16]. The number of polyQ diseases has expanded, adding up to at least nine dominant inheritable neurodegenerative diseases that are all caused by triplet repeat expansions in coding regions, which give rise to proteins with pathologically long polyQ repeats. Notably, the triplet repeats encoding polyQ are unstable once the pathologic threshold has been reached, due to

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which the repeat length tends to expand with every generation [17]. Since there is an inverse correlation between the length of the repeat and the age when the first symptoms occur, the ageof-onset typically tends to decline while the severity tends to increase with every generation, a phenomenon known as anticipation [17]. Despite the many similarities, the causative repeat expansion in ataxin-3 differs in some aspects from the repeats in other polyQ diseases. First, while the repeat typically resides close to the amino termini of the proteins in other polyQ diseases, it localizes not far from the carboxy terminus in ataxin-3. Second, the smallest pathological repeats in ataxin-3 are around 60 amino acids, which is considerably larger than the wildtype repeats ranging from 10 to 40 amino acids, whereas a more gradual increase from wild-type to pathological repeat length has been observed in other polyQ diseases. Third, the well-defined enzymatic activity by which native ataxin-3 fulfills its many cellular functions is exceptional in the sense that other polyQ-containing proteins are typically scaffolding proteins that execute their function by establishing protein–protein interactions while lacking intrinsic enzymatic activity. It is also by means of its enzymatic activity that ataxin-3 plays a pivotal role in protecting cells from the type of stress conditions and cellular insults, that are inflicted by polyQ proteins and other neurodegeneration-associated proteins [18]. Hence, the functional roles of ataxin-3 deserve a closer look if we want to understand the complex interplay between mutant ataxin-3 and cellular stress.

10.2

Ataxin-3

Ataxin-3 is the founding member of the Josephin family of deubiquitylating enzymes (DUBs). Besides the Josephin family, there are five other families of ubiquitin proteases: the Ubiquitin Specific Proteases (USP) family, the Ubiquitin C-terminal Hydrolases (UCH) family, the Ovarian Tumor (OTU) family, the ZUFSP/Mug105 family, and the Jab1/Mov34/Mpn (JAMM) family [19–21]. With the exception of the few

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members that belong to the JAMM family, which are metalloproteases, DUBs are cysteine proteases that specifically cleave the isopeptide bond that covalently attaches the ubiquitin to its target protein. Removal of this post-translational modification from substrates alters their behavior and, at the same time, replenishes the intracellular pool of free ubiquitin [22]. The human genome encodes around one hundred DUBs that display different specificities toward substrates and various ubiquitin modifications. Ubiquitin is a small protein that is covalently linked to the ε-amino group of lysine residues or, less frequently, to the free α-amino group of substrates [23]. The process of modifying a protein with ubiquitin is known as ubiquitylation while the reverse reaction resulting in the removal of ubiquitin is referred to as deubiquitylation. An enzymatic cascade, involving a large number of proteins that belong to the classes of ubiquitin activases, ubiquitin conjugases, and ubiquitin ligases, executes ubiquitylation of proteins [24]. Ubiquitin contains seven lysine residues, all of which are also targets for additional rounds of ubiquitylation, giving rise to the formation of polyubiquitin chains. These chains can consist of ubiquitin moieties connected by the same type of lysine-linkage, a mixture of lysine-linkages, and/or branched connections with multiple lysine residues in a single ubiquitin being modified [25]. Since a single ubiquitin chain can be composed of combinations of these alternatives, an astronomical number of structurally different polyubiquitin modifications can be generated through the process of ubiquitylation. Although the field is gaining ground in deciphering the resulting ubiquitin code, it is presently hard to predict how certain modifications will affect the behavior or fate of proteins [26]. Best studied are Lys48-linked ubiquitin chains, which are the canonical chains for targeting proteins for proteasomal degradation, thereby shortening their half-lives [27], and Lys63-linked ubiquitin chains, which do not target for proteasomal degradation but instead change the behavior of modified proteins in different ways [28, 29]. A number of proteins that share structural features with ubiquitin also serve as post-translational

N. P. Dantuma and L. K. Herzog

modifications, each requiring their own enzymatic cascade for conjugation. These include, among others, the small ubiquitin-like modifiers (SUMOs), NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8), and ISG15 (Interferon-stimulated gene 15) [30]. Modifications of proteins with these ubiquitin-like proteins have distinct outcomes. The interconnection of ubiquitylation and related modifications adds another layer of complexity to the code, further challenging our understanding of these complex signaling pathways. The first suspicion that ataxin-3 was directly involved in ubiquitin-dependent processes was raised when ubiquitin-interacting motifs (UIMs) were identified in ataxin-3 [31]. These UIMs are related to the ubiquitin-binding domains that had previously been found in one of the subunits of the proteasome [32]. The originally identified ataxin-3 sequence encoded two UIMs in its C-terminus followed by the polyQ repeat [14] (Fig. 10.1). However, shortly after, another variant containing an alternative C-terminus with a third UIM downstream of the polyQ repeat was identified [33]. The 2UIM and 3UIM variants, as they are referred to, are derived from alternatively spliced mRNA transcripts with the 3UIM variant being most widely expressed and the predominant isoform in the brain. Accordingly, the 3UIM variant was found in intranuclear inclusions in the brains of MJD patients [34]. The significance of the 2UIM variant is less clear but it appears to be expressed at lower levels, has an increased propensity to aggregate and shorter half-life due to faster proteasomal degradation [35]. Biochemical analysis indeed confirmed that the predicted UIMs interacted directly with ubiquitin and suggested that ubiquitin chains need to consist of at least four ubiquitin moieties to bind efficiently to ataxin-3 [31]. The UIMs also facilitate the translocation of wild-type ataxin-3 to aggregates containing polyQ fragments, which was in line with its ubiquitin-binding activity and suggested a role of ataxin-3 in the regulation of inclusion bodies [36]. Since the formation of these cytosolic inclusion bodies, also known as aggresomes [37], is a protective response of cells against aggregation-prone proteins [38], this

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

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≤ 40 wild-type 41-59 intermediate (rare) ≥ 60 pathogenic

Fig. 10.1 Schematic illustration of the ATXN3 gene and the primary structure of the protein product. The ATXN3 gene is located on chromosome 14q32.1 and contains 11 exons. Two alternatively spliced mRNA transcripts are depicted. The first splice variant is terminated after exon 10, resulting in a protein with only two UIM motifs and a short C-terminal hydrophobic stretch downstream of the polyQ repeat. The alternative splice variant includes exon 11, giving rise to a protein containing a third UIM after the polyQ repeat. Both ataxin-3 protein variants contain the catalytic Josephin domain, which harbors the catalytic activity, catalyzed by cysteine 14 at its N-terminus. Additionally, two ubiquitin-binding sites (UbS1 and UbS2) are located in the Josephin domain

and mediate interaction with ubiquitin and hHR23B, respectively. Ataxin-3 is also able to bind the ubiquitinlike modifier SUMO-1 via a SUMO-interacting motif (SIM) in the C-terminal part of the Josephin domain. UIM1 and UIM2 are implicated in binding to K48-linked ubiquitin chains, while UIM3 seems to be required for K63 selectivity. Additionally, UIM2 and UIM3 are required for ataxin-3 interaction with the UbL domain of the E3 ligase Parkin. Interaction with VCP is mediated by a VCP-binding motif (VBM), which is located in between the UIM2 and polyQ repeat. The length of the polyQ repeat ranges from up to 40 repeats in healthy individuals to expansions of 60 repeats and more in MJD patients. Intermediate repeat lengths are rarely observed

foresighted a possible role of ataxin-3 in the molecular defense mechanisms against misfolded proteins. The ubiquitin-like (UbL) N-terminal domain of the ubiquitin ligase Parkin also binds to ataxin-3 in a UIM-dependent fashion [39]. This is of particular interest as mutations in Parkin are responsible for an autosomal recessive juvenile onset form of PD [40], a disease that shares symptoms with MJD, suggesting that a common pathway may be dysfunctional in these diseases. While UIM1 and UIM2 are located directly adjacent to each other, a spacer separates UIM2 and UIM3 [14]. The spacer contains two interesting features as it is not only the place where the

polyQ repeat resides but it also harbors a binding motif for the valosin-containing protein (VCP), also known as Cdc48 or p97, which is a ubiquitinselective segregase involved in protein degradation [41]. VCP is, like ataxin-3, genetically linked to late-onset neurodegenerative disease since mutations in the gene encoding for VCP give rise to familial frontotemporal dementia [42] and amyotrophic lateral sclerosis (ALS) [43]. Bioinformatic analysis revealed that the N-terminal half of ataxin-3, which had been coined the Josephin domain, shared sequence characteristics with cysteine proteases and remotely resembled the catalytic domain of certain

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DUBs [44]. Based on this in silico finding, it was speculated that ataxin-3 might display hydrolyzing activity toward the ubiquitin conjugates that interact with its UIMs. Indeed, this was confirmed by the finding that ataxin-3 reduced ubiquitylation of model substrates in vitro in a UIM-dependent fashion [31] (Fig. 10.2). Ataxin-3 preferentially trims polyubiquitin chains consisting of at least six ubiquitin moieties and displays little activity to shorter chains. While both proteasomal targeting K48-linked polyubiquitin chains and non-proteasomal K63-linked chains are hydrolyzed by the isolated Josephin domain, fulllength ataxin-3 has a clear preference for K63-linked ubiquitin chains or ubiquitin chains that consist of a mixture of K63- and K48-linkages [45]. Inactivation of the UIMs converts ataxin-3 from a DUB with a K63-ubiquitin chain preference to a DUB that acts primarily on K48-ubiquitin chains, suggesting that the UIMs and not the catalytic domain dictates its chain specificity [46]. Notably, the tandemlyorganized UIM1 and UIM2 specifically interact with K48-ubiquitin chains, implying that UIM3 is required for the observed preference for K63-linked ubiquitin chains [47]. With the identification of the DUB activity executed by its Josephin domain, ataxin-3 became the founding member of the Josephin family of DUBs. The Josephin domain contains two additional ubiquitin-binding sites annotated as UbS1 and UbS2 [48]. While the UIMs in the C-terminal part are presumably responsible for capturing polyubiquitylated substrates, the UbS1 and UbS2 in the Josephin domain are involved in the catalytic reaction that results in the cleavage of ubiquitin [49]. The UbS1 motif, which is located close to the active site of ataxin-3, is critical for the DUB activity and mutations in this site render the Josephin domain inactive. A Josephin domain with a mutant UbS2 motif retained DUB activity but its ability to cleave K48-linked ubiquitin chains was strongly reduced, while K63-linked ubiquitin chains were still efficiently cleaved [49]. The UbS2 motif is also the site that mediates interaction with the UbL domain of hHR23A/B

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(human Homologue of Rad23A/B) [49], one of the ubiquitin shuttle factors that delivers ubiquitylated substrates to the proteasome [50]. Interestingly, the interaction between hHR23A/B and ataxin-3 results in stabilization of the latter by decelerating ubiquitinindependent proteasomal degradation of ataxin-3 [51]. Since this also holds true for the mutant ataxin-3 responsible for MJD, tampering with the interaction between hHR23A/B and ataxin-3 could have therapeutic potential in MJD. The effect of hHR23A/B on ataxin-3 turnover is somewhat similar to the well-established role of hHR23A/B in nucleotide excision repair (NER), where hHR23A/B prevents proteasomal degradation of the NER sensor XPC (Xeroderma pigmentosum complementation group C) through a direct interaction [52]. Also NEDD8 [53] and SUMO1 [54, 55], two other ubiquitin-like modifiers, interact with the Josephin domain, which may be indicative for a more promiscuous activity of ataxin-3 towards ubiquitin-like modifiers (Fig. 10.2). The precise location of the NEDD8-interaction site is unknown but it is plausible that it shares interaction surfaces with ubiquitin since ataxin-3 is able to cleave also NEDD8 fusions in vitro, although the functional relevance of this activity in cells remains elusive [53]. In silico analysis predicted the presence of a SUMO-interacting motif (SIM) in the C-terminus of the Josephin domain and, in having so, ataxin-3 shares this feature with a selected group of proteins in which the presence of UIMs and SIMs are combined [56]. For SIM/ UIM-containing RAP80 (Receptor-associated protein 80), a protein involved in DNA repair, this combination of UIMs with a SIM is important for its selective recruitment to hybrid SUMO– ubiquitin chains [57]. While a possible interaction of ataxin-3 with hybrid chains remains to be explored, it has been confirmed that the SIM endows ataxin-3 with SUMO-binding properties [54, 55]. However, in contrast to ubiquitin and NEDD8, SUMO fusions are not hydrolyzed by ataxin-3 in vitro but, interestingly, SUMO1 was found to stimulate the protease activity of ataxin3 toward ubiquitin chains [55].

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binding

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Fig. 10.2 Binding and hydrolysis of ubiquitin and ubiquitin-like modifications by ataxin-3. Ataxin-3 is a deubiquitylating enzyme that can hydrolyze both K48and K63-linked as well as mixed ubiquitin chains with these linkages. It preferentially targets chains consisting of four or more ubiquitin moieties. In addition to ubiquitin,

10.3

Combating Proteotoxic Stress

Ataxin-3 has been implicated in a variety of ubiquitin-dependent mechanisms that are directly or indirectly linked to cell survival. In spite of the variety in the mechanisms that involve ataxin-3, a common denominator in most of these processes is their connection to the cell’s ability to deal with proteotoxic and genotoxic insults. Ataxin-3 is present in the cytosolic and nuclear compartments of cells and functions related to both localizations of ataxin-3 have been revealed in a number of studies. The reported activities of ataxin-3 in counteracting proteotoxic stress primarily take place in the cytosolic compartment of the cells. It is, however, unclear if the nuclear pool of ataxin-3 is also engaged in protein quality control but, given ataxin-3’s role in the regulation of proteasomal degradation, which takes place in both compartments, it is likely that the realm of ataxin-3 in proteotoxic stress includes the nuclear compartment. Ataxin-3 is itself modified by ubiquitin and this ubiquitylation event is stimulated by insults that cause proteotoxic stress [46]. By a yet unknown molecular mechanism, stress-induced ubiquitylation of ataxin-3

NEDD8

SUMO1

ataxin-3 is also able to bind the ubiquitin-like modifier NEDD8 and possesses deneddylase activity. The small ubiquitin-like modifier SUMO1 is also recognized and bound by ataxin-3. While ataxin-3 does not hydrolyze this modification, the interaction with SUMO1 increases its deubiquitylating activity

enhances its catalytic activity, suggesting that deubiquitylation of substrates by ataxin-3 is an integrated part in the cellular response against proteotoxic stress. There are three primary mechanisms at the cell’s expense to deal with the continuous threat of misfolded proteins, which, if left uncontrolled, may jeopardize the delicate protein homeostasis in the intracellular environment. First, dedicated ubiquitin ligases recognize aberrant proteins, sometimes with the help of chaperones, and target them for rapid destruction by the proteasome [18]. The ubiquitin/proteasome system (UPS) is a fast and efficient way to instantly neutralize aberrant proteins but becomes toothless when the proteins have had the opportunity to precipitate into insoluble aggregates [58]. This is probably a consequence of the limitation that proteasomes require unfolding of the substrates to allow their translocation into the proteolytic chamber, which may be hard to accomplish for protein substrates residing in aggregates [59]. When it comes to protein aggregates and other large complexes or even subcellular structures, the second protective mechanism comes in play: macroautophagy, here referred to as autophagy. Autophagy encompasses the

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capturing of cytosolic content in newly formed double-membrane vesicles, known as autophagosomes, which fuse with lysosomes, where there macromolecular cargo is hydrolyzed [60]. Since autophagy does not require the unfolding of the substrate, it is equally suited for degradation of soluble and aggregated proteins. If clearance of the aberrant proteins falls short, a third option remains to temporarily relieve the burden of misfolded proteins. This strategy involves sequestration of aggregation-prone proteins followed by their dynamin-dependent transport to the microtubule-organizing center (MTOC), where they are stored in inclusion bodies that are confined by a vimentin cage [37, 61]. Although these deposits of inherently toxic proteins in inclusion bodies may persist over years, as evidenced by their presence in affected tissues in many neurodegenerative as well as nonneuronal protein misfolding diseases [62], they do not have to be a final destiny

for protein aggregates as autophagy can also facilitate their clearance [63]. This is likely to be important for cell survival on the longer perspective since the presence of inclusion bodies does not seem to be without negative consequences for the cell [64]. It should be emphasized though that their overall mode of action appears to be protective, at least in the initial phase, as illustrated by the fact that cells that fail to form these structures are more susceptible to the negative impact of aggregationprone proteins on cell viability [38]. Interestingly, ataxin-3 appears to be implicated in each of these protective measures, underscoring its intricate and multifunctional role in the maintenance of protein homeostasis (Fig. 10.3). The interaction of ataxin-3 with the ubiquitinselective segregase VCP triggered investigations that explored a possible role of ataxin-3 in the ubiquitin-dependent proteasomal degradation of substrates derived from the endoplasmic

Cytoplasm ERAD

Autophagy regulation

prevents proteasomal degradation of beclin-1

Ataxin-3

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ATG14L

p97/ VCP

promotes extraction of ERAD substrates

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promotes aggresome formation HDAC6

proteasome Autophagy initiation

Fig. 10.3 Cytosolic functions of ataxin-3. Ataxin-3 is involved in the regulation of several cytoplasmic processes that prevent or counteract proteotoxic stress. Interaction of ataxin-3 with the autophagy-regulator beclin-1 is mediated by the polyQ repeat and results in removal of K48-linked ubiquitin chains from beclin-1 leading to its stabilization and stimulation of autophagy. Ataxin-3 is recruited to and promotes the formation of aggresomes,

aggresome

likely by generation of free ubiquitin chains, that are recognized by HDAC6 and required for HDAC6/dyneinmediated transport of misfolded proteins to the aggresome. Ataxin-3 is also required for efficient endoplasmic reticulum (ER)-associated degradation (ERAD), assisting the AAA-ATPase VCP in the extraction and unfolding of proteins for subsequent delivery to the proteasome

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

reticulum (ER). ER-associated degradation (ERAD) plays a central role in governing protein homeostasis in the ER compartment [65]. A critical point in the sequence of events that leads to the degradation of ER substrates by the cytoplasmic UPS is their retrotranslocation from the ER to the cytosol. This event involves the hexameric VCP complex, which dislocates ubiquitylated proteins from the ER membrane by means of its AAA-ATPase activity [66, 67]. This pathway consists of the recruitment, unfolding, and translocation of ubiquitylated proteins similar to what occurs with proteins deemed for degradation at the proteasome. Since deubiquitylation of proteins at the entrance of the proteasome, which is composed of an AAA-ATPase complex related to VCP, is crucial to facilitate their translocation and degradation [68], it may not be surprising that VCP also requires the activity of associated DUBs to execute the segregation of proteins from membranous structures. Indeed, ataxin-3 was found to play a stimulatory role in this process as overexpression of catalytic dead ataxin-3 delayed the degradation of ERAD substrates [69]. In further support of this hypothesis, it was shown that a ubiquitin-binding protein that prevents deubiquitylation of VCP-associated substrates by ataxin-3 [70] or administration of a chemical compound that inhibits this process [71] delays the degradation of ERAD substrates, while having no effect on substrates that do not require VCP for degradation. It should be noted that in another study a model has been proposed in which deubiquitylation of substrates by ataxin-3 does not stimulate but rather prevents VCP-mediated degradation [72]. The latter is primarily based on the observation that overexpression of wild-type axatin-3 stabilizes ERAD substrates, which can also be the result of a stoichiometric imbalance between VCP and ataxin-3. An interesting twist and complicating factor is the finding that ataxin-3 is not only involved in ERAD but is also itself a substrate for the ERAD ubiquitin ligase Gp78 leading to its proteasomal degradation [73]. Overall a picture has emerged in which ataxin3 plays a stimulatory role in ERAD leading to the counterintuitive situation where protein

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deubiquitylation stimulates ubiquitin-dependent proteasomal degradation [74]. It remains to be seen whether the activity of ataxin-3 in VCP-mediated segregation and degradation is confined to ERAD. Since the recognition of the central role of VCP in ERAD, a number of other processes involving protein extraction at other subcellular localizations, such as mitochondria and chromatin, have been shown to depend on VCP [75, 76]. Moreover, VCP is required for degradation of some soluble proteins, probably through its ability to generate the unstructured initiation sites that the proteasome needs to process substrates [77, 78]. Thus, the reach of the concerted action of ataxin-3 and VCP may be more global than originally anticipated and dysfunction of this mechanism may therefore have far-reaching consequences. The fact that both ataxin-3 and VCP are genetically linked to neurodegenerative diseases involving disturbances of protein homeostasis makes this functional connection particularly interesting [42, 43]. Another interaction partner of ataxin-3 that is of interest in the context of autophagy is Parkin. Mutations in Parkin are causative in autosomal recessive juvenile PD (AR-JP), which, like nonfamilial forms of PD, is characterized by a progressive loss of dopaminergic neurons in the substantia nigra of affected individuals [40]. Parkin is the functional opposite of ataxin3: it is a RING-Between-RING (Really Interesting New Gene) (RBR) ubiquitin ligase with a RING/Hect hybrid mode of action, which accepts activated ubiquitin and conjugates ubiquitin chains of various linkages to substrates [79]. Notably, early studies linked Parkin to degradation of the Pael receptor [80], which is an ER substrate that tends to accumulate in an unfolded state, inducing ER stress and cell death in dopaminergic neurons [81]. However, nowadays Parkin’s involvement in mitochondrial homeostasis has been studied in most detail [82]. The majority of characterized substrates of Parkin are residing at the mitochondria, where they are modified by Parkin with a variety of ubiquitin chains. While the K48-linked ubiquitylation of mitochondrial proteins results in VCP-assisted extraction from mitochondria and subsequent proteasomal

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degradation [83], K63-linked ubiquitylation at the mitochondria triggers substrate clearance by selective autophagy, i.e., “mitophagy” [84]. Destruction of mitochondria by mitophagy is an important mechanism for cells to remove dysfunctional mitochondria that can otherwise become a source for oxidative stress. The N-terminal UbL domain of Parkin directly interacts with the UIM domains in ataxin-3, which regulates the ubiquitylation status of Parkin. Parkin is subject to autoubiquitylation, as has been found for many ubiquitin ligases, and modifies itself with K6-, K27-, K29-, and K63-linked ubiquitin chains [39]. Ataxin-3 interferes predominantly with K27and K29-linked ubiquitylation of Parkin and prevents targeting of Parkin for degradation by autophagy. At least K27-linked ubiquitin chains have indeed been linked to degradation of substrates by autophagy, which may explain how ataxin-3 counteracts this process [85]. Even though this process is dependent on the catalytic activity of ataxin-3, it does not involve active deubiquitylation of Parkin since ataxin-3 is unable to remove ubiquitin chains from Parkin [86]. Instead, it stabilizes the interaction of Parkin with its partner ubiquitin conjugating enzyme, hindering the transient interaction between these proteins that is required for efficient transfer of ubiquitin to Parkin. Expression of mutant ataxin-3 with an expanded polyQ results in destabilization of Parkin, which may be of relevance for the molecular mechanisms underlying MJD [39]. Recent work revealed a more direct stimulatory function of ataxin-3 in autophagy. In this study, it was shown that ataxin-3 interacts with beclin-1 through its polyQ domain, resulting in deubiquitylation and stabilization of beclin-1 [87]. Since beclin-1 stimulates autophagy, its stabilization by ataxin-3 is accompanied by induction of autophagy. Interestingly, polyQ-expanded ataxin-3 as well as huntingtin and atrophin-1, which are causative for HD and DRPLA, respectively, showed increased binding to beclin-1, outcompeting wild-type ataxin-3 and thereby providing a potential explanation for the impairment in autophagy observed in patient fibroblasts. It is noteworthy that genetic tampering with autophagy in the brain results in

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neurodegenerative disorders with accumulation of misfolded proteins in ubiquitin-positive inclusions, suggesting a possible role for defective autophagy in these conditions [88, 89]. Consistent with this model, several neurodegeneration-associated proteins have been implicated in autophagy but the relevance of autophagy impairment for the etiology of neurodegenerative diseases in general, and MJD in particular, remains to be confirmed [90]. Owing to its ability to trim polyubiquitin chains, ataxin-3 is also implicated in the sequestration of misfolded proteins in aggresomes [91]. These cytosolic inclusions are another cellular measure to combat proteotoxic stress as they enable cells to minimize the interference of aggregation-prone proteins with critical cellular processes through their confinement in a single subcellular structure. This sequestration mechanism is probably a final resort for cells that are overwhelmed by aberrant proteins and is typically activated in cells that are exposed to stress conditions or produce mutant proteins, as is the case in many neurodegenerative diseases [92]. Aggresome formation involves the microtubule-dependent transport of ubiquitylated proteins to the MTOC by a cytosolic protein complex including the histone deacetylase 6 (HDAC6), which binds simultaneously ubiquitin conjugates and the dynamin motor complex [93]. The balance between the ubiquitinbinding proteins VCP and HDAC6 is critical in determining whether the fate of a ubiquitylated protein will be VCP-mediated protein degradation or HDAC6-mediated aggresome formation [94]. Surprisingly, the ubiquitin-binding domain of HDAC6 binds only free ubiquitin chains with unanchored carboxy termini [95]. Wild-type ataxin-3 localizes not only to aggresomes by means of its UIM domains, as mentioned above, but also to pre-aggresomal protein aggregates that upon translocation to the MTOC build the aggresome. It has been shown that the presence of ataxin-3 in protein aggregates is important for efficient recruitment of HDAC6 and it has been proposed that ataxin-3 generates the free ubiquitin chains required for HDAC6/dynamindependent transport of the aggregates [95].

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

Altogether, there is a large body of evidence supporting functional roles of ataxin-3 in the three major cellular defense mechanisms that protect cells against the toxic properties of aggregationprone proteins.

10.4

Combating Genotoxic Stress

Transport of ataxin-3 into the nucleus and its association with the nuclear matrix was already recognized soon after the discovery of ataxin-3 [96]. However, insights in its role in the maintenance of genome integrity have only recently emerged. The fact that the function of nuclear ataxin-3 has been largely ignored for many years does not dispel, however, the importance of nuclear ataxin-3 in the etiology of MJD as the presence of nuclear inclusions is tightly linked to the disease pathology and toxic properties of mutant ataxin-3 [97]. Ataxin-3 contains one nuclear localization signal (NLS) and two nuclear export signals (NESs), resulting in active shuttling of ataxin-3 between the cytosolic and nuclear compartments [98]. Heat shock- and oxidative stress-induced proteotoxic stress results in NLS-independent translocation of ataxin-3 to the nucleus, changing its distribution from a predominantly cytosolic to a predominantly nuclear protein [99]. Even though the significance of this redistribution remains obscure, it suggests that nuclear import may be regulated and that additional nuclear roles of ataxin-3 in the acute stress response may exist. Several reports have indicated a role of ataxin3 in DNA damage response (DDR) signaling and DNA repair (Fig. 10.4). Ataxin-3 interacts with the polynucleotide kinase/phosphatase (PNKP) [100], a bifunctional enzyme that compasses both 30 -phosphatase and 50 -kinase activity. These activities are required for generating DNA-end compatibility during DNA singlestrand break (SSB) repair as well as the repair of DNA double-strand break (DSB) by non-homologous end joining (NHEJ) [101]. While recombinant wild-type ataxin-3 was able to stimulate PNKP phosphatase activity in vitro, mutant polyQ-expanded ataxin-3

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inhibited phosphatase activity [100]. Additionally, depletion of ataxin-3 in cultured cells resulted in decreased PNKP activity, which coincided with accumulation of DSBs. Ataxin-3 directly interacts with PNKP and PNKP was found enriched in intranuclear inclusions in MJD brain. In line with this, PNKP activity was decreased in a mouse model expressing pathologically expanded ataxin-3 [102]. Notably, PNKP is not the only DNA repair protein that has found to be enriched in intranuclear inclusions in MJD patients, since the ubiquitin receptor hHR23 involved in nucleotide excision repair also accumulates in these structures [103, 104]. Although this is accompanied by destabilization of its binding partner XPC [104], which functions as an NER sensor, it does not affect the capacity of this DNA repair pathway [103]. The functional involvement of ataxin-3 in DNA damage repair was further corroborated when ataxin-3 was found to ensure and reinforce efficient DDR signaling by counteracting the ubiquitin-mediated extraction of the MDC1 (Mediator of DNA damage checkpoint protein 1) by the SUMO-targeted ubiquitin ligase RING finger protein (RNF)4 [55]. Ataxin-3 rapidly accumulates at DSBs in a SUMOylationdependent manner involving the SIM in its catalytic Josephin domain, which directly interacts with SUMO1. Interaction with SUMO1 stimulated ataxin-3 deubiquitylating activity in vitro, suggesting that SUMO-dependent recruitment may be coupled to substrate deubiquitylation at the damage site. This finding was further supported by the observation that ataxin-3 depletion increased MDC1 ubiquitylation in cells, resulting in a decreased retention time at DSBs [55]. The reduced retention time of MDC1 at DSBs is accompanied by impaired recruitment of downstream factors involved in HR and NHEJ, such as RNF8, RNF168, 53BP1 (p53 binding protein 1) and BRCA1 (Breast Cancer gene 1) [55]. Accordingly, depletion of ataxin-3 results in impairment of these DSB repair mechanisms and increased sensitivity of cells to DNA damage. A number of other DDR and DNA repair proteins have been shown to be modified in a SUMO-targeted

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Nucleus Transcription

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Cell cycle progression and replication

CBP/ p300 Ac Ac

Ac Ac

RPA-ssDNA

ON

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DNA damage or replication stress

Ac Ac

P

stabilization of Chk1

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DNA damage signaling and repair Ataxin-3

promotes phosphatase activity Ataxin-3

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Single-strand break repair

prolongs MDC1 retention

shortens MDC1 retention

proteasomal targeting

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Fbox/DWD Skp1/ DDB1 CUL1/CUL4

MDC1-dependent signaling (53BP1/BRCA1) and repair (NHEJ/HR)

Checkpoint signaling

Checkpoint termination

Fig. 10.4 Nuclear functions of ataxin-3. The nuclear pool of ataxin-3 is engaging in processes counteracting genotoxic insults and in transcriptional regulation. Ataxin-3 directly interacts with the transcriptional coactivators p300/CBP and prevents histone acetylation and transcriptional activation. During normal conditions as well as DNA damage and replication stress, ataxin-3 promotes Chk1-mediate checkpoint signaling and genome integrity by antagonizing the two ubiquitin ligase complexes DDB1/CUL4 and FBXO6/CUL1, that target Chk1 for proteasomal degradation. Interaction of ataxin-3 with the bifunctional enzyme PNKP stimulates its

phosphatase activity and thereby promotes the generation of DNA-end compatibility during DNA single-strand break (SSB) repair as well as the repair of DNA doublestrand breaks (DSB) by nonhomologous end joining (NHEJ). Additionally, ataxin-3 was shown to promote robust activation of the DNA damage response (DDR) signaling following DSBs by counteracting the SUMOtargeted ubiquitin ligase RNF4, thereby increasing the retention time of MDC1 at DNA breaks. MDC1 is required for efficient recruitment of downstream DNA signaling and repair factors

fashion by RNF4 leading to their extraction and subsequent proteasomal degradation, suggesting that the opposing activities of RNF4 and ataxin-3 may have a broader impact [105]. Adding another layer of complexity to the connection between ataxin-3 and SUMO1, ataxin-3 is also itself modified by SUMO1 but this does not affect its ubiquitylation status or, in case of mutant ataxin3, its propensity to aggregate [106]. Proteomic analysis for potential substrates of ataxin-3 in the DDR identified Checkpoint kinase (Chk)1 together with the ubiquitin ligase components DDB1 (DNA damage-binding protein 1), Cullin (CUL)4A and CUL1 as novel interactors [107]. Chk1 levels are tightly regulated during cell cycle progression and in response to replication

stress. The two E3 ubiquitin ligase complexes consisting of DDB1 and CUL4A as well as CUL1 in complex with FBXO6 (F-box only protein 6) were found to mediate polyubiquitylation of Chk1, thereby targeting it for proteasomal degradation to promote checkpoint termination and completion of DNA repair [108, 109]. Ataxin-3 antagonizes the actions of DDB1/CUL4A and FBXO6/CUL1, stabilizing Chk1 under normal conditions as well as DNA damage to promote Chk1-mediated checkpoint signaling, DNA repair, and genome integrity. Consistent with a central role of ataxin-3 in the cellular response to DNA damage, accumulation of DNA damage was confirmed in affected brain regions of MJD patients [100]. Although defects in DNA repair proteins typically give rise to cancer

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

susceptibility syndromes, a large study on the incidence of malignancies in patients with polyQ diseases revealed a reduced occurrence of cancer in this population [110]. This has been interpreted as the possible activation of a common mechanism that protect against cancer, which may be a consequence of adaptive responses against the cellular stress inflicted by the polyQ repeats. However, it should be pointed out that the study used hereditary ataxia as a proxy for polyQ ataxias and was not able to discriminate between MJD patients and individuals suffering from other hereditary ataxias [110]. Together, these pleiotropic roles of ataxin-3 in DNA repair and DDR pathways implicate ataxin-3 in the safeguarding of cells from genotoxic insults and in the maintenance of genomic integrity. In addition to its role in combating genotoxic insults, ataxin-3 has also been implicated transcriptional regulation by two independent corepressor activities. The C-terminus, containing the polyQ stretch, inhibits coactivator-dependent transcription by interaction with the histone acetyl transferases PCAF and p300 and the p300associated cofactor CBP [111]. The N-terminal Josephin domain was found to inhibit histone acetylation by direct binding to histones and blocking access of coactivators to the acetylation sites. Interestingly, several other proteins with expanded polyQ repeats cause dysregulation of transcription [112]. Despite the otherwise largely divergent functions of the polyQ proteins, dysregulated gene expression may present another common key feature of these diseases.

10.5

Targeting Mutant Ataxin-3 in MJD

The dominant inheritance and the striking similarities between various polyQ diseases suggest that these diseases are caused primarily by toxic gain-of-function mechanisms that are a direct consequence of the repeat expansion. The fact that the switch from non-pathological to pathological repeat length correlates with the formation of inclusions by the mutant protein suggests that the propensity of the mutant protein to aggregate is a critical determinant for this gain-of-

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function. To what extent the gain-of-function by the aggregation-prone repeats are truly novel features of the protein or, alternatively, enhance an existing function of the protein is not clear. Distinguishing between these two possibilities is of importance since for the latter it will be critical to understand the function of the wild-type protein whereas many studies focus on the behavior of the pathologic mutant protein while disregarding native functions. The emerging understanding that low-complexity domains, including polyQ repeats, are important for compartmentalization of proteins in membrane-less structures by phase separation hints toward biological functions for domains consisting of repetitive sequences [113]. Despite the predominant role of toxic gain-offunctions attributed to the mutant polyQ proteins, it is likely that partial loss-of-function of these proteins also contributes to the complex cellular pathology observed in these diseases. In this respect, the role of ataxin-3 in controlling proteotoxic and genotoxic stress is highly relevant as the presence of mutant ataxin-3 may challenge, at the same time, the very same quality control systems, whose functions are jeopardized due to ataxin-3 deficiency. It is, in that respect, comforting that genetic deletion of ataxin-3 does not result in striking abnormalities in mice [114]. Moreover, neither depletion nor overexpression of ataxin-3 did have a dramatic effect on the brain pathology in a rat model for MJD [115]. It remains, however, to be seen whether absence of ataxin-3 triggers compensatory mechanisms involved in protein quality control. In line with this possibility, nematodes that lack the ataxin-3 orthologue have an overactive stress response with enhanced expression of several molecular chaperones resulting in increased thermotolerance [116]. Additionally, acute depletion of ataxin-3 may have a different impact on the cellular physiology as compared to chronic deficiency. Indeed, acute depletion of ataxin-3 by RNA interference causes increased sensitivity of human cells to ionizing radiation [55]. Possible loss-of-functions or triggering of adaptive response, which could be beneficial or detrimental, should be kept in mind when exploring

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therapeutic approaches aimed at targeting mutant ataxin-3. Fortunately, patients express in addition to the mutant protein also wild-type ataxin-3 from the unaffected allele, opening the possibility to eliminate the mutant protein without generating full depletion of ataxin-3, although haploinsufficiency remains an unavoidable concern even with these approaches. There are presently no therapies or drugs available that can halt or reverse the pathology in MJD patients. It is easy to be misled by the fact that polyQ diseases can be attributed to a singlemutant protein in thinking that MJD, in particular, and polyQ diseases, in general, are “simple” diseases as the cellular etiology in these disorders is still complex. The number and variety of processes that are dysfunctional in cells expressing these mutant proteins is tantalizing and hence it is hard to picture that an effective therapeutic effect will be feasible by restoring functionality of the mechanisms that fail in the face of accumulated levels of the mutant ataxin-3. Therefore, most experimental therapeutic strategies for polyQ diseases that are currently being explored target the root of the problem and aim at reducing the levels of the culprit proteins. It is promising that stalling expression of the mutant ataxin-3 in transgenic mice has been shown to reverse the pathology [117], a phenomenon that had been earlier reported for a murine HD model [118]. A cautionary point is that we do not know how general this phenomenon is and whether it applies to all cells affected by mutant ataxin-3. A recent study suggested that this does not hold true when the mutant protein is expressed in the forebrain of mice [119]. Despite the fact that the nuclear inclusion disappeared upon switching off the expression of the transgene encoding mutant ataxin-3, the behavioral symptoms remained in this mouse model. Nevertheless, the safest bet for therapeutic interference appears to be elimination of the mutant protein responsible for the disease, preferably prior to disease onset. The enormous potential of the recently emerged genome editing tools for treatment of MJD and other polyQ diseases has drawn considerable attention in recent years as this may potentially allow to directly correct the source of the

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problem: the mutant gene itself [120]. CRISPR/ Cas9 genome editing has been successfully used to inactivate the mutant huntingtin allele in cellbased HD models [121, 122] and a HD mouse model [123, 124]. For MJD, it has been shown that CRISPR/Cas9 can be used to correct the mutant allele by selectively deleting the repeat expansion in induced pluripotent stem cells derived from an MJD patient [125] (Fig. 10.5a). While the therapeutic use of CRISPR/Cas9mediated genome editing is still in an early exploratory phase, other attempts to reduce the levels of the mutant ataxin-3 in experimental models have already been ongoing for over a decade. An arsenal of different techniques has been used to target the mutant ataxin-3 at the mRNA or protein level. Whenever possible, these strategies have been tweaked to selectively reduce the levels of the mutant protein while leaving expression of the wild-type ataxin-3 unaffected. However, in most cases the intervening methods are unable to distinguish between wildtype and mutant ataxin-3, which may increase the chances of side effects due to ataxin-3 deficiency. Since the only difference between the wild-type and mutant protein is the presence of the expanded polyQ repeat, it is not at all trivial to selectively target expression of the mutant protein. In some instances, the presence of single nucleotide polymorphisms (SNPs) can be exploited. With those strategies that indiscriminately target wild-type and mutant ataxin-3, the potential presence of loss-of-function effects may motivate long-term follow-up of their effect in animal models prior to implication of these techniques in patients. A premium choice for reducing the levels of mutant ataxin-3 in MJD has been various RNA interference strategies (Fig. 10.5b). First attempts to target ataxin-3 transcripts were based on lentiviral-delivered small hairpin RNAs (shRNA) directed against a common SNP in MJD patients. This confirmed that specific targeting of mutant human ataxin-3 reduced the brain pathology in a rat model for MJD and provided at the same time a proof-of-principle that SNPs can be used for allele-specific silencing of mutant ataxin-3 [126]. Injection of this

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

Fig. 10.5 Therapeutic approaches for targeting ataxin-3 in MJD. (a) Guided cleavage of the mutant ATXN3 gene using CRISPR/Cas9-mediated gene editing allows modification of the mutant allele to delete the CAG repeat or decrease repeat number below the pathological threshold. (b) The levels of ATXN3 mRNA can be reduced by using small hairpin RNA (shRNA) or stable nucleic acid lipid particles (SNALPs) to induce Dicer/RISC-dependent mRNA decay or by antisense oligonucleotides (ASOs) to induce RNAse H-dependent degradation. These methods allow exploitation of the presence of SNPs in the mutant ATXN3 allele to specifically target mutant ATXN3 mRNA, leaving the mRNA encoding native ataxin-3 unaffected. (c) ASOs can also be used to induce exon skipping during mRNA processing, to give rise to modified or truncated versions of ataxin-3 lacking the polyQ repeat or regions containing calpain-cleavage sites that have been described to play a role in the disease pathogenesis to prevent proteolytic cleavage and generation of shorter polyQ-containing fragments. (d) To decrease the levels

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of mutant ataxin-3, its degradation by cellular degradation pathways such as the ubiquitin/proteasome system (UPS) or autophagy can be stimulated. Both, pharmacological inhibition of USP14 and overexpression of the GTPase CRAG enhance proteasomal turnover of ataxin-3. The degradation of aggregated species of ataxin-3 can be stimulated by enhanced autophagy, induced by increased expression of beclin-1 or by stimulating its activity using autophagy inducing peptides. (e) Similar to (c), the cleavage of mutant ataxin-3 can also be prevented at the protein level by inhibition of calpains using calpastatin expression or calpain inhibitors such as calpeptin or BDA-410. Additionally, calpain-mediated cleavage can be prevented by inhibition of calcium release, which is required for the activity of these proteases, by Dantrolene. (f) As nuclear aggregates of mutant ataxin-3 have been implicated in the etiology of MJD and are particularly toxic, prevention of nuclear localization of ataxin-3 by deletion of the nuclear import complex protein KPNA3 is another approach that has been explored for its therapeutic potential

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lentiviral vector into the cerebellum of transgenic mice expressing human mutant ataxin-3 not only reduced the neuropathology but also improved the performance of the mice in various behavioral tests for cerebellar function [127]. Like other nucleotide-based approaches, delivery of the active compound is, however, a critical limiting factor since direct administration in the brain or spinal fluid to bypass the blood–brain barrier is problematic in a therapeutic setting. The development of stable nucleic acid lipid particles (SNALPs) containing siRNAs directed against mutant ataxin-3, which upon intravenously administration effectively reduce levels of mutant ataxin-3 and alleviate neurological defects in MJD mouse models, shows that more practical administration regimens are possible [128]. Another way of targeting ataxin-3 transcripts are antisense oligonucleotides (ASOs), which are short, single stranded oligonucleotides that bind to complementary mRNA and result in altered RNA function [129] (Fig. 10.5c). This technique has been improved enormously since its discovery four decades ago, making it suitable for therapeutic purposes [130] and of potential interest for the treatment of inheritable neurodegenerative disorders [129, 131]. A major advantage of this technique is its high tolerability, widespread distribution, and relatively efficient delivery to the brain as well as its long-lasting effects. Treatment with non-allele specific ASOs resulted in downregulation of wild-type and mutant ataxin3 in MJD fibroblasts and in transgenic hemizygous mice expressing human mutant ataxin-3 from the full-length ATXN3 gene [132]. Furthermore, the ASOs resulted in a reduction of aggregated, high-molecular weight ataxin-3 species. Contrary to these promising results, the same treatment failed to reduce ataxin-3 levels in a mouse model expressing mutant ataxin-3 from cDNA-based transgene, potentially due to differences in RNA processing and accessibility due to the lack of intronic sequences and the expression of a single isoform [132]. ASOs have also been used to modify the processing of ataxin-3-encoding mRNAs in order to generate transcripts that encode proteins lacking the polyQ repeat. ASO-induced exon skipping

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results in the generation of an alternatively spliced transcript that encodes a polyQ-less ataxin-3 that is similar, but not identical, to wild-type ataxin-3 as it also lacks the third UIM and has an alternative carboxy terminus [133]. Treatment of MJD mice possessing the full-length human ATXN3 gene, including introns and flanking regions, resulted in significant reduction of insoluble ataxin-3 species in the cerebellum and largely prevented nuclear accumulation of mutant ataxin-3 in the striatum, a condition that has been shown to aggravate aggregation in vivo and is suggested to be a prerequisite for MJD pathogenesis [133]. A similar approach to generate transcripts that lack a proteolytic cleavage site that contributes to the toxic effect of ataxin-3 was, however, less successful as it affected the ubiquitin-binding property of ataxin-3 and targeted both the wild-type and mutant allele [134]. A particularly elegant strategy has been the generation of an oligomer that is specific for expanded polyQ repeats [135]. Considering the large number of SNP-specific ASOs required to cover the patient population and the possible absence of allele-specific SNPs, selectively targeting the mutant transcripts based on the repeat expansion is a tempting but technically challenging alternative. The rational in this study was that, due to the repeat expansion, transcripts encoding mutant ataxin-3 will bind more nucleotide oligomers directed against the repeat than the wild-type allele and therefore a more pronounced effect of CAG-specific oligomers on the mutant transcript was anticipated. Peptide nucleic acid (PNA) conjugates that contained a 19-base nucleic acid indeed resulted in a selective inhibition of mutant ataxin-3 and huntingtin in patient-derived fibroblasts [135]. Not only can this approach circumvent the need for using SNPs for allele-specific targeting, but it may also provide a single vehicle that can be effectively used for treatment of different polyQ diseases. Instead of targeting the synthesis of mutant ataxin-3, its steady-state levels can also be reduced by enhancing the clearance of the mutant protein. An attractive approach to accomplish this is the mobilization of the cell intrinsic mechanisms for destroying these toxic proteins, which are the UPS

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

and autophagy (Fig. 10.5d). A complicating factor is that autophagy is impaired in several neurodegenerative diseases, including MJD [136], which may be attributed to the effect of ataxin-3 on beclin1 [87]. Boosting autophagy by overexpression of beclin-1, using lentiviral delivery, decreased the number of ataxin-3 inclusions and had a beneficial effect on the neuropathology and symptoms in MJD mouse models [137]. Even though lentiviral expression of beclin-1 may not be realistic in a therapeutic setting, it is noteworthy that autophagy-inducing peptides have been developed that specifically stimulate beclin-1 [138]. While aggregation-prone proteins can compromise the UPS when acutely overexpressed [139, 140], several studies have shown that the activity of the UPS is relatively well conserved in mouse models for polyQ diseases [141–143]. Thus, it may be possible to take advantage of this pathway in the elimination of the causative mutant proteins in polyQ diseases [144]. A small drug-like compound that inhibits the proteasome-associated deubiquitylating enzyme USP14 and enhances the degradation of aggregation-prone proteins was found to reduce the levels of ectopically expressed ataxin-3 in murine fibroblasts [145]. Moreover, expression of the GTPase CRAG stimulates the clearance of polyQ proteins through the UPS by a poorly defined mechanism [146], involving the antioxidant response [147]. These examples show that the UPS and autophagy can be exploited to promote clearance of mutant ataxin-3. Two other features that are directly linked to the toxic effects of mutant ataxin-3 are proteolytic cleavage and nuclear translocation. Both events have also been subject to attempts to modulate the toxic properties of ataxin-3. Proteolytic cleavage of mutant proteins gives rise to truncated, polyQ-containing fragments with increased toxicity and propensity to aggregate [148]. For ataxin-3, this phenomenon has been attributed to caspaseand calpain-dependent cleavage of the full-length ataxin-3 [149, 150] (Fig. 10.5e). Interestingly, cleavage of ataxin-3 as well as aggregation of ataxin-3 were enhanced in patient-derived neuronal cells upon stimulation [151]. The excitationinduced cleavage of ataxin-3 that is responsible for this effect was dependent on calpain as well as Ca2

253

+

influx in neurons, which may relate to the fact that calpain is a calcium-dependent protease. Inhibition of calpains by calpastatin expression alleviated ataxin-3 toxicity in cultured cells [152] and reduced symptoms in vivo in an MJD mouse model [153]. While these studies rely on genetic modification of the disease models, pharmacological inhibition of calpains and calcium-signaling has also provided encouraging results. Calpeptinmediated inhibition of calpain in a zebrafish model for MJD inhibited cleavage of the mutant protein and prevented motor defects [154]. Similarly, the calpain inhibitor BDA-410 decreased the levels of both cleaved and full-length ataxin-3 and alleviated neurodegenerative phenotypes both on a cellular and behavioral level in MJD mice [155]. Targeting calcium homeostasis with Dantrolene, a calcium-signaling stabilizer used in the clinic to alleviate malignant hyperthermia and muscle spasticity, also prevented motor defects and neuronal loss in MJD mice [156]. Nuclear localization of mutant ataxin-3 is a critical determinant for toxicity [97]. Hence, modulating the localization of the mutant protein presents another therapeutic angle in MJD (Fig. 10.5f). Karyopherin α-3 (KPNA3), also known as importin subunit α-3, promotes nuclear import of full-length and truncated polyQexpanded ataxin-3 [157]. Depletion of KPNA3 in fruit flies expressing mutant ataxin-3 resulted in decreased aggregation and rescued neurodegeneration. Moreover, genetic ablation of KPNA3 in an MJD mouse model alleviated both molecular and behavioral defects underscoring the potential of targeting nuclear import [157]. A caveat with this approach is that the exclusion of ataxin-3 from the nucleus is likely to have a negative impact on native functions of ataxin-3 that require nuclear localization, such as DNA damage signaling and repair.

10.6

Concluding Remarks

Regardless of the many hurdles that still need to be taken, MJD remains an intriguing candidate for development of novel therapeutics aimed at reducing expression of a mutant protein. Its strict

254

monogenetic nature, the important role of a toxic gain-of-function mechanism, and the possibility to identify gene-carriers before disease onset, allowing initiation of the treatment already in clinically presymptomatic patients, are features that motivate the development of therapeutics. Last but not least, the lack of curative therapies is a strong incentive to explore innovative, unprecedented ways to halt or reverse the progression of the disease. Fortunately, absence of ataxin-3 is relatively well tolerated even though we do not know currently how this will affect neuronal viability in the long term. It is therefore desirable that the development of therapeutic strategies aimed at decreasing ataxin-3 levels will take place parallel to thorough basic research aiming at a better understanding of the native functions of ataxin-3 as this may help us to anticipate and counter potential side effects caused by ataxin-3 deficiency. Even though challenging, we anticipate that selectively targeting the expression of the mutant protein can have a dramatic beneficial effect and slow down disease progression. This objective may be harder to reach for related, more common neurodegenerative diseases, such as nonfamilial AD and PD, where the early events leading to the disease are more multifactorial in nature. Importantly, because of the similarities between neurodegenerative diseases caused by misfolded proteins, it is feasible that development of successful therapies for MJD will aid, in the long run, also the tackling of these more common age-related neurodegenerative diseases. Note After the acceptance of this paper, two studies have been published that reported new functions of ataxin-3. Singh et al. reported that ataxin-3 together with VCP stimulates the chromatin extraction of the ubiquitin ligase RNF8, which is important for regulation of the DNA damage response [158]. We reported that ataxin3 interacts with the ubiquitin-like autophagy proteins LC3C and GABARAP and stimulates autophagy independent of beclin-1 [159]. Acknowledgments The Dantuma lab is financially supported by grants from the Swedish Research Council, the Swedish Cancer Society, the EU Joint Progamme— Neurodegenerative Disease Research (JPND) (CureALS)

N. P. Dantuma and L. K. Herzog and the Karolinska Institute (StratNeuro grant). L.K.H. is supported by a KID PhD grant awarded by the Karolinska Institute.

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Machado–Joseph Disease: A Stress Combating Deubiquitylating Enzyme Changing Sides

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Part III Infection, Inflammation and Developmental Disorders

SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends

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Ahmed El Motiam, Santiago Vidal, Rocío Seoane, Yanis H. Bouzaher, José González-Santamaría, and Carmen Rivas

Abstract

SUMO is a ubiquitin-like protein that covalently binds to lysine residues of target proteins and regulates many biological processes such as protein subcellular localization or stability, transcription, DNA repair, innate immunity, or antiviral defense. SUMO has a critical role in the signaling pathway governing type I interferon (IFN) production, and among the SUMOylation substrates are many IFN-induced proteins. The overall effect of IFN is increasing global SUMOylation, A. El Motiam · S. Vidal · R. Seoane · Y. H. Bouzaher Centro Singular en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidad de Santiago de Compostela, Santiago de Compostela, Spain Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago de Compostela, Spain J. González-Santamaría Grupo de Biología Celular y Molecular de Arbovirus, Instituto Conmemorativo Gorgas de Estudios de la Salud, Panamá, Panama

pointing to SUMO as part of the antiviral stress response. Viral agents have developed different mechanisms to counteract the antiviral activities exerted by SUMO, and some viruses have evolved to exploit the host SUMOylation machinery to modify their own proteins. The exploitation of SUMO has been mainly linked to nuclear replicating viruses due to the predominant nuclear localization of SUMO proteins and enzymes involved in SUMOylation. However, SUMOylation of numerous viral proteins encoded by RNA viruses replicating at the cytoplasm has been lately described. Whether nuclear localization of these viral proteins is required for their SUMOylation is unclear. Here, we summarize the studies on exploitation of SUMOylation by cytoplasmic RNA viruses and discuss about the requirement for nuclear localization of their proteins. Keywords

SUMO · RNA viruses · Interferon

Dirección de Investigación, Universidad Interamericana de Panamá, Panamá, Panama C. Rivas (*) Centro Singular en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidad de Santiago de Compostela, Santiago de Compostela, Spain Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago de Compostela, Spain Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología (CNB), CSIC, Madrid, Spain e-mail: [email protected]

11.1

SUMO Conjugation

SUMOylation consists in the covalent attachment of the small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of a target protein. This conjugation is an enzymatic process that occurs in cascade. The precursor SUMO proteins are proteolytically processed to the

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_11

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mature forms, exhibiting their C-terminal diglycine motifs, by sentrin/SUMO-specific proteases (SENPs). The resulting mature forms of SUMO are then activated by the SUMOactivating E1 enzyme (SAE1/SAE2). Subsequently, the SUMO molecule is transferred to the E2-conjugating enzyme Ubc9, the only E2 SUMOylation enzyme discovered to date. Finally, the carboxyl group of the glycine residues at the SUMO carboxy terminus forms an isopeptide linkage with the ε-amino group of a lysine residue on the target protein, a step that can be facilitated by SUMO E3 ligases. SUMOylation can be then reversed by SENPs. Usually, the target lysine for SUMO is located in the consensus sequence ψKxE (where ψ is a hydrophobic residue and x any residue) in the target protein, although SUMO can be also conjugated to lysine residues located in non-consensus sequences. Four different SUMO isoforms have been described in mammals (SUMO1 to SUMO4). SUMO1 protein is found mainly conjugated to targets due to its limited quantity and only shares 50% identity with SUMO2/3. SUMO2 and SUMO3 share 97% sequence identity and often are referred to as SUMO2/3, and its conjugation is stress-inducible [1]. SUMO4 remains poorly characterized and its physiological relevance is still unclear [2, 3]. The main function of SUMO proteins is to regulate protein–protein interaction, which ultimately results in changes in protein subcellular localization or stability, transcription, DNA repair, innate immunity, or antiviral defense. Interestingly, many SUMO-modified proteins contain SUMOinteracting motifs (SIMs), allowing the noncovalent interaction of SUMO substrates with SUMO or non-SUMOylated proteins [4, 5].

11.2

Regulation of the Type I Interferon (IFN) Production by SUMO

The first line of defense against viruses is the induction of type I interferons (IFNs). During RNA virus infection, the viral genomic RNA and replication intermediates produced by the

virus are recognized as pathogen-associated molecular pattern (PAMP) by pattern-recognition receptor (PRR) proteins such as Toll-like receptors, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetic and physiology 2 (LGP2), initiating a signaling cascade that results in the production of type I IFN. SUMO interacts with different intermediates along this signaling cascade modulating the transcriptional transactivation of type I IFN (Fig. 11.1). SUMOylation of MDA5 and RIG-I, promoted by the SUMO E3 ligase TRIM38, has been shown to be required for its dephosphorylation and activation upon virus infection [6]. Once RIG-I is activated, it recruits the inhibitor of kB kinase (IKK) α, β, and ε through its interaction with MAVS, leading to the activation of NF-kβ, interferon regulatory factor (IRF)3, and IRF7, and consequently resulting in IFNβ and NF-kβ promoter activation. Frequently, the activation of both NF-kβ and IRF3/7 pathways by RIG-I in response to virus infection requires the NF-kβ essential modulator (NEMO), an adaptor protein that organizes the assembly of IKKs into activated high-molecular-weight complexes. Conjugation of SUMO2/3 to NEMO, a process downmodulated by SENP6, inhibits its interaction with the deubiquitinase CYLD strengthening the activation of IKK [7]. The noncanonical IKK kinase TANK-binding kinase 1 (TBK1), which mediates the activation of IRF3, has also been shown to be a pivotal player in antiviral innate immunity, and its modification by SUMO supports its antiviral function, likely through contributing to adaptor binding for the transduction of antiviral signaling [8]. In addition to its role as a modulator of the RIG-I phosphorylation, SUMO1 conjugation to RIG-I also inhibits its K48-linked polyubiquitination and degradation [6] and promotes its interaction with MAVS [9], inducing activation of the IFNβ promoter. In addition, upregulation of IFN expression has been observed as a result of the modification of the virus sensor MDA5 by SUMO1 after Ubc9 or PIAS2β overexpression [10].

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SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends

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Fig. 11.1 Transactivation of type I IFN upon RNA virus infection by SUMO. Detection of the viral RNA by RIG-I and MDA5 led to activation of IFN production. SUMO, by conjugating MDA5 and RIG-I, inhibits their

phosphorylation and induces their stabilization [6]. Conjugation of SUMO to NEMO inhibits its interaction with the deubiquitinase CYLD strengthening the activation of IKK and inducing an increase of type I IFN production [7]

SUMOylation can also have a negative impact on IFN production. SUMO1 can inactivate NF-kB by modifying IkBα and inhibiting its degradation [11]. In addition, SUMOylation of IRF3/ 7 in response to Toll-like receptor (TLR) and RIG-I activation can act as a transcriptional repressor of IRF3 and IRF7, a mechanism that has been postulated to be part of the negative feedback loop of normal IFN signaling [12]. However, conjugation of SUMO to IRF3 not necessarily inhibits IRF3. Ran and colleagues found that SUMO can conjugate to K87 in IRF3, a lysine residue involved in IRF3 K48-ubiquitin

conjugation and degradation [13]. Consequently, I IFN downmodulation of IRF3 SUMOylation induced by SENP2 promotes its ubiquitination, negatively regulating the virus-triggered type induction and cellular antiviral response [13]. Whereas PIAS1 has been reported to work as E3 SUMO ligase for both IRF3 and IRF7 [14], the repression of the ability of IRF7 to induce type I IFN transcription by SUMOylation has been shown to be promoted by TRIM28 [15]. Together, these studies show that SUMO plays an important role in the type I IFN expression in response to RNA virus infection.

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SUMO and Type I IFN Responses

IFN binding to specific receptors at the cell surface activates the receptor-associated protein tyrosine kinases Janus kinase 1(JAK1) and tyrosine kinase 2 (TYK2), which phosphorylate the signal transducer and activator of transcription 1 (STAT1) and STAT2. These two proteins form dimers and associate with IRF9 to form a transcriptionally active IFN-stimulated gene factor 3 (ISGF3). ISGF3 binds to IFN-stimulated response elements (ISREs) leading to transcription of IFN-stimulated genes (ISGs). This canonical type I IFN signaling can be modulated by SUMO (Fig. 11.2). SUMO has been shown to interact with STAT1 in a covalent manner, and this interaction

Fig. 11.2 Effect of SUMO on type I IFN response. Activation of STAT1 in response to IFN is downmodulated by SUMO conjugation. However, SUMO does not alter the IFNα signaling, likely because STAT2 is not affected by SUMO and it can compensate for the reduction in STAT1

has been proposed to inhibit STAT1 activity, since a STAT1 SUMOylation mutant is hyperphosphorylated and exhibits increased DNA binding on STAT1 responsive gene promoters [16–18], and phosphorylation of STAT1 induced by IFNβ treatment inhibits its SUMOylation [19]. In addition, SUMO overexpression has been shown to reduce the IFN-induced STAT1 phosphorylation and downmodulate IFNγ transcriptional responses [20]. However, SUMO does not alter IFNα signaling, likely because STAT2 is not affected by SUMO and it can compensate for the reduction in STAT1 phosphorylation [20]. IFN establishes an antiviral state in cells by inducing a number of proteins with antiviral activity. Interestingly, many of these IFN-induced proteins are SUMO substrates and

phosphorylation [16–20]. Conjugation of SUMO to p53, PML, and MxA promotes their antiviral activities. The regulation of the antiviral activity of PKR by SUMO may depend on the SUMO isoform [21–23]

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SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends

their activities are regulated by SUMOylation and/or noncovalent interaction with SUMO. The GTPases Mx exert antiviral activity against a variety of RNA and DNA viruses. MxA protein has been reported to interact with SUMO1, SAE2, and Ubc9 [24, 25]. In addition, a recruitment of Mx to PML-NBs and its modification by SUMO2/3 have also been reported [24]. Interestingly, noncovalent SUMO interaction but not SUMOylation has been found to be essential for the antiviral effect exerted by MxA on vesicular stomatitis virus (VSV), since Ubc9 depletion does not affect the protective effect of MxA on VSV replication [26]. The Tumor Suppressor Protein p53 can interfere with the replication of several viruses, and activation of p53 has been proposed as a potential therapy against viral infections [27]. IFN treatment induces the transactivation of p53 [28], but it also induces its SUMOylation, modification that contributes to the antiviral functions of IFN [29]. In addition, SUMOylation of p53 induced by IFN has also been shown to contribute to senescence induction, a process that helps to control virus replication [29, 30]. The Double-Stranded RNA (dsRNA)-Dependent Protein Kinase (PKR) is an IFN-inducible protein that can phosphorylate the alpha subunit of the protein synthesis initiation factor eIF2α, resulting in a shut-off of protein translation, apoptosis induction, and inhibition of virus replication [31]. PKR is regulated by covalent and noncovalent interaction with SUMO [21– 23]. SUMOylation, as well as noncovalent SUMO interaction, is required for efficient PKR–dsRNA interaction, PKR dimerization, phosphorylation of eIF-2α, and inhibition of VSV replication [21, 22]. Although virus infection promotes the modification of PKR by SUMO1 and SUMO2/3, recently, a specific role of SUMO3 counteracting PKR activation and stability upon virus infection has been proposed [23].

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PML is an IFN-induced protein that plays a key role in the establishment of innate antiviral responses. Thus, PML KO mice are more sensitive than WT to infection with different viruses such as lymphocytic choriomeningitis virus (LCMV), encephalomyocarditis virus (EMCV), or VSV, and cells from these mice are also more sensitive to infection with rabies virus [32–34]. In addition, overexpression of specific PML isoforms confers resistance to some virus infections. Thus, PMLIII protects against human foamy virus (HFV) [35], poliovirus [36], VSV, or influenza A virus [37], and PMLIV confers resistance to rabies virus and EMCV infection [34]. PML interacts with SUMO in a covalent and noncovalent manner. SUMOylation of PML is required for PML-NB formation and for promoting SUMOylation of specific substrates recruited on PML-NBs [38–40]. Moreover, modification of PMLIV by SUMO positively regulates IFNβ synthesis through IRF3 activation, and it is required for protecting cells from virus infection [41]. PML-III and PML-IV also recruit Ubc9 to PML-NBs, and this recruitment has been shown to be essential for the increase in global SUMOylation induced by IFN [42]. This is not the unique mechanism that contributes to the increase in global SUMOylation upon IFN treatment. Among the IFN-regulated proteins is Lin28B, a RNA binding protein that represses the expression of miRNAs from let-7 family [43–47] which targets SUMO transcripts [48]. The IFN-induced global SUMOylation, as well as the SUMOylation of viral restriction factors induced by IFN, is likely part of the cellular antiviral defense mechanisms of the host since some viruses have developed strategies to inhibit those processes. In addition, some viruses have evolved to hijack the SUMOylation machinery of the cell to regulate their own proteins. SUMO and Ubc9 proteins are mainly located at the cell nucleus. Therefore, for many years it has been suggested that SUMOylation was relevant mainly for infection of nuclear replicating viruses. In agreement with this hypothesis, exploitation of the SUMO pathway has been

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reported to be a common strategy of DNA viruses and of those few RNA virus replicating in the nucleus, such as influenza virus or human immunodeficiency virus (HIV)-1 [49, 50]. Lately, numerous reports demonstrate that the SUMO pathway is also relevant for cytoplasmic RNA viruses.

11.4 11.4.1

SUMO and RNA viruses Reoviridae

Viruses of the Reoviridae family are non-enveloped icosahedral virus of around 60–80 nm that contain from 9 to 12 segments of linear double-stranded RNA. Although reovirus replication occurs in the cytoplasm, several viral proteins have been detected in the cell nucleus. Thus, the nonstructural sigma 1 protein, a determinant of reovirus virulence, contains a functional nuclear localization signal (NLS) [51], and it can be detected in the nucleus during reovirus infection [52, 53]. The avian reovirus core protein sigma A accumulates in the nucleoplasm of mammalian cells or in the nucleolus and cytoplasm of avian cells [54], and the reovirus minor capsid protein mu 2 of specific strains localizes to nuclear speckles [55]. The Reoviridae family is constituted by several genera, whose members have a varied host range. Rotavirus is a genus in the Reoviridae family that is recognized as the single most important cause of severe gastroenteritis in infants of a wide range of mammals. Rotavirus genome is constituted by 11 dsRNA segments encoding six structural (VP1–4, VP6, and VP7) and six nonstructural (NSP1–6) proteins. Early stages of viral assembly and viral RNA replication take place in virus-induced inclusion bodies called viroplasms localized in the infected cell cytoplasm. Viroplasms are formed by VP1, VP2, VP3, VP6, NSP2, and NSP5. Five out of six of the viroplasms components, VP1, VP2, NSP2, VP6, and NSP5 proteins, are modified by SUMO; and three of them, VP1, VP2, and NSP2

proteins, also interact in a noncovalent manner with SUMO. SUMOylation of NSP5 has been shown to be essential for the formation of viroplasm-like structures (VLS) generated by overexpression of VP2 (VLS-VP2i). In addition, upregulation of SUMOylation positively modulates rotavirus replication and viral protein production, whereas interference of Ubc9 produces a marked decrease in the synthesis of viral proteins and virus titer [56]. The extensive exploitation of the SUMOylation machinery by rotavirus evokes to that described for influenza virus [57]. However, in contrast to influenza virus, rotavirus conducts its life cycle in the cytoplasm and, so far, nuclear localization of rotavirus proteins has not been reported. Members of the Orthoreovirus genus have been evaluated as putative anticancer agents and, as reported for rotavirus, Ubc9 also contributes to their efficient replication. Ubc9 has been shown to interact with the outer fiber protein VP55 from the grass carp reovirus (GCRV)-104 or the type II GCRV, with sigma C from avian reovirus (ARV) and with sigma 1 from mammalian reovirus (MRV) by using yeast two-hybrid system. Furthermore, a positive correlation between Ubc9 levels and (GCRV)-104 replication has been reported [58]. Therefore, SUMOylation has been proposed as a tool to improve the therapeutic efficacy of oncolytic reoviruses. Although it has been hypothesized that SUMOylation of the outer fiber proteins may increase tropism for host cells, so far, no SUMOylation of orthoreovirus proteins has been demonstrated.

11.4.2

Paramyxoviridae

Paramyxoviridae family is constituted by singlestranded negative-sense RNA genome viruses. Paramyxovirus replication takes place in the cytoplasm; however, several paramyxoviral proteins have been detected in the nucleus in infected cells. So far, there is just one example of exploitation of SUMOylation machinery by paramyxovirus.

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SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends

Parainfluenza virus 5 (PIV5) is a prototypic member of the Rubulavirus genus. The genome of PIV5 encodes eight known proteins. After evaluation of the putative SUMOylation of four proteins of the virus (the two components of the viral RNA-dependent RNA polymerase L and P, the nucleocapsid NP protein, and the nonstructural V protein), only the P protein was found to be SUMOylated and only by SUMO1 and not by SUMO2/3 [59]. Analysis of a recombinant PIV5 containing a mutant of the P protein in the SUMOylation motif revealed a reduction in the titer, viral RNA synthesis, and protein expression relative to the WT PIV5. Therefore, SUMOylation was proposed to regulate PIV5 gene expression through the regulation of viral RNA transcription [59]. Whether the P protein of PIV5 can enter into the nucleus is unknown.

11.4.3

Pneumoviridae

Pneumoviridae is a family of large enveloped single-stranded negative-sense RNA virus. One of the members of the family that affects humans is the respiratory syncytial virus (RSV). Although RSV is a cytoplasmic virus able to replicate in enucleated cells, its matrix (M) protein has been found inside the nucleus of infected cells [60]. Different reports reveal a relationship between RSV and the host cell SUMOylation machinery. Thus, an association between a cluster of host proteins involved in the SUMOylation process and the phosphoprotein (P) of the virus has been found after integration of proteome and transcriptome datasets [61]. In addition, SUMO seems to play a role in the pathogenesis of the virus [62]. The cellular damage and lung inflammation caused by RSV infection are associated with the generation of reactive oxygen species (ROS) and oxidative stress, via degradation of the transcription factor NF-E2-related factor (NRF2), which occurs in a SUMO-specific E3 ubiquitin-ligase-RING finger protein (RNF4)dependent manner [62]. However, so far, no SUMOylation of pneumovirus proteins has been demonstrated.

11.4.4

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Nodaviridae

Nodavirus are non-enveloped viruses with a genome consisting in two molecules of singlestranded positive-sense RNA. The family includes two genera, Alphanodavirus and Betanodavirus. Viruses belonging to the first genus infect insects, whereas betanodaviruses infect fishes. Nodavirus replicate in the host cell cytoplasm. However, the capsid protein of nodavirus contains a nuclear localization signal that targets the viral capsid to the nucleus of infected cells [63]. Infection with the betanodavirus red-spotted grouper nervous necrosis virus (RGNNV) induces the transcriptional upregulation of SUMO3, and SUMO3 overexpression increases IFN and ISRE promoter activity, suggesting that SUMOylation may play a role in the immune response of fish to viral infection [64]. However, SUMO3 has been shown to enhance RGNNV replication in vitro [64], suggesting that RGNNV is able to exploit the host SUMOylation machinery. So far, no SUMOylation of nodavirus proteins has been demonstrated.

11.4.5

Picornaviridae

Picornaviridae is a family of non-enveloped viruses with single-stranded positive-sense RNA genome containing usually a single open reading frame encoding a precursor protein that can be processed by viral proteinases. It contains more than 30 genera and more than 75 species. Although the replication of picornaviruses occurs in the cytoplasm, some viral 3C proteases have been found to enter the nucleus using a NLS present in the RNA-dependent RNA polymerase (3D), in order to prevent host transcription and cap-dependent translation to provide cellular resources for viral replication [65]. In addition to 3C and 3D, EMCV nonstructural 2A and 3B proteins have been also found in the cell nucleus [66]. The Enterovirus 71 (EV71) belongs to the Enterovirus genera within the Picornaviridae

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family, and it is the common cause of hand, foot, and mouth disease. The polymerase 3D of EV71 is modified by SUMO1 and SUMO3, and this modification promotes its K63-linked ubiquitination enhancing the stability of the viral protein. Consequently, SUMOylation is required for 3D polymerase activity and virus replication, and this may explain why SUMO1 overexpression enhances viral replication [67]. Interestingly, a SUMO-dependent ubiquitination of the protease 3C has also been described. SUMO modification of 3C has been proposed to be a host cell defense mechanism against virus replication and apoptosis induction during EV71 infection because SUMOdependent ubiquitination of 3C was associated with reduced protease activity of 3C in vitro, increased degradation, decreased apoptosis induction and virus replication, and reduced EV71-elicited neurovirulence in a mouse model [68]. A connection between the human enteroviruses Coxsackievirus B5 (CVB5) or CVB3 and the host cell SUMOylation system has also been proposed. CVB5 infection induces redistribution of SUMO1 and Ubc9 all over the cell, which has led to the hypothesis that CVB5 might interfere with, and perhaps destabilize, protein SUMOylation [69]. In addition, CVB3 infection has been shown to induce the degradation of p53, likely through the promotion of the SUMOylation of the proteasome regulator REG to promote viral replication [70]. However, so far, no SUMOylation of CVB5 or CVB3 proteins has been demonstrated. Another picornavirus that exploits the host cell SUMOylation machinery to modulate the activity of restriction factors is EMCV, the prototype of the Cardiovirus genus. EMCV, likely through the protease 3C, induces proteasome and SUMOdependent degradation of PMLIII [33].

Orthobunyavirus, and Tospovirus. Although bunyavirus replication occurs in the cytoplasm, virus production is blocked in enucleated cells. Therefore, the role of the nucleus is unclear. So far, a role of SUMO in the viral life cycle of viruses belonging to this family has only been reported for Hantavirus. The hantaviruses genome consists of three RNA segments encoding the nucleocapsid protein NP, two surface glycoproteins G1 and G2, and the viral polymerase L [71]. Interaction between the nucleocapsid protein of different hantaviruses, including Hantaan virus (HTVN), Seoul virus (SEOV), Tula virus (TULV), Puumala virus, and Andes virus (ANDV), and SUMO1 or SUMOylation pathway proteins, such as Ubc9, PIAS1, PIASxβ, or RanGAP, has been reported [72–74]. However, so far, no SUMOylation of NP was observed and the function of these interactions in the viral life cycle remains unknown.

11.4.6

11.4.8

Bunyaviridae

Bunyaviridae is a large family of enveloped single-stranded RNA viruses. It is constituted by 5 genera: Phlebovirus, Nairovirus, Hantavirus,

11.4.7

Coronaviridae

Coronaviridae is a family of enveloped singlestranded positive-sense RNA viruses. It has two subfamilies, Coronavirinae and Torovirinae. The entire replication cycle takes place in the cytoplasm. However, the multifunctional nucleocapsid N protein of coronavirus can be detected in the nucleolus [75, 76], although this ability varies between N proteins of different coronaviruses. The severe acute respiratory syndrome-related coronavirus (SARS-Cov) N protein can directly interact with human Ubc9 [77] and is modified by SUMO [78]. Analysis of an N protein SUMOylation mutant revealed that SUMOylation promotes N homo-oligodimerization and nucleolar localization and plays certain roles in the Nprotein-mediated interference of host cell division.

Arteriviridae

Arteriviridae is a family of enveloped, singlestranded positive-sense RNA virus that includes only the Arterivirus genus. One of the members

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SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends

of the family is the porcine reproductive and respiratory syndrome virus (PRRSV). The genome of PRRSV contains several overlapping open reading frames. The ORF1a and 1b encode the viral nonstructural proteins that play important roles in replication and transcription of the viral genome, in modulation of the host innate immunity, and in pathogenesis and virulence. The remaining ORFs encode the structural proteins of PRRSV including the viral nucleocapsid N protein. The N protein is one of the most abundant PRRSV proteins, highly immunogenic in pigs and with important roles in PRRSV replication and immune evasion. PRRSV replicates in the cytoplasm of infected cells. However, the N protein localizes specifically in the nucleus and nucleolus of virus-infected cells, localization that modulates the pathogenesis of the virus in pigs [79]. Wang and collaborators found many conserved lysine residues in various proteins of PRRSV that were predicted to be SUMOylated using in silico prediction analysis of the amino acid sequences of PRRSV structural and nonstructural proteins [80]. Furthermore, the authors demonstrated that the PRRSV N protein can be modified by SUMO and that the nonstructural PRRSV proteins NSP1β, NSP4, NSP9, NSP10, and N colocalize and interact with Ubc9 [80]. Altogether these data suggest that the virus can exploit the cell host SUMOylation pathway. However, the overall effect of SUMO on PRRSV seems to be negative, as knockdown of Ubc9 promotes virus replication and overexpression of Ubc9 inhibits viral genomic replication [80]. The role of PRRSV N protein SUMOylation, the consequences of the interaction between Ubc9 and the viral proteins, or the molecular mechanisms by which Ubc9 modulates PRRSV replication are still unknown.

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are three Filoviridae genera: Ebolavirus, Marburgvirus, and Cuevavirus. So far, only one of the virus of the family, Ebola virus (EBOV), has been probed to exploit the SUMOylation pathway. EBOV encode seven structural proteins: the nucleoprotein NP, the polymerase cofactor VP35, the matrix protein VP40, the transcription activator VP30, the minor matrix protein VP24, the glycoprotein GP, and the RNA-dependent RNA polymerase L. The GP transcript can be edited giving rise to four alternative forms of gene products. In addition to serve as structural components, EBOV proteins play multiple roles in the virus life cycle. SUMO has been proposed to be critical for EBOV life cycle, and modulation of the interaction between SUMOylation pathway components and viral proteins may represent a novel target for therapeutics to block EBOV infection. VP35 has been reported to interact with Ubc9 and PIAS1 and induce a PIAS1-mediated SUMOylation of IRF7 and IRF3, a mechanism through which EBOV VP35 disrupts antiviral responses [14]. In addition, EBOV hijacks the cellular SUMOylation system in order to modify its own proteins. Thus, the multifunctional VP40 protein, involved in regulating virus budding, nucleocapsid recruitment, virus structure and stability, and viral genome replication and transcription, is modified by SUMO [81]. Interestingly, SUMO was also found to be included into the viral particles formed by VP40. One consequence of VP40 SUMOylation is the regulation of its ubiquitination [81]. Although EBOV replication takes place in the cytoplasm of the infected cells, EBOV VP40 protein has been detected in the cell nucleus [82].

11.4.10 Flaviviridae 11.4.9

Filoviridae

Filoviridae is constituted by enveloped virus containing a non-segmented negative-strand RNA genome. This family contains some of the deadliest pathogens known to date. There

Flaviviridae is a family of small enveloped viruses with non-segmented positive-sense RNA genomes. Flaviviridae genome encodes a single polyprotein that is cleaved into three structural proteins (capsid (C), premembrane (prM), and envelope (E)), and seven nonstructural

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(NS) proteins. Although flavivirus replication occurs in the cytoplasm, some viral proteins translocate to the nucleus during infection contributing to viral replication [83]. The Flaviviridae family includes four genera: Flavivirus, Pestivirus, Hepacivirus, and Pegivirus. The Flavivirus genus consists mainly of arthropod-borne viruses and includes important human pathogens such as dengue virus (DENV), Zika virus (ZIKV), or yellow fever virus (YFV). Several studies indicate that SUMO plays an important role in flavivirus replication and suggest that SUMO inhibitors may be a broad spectrum anti-flaviviral strategy. Thus, treatment with the SUMOylation inhibitor 2-D08 has been reported to significantly reduce the replication of ZIKV in vitro in different cell types and in a doseresponse manner [84]. How SUMOylation favors ZIKV replication is not clear. The ZIKV RNA-dependent RNA polymerase NS5 has a putative SIM domain that is highly conserved among Zika strains that is required for the NS5-mediated suppression of type I IFN signaling [84]. However, functionality of the SIM domain in the interaction between ZIKV NS5 and Ubc9 or SUMO or SUMOylation of the viral protein has not been demonstrated. 2-D08 treatment, as well as Ubc9 interference, also reduces replication of DENV [84, 85]. Several DENV proteins might interact with the SUMOylation machinery. Ubc9 has been predicted to bind to different DENV proteins by using a computational approach [86]. Furthermore, interaction between Ubc9 and the nonstructural proteins NS2B, NS4B, and NS5, as well as with the envelope protein E, was probed using a yeast two-hybrid assay [87, 88]. However, after evaluation of the putative SUMO1 modification of ten different DENV proteins, only SUMOylation of NS5 was demonstrated [85]. An intact SIM domain in NS5 was shown to be required for its SUMOylation, which protected DENV NS5 against proteasome degradation, supporting virus replication [84]. Interestingly, NS5 protein contains two functional NLS and is one of the DENV proteins that can be found inside the nucleus, where it interferes with cellular splicing [89]. Although Ubc9 was

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required for efficient replication of DENV, a study on type 2 dengue virus (DV-2) showed that overexpression of Ubc9 reduces the viral plaque formation in mammalian cells, likely due to the involvement of Ubc9 in the host defense system to prevent virus propagation [87]. SUMOylation inhibitor treatment also significantly inhibited the replication of other flavivirus such as Japanese encephalitis virus (JEV), West Nile virus (WNV), or YFV. In addition, in silico analysis of 78 representative flaviviruses revealed the presence of a putative SIM domain at the NS5 protein in 92.3% of the analyzed viruses [84]. Interestingly, the authors found that only the insect-specific flavivirus lacked putative SIMs. Therefore, they proposed that it can be an evolutionarily conserved modification process among flaviviruses to enhance virus replication and suppress host antiviral response. The SUMO system has been shown to be also required for the replication of hepatitis C virus (HCV), the best characterized member of the Hepacivirus genus of the Flaviviridae family with several proteins containing functional NLSs and nuclear export signals (NESs) including the core, NS2, NS3, and NS5 proteins [90]. HCV infection upregulates SUMO1 expression, a host factor essential for HCV replication [91]. Ubc9 has also been shown to be required for HCV RNA replication [92]. The requirement of the host cell SUMOylation machinery for HCV replication is probably linked to the SUMOylation-dependent stability of NS5A [92] and to the SUMO-mediated regulation of the expression of the viral core protein [91]. The importance of NS5 SUMOylation for HCV was also supported by the constant reversion of the SUMO-defective mutant K348R of HCV NS5 to the WT sequence during virus replication [92]. In addition, SUMO1 is also required for lipid droplet accumulation, organelles that work as a platform for virus replication, assembly, and production [91]. In contrast to the essential role of SUMO1 and Ubc9 in HCV replication, PIAS2-mediated SUMOylation has been identified as a restriction factor against HCV replication [93]. Thus, an enhancement of HCV core, NS3, and NS5A

11

SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends

protein expression in infected cells, as well as of viral assembly and budding efficiency, was observed after knockdown of PIAS2 [93]. In contrast, exogenous overexpression of PIAS2 decreased HCV core, NS3, and NS5A expression and the viral assembly and budding efficiency. When expressed together with SUMO1, PIAS2 induced the degradation of HCV core, NS3, and NS5A proteins expressed from individual plasmids and after treatment with the proteasome inhibitor MG132, PIAS2 interacts with and enhances SUMOylation of the core protein. Therefore, PIAS2 has been proposed to mediate the degradation of the core, NS5A, and NS3 proteins through a ubiquitin-independent proteasomal pathway [93]. Interestingly, SUMO1 may have a role in the promotion of HCV replication by alcohol exposure. Thus, alcohol treatment has been shown to induce upregulation in PIASγ expression which induces autophagy, at least partially through promoting the accumulation of SUMO1 conjugated proteins, and consequently promotes HCV replication [94]. SUMO has also been demonstrated to be relevant for members of the Flaviviridae family not pathogenic for humans such as the classical swine fever virus (CSFV). CSFV belongs to the genus Pestivirus within Flaviviridae family. It has three structural components, including the core protein. Core protein of CSFV interacts with SUMO1 and Ubc9, and mutant viruses with deletions in the putative SUMOylation sites of the core protein show attenuated phenotypes and limited spreading within infected swine, suggesting that SUMOylation pathway may contribute to curtailing viral clearance [95]. Only mutation for both SUMO1 and Ubc9 interaction led to complete attenuation. However, whether the core protein is indeed modified by SUMO or whether the attenuation is related with reduced SUMOylation is not known.

11.4.11 Rhabdoviridae Viruses from the Rhabdoviridae family are single-stranded negative-sense RNA viruses.

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The prototypes of this family are VSV and rabies virus (RABV). Although VSV infection does not induce global SUMOylation [26, 49], it induces the SUMOylation of specific proteins, promoting their antiviral activity, such as the tensin homolog deleted for chromosome 10 (PTEN) [96], the double-stranded RNA-dependent protein kinase (PKR) [22], p53 [29], and MxA [26]. Based on these reports, an upregulation of SUMOylation will likely be negative for VSV replication. In fact, although SUMO expression reduced IFN synthesis upon VSV infection, stable expression of SUMO1 or SUMO3 resulted in resistance to VSV infection [26]. However, whether the control of VSV replication by SUMO overexpression is mediated by SUMOylation is not so clear, since the synthesis of VSV proteins is not affected by Ubc9 knockdown [26]. Whether SUMOylation of p53, PKR, or PTEN protects against RABV infection is unknown. What it has been reported is that SUMO3 overexpression has a positive effect on RABV infection, likely due to the inactivation of IRF3 caused by its SUMOylation in response to RABV infection [26].

11.5

Conclusions

Type I interferon induces the transcription of multiple genes leading to immunomodulatory effects. Among these IFN upregulated genes, there are SUMO proteins as well as multiple targets of SUMO, suggesting that SUMO may play a role in immunity. Viruses have evolved to exploit the SUMOylation machinery of the cell to improve their replication. Many viral proteins are SUMOylated or influence the SUMOylation of cellular proteins. Initially, most of the viral proteins evaluated as putative SUMO substrates were nuclear proteins of DNA viruses or RNA viruses replicating in the nucleus because SUMO and SUMOylation pathway components are localized mainly at the cell nucleus. However, in recent years numerous studies have demonstrated that cytoplasmic RNA viruses also exploit the cellular SUMO pathway. Although SUMOylation may contribute to the control of

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virus replication, SUMO seems to have an overall positive effect in most of the cytoplasmic RNA viruses. Thus, SUMO proteins or components of the SUMOylation pathway such as Ubc9 are required for the replication of some picornavirus, reovirus, nodavirus, and flavivirus and play a role in pnemovirus or flavivirus pathogenesis. In addition, SUMO conjugates to viral proteins from reovirus, paramyxovirus, picornavirus, coronavirus, arterivirus, filovirus, and flavivirus, and only a few examples of a negative effect of SUMO have been reported for members of the Flaviviridae, Arteriviridae, and Rhabdoviridae families. Interestingly, many of the viral proteins from cytoplasmic RNA viruses that are SUMO substrates can be detected at the cell nucleus during the viral cell cycle. Additional information will be required in order to determine whether there is a relationship between these two events. Acknowledgments Funding at the laboratory of CR is provided by the Ministry of Science, Innovation and Universities (MICINN) and FEDER (BFU-2017-88880P) and by GRC GI-2119 (Xunta de Galicia). SV is a predoctoral fellow funded by Xunta de Galicia. AEM is a recipient of a FPI fellowship from the Spanish MICINN. JGS laboratory is supported by grants from Secretaría Nacional de Ciencia, Tecnología e Innovación de Panamá (SENACYT) (FID0172016), Ministerio de Economía y Finanzas (009044.060), and Dirección de Investigación, Universidad Interamericana de Panamá (DI-UIP633800). JMP and JGS are members of Sistema Nacional de Investigación (SNI from SENACYT).

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The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication Daniela Batista-Almeida, Tania Martins-Marques, Teresa Ribeiro-Rodrigues, and Henrique Girao

Abstract

Given the low mitotic activity of cardiomyocytes, the contractile unit of the heart, these cells strongly rely on efficient and highly regulated mechanisms of protein degradation to eliminate unwanted potentially toxic proteins. This is particularly important in the context of disease, where an impairment of protein quality control mechanisms underlies the onset and development of diverse cardiovascular maladies. One of the biological processes which is tightly regulated by proteolysis mechanisms is intercellular communication. The different types of cells that form the heart, including cardiomyocytes, endothelial cells, fibroblasts, and macrophages, can communicate directly, through gap junctions (GJ) or tunneling nanotubes (TNT), or at long distances, via extracellular vesicles (EV) or soluble factors. The direct communication between cardiomyocytes is vital to ensure the anisotropic propagation of the electrical impulse, which allows the heart to beat in a coordinated and synchronized manner, as a functional syncytium. The rapid and efficient propagation of D. Batista-Almeida · T. Martins-Marques · T. Ribeiro-Rodrigues · H. Girao (*) Faculty of Medicine, Coimbra Institute for Clinical and Biomedical Research (iCBR), Center for Innovative Biomedicine and Biotechnology (CIBB), Clinical Academic Centre of Coimbra (CACC), University of Coimbra, Coimbra, Portugal; [email protected]

the depolarization wave is mainly conducted by low resistance channels called GJ, formed by six subunits of a family of proteins named Cxs. Dysfunctional GJ intercellular communication, due to increased degradation and/or redistribution of connexin43 (Cx43), the main Cx present in the heart, has been associated with several cardiac disorders, such as myocardial ischemia, hypertrophy, arrhythmia, and heart failure. Besides electrical coupling, a fine-tuned exchange of information, namely proteins and microRNAs, conveyed by EV is important to ensure organ function and homeostasis. Disease-induced deregulation of EV-mediated communication between cardiac cells has been implicated in diverse processes such as inflammation, angiogenesis, and fibrosis. Therefore, a better understanding of the mechanisms whereby proteolysis modulates the cross talk between cardiac cells is of utmost importance to develop new strategies to tackle diseases caused by defects in intercellular communication. Keywords

Proteostasis · Intercellular communication · Cardiovascular diseases · Gap junctions · Extracellular vesicles · Tunneling nanotubes

The heart is the muscle responsible for pumping blood throughout the organism to supply the energetic and metabolic needs of organs and tissues.

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_12

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The maintenance of the integrity and proper function of the heart relies on a tightly regulated proteostasis network that ensures the fitness of the proteome and a fine-tuned communication between the different cells that constitute the heart [1]. Not surprisingly, disturbances of protein homeostasis and/or intercellular communication mechanisms are broadly implicated in cardiovascular diseases, which remain the first cause of morbidity and mortality worldwide [2, 3]. Therefore, it is crucial to better understand the molecular mechanisms and signaling pathways responsible for the maintenance of cardiac homeostasis. In this review, a particular focus will be given to the interplay between proteostasis and intercellular communication in cardiovascular pathophysiology. A common feature of many cardiac diseases is the decline of proteostasis network efficiency and loss of proteome integrity, due to acute or chronic damaging insults. In both cases, the characteristic accumulation of misfolded and damaged proteins and organelles can be ascribed to an abnormal excessive production of toxic species and/or to a decreased proteostasis activity, namely chaperone reduction and impairment of UPS or autophagy systems [4]. Therefore, different outcomes in cellular proteotoxicity are implicated in cardiac pathologies of diverse etiologies and progress profile, including myocardial infarction (MI), hypertrophic cardiomyopathy, atrial fibrillation (AF), and heart failure (HF) [1, 2]. A paradigmatic example of a transient acute proteotoxic condition is MI, the leading cause of death in Western countries, which is generally secondary to a partial or total coronary arterial blockage that results in a shortage of oxygen and nutrients supply to a given area of the myocardium [5]. Here, a fast readjustment of intercellular communication and proteostasis is required to permit cardiac cells to cope with these adverse conditions. Despite the remarkable scientific and clinical advances in the last few years, long-term cumulative structural and functional alterations following MI remain the major driver of HF, which is ultimately characterized by a reduced cardiac output and insufficient blood supply to peripheral organs and tissues [6]. In this case, HF, along with AF, caused by defects in the electrical conduction system leading to persisting arrhythmia,

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and some forms of hypertrophic cardiomyopathy, resulting from either sarcomere gene mutations or pressure overload, can be considered progressive cardiac diseases [7, 8]. These chronic conditions typically encompass two consecutive stages: an initial compensated and likely reversible form, followed by a maladaptive and decompensated stage, in which therapeutic interventions are usually unsuccessful [7]. The accumulation of toxic protein aggregates is often a main feature of cardiac diseases associated with proteostasis deregulation; however, the activation of the proteolytic systems can be either protective or detrimental, depending on the stage of the disease [9]. Hence, a correct and fine-tuned regulation of these mechanisms is essential to ensure an effective and successful therapeutic strategy. One of the biological processes that is tightly regulated by protein degradation is intercellular communication. Indeed, different forms of communication, mediated by GJ or EV, can be directly affected by changes in the proteostasis network [10]. For example, given the very unusual short half-life of the GJ protein Cx43, both proteasomal and lysosomal degradation have been shown to play an important role in modulating cellular cross talk [5]. Accordingly, defects in cardiac cell–cell communication, with a direct impact on heart function, have been also associated with the onset and the development of cardiac diseases, including MI, cardiac hypertrophy, and HF [11–14]. However, as with proteostasis, intercellular communication changes can be either a cause or consequence, depending on the cardiac pathology and its stage. Altogether these observations demonstrate that the players involved in the maintenance of cardiac proteostasis and intercellular communication can constitute promising and attractive therapeutic targets to different cardiac diseases [2, 4, 15, 16].

12.1

The Cardiac Proteostasis Network

Overall, proteome homeostasis is primarily achieved by the orchestrated action of players and mechanisms involved in protein synthesis,

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The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication

folding, post-translational processing, and clearance [1, 17]. It is estimated that around 30% of newly synthesized polypeptides never attain their mature folded conformation and reach their final destination. Here, the role of protein quality control (PQC) members, which assist in accurate folding and degradation of misfolded proteins, is vital to ensure a healthy proteome and to preserve cellular fitness [17]. During cell division, asymmetric distribution of damaged proteins and aggregates to one of the daughter cells contributes to restrain proteotoxicity through a dilution effect [18]. Given that cells with low mitotic activity, including cardiomyocytes, cannot benefit from this protective process, they are particularly susceptible to the accumulation of damaged components, ascribing a crucial importance to PQC mechanisms [1]. Recently, a large-scale in vivo protein dynamics dataset was published, revealing that 9.4 cardiac proteins are replaced each 100 days (median half-life of 7.3 days), with more abundant proteins displaying slower turnover rates [19]. Interestingly, global protein degradation in zebrafish hearts was shown to decline in response to stress [20]. Nevertheless, specific protein subsets exhibited decreased turnover, such as proteins involved in glucose metabolism and hypoxic response, suggesting that selective protein degradation sustains the activation of pro-survival mechanisms in the stressed myocardium [20]. PQC mechanisms are mainly ensured by the action of chaperones and of three main proteolytic systems—the ubiquitin-proteasome system (UPS), autophagy, and calpain proteases. Not surprisingly, failure to properly regulate protein degradation has been associated both with cardiac aging and with a wide variety of cardiac pathologies, including HF and hypertrophic cardiomyopathy [2].

12.1.1

Ubiquitin-Proteasome System (UPS)

Protein chaperones, including heat shock proteins (HSPs) and chaperonins, are master regulators of folding, refolding, and stability of nascent

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polypeptides. In striated muscles, including the heart, several members of the subfamily of small HSPs are highly expressed, which has been associated with important functions upon specialized PQC of the sarcomere and resistance to stress [21]. Accordingly, overexpression of αBcrystallin, the major small HSP in cardiomyocytes, has been reported to increase resistance to ischemia/reperfusion (I/R) injury and pressure overload [22, 23]. Consistent with a vital role of chaperones in maintaining the cardiac proteome, mutations in αB-crystallin, heat shock protein beta (HSPB)-8, and the co-chaperone Bcl-2-associated athanogene 3 (BAG3) have been implicated in the development of cardiomyopathies and HF [24–26]. Besides cytosolic proteins, chaperones detect and bind newly synthetized misfolded proteins of the secretory pathway, including plasma membrane proteins, thereby preventing its aggregation and directing proteasomal degradation via endoplasmic reticulum (ER)-associated degradation (ERAD) [27, 28]. The proteasome is a barrel-shaped multiprotein complex, responsible for the degradation of ~90% cellular proteins, which are targeted to the proteasome after covalent attachment of ubiquitin to lysine residues on target proteins, through the sequential action of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3) [29]. E3-ligases are substrate-specific and catalyze the addition of single ubiquitin moieties or ubiquitin chains to target proteins, thus regulating selectivity in UPS degradation [30]. In the heart, several muscle-specific E3 ligases, including atrogin-1 and muscle ring finger-1 (MuRF-1), sustain ubiquitination and proteasomal degradation of long-lived sarcomeric proteins, such as desmin, myosin heavy chain, and cardiac troponin I [31, 32]. Consistent with an important regulatory role of these enzymes upon post-infarction cardiac remodeling, a reduced expression of both atrogin-1 and MuRF-1, with a concomitant increase in troponin I, was found in human heart biopsies from MI patients [33]. Despite being counterintuitive, enhanced proteasomal activity was found in hypertrophied hearts, acting in concert with protein synthesis following pressure overload, contributing to an

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increased ventricular mass [34]. Strikingly, decreased proteasome function was demonstrated to drive further transition into HF [34]. In addition, UPS has been implicated in the regulation of cardiac signaling pathways. For example, calcineurin/nuclear factor of activated T cell (NFAT)-dependent signaling can be regulated by atrogin-1, which ubiquitinates and targets proteasomal degradation of calcineurin, ultimately inhibiting stress-induced cardiomyocyte hypertrophy [35]. Accordingly, it was found that UPS impairment results in enhanced calcineurin/ NFAT activation in a mouse model of desminrelated cardiomyopathy (DRC), thus promoting maladaptive remodeling in diseased hearts [36]. Remarkably, DRC can be caused not only by mutations in desmin but also in αB-crystallin, resulting in a major accumulation of intracellular ubiquitinated aggregates that exacerbates UPS dysfunction [37]. In these conditions, it has been proposed that clearance of large inclusion bodies is ensured by autophagy. Here, the co-chaperone/ E3 ligase C-terminal of HSP70-interacting protein (CHIP), heat shock protein 70 (HSP70), and members of the BAG family were reported to cooperatively act upon aggresome formation and further interaction with autophagy receptors for its degradation, acting as important modulators of the cross talk between different degradation pathways [2, 38]. Although BAG-1 has been classically described as an adaptor for UPS degradation of CHIP-ubiquitinated substrates, more recent data have shown that, similarly to BAG-3, BAG-1 interacts with the autophagy machinery, playing an important role in autophagy-mediated cardioprotection during ischemic preconditioning [39, 40]. Furthermore, infarct size and arrhythmogenesis were increased in CHIPdeficient mice, reinforcing its important role upon coordination of stress responses [41].

12.1.2

Autophagy

Autophagy is a term used to collectively define the mechanisms of lysosomal-dependent degradation of cellular components, classically via three main pathways—macroautophagy,

microautophagy, and chaperone-mediated autophagy (CMA) [42]. In the following sections, we will discuss the implications of the (dys)function of some of these pathways upon cardiac pathophysiology.

12.1.2.1 Macroautophagy Macroautophagy (hereafter referred to as autophagy) is the most well-studied lysosomal catabolic pathway, whereby obsolete long-lived proteins, aggregates, or organelles are engulfed by a double-membrane vesicle, the autophagosome [29]. Autophagy is mainly conducted by the sequential action of autophagy-related proteins (Atgs) that control the different stages of the autophagic process, namely the phagophore formation, expansion, and autophagosome maturation before fusion with the lysosomes [42, 43]. Selectivity in autophagy is mainly conferred by substrate ubiquitination, which drives recognition by specific cargo receptor proteins, such as sequestosome-1 (p62/SQSTM1) and neighbor of BRCA1 gene 1 (NBR1), which function as physical links between ubiquitinated cargo and the autophagosome [44]. In the heart, autophagy has been primarily described as a double-edged sword mechanism. On the one hand, autophagy activation ensures cardiac homeostasis, promoting proteome integrity and cell survival in basal conditions and under energy stress, whereas other studies correlate autophagy upregulation with exacerbated tissue damage [9]. Both the identity of the autophagy players involved and the nature and duration of the stress stimuli have been proposed to underlie the differential roles attributed to autophagy activation [29]. MI is a paradigmatic example of this controversy, with a substantial amount of data showing that autophagy is upregulated in response to ischemia and I/R in vitro and in vivo [9]. Interestingly, hypoxia and glucose deprivation during ischemia were found to induce activation of AMP-activated protein kinase (AMPK), which sustains autophagy activation and energy homeostasis [45, 46]. Conversely, Beclin-1-mediated autophagy during the reperfusion phase was related to larger infarct sizes and cardiomyocyte

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The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication

apoptosis [45]. In line with these results, degradation of the GJ protein Cx43 by autophagy was differentially modulated by AMPK and Beclin-1 during ischemia and I/R, respectively, suggesting that Cx43 degradation during ischemia restrains cell injury propagation from the affected area to the healthy tissue, whereas during reperfusion, exacerbated degradation of GJ reflects uncontrolled autophagy activation, contributing to cell death [5, 47]. During reperfusion, it was also demonstrated that autophagosome clearance is impaired, likely due to a downregulation of lysosome-associated membrane protein2 (Lamp2) that is critical for autophagosomelysosome fusion [48]. Here, the observed phenotype closely resembled the clinical features of Danon disease, an X-linked condition caused by loss-of-function mutations in the gene encoding for Lamp2 that results in hypertrophic cardiomyopathy in humans [49]. Besides obsolete proteins and aggregates, autophagy mediates selective degradation of organelles, namely mitochondria, by mitophagy, which assumes particular importance in the setting of myocardial ischemia, where it governs selective degradation of damaged mitochondria [50]. Recently, a Rab9-dependent mitophagy mechanism was uncovered in cardiomyocytes, independently of canonical autophagy activation [51]. Importantly, in a mouse model of DRC, overexpression of Atg7 reduced the accumulation of proteotoxic aggregates and ameliorated cardiac dysfunction and hypertrophy, thereby increasing overall survival in these animals [52]. In basal conditions, cardiac-specific loss of Atg5 resulted in marked structural alterations, accumulation of ubiquitinated proteins, and cardiac hypertrophy in mice [53]. Strikingly, pressure overload in Atg5depleted animals exacerbates contractile dysfunction, implying autophagy as an important modulator of the cardiac adaptive response to hemodynamic stress [53]. Nonetheless, downregulation of Beclin-1 mitigated pathological remodeling in a mouse model of cardiac hypertrophy, reinforcing the player- and context-specific role of autophagy activation under stress conditions [54]. It is important to notice that besides

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autophagosome formation, Beclin-1 is required for autophagosome–lysosome fusion and clearance [42, 43]. Therefore, it is conceivable that, under particular circumstances, Beclin-1 inhibition restores autophagosome processing, thus alleviating proteotoxicity and preventing progression to HF [54]. In the context of AF, autophagy has been mainly associated with enhanced electrical remodeling and cardiomyocyte damage [55]. Accordingly, increased expression of Atg7 and p62, as well as markers of ER stress, was found in AF patients, where it has been correlated with an impaired proteostasis and structural remodeling [55, 56]. Furthermore, it was demonstrated that ubiquitindependent degradation of the L-type calcium channel (Cav1.2) by autophagy contributed to the development of AF in animal models [55, 56].

12.1.2.2 Autophagy Mediated by Chaperones CMA participates in selective degradation of individual proteins containing a pentapeptide motif (KFERQ-like) that are recognized by heat shock cognate 70 (Hsc70) and targeted to the lysosome via the Lamp2a receptor [57]. Although CMA activation has been described in hearts from fasted animals, more compelling data are required in order to confirm the importance of CMA-mediated degradation upon the pathophysiology of heart diseases [58]. In fact, despite cardiac ryanodine receptor 2 (RyR2) has been identified as a CMA substrate, depletion of Lamp2a did not prevent RyR2 degradation during I/R, implying that other pathways are involved in these specific conditions [59]. In skeletal muscle, a different type of autophagy, named chaperone-assisted selective autophagy (CASA), involving the action of multiple chaperones, such as CHIP and BAG-3, was implied in tension-induced degradation of the Z-disc protein filamin, thus contributing to preserve muscle function [60]. Although the role of CASA in the heart has not yet been elucidated, downregulation of BAG-3 was reported in end-stage failing human hearts, and BAG-3 mutations were identified in patients with idiopathic dilated cardiomyopathy, suggesting a role

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for BAG-3-mediated proteolysis during cardiac stress [61].

and mitigated apoptotic cell death in atrial pacing models [67].

12.1.3

12.1.4

Calpain Proteases

The calpain system comprises a family of calcium-dependent proteases, localized in the muscular Z-discs, whose function is tightly regulated by its endogenous inhibitor calpastatin [62]. In contrast to other proteolytic systems, calpains perform proteolytic processing, but not degradation, of specific substrates [63]. In the context of various heart disorders, multiple cytoskeletal and myofibrillar proteins have been described as substrates of calpains, ultimately impacting sarcomeric structure and cardiac function [64]. In basal conditions, it was demonstrated that calpain-1 could be activated by physiological calcium concentrations, which resulted in fragmentation of desmin and protein kinase Cα (PKCα) and facilitated ubiquitination of cardiac proteins for further proteasomal degradation [65]. Importantly, calpain activity inhibition resulted in the accumulation of misfolded proteins and autophagosomes in the heart, ultimately resulting in a dilated cardiomyopathy phenotype [65]. During I/R, the increase in intracellular calcium levels induces calpain activation, which has been implicated in reperfusion injury, thus posing calpains as important therapeutic targets for myocardial infarction [64]. Furthermore, calpain inhibition in murine failing hearts prevented cleavage of junctophilin-2, preserving the ultrastructural integrity of T-tubules [66]. Besides, calpain activity was enhanced in response to stress hormones and stretch, resulting in activation of inducible nuclear factor-κB (NF-κB)-mediated gene transcription, thereby participating in the pathophysiology of cardiac hypertrophy, likely via calpain-dependent cleavage of the cytosolic NF-κB inhibitor [62]. Hyperactivation of calpains was also reported in AF patients, likely due to abnormal calcium handling [67]. Consistent with a detrimental role of calpains in the development of AF, calpain inhibition in vitro rescued Cav1.2 degradation

Proteolysis Targets in Cardiovascular Diseases

The huge number of molecular players involved in proteolysis and in PQC in the heart and their subsequent downstream effectors offer a large and attractive set of possible targets. Several studies have tried to identify pharmacological approaches targeting proteostasis players that can be beneficial for cardiac diseases [2]. For example, Sildenafil, a drug commonly used for the treatment of HF and pulmonary hypertension, has been shown to prevent cardiac remodeling in DRC, by cyclic GMP-dependent protein kinase activation, through a mechanism that is mediated by UPS enhancement [68]. Nevertheless, it was also reported that proteasome inhibition prevents ventricular hypertrophy in a mouse model of HF by chronic administration of isoprenaline [69]. These apparent contradictory effects could be explained by differences in the expression and activity of other proteins involved in UPS-mediated degradation in each disease model. Similarly, activation of autophagy can also be either beneficial or detrimental, depending on the extent and severity of proteotoxicity and pathological condition. Accordingly, autophagy is beneficial in the context of DRC; however, it promotes the development of pressure overloadinduced HF in aortic-banding mice [2]. Rapamycin, an autophagy inducer via mammalian target of rapamycin (mTOR) inhibition, or AMPK activators, such as metformin, were shown to improve cardiac function, reverse cardiac hypertrophy, and protect against MI-induced HF [70, 71]. Currently, some pharmacological drugs targeting PQC players are being tested in clinical trials. Geranyl-geranylacetone (GGA), known to induce HSP expression, has shown to promote the refolding and/or degradation of damaged proteins and, as a result, alleviated proteotoxicity in cardiomyocytes [72]. Moreover, in a mouse model of DRC, this compound was able to

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hamper the progression of HF, through the upregulation of HSPB1 and HSPB8 expression. By reducing aggregate formation, GGA also reduced fibrosis with a concomitant improvement of heart function [72, 73]. The chemical chaperone 4-phenylbutyrate (4PBA), already available for the treatment of urea cycle disorders, is another promising molecule to attenuate proteotoxicity in cardiomyocytes. In fact, 4PBA is an inhibitor of ER stress and promotes the refolding of damaged and misfolded proteins, being currently tested in clinical trials for other misfolded protein-associated diseases including Parkinson and Huntington’s [56, 74]. As described before, a myriad of elements involved in the proteostasis network that failed to accomplish their mission could represent valid therapeutic targets, limiting proteotoxicity in cardiac cells and consequently the development of heart diseases. For example, improving PQC by inducing proteosomal or autophagic degradation has been found to delay the onset of cardiomyopathy in mice with the CRYABR120G mutation, an animal model that recapitulates some of the cardinal features of human DRC. Moreover, in most patients with hypertrophic cardiomyopathy, a significant accumulation of autolysosomes and autophagosomes is observed, suggesting excessive degradation of sarcomeric proteins. In these patients, the modulation of PQC system, namely by inhibiting amyloid oligomer formation, might also be beneficial [52, 75]. In agreement, it was demonstrated that overexpression of Atg7 decreased amyloid oligomer accumulation in cardiomyocytes from CRYABR120G mice, thus improving cardiac performance [52, 76]. Another cardiac pathology that is associated with derailment of proteostasis is AF, characterized by contractile dysfunction due to remodeling of atrial cardiomyocytes and degradation of the sarcomeric cytoskeleton [8]. Reduction of chaperones, increased proteolytic processes via calpains, and ER stress-induced autophagy are some of the proteostasis-related alterations observed [77]. It has been shown that exacerbated histone deacetylate 6 (HDAC6) activation leads to microtubule network disintegration and consequent calpain-mediated

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degradation. Accordingly, the HDAC6 inhibitor ricolinostat (ACY1215), prescribed for myeloma treatment, could also constitute a promising therapeutic option in AF [78]. Similar to AF, it has been reported that cardiomyocytes from patients with HF associated with MI or valvular disease have enhanced autophagic degradation activity, which can result in an excessive degradation of the sarcomeres, contractile dysfunction, and disease progression [79, 80]. Furthermore, patients with risk factors for HF and arrhythmia, namely obesity or type 2 diabetes, displayed both altered chaperone expression and enhanced autophagic flux, which can contribute to derailed PQC system at the onset of these heart diseases [80]. Besides all the potential of these pharmacological compounds as promising therapeutic options in different cardiac diseases, exercise and caloric restriction have also been shown to provide cardiac beneficial effects by improving PQC and delaying the development of cardiac diseases [2].

12.2

Intercellular Communication in the Heart

The heart is formed by different types of cells, including cardiomyocytes, the contractile unit of the heart, endothelial and smooth muscle cells that form the blood vessels that ensure cardiac blood supply, fibroblasts that produce extracellular matrix and confer support, and immune cells [81]. Expectedly, well-balanced intercellular communication in the heart is essential to support both electrical and metabolic coupling. This communication can be direct, between adjacent cells, by GJ, or at longer distances via extracellular vesicles (EV) [82]. More recently, a novel type of intercellular communication has been described between near cells, through thin structures called TNT [83].

12.2.1

Gap Junctions

GJ are specialized cell–cell contacts formed by membrane proteins called Cx, which provide direct intercellular communication between

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eukaryotic cells, allowing the cytoplasmic exchange of small metabolites, ions, second messengers, linear peptides, or small RNA molecules [82]. Communication mediated by low resistance GJ channels is of particular importance in the heart, where these channels localize preferentially in discrete regions of cardiomyocyte– cardiomyocyte coupling, called intercalated discs. This specific localization allows the rapid and anisotropic propagation of the electrical impulse generated in the cardiomyocytes localized at the sinoatrial and atrioventricular nodes, throughout all the heart muscle cells, ensuring its coordinated and synchronized contractility [5]. GJ channels are formed by the docking of two Cx-containing hexameric structures—hemichannels—provided by each of the two adjacent cells [84]. Of the 21 different Cx genes encoded by the human genome, three main isoforms are expressed in the adult heart: Cx40, Cx43, and Cx45 [85]. Of those, Cx43 is the most abundant, being expressed mainly in ventricular cardiomyocytes [86]. Several studies demonstrated that GJ-mediated intercellular communication (GJIC) can be regulated at different levels including channel synthesis, gating, and degradation. Not surprisingly, disturbances in these processes can compromise intercellular communication, thereby contributing to cardiovascular diseases, including MI, HF, hypertrophic cardiomyopathy, or AF [84]. At the onset of myocardial ischemia, which is associated with an increase in cytosolic calcium and proton concentration, reduced ATP concentration, and accumulation of amphipathic lipid metabolites, there are marked changes in protein kinases and phosphatases activity that ultimately result in alterations in Cx43 phosphorylation status and GJ closure [86]. Besides the impact on gating of the GJ channels, rapid and reversible phosphorylation/dephosphorylation events modulate various stages in Cx43 life cycle, namely its stabilization at the plasma membrane. For example, ischemia-induced phosphorylation of Cx43 at Ser373 affects the interaction of Cx43 with zonula occludens-1 (ZO-1) and 14-3-3 proteins, upstream of phosphorylation at Ser368 and Ser255, the latter being crucial to Cx43 internalization and cell–cell communication regulation

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[87, 88]. Moreover, dephosphorylation of Ser365 (the GJIC “gatekeeper”) was described to precede Ser368 phosphorylation upon ischemia. In addition to phosphorylation, ischemiainduced ubiquitination of Cx43 has been shown to regulate internalization and further degradation of Cx43 by autophagy in cardiomyocytes, which directly associates with a decreased intercellular communication [5, 47]. Interestingly, degradation-dependent regulation of intercellular communication can occur at the level of matrix metalloproteinases (MMPs). Indeed, it has been reported that hypoxia/ischemia-induced degradation of Cx43 can be mediated by MMPs, namely MMP-7 and MMP-9, with a deleterious impact on electrical conduction and overall survival in post-MI [89, 90]. Ischemia can also result in the accumulation of Cx43 at the lateral membranes of cardiomyocytes, where it has been associated with a decrease in electrical coupling, likely contributing to arrhythmogenesis, a common complication of myocardial ischemia and infarction in humans [91, 92]. Importantly, defects in Cx43 localization have been shown to impact the distribution and trafficking of other cardiac proteins. In agreement, downregulation of Cx43 reduced the surface expression of voltage-gated sodium channel (Nav1.5) and the cardiac sodium current, which contributes to arrhythmia susceptibility [93]. Mounting evidence indicates that the role of GJ channels goes beyond electrical coupling. In fact, it has been proposed that GJ mediate metabolic coupling between cardiac cells, leading to the propagation of cellular damage signals from the ischemic area to distant cells [82]. Furthermore, unopposed Cx43 channels, i.e., hemichannels, which were initially viewed as non-functional intermediate forms of GJ, have been described as pathologically relevant, opening in response to increased intracellular calcium, pH imbalance, oxidative stress, or membrane depolarization during ischemia, leading to the release of important metabolites, such as ATP, and loss of ionic gradients, calcium overload, cell swelling, and ultimately irreversible cellular damage [84, 94].

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The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication

Similarly to what has been described in ischemia, Cx43 expression is reduced in diverse animal models of HF. It was reported that overexpression of Cx43 in cardiomyocytes of an arrhythmogenic HF model improved not only cell–cell coupling, but also cardiac function, demonstrating the association between Cx43 degradation and the development of arrhythmia and HF [85, 91, 92]. Accordingly, besides alterations on the total levels of this protein, analysis of hearts from patients with HF showed a mislocalization of Cx43 to the lateral membrane of myocytes [85, 95, 96]. Additionally, inhibition of the UPS, as well as MMP-9 activity, was described to prevent downregulation of Cx43 in a rat model of HF and to hamper the progress of the disease [97– 99]. It is important to acknowledge that UPS function is vital in the context of ERAD, ensuring that only properly folded membrane proteins are trafficked to the plasma membrane. However, if this quality control mechanism is disrupted, misfolded proteins can reach the plasma membrane. In agreement, despite it was demonstrated that inhibition of ERAD results in the stabilization of Cx43, it is conceivable that the impact of disease-associated impairment of ERAD upon Cx43 levels and distribution has important consequences regarding intercellular communication [100, 101]. Several studies demonstrate that cardiac hypertrophy is associated with alterations in the levels and/or distribution of Cx43, with impact on the number and size of GJ. As with many other cardiac pathologies, a redistribution of Cx43 from the intercalated discs to the cardiomyocyte lateral membranes has been observed in hypertrophic cardiomyopathy, affecting GJIC and electrical conduction. However, it remains to be defined whether a Cx43 decrease represents a cause or a consequence of disease [102, 103]. Furthermore, the relationship between Cx43 degradation and hypertrophy appears to change with the stage and severity of the disease. In general, in an initial and compensated stage of hypertrophy, Cx43 is found at the lateral membrane of cardiomyocytes, with no detectable changes in Cx43 mRNA or protein levels [79, 102, 104]. With the progress of the

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disease into a maladaptive and decompensated stage, leading to HF, it is observed not only a mislocalization of Cx43 but also a downregulation of Cx43 mRNA and protein and an upregulation of Cx45 [105–107]. It has been suggested that the formation of heterotypic GJ channels of Cx43 and Cx45, characterized by a lower conductance than homotypic Cx43 channels, contributes to the rise of ventricular arrhythmias [107]. Cardiac hypertrophy has also been associated with the activity of WW domain-containing E3 ubiquitin ligase 1 (WWP1), a ubiquitin ligase whose expression is enhanced in age-associated disorders. In a mouse model of WWP1 overexpression, which develops spontaneous left ventricular hypertrophy, it was suggested that the increased ubiquitination and degradation of Cx43 underlie the incidence of lethal arrhythmias [108]. Furthermore, a substantial amount of data highlights the importance of Cxs in the development of AF. In agreement, the two most abundant atrial Cxs, Cx40 and Cx43, have been described to mislocalize to the lateral cardiomyocyte membranes at the onset of AF [109]. Although the association between Cx levels and AF is not entirely consensual, even with the same experimental models, most studies report a decrease in Cx40/Cx43 ratio rather than significant changes in the total levels of Cx43, which likely impacts intercellular communication [110–112]. Moreover, several mutations and genetic variants, such as single nucleotide polymorphisms (SNPs) in the gene encoding for Cx40, have been related to impaired trafficking, inability to form GJ, abnormal channel function, or increased degradation that might contribute to the pathophysiology of AF [113, 114]. Gene mutations and SNPs in the Cx43-encoding gene were also described and correlated with impaired intracellular trafficking of the protein, likely explaining the predisposition for the development of AF [115, 116].

12.2.2

Extracellular Vesicles

Another form of intercellular communication is through EV. Even though initially considered as vehicles of unwanted cellular content, EV are

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crucial players in conveying information between cells, tissues, and organs, vital to ensure organism homeostasis [82]. In a general definition, EV are lipid bilayer-delimited particles naturally released from cells [117]. According to their subcellular origin, EV can be further classified into three main subtypes: (1) exosomes, which originate from the fusion of multivesicular bodies (MVBs) with the plasma membrane; (2) ectosomes, also denominated microparticles or microvesicles, which result from the outward budding of the plasma membrane; and (3) apoptotic bodies, by-products of cell apoptosis [117]. Due to the lack of consensus on isolation techniques and specific markers to clearly discriminate each subtype of vesicles, the use of the term EV is recommended [118]. Potentially produced by every cell type and present in almost every biological fluid, EV support intercellular communication at short distances, within the same organ, or travel and elicit a biological response in a receptor cell at distant tissues or organs. The EV-elicited biological response can be mediated not only through the way EV interact with target cells, but also by the transfer and processing of intraluminal EV content within recipient cells, i.e., cytoplasmic and membrane-associated proteins, sugars, lipids, infectious particles, and nucleic acids [119]. In fact, different models have been used to describe the interaction of EV with acceptor cells: (1) docking through specific interactions between exosomal surface proteins and plasma membrane receptors, triggering signaling cascades; (2) internalization of EV followed by intracellular release of the vesicle content; (3) fusion of the EV with the plasma membrane and cargo release directly into the cytosol; and (4) coupling of Cx43-containing channels localized both at the cell and EV surface mediating the vesicle unloading into the target cells [117, 120]. Since the amount and composition of EV can vary according to the cell status or microenvironment at the time of biogenesis, EV have been broadly explored as potential diagnostic and prognostic tools, pharmacological targets, or therapeutic vehicles [12, 121, 122]. One of the areas where EV have deserved more attention is in the context of cardiac

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ischemia, with several studies showing that the involvement of EV in this pathological condition is highly dependent on the type of cells involved. Numerous studies demonstrated that EV have a protective effect after ischemia and I/R injury, modulating different cellular processes and molecular players including cell death and survival, angiogenesis, inflammatory response, fibrosis, and proliferation of the resident stem/ progenitor cells. In fact, it has been reported that EV from different sources, namely induced pluripotent stem cells (iPSCs), cardiosphere-derived cells (CDCs), cardiac progenitor cells (CPCs), mesenchymal stem cells (MSCs), and circulating blood increase cardiomyocyte survival, decrease infarct size, and improve cardiac function [123– 128]. For example, MSC-derived EV were reported to decrease oxidative stress, inhibit inflammation, and activate the insulin-like growth factor 1 (IGF1)-phosphoinositide 3-kinase (PI3K)-Akt pathway reducing cardiomyocyte apoptosis, promoting angiogenesis, and preventing cardiac dysfunction upon myocardial infarction [123, 129]. Additionally, circulating EV were described to deliver protective signals to the myocardium by activating a pro-survival signaling pathway dependent on toll-like receptor 4 (TLR4) and the cardioprotective HSP27 (also known as HSPB1) in cardiomyocytes [128]. As stated above, cardiomyocyte survival and infarct size can be modulated by proteostasis mechanisms, such as autophagy and proteasome-dependent degradation. It has been shown that changes in cellular proteolytic activity can impact vesicle biogenesis and secretion, thus affecting intercellular communication mediated by EV. In agreement, it has been reported that EV can regulate autophagy activity in cardiomyocytes through the activation of mTOR complex 1 or efficient paracrine transfer of miR-30a with a consequent decrease in autophagy-related proteins Beclin-1 and Atg12 and decreased microtubule-associated protein 1A/1B light chain 3B (LC3)-II/LC3-I ratio [125, 130, 131]. Additionally, it was demonstrated that EV carrying functional 20S proteasome were able to reduce misfolded proteins in the heart after I/R injury, thereby

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supporting the emergent concept that EV convey proteostasis players able to mount an efficient protective response in target cells [126]. Furthermore, an efficient and coordinated inflammatory response can determine cardiomyocyte survival and disease outcome post-MI. Strikingly, CDC-derived EV were reported to contain Y RNA fragments that when actively transferred to macrophages are able to induce transcription and secretion of the antiinflammatory interleukin (IL)-10, which is cytoprotective toward oxidatively stressed cardiomyocytes and reduced infarct size following I/R [132]. Moreover, it was recently shown that cardiomyocyte-derived EV modulate the pro-inflammatory profile of macrophages. It was suggested that under basal conditions, EV released by cardiomyocytes are important to maintain macrophages in an “alert state,” vital to underpin immune surveillance of the heart [133]. Furthermore, EV derived from dendritic cells exposed to post-MI environment activate CD4+ T cells, resulting in increased expression of chemokines and the inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF), ultimately improving cardiac function [127]. MI also leads to an increase in local generation of EV, mainly derived from cardiomyocytes and endothelial cells, which promote the release of chemokine ligand (CCL)-2, CCL 7, and IL-6 from cardiac monocytes helping in the regulation of local inflammatory responses [134]. EV are also important to prevent cardiac remodeling during ischemia, which involves fibroblast proliferation and deposition of extracellular matrix (ECM) components in a process called fibrosis. Remarkably, EV have an inhibitory effect on fibrosis-related genes and proteins, including methylated CpG-binding protein 2 (MeCP2) and collagens type I and II mostly due to the transfer of microRNAs (miRs) to fibroblasts [135, 136]. The formation of new vessels from preexisting ones, or angiogenesis, is a stepwise process that includes degradation of the basement membrane and activation of endothelial cells to a proliferative, motile, and invasive phenotype which leads to the new vascular structure

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[137]. Several studies indicate that EV derived from cardiomyocytes, iPSCs, CPCs, or MSCs promote survival, proliferation, and migration of endothelial cells in a ischemia and I/R context, by the transfer of pro-angiogenic microRNAs (miRs) and proteins, or by activating pro-angiogenic pathways [123, 138–143]. In fact, EV derived from ischemic cardiomyocytes demonstrated to be proangiogenic by increasing endothelial cell proliferation and sprouting, as well as the capillary density in the infarct border zone, a mechanism that is partially mediated by the transfer of miR-143 and -222 [143]. Another example is the transfer of Jagged1 via MSCs-derived EV to endothelial cells that increases in vitro and in vivo angiogenesis [140]. Besides its role in the modulation of cellular responses to ischemia, miRs present in EV can be useful for the evaluation of myocardial damage. Example of that is the detection of increased levels of ischemia-responsive miRs (miR-1, miR-133a, and miR-133b) in circulating EV of patients with acute MI lacking elevation of cardiac troponin or serum creatine phosphokinase [144]. In addition, Cx43-encoding gene was described as one of the multiple targets of miR-1, and overexpression of miR-1 was reported to enhance RyR2 activity [145– 149]. Therefore, it is conceivable that decreased Cx43 levels and impaired calcium handling further exacerbate arrhythmogenesis after MI. Several studies demonstrate that EV can also be regulators of cardiac hypertrophy. For example, it was demonstrated that cardiomyocyte hypertrophy induced by EV secreted by cardiac fibroblasts relies on vesicular enrichment in miR passenger strands or star miRs, namely miR-21 [150]. Furthermore, it has been described that activation of renin-angiotensin feedback with consequent increase in the levels of angiotensinII (AngII) induces pathological cardiac hypertrophy. However, the mechanisms that regulate these processes are not fully understood. Data show that AngII stimulates cardiac fibroblasts to release EV, which increase both the production of AngII by target cardiomyocytes and the expression of its receptor leading to cardiac hypertrophy [151]. Besides, PPARγ activation in adipocytes

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was shown to increase expression and secretion of miR-200 in EV that upon delivery to cardiomyocytes promote mTOR activation, triggering subsequent cardiomyocyte hypertrophy [152]. More recently, it was demonstrated that levels of circulating miR-424 are increased in pulmonary hypertension patients, being associated with right ventricular hypertrophy [153]. Additionally, EV secreted by pulmonary arterial endothelial cells in an in vitro model of disease are enriched in this miR and can trigger a disease-associated response on cardiomyocytes [153].

the latter [155, 156]. It has been suggested that TNT-mediated communication plays a cardioprotective role upon cardiomyocyte stress; however, the mechanisms associated remain elusive [154, 158, 160]. The presence of TNT-like structures has also been described in the heart tissue, between myocytes and non-myocyte cells suggests that these structures can be involved in cardiac health and disease [154, 157, 161].

12.2.3

Given the importance of GJIC for proper function of the heart, impairment of this biological process has been often implicated in various cardiac diseases. Therefore, GJIC manipulation or specific modulation of Cxs has been proposed as potential therapeutic options for these disorders [162]. However, a manageable and efficient pharmacological manipulation of GJIC is still lacking, probably due to the number of different variants of heterotypic Cx channels and the amount of players and pathways involved in the regulation of GJIC [85]. Moreover, when designing a therapeutic approach targeting GJ, it is important to discern between electrical and metabolic coupling. For example, it has been demonstrated that strategies preserving GJ-mediated electrical coupling are beneficial to maintain electrical conduction, whereas the metabolic communication can be detrimental, as it can disseminate “dead signals” away from the injured area [163]. Therefore, depending on the particular pathologic situation, the impact of GJIC modulation can be different. Despite some controversy, enhancement of GJIC by using antiarrhythmic peptides is, up to now, the most efficient strategy against different types of arrhythmias. These peptides were shown to increase both GJ-mediated electrical and metabolic coupling and to reduce ischemia and I/R-induced arrhythmias [164, 165]. EV also constitute attractive targets that can be easily isolated and manipulated, thus posing as very powerful tools as therapeutic and diagnostic entities. Indeed, EV can be used to convey

Tunneling Nanotubes

Interestingly, TNT were recently described as a novel type of long-distance communication. TNT are characterized for being long and thin membrane structures, non-attached to the substrate, with a typical diameter of 50–700 nm and a length up to 100 μm, and the presence of F-actin (microtubules were also detected in the ticker TNT). These fragile structures mediate cell–cell communication through the transfer of diverse molecules, such as proteins, signaling mediators, pathogens, and organelles [82]. TNT have been identified in many in vitro systems, including immune, epithelial, neural, fibroblasts, and cardiac cells, and have been associated with numerous biological processes such as immune defense, spread of pathogens, development, transdifferentiation, cancer progression, and resistance to stress [83]. Similar membrane extensions were also shown in vivo, where they have been implicated in cell migration, wound healing, electrical coupling, stem cell differentiation, and embryogenesis [82, 83]. In the cardiovascular system, TNT connections were described to mediate the transfer of mitochondria and the propagation of calcium signals bursting between cardiomyocytes, fibroblasts, cardiomyocytes and fibroblasts, stem cells and endothelial progenitor cells [154– 159]. The communication between cardiomyocytes and stem cells was also suggested to contribute to the differentiation of

12.2.4

Intercellular Communication Targets in Cardiovascular Diseases

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therapeutic molecules to specific target cells. Moreover, their presence in virtually every biological fluid of the body allied to the use of easy and fast isolation techniques renders EV as excellent candidates as biomarkers and promising targets for pharmacological strategies [81, 119, 166]. Several studies have suggested EV as therapeutic options for different cardiac diseases, including ischemic heart disease, atherosclerosis, and hypertrophic cardiomyopathy. It was demonstrated that cardiomyocyte-derived EV can ameliorate heart dysfunction in ischemic heart disease, by decreasing the damage associated with ischemia and I/R injury, limiting myocardial fibrosis, and stimulating angiogenesis [167]. Interestingly, EV have been identified as important players in promoting angiogenesis in different physiological and pathological circumstances. In a cardiac context, it was shown that EV secreted by cardiomyocytes subjected to ischemia have an impact upon endothelial cells, promoting cardiac angiogenesis which can pave the way toward the development of new approaches aiming at promoting the formation of new vessels [143]. It was also shown that EV released by endothelial cells subjected to shear stress are able to reduce atherosclerotic plaque formation in a mouse model of atherosclerosis [168, 169]. The majority of the studies in this field tried to identify the vesicle players responsible for the effect on the target cells, in order to find new biomarkers of diseases and/or molecules with therapeutic value, with miRs being the most studied EV cargo. Moreover, EV can be manipulated and used as therapeutic drug delivery vehicles [170–172]. This can be an efficient approach to increase the efficiency and selectivity of drug delivery, thus limiting the side effects caused by the administration of free drug. In fact, it was found that the presence of Cx43 in EV loaded with doxorubicin has the same therapeutic outcome compared with the administration of free doxorubicin; however, doxorubicin-loaded EV decrease the cardiotoxicity provoked by the drug [173]. Increasing findings highlight the cardioprotective effects of EV, namely in I/R injury [174]. More recently, TNT have emerged as important players for intercellular communication

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implicated in different biological processes associated with both physiologic and pathologic conditions. Since these structures mediate the cell-to-cell transfer of diverse materials, including miRs, proteins, vesicles, organelles, prion proteins, bacteria, and virus between connected cells, they can have opposite roles [83]. On the one hand, they can participate in the spreading of unwanted material, including infectious particles and damaged or obsolete organelles, and on the other hand, they can mediate the transfer of “healthy” organelles and therapeutic drugs. This means that inhibition of the formation of TNT in certain pathologies could be a therapeutic strategy to avoid the spread of infection, whereas in others TNT can be useful to help delivering drugs or functional molecules or organelles [175–177]. In this context, TNT manipulation could be an option for the treatment of mitochondrial and lysosomal-related disorders [178]. Although TNT occur under physiological conditions, studies have shown that stressful environments, such as inflammation, oxidative stress, and toxins, can enhance TNT formation between cells although the mechanisms and biological relevance have not been clarified so far, including in the context of cardiac diseases [14, 83]. However, many aspects of TNT biology remain largely unknown and, therefore, more studies are needed to understand the mechanisms involved in the regulation of TNT-mediated intercellular communication in health and disease in order to develop TNT-based therapeutic strategies [178].

12.3

Concluding Remarks and Future Perspectives

Despite the noteworthy progresses witnessed in the last few years, cardiovascular diseases remain the leading cause of death worldwide. Among the various mechanisms and signaling pathways that have been implicated in the onset and development of these disorders, derailed proteostasis and intercellular communication impairment have been widely and consistently reported. However, in many cases the involvement of these processes depends not only on the type of disease but also

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on its severity or progressive stage. Furthermore, it remains often unclear whether proteostasis and/or intercellular communication deregulation is a cause or consequence of the disorder. For example, activation of autophagy can be cardioprotective in early stages of several diseases, whereas in later maladaptive stages autophagy is commonly detrimental and contributes to worsen the clinical phenotype. Concerning GJ-mediated intercellular communication it can be useful to preserve electrical coupling, to maintain contractile unit, but metabolic coupling is disadvantageous since it can support damage spreading. Long-distance communication, mediated by EVs, can convey either protective or harmful signals, depending on the impact they have on the nature of recipient cells. Therefore, since in either circumstance one strategy does not fit all, a better understanding of the mechanisms whereby proteostasis or cell–cell communication defects contribute to cardiovascular diseases is vital to implement tailored approaches, which will pave the way toward more efficient therapeutic strategies. In conclusion, more mechanistic insights are needed to identify putative druggable targets. Acknowledgments We apologize to all colleagues whose work could not be cited due to space limitations. This work was supported by the European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) [under the projects PAC “NETDIAMOND” POCI-01-0145-FEDER016385, HealthyAging2020 CENTRO-01-0145-FEDER000012-N2323, POCI-01-0145-FEDER-007440, CENTRO-01-0145-FEDER-032179, CENTRO-01-0145FEDER-032414, and FCTUID/NEU/04539/2013 to CNC.IBILI]. DBA was supported by SFRH/BD/115003/ 2016, TMM by PD/BD/106043/2015, TRR by PD/BD/ 52294/2013 from Fundação para a Ciência e a Tecnologia (FCT).

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By the Tips of Your Cilia: Ciliogenesis in the Retina and the Ubiquitin-Proteasome System

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Vasileios Toulis and Gemma Marfany

Abstract

Primary cilia are microtubule-based sensory organelles that are involved in the organization of numerous key signals during development and in differentiated tissue homeostasis. In fact, the formation and resorption of cilia highly depends on the cell cycle phase in replicative cells, and the ubiquitin proteasome pathway (UPS) proteins, such as E3 ligases and deubiquitinating enzymes, promote microtubule assembly and disassembly by regulating the degradation/availability of ciliary regulatory proteins. Also, many differentiated tissues display cilia, and mutations in genes encoding ciliary proteins are associated with several human pathologies, named ciliopathies, which are multi-organ rare diseases. The retina is one of the organs most affected by ciliary gene mutations because photoreceptors are ciliated

cells. Photoreception and phototransduction occur in the outer segment, a highly specialized neurosensory cilium. In this review, we focus on the function of UPS proteins in ciliogenesis and cilia length control in replicative cells and compare it with the scanty data on the identified UPS genes that cause syndromic and non-syndromic inherited retinal disorders. Clearly, further work using animal models and gene-edited mutants of ciliary genes in cells and organoids will widen the landscape of UPS involvement in ciliogenesis and cilia homeostasis. Keywords

Cilia · Ciliogenesis · Ciliopathy · DUBs · Ubiquitin-proteasome system · Photoreceptor

13.1 V. Toulis Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Barcelona, Spain CIBERER, ISCIII, Universitat de Barcelona, Barcelona, Spain G. Marfany (*) Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Barcelona, Spain CIBERER, ISCIII, Universitat de Barcelona, Barcelona, Spain Institut de Biomedicina (IBUB-IRSJD), Universitat de Barcelona, Barcelona, Spain e-mail: [email protected]

The Retina and Photoreceptors

The retina is a multilayer neurosensory tissue that covers the inner surface of the eye. In vertebrates, the retina is formed by at least six different highly specialized neuronal cell types [1]. The lightsensitive photoreceptor cells are responsible for absorption of the light stimuli (capturing photons) and initiation of the phototransduction cascade, which eventually transmits the electric signal to the visual centers in the brain [2]. There are two types of photoreceptors, rods and cones. Cones are responsible for visual acuity and color

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_13

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perception in photopic conditions, while rods are sensitive in dim light and are responsible for scotopic and non-color vision [3]. The proteins that are implicated in photoreception and phototransduction are localized in a specialized photoreceptor compartment, the outer segment (OS), which is a highly specialized sensory cilium that contains ordered stacks of membranous disks. However, the OS lacks the protein synthesis machinery and, thus, all the components of the OS are synthesized in the inner segment (IS) of photoreceptors and transported to the OS through a microtubule ciliary gate, known as the connecting cilium [4]. The tips of photoreceptor OS are physically in contact with the retinal pigment epithelium (RPE), a single layer of pigmented cells, which participates in maintaining the visual cycle [5] as well as in the daily shedding of OS disks by phagocytosis of the photoreceptor tips [6]. Dysregulation by either genetic mutations or external factors of such a regulated morphological and functional organization triggers apoptosis of photoreceptor and related neurons, thus leading to retinal dystrophy and blindness [7].

13.2

Photoreceptor Cilia

Primary cilia are microtubule-based extensions of the apical plasma membrane that act as cell sensors of external cues. Cilia are signaling receptor hubs that modulate developmental signaling, such as sonic hedgehog, Wnt, and platelet-derived growth factor pathways. Cilia are also receptors of external stimuli and transducers of sensory perception and are involved in chemosensation, olfaction, mechanosensation, and photoreception [4]. It is well known that many ciliary proteins are associated with several human rare diseases, named ciliopathies, which include cystic kidney disease and retinal degeneration traits, such as Bardet–Biedl syndrome (BBS), Usher syndrome, Joubert syndrome, Senior-Locken syndrome, or Meckel–Gruber syndrome, among others [8, 9]. Ciliopathies are usually syndromic disorders since many different tissues and organs display ciliated cells, among them the retina, cochlea, and kidney.

The cilium is composed of a long microtubule doublet, called axoneme, surrounded by an external membrane that is continuous with the plasma membrane of the cell. The axoneme is grown directly from the distal end of a mother centriole (or basal body) through a multistep process, named ciliogenesis or cilia formation, which requires microtubule polymerization and intraflagellar transport (IFT) for cilium elongation [10]. IFT is a bidirectional transport of cargo proteins from the base of the cilium to the tip (anterograde transport) and vice versa, from the tip to the base of the cilium (retrograde transport). Different molecular motors facilitate trafficking: for instance, kinesin-II and cytoplasmic dynein 2, respectively involved in anterograde and retrograde transport, associate with specific IFT proteins which are organized into two major complexes for cargo transport. The IFT-B complex is involved in anterograde trafficking whereas IFT-A is predominantly involved in retrograde trafficking [11]. In photoreceptors, the microtubule region that connects the IS, where all the proteins are synthesized, with the membranous disks of the OS, that is, the region between the basal body and the base of the cilium, receives several names, i.e., transition zone, ciliary gate, or connecting cilium. The transition zone (where the axoneme transitions from the triplet to the doublet microtubule conformation) serves at least two functions, as a docking module of the cilium to the membrane as well as a regulated and restrictive gate through which the cargo proteins are transported to and from the OS using the IFT machinery [4].

13.3

DUBs and the UbiquitinProteasome System (UPS)

The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin-proteasome system. Ubiquitination is a post-translational modification that consists in the attachment of ubiquitin (Ub) to a protein substrate [12]. For many proteins, the attachment of ubiquitin and growth of polyubiquitin chains is

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By the Tips of Your Cilia: Ciliogenesis in the Retina and the Ubiquitin-Proteasome System

an obligatory step in their degradation [13]. Ubiquitination is a highly dynamic and reversible reaction where ubiquitin is conjugated by the serial action of specific ubiquitin ligases and cleaved off from substrates by deconjugating proteases, also named deubiquitinating enzymes (DUBs) [13]. Ubiquitin conjugation relies on a hierarchically consecutive activity of E1, E2, and E3 ligases. By far, the largest family is that of E3 ligases, which are subclassified into three main groups according to their mode of ubiquitin ligation [14]. Concerning DUBs, most are cysteine proteases, but according to their structure and catalytic motifs they are also subclassified into six different families [15]. The human genome contains more than 600 hundred E3 ligases and close to 100 DUBs. The world of posttranslational peptide conjugation has also expanded to include other ubiquitin-like peptides (e.g., SUMO, NEDD8, ISG15, and Atg5) [16], all of which are molecular tags that regulate protein fate. Post-translational ubiquitin and ubiquitin-like modifications play an important role during photoreceptor differentiation and ciliogenesis. For instance, the key transcription factor for the determination of the photoreceptor cell fate, Nr2e3, is post-translationally modified via SUMOylation by Pias3, turning it into both a potent repressor of cone-specific gene expression and an activator of rod-specific genes in rods [17]. Moreover, previous studies have analyzed the expression levels and drawn the expression map of genes related to SUMO and Ub pathways in the mouse retina, thus indicating that some of them could be possible regulators of photoreceptor differentiation and/or candidate genes for causing retinal dystrophies (e.g., Cbx4, Tls, Hdac4, Uchl-1, Atxn3, Usp45, Usp53, Usp54) [18, 19].

13.4

UPS and Ciliogenesis in Replicative Cells

In vertebrates, ciliogenesis and cell division are mutually exclusive because the centrioles must be released from the plasma membrane to function in the mitotic apparatus. Therefore, in replicative

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cells, ciliogenesis and cilia resorption are highly regulated processes that depend on the cell cycle phase, the microtubule network organization, cellular proteostasis, and cilia-mediated signaling cues. On the other hand, many cells produce cilia after escaping cell cycle and entering into differentiation. The proteins and signals that regulate ciliogenesis and cilium disassembly in replicative cells might be common to those involved in cilia maintenance in quiescent and differentiated cells, such as photoreceptors, although some partners might be cell type- or organ- specific. Since microtubule polymerization and depolymerization are highly dynamic, any alteration in the equilibrium of these two processes will directly affect cilium formation or resorption. Although the mechanistic details are not yet fully understood, post-translational modifications of centrosomal and ciliary proteins are key to ciliogenesis [20]. Previous studies have identified UPS proteins that localized at the cilium and might regulate cilium formation, for instance, the chaperone VCP (valosin-containing protein) (involved in ubiquitin signaling quality control and positive regulator of misfolded protein degradation), the ubiquitin-activating enzymes UBA1 and UBA6, and the E3 ubiquitin ligases NEDD4L (neural precursor cell-expressed, developmentally downregulated 4-like) and MYCBP2 (MYC-binding protein 2) [21]. Other studies have identified UPS factors as positive or negative regulators of ciliogenesis and cilium length [22, 23]. Indeed, instrumental UPS proteins involved in cell cycle regulation, such as the anaphase-promoting complex (APC), are also regulating cilia assembly/disassembly. APC is recruited to basal bodies in quiescent cells where it promotes the degradation of KIF2A (a microtubule depolymerase), but different subunits of APC may serve different regulatory functions concerning the cilia, and APC’s role is most probably that of a modulator of ciliary microtubule depolymerization depending on the cell phase and requirements [24]. For instance, CUL3 is an E3 ubiquitin ligase that participates in the ubiquitination of many proteins. Interaction of CUL3 with one of its

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substrate adaptors, KCTD17, is required to polyubiquitinate trichoplein, a negative regulator of ciliogenesis, and remove it from mother centrioles. Therefore, CUL3-KCTD17 is a positive regulator of ciliogenesis, since it targets trichoplein, thereby inactivating Aurora A and allowing axoneme extension [25]. Similarly, VHL (von Hippel-Lindau) is a tumor suppressor that enhances primary cilia formation. VHL inactivation induces Aurora kinase A activity, thus causing regression of the primary cilium by promoting histone deacetylase-dependent tubulin depolymerization of the ciliary axoneme [26]. Another example is the E3 ubiquitin ligase MIB1, a component of centriolar satellites that acts as a negative regulator of ciliogenesis by ubiquitinating key ciliogenesis-promoting factors, targeting them for degradation and, as a consequence, suppressing primary cilium formation [23]. Furthermore, another E3 ligase important for the correct cilia formation is UBR5, which ubiquitinates CSPP, a centrosomal protein essential for ciliogenesis [27]. In addition, two other E3 ligases, BBS11/TRIM32 and MARCH7, are localized in the centrosome and ubiquitinate ephrocystin-5 (NPHP5), protein involved in the control of ciliogenesis [28]. Concerning DUBs, depletion of USP21 compromises the reestablishment of the microtubule network after depolymerization and, thus, reduces primary cilium formation [29]. Also, USP14 controls ciliogenesis and cilia elongation through the downregulation of Hedgehog signal transduction, since USP14 inhibition positively affects the Hedgehog pathway [30]. On the other hand, CYLD, a tumor suppressor gene that encodes a deubiquitinating enzyme, causes cylindromatosis and is implicated in various signaling pathways. CYLD shows a specific localization at the centrosomes and the basal bodies, where it promotes ciliogenesis, and mutations in this gene cause cilia formation defects due to impaired basal body migration and docking [31]. Another DUB regulator is USP8, which deubiquitinates and stabilizes trichoplein, thus favoring ciliogenesis and counteracting the previously mentioned CUL3-KCTD17 ubiquitin ligase activity [32].

V. Toulis and G. Marfany

Not only the substrate/interacting partners of each protein but also the localization within the cilium organelle (whether at the basal body, the connecting cilium/transition zone, the axoneme, or the ciliary tip) (Fig. 13.1) and the precise cell cycle phase where the protein is being expressed are relevant for function [24]. The role of regulatory cilium proteins is different whether involved in anchoring the mother centriole to the membrane, microtubule organization and polymerization, ciliary trafficking, or cilium resorption.

13.5

UPS and Retinal Dystrophies

Several ubiquitin and SUMO pathway proteins participate in retinal development and photoreceptor differentiation, and mutations in the corresponding coding genes are causative of inherited retinal dystrophies (IRDs). A comprehensive analysis of the expression of DUB genes has been performed in the adult retina [19] and in fetal retinas of mouse and humans [33]. Moreover, and as already mentioned, many components of the ubiquitin-proteasome system are involved in the control of ciliogenesis and regulate cilium formation in photoreceptor cells, being potential candidates for causing a wide spectrum of ciliopathies as well as other disorders restricted to the retina. Remarkably, the UPS genes involved in the regulation of ciliogenesis in cycling cells have been mostly associated with cancer but not to retinal disorders yet, most probably because mutations in these genes alter centrosome function and microtubule network organization, thus affecting multiple cell types and organs. Some clues on ubiquitin and SUMO pathway genes that particularly participate in retinal development and photoreceptor differentiation have been described in animal models. For instance, fat facets (FAF/USP9X) is a deubiquitinating enzyme that controls cell-to-cell communication and clathrin endocytosis in Drosophila photoreceptors. The FAF/USP9X mutant shows endocytosis dysregulation and ectopic photoreceptor determination and, thus, displays severe

13

By the Tips of Your Cilia: Ciliogenesis in the Retina and the Ubiquitin-Proteasome System

307

Fig. 13.1 Identified UPS proteins that regulate ciliogenesis in replicative and differentiated cells. Schematic representation of the localization of various UPS components into the cilium in replicative versus differentiated cells (e.g., photoreceptors). Many of these proteins display different roles during ciliogenesis and,

thus, show specific localization into the centrioles and/or axoneme of the cilium. In the sensory cilium, only E3 ligases and DUBs with a specific function in the retina are localized in the cilium, in contrast to replicative cells, where proteins show cell cycle-dependent localization reflecting cilia formation/resorption

defects in photoreceptor differentiation [34]. Usp5 deficiency in Drosophila eyes causes impairment in eye development. Loss of Usp5 results in upregulation of Notch signaling and downregulation of RTK (receptor tyrosine kinase) signaling, leading to impaired photoreceptor development [35]. UCH-L1 is a DUB that participates in multiple pathways during eye development in Drosophila. Its overexpression in the eye imaginal disks induces a rough eye phenotype in the adult fly by downregulating the MAPK (mitogen-activated protein kinase) pathway [36]. On the other hand, the knockdown of DUB genes by morpholino injection in zebrafish embryos has identified usp45 [37] and atxn3 (Toulis et al. unpublished data) as causative of moderate to severe eye morphological defects, with defective formation of the retinal structures. Apart from the role of UPS genes’ role in retinal development and photoreceptor

differentiation in animal models, mutations in several genes related to UPS in humans cause retinitis pigmentosa (RP), the most prevalent inherited retinal dystrophy (1:4000 people worldwide), and other inherited retinal disorders. Moreover, dysfunction of proteins of the UPS has been also associated with multifactorial retinal disorders, such as age-related macular degeneration, glaucoma, diabetic retinopathy, and retinal inflammation [38]. Among the UPS genes mutated in retinal disorders in humans, KLHL7 encodes an E3 ubiquitin ligase of BTB-Kelch subfamily widely expressed in the retina, especially in rod photoreceptors. Different mutations in KLHL7 have been associated with a late-onset form of autosomal dominant retinal degeneration that preferentially affects the rod photoreceptors, affecting both rod and cone electrophysiology [39–41]. On the other hand, biallelic mutations

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in this gene cause a much severe recessive phenotype, the Crisponi syndrome (CS)/coldinduced sweating syndrome type 1 (CISS1)-like phenotype, with high neonatal lethality due to a developmental multi-organ disorder including early-onset retinal neurodegeneration [42]. The substrates of KLHL7 have not been determined, but its interaction with CUL3 suggests a direct or indirect proteostasis regulation of many CUL3 substrates related to ciliogenesis. TOPORS stands out as one of the first genes related to UPS identified as a causative of inherited retinal dystrophies. TOPORS is a RING domain-containing E3 ubiquitin and SUMO dual ligase that localizes in the nucleus in speckled loci associated with promyelocytic leukemia bodies. Most notably, TOPORS localizes primarily to the basal bodies of photoreceptor sensory cilium connecting cilium and in the centrosomes and plays an important role in the regulation of primary cilia-dependent photoreceptor development and function, since its knockdown in zebrafish results in defective retinal development photoreceptor outer segment formation [43]. Point mutations, insertions, or deletions in TOPORS have been identified in different families explaining approximately 1% of autosomal dominant RP [44, 45], and it can be considered as a potential ciliopathy gene. However, no relevant function in non-retinal cilia has been reported yet for TOPORS. Even though not directly related to ciliary function, mutations in the PRPF8 gene in heterozygosis have been identified in Spanish families to cause adRP, most probably by haploinsufficiency [46]. PRPF8 is a pre-mRNA splicing factor participating in the dynamic assembly and dissociation of the spliceosome. PRPF8 displays the motifs of JAMM deubiquitinating zinc proteases and is usually grouped within this DUB group, but it is a catalytically inactive protein, since it lacks the residues that bind the metal ion required for activity. In agreement with the retinal degeneration phenotype previously observed in the knockdown of usp45 in zebrafish embryos [37], mutations in USP45 have been also associated with retinal dystrophies in human patients. Whole-exome

V. Toulis and G. Marfany

sequencing (WES) in Chinese families identified biallelic mutations within this gene implicated in the occurrence of Leber congenital amaurosis (LCA), an early and severe form of inherited retinal disorders, thus confirming the importance of USP45 in the maintenance of correct photoreceptor function [47]. The authors suggest a possible relation with ciliogenesis, even though no data are provided to support this hypothesis. Again the localization of these proteins along the cilium organelle is extremely relevant for their function (Fig. 13.1). In the photoreceptor outer segment, which is a highly specialized cilium, intraciliary trafficking of the large amount of phototransduction and structural cargo proteins requires a highly regulated anterograde and retrograde transport. Therefore, a finely tuned control at the ciliary gate in the transition zone is paramount in photoreceptors; any transport disturbance may disrupt the cell homeostasis and, thus, trigger photoreceptor apoptosis. However, no UPS-related genes have been yet reported to regulate this key target for correct photoreceptor function. It is remarkable that, although the retina is an extremely common organ affected in ciliopathies and many UPS proteins regulate ciliogenesis, in humans, mutations in only two UPS genes, which encode E3 ubiquitin ligases: TOPORS, for non-syndromic adRP, and TRIM32 (BBS11), for syndromic recessive BBS [48], have been undeniably associated with both ciliogenesis and retinal defects. Many research groups have addressed efforts in dissecting the relevance of proteins in controlling cell cycle and how their alteration results in cancer, whereas the identification of relevant proteins in neurosensory cilia mostly relies on mutations in human patients of rare diseases, which are clearly limited in number. Since proteomics and genetic analyses have identified more than 100 proteins involved in the formation of functional sensory cilia in the retina, and many of them are potentially controlled by ubiquitin and SUMO post-translational modifications, we hypothesize that the regulation landscape of photoreceptor ciliogenesis is still devoid of key UPS regulatory players (Fig. 13.1). Further work is required to unveil

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By the Tips of Your Cilia: Ciliogenesis in the Retina and the Ubiquitin-Proteasome System

novel E3 ligases and DUBs involved in cilia formation and ciliary trafficking and elucidate their precise function in photoreceptors, but with the application of highly precise gene editing techniques to generate specific mutants in cell and animal models, as well as in human organoids, we foresee a burgeoning field in the study of UPS in the regulation of ciliogenesis and its implications for elucidating the molecular basis of human disease.

13.6

Concluding Remarks

The retina is a complex neuronal tissue that requires a fine regulation at the transcriptional and protein levels. The ubiquitin-proteasome system participates in this regulation and we postulate that post-translational modifications, such as ubiquitination and SUMOylation, are implicated in the determination of photoreceptor cell fate, as well as retina development and ciliogenesis. Several reports have shown that some components of the UPS regulate the correct retinal function, especially in photoreceptors and sensory cilia. We postulate that further work will posit new E3 ligase and DUB genes as excellent candidates for either syndromic ciliopathies or non-syndromic retinal dystrophies. Acknowledgments Work by VT and GM was supported by grants SAF2013-49069-C2-1-R and SAF2016-80937R (Ministerio de Economía y Competitividad/FEDER), 2017 SGR 738 (Generalitat de Catalunya), and La Marató TV3 (Project Marató 201417-30-31-32) to GM. VT is fellow of the MINECO (BES-2014-068639, Spain).

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310 24. Hossain D, Tsang WY (2018) The role of ubiquitination in the regulation of primary cilia assembly and disassembly. Semin Cell Dev Biol 93:145–152 25. Kasahara K, Kawakami Y, Kiyono T et al (2014) Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nat Commun 5:5081 26. Xu J, Li H, Wang B et al (2010) VHL inactivation induces HEF1 and Aurora kinase a. J Am Soc Nephrol 21:2041–2046 27. Shearer RF, Frikstad KM, McKenna J et al (2018) The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia. Mol Biol Cell 29:1542–1554 28. Das A, Qian J, Tsang WY (2017) USP9X counteracts differential ubiquitination of NPHP5 by MARCH7 and BBS11 to regulate ciliogenesis. PLoS Genet 13: e1006791 29. Urbé S, Liu H, Hayes SD et al (2012) Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions. Mol Biol Cell 23:1095–1103 30. Massa F, Tammaro R, Prado MA et al (2019) The deubiquitinating enzyme Usp14 controls ciliogenesis and hedgehog signaling. Hum Mol Genet 28:764–777 31. Eguether T, Ermolaeva MA, Zhao Y et al (2014) The deubiquitinating enzyme CYLD controls apical docking of basal bodies in ciliated epithelial cells. Nat Commun 5:4585 32. Kasahara K, Aoki H, Kiyono T et al (2018) EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase. Nat Commun 9:758 33. Esquerdo-Barragán M, Brooks MJ, Toulis V, Swaroop A, Marfany G (2019) Expression of deubiquitinating enzyme genes in the developing mammal retina. Mol Vis 25:800–813 34. Cadavid AL, Ginzel A, Fischer JA (2000) The function of the Drosophila fat facets deubiquitinating enzyme in limiting photore-ceptor cell number is intimately associated with endocytosis. Development 127:1727–1736 35. Ling X, Huang Q, Xu Y et al (2017) The deubiquitinating enzyme Usp5 regulates Notch and RTK signaling during Drosophila eye development. FEBS Lett 591:875–888 36. Thao DTP, An PNT, Yamaguchi M, LinhThuoc T (2012) Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye development in Drosophila melanogaster. Cell Tissue Res 348:453–463

V. Toulis and G. Marfany 37. Toulis V, Garanto A, Marfany G (2016) Combining zebrafish and mouse models to test the function of deubiquitinating enzyme (dubs) genes in development: role of USP45 in the retina. Methods Mol Biol 1449:85–101 38. Campello L, Esteve-Rudd J, Cuenca N, Martín-Nieto J (2013) The ubiquitin-proteasome system in retinal health and disease. Mol Neurobiol 47:790–810 39. Friedman JS, Ray JW, Waseem N et al (2009) Mutations in a BTB-Kelch protein, KLHL7, cause autosomal-dominant retinitis pigmentosa. Am J Hum Genet 84:792–800 40. Hugosson T, Friedman JS, Ponjavic V et al (2010) Phenotype associated with mutation in the recently identified autosomal dominant retinitis pigmentosa KLHL7 gene. Arch Ophthalmol 128:772–778 41. Wen Y, Locke KJ, Klein M et al (2011) Phenotypic characterization of 3 families with autosomal dominant retinitis pigmentosa due to mutations in KLHL7. Arch Ophthalmol 129:1475–1482 42. Angius A, Uva P, Buers I et al (2016) Bi-allelic mutations in KLHL7 cause a Crisponi/CISS1-like phenotype associated with early-onset retinitis pigmentosa. Am J Hum Genet 99:236–245 43. Chakarova CF, Khanna H, Shah AZ et al (2011) TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein. Hum Mol Genet 20: 975–987 44. Chakarova CF, Papaioannou MG, Khanna H et al (2007) Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy. Am J Hum Genet 81:1098–1103 45. Bowne SJ, Sullivan LS, Gire AI et al (2008) Mutations in the TOPORS gene cause 1% of autosomal dominant retinitis pigmentosa. Mol Vis 14:922–927 46. Martínez-Gimeno M, Gamundi MJ, Hernan I et al (2003) Mutations in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 in Spanish families with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci 44:2171–2177 47. Yi Z, Ouyang J, Sun W et al (2019) Biallelic mutations in USP45, encoding a deubiquitinating enzyme, are associated with Leber congenital amaurosis. J Med Genet 56:325–331 48. Chiang AP, Beck JS, Yen HJ et al (2006) Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11). Proc Natl Acad Sci U S A 103: 6287–6292

TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders

14

Germana Meroni

Abstract

The TRIM family comprises proteins characterized by the presence of the tripartite motif composed of a RING domain, one or two B-box domains and a coiled-coil region. The TRIM shared domain structure underscores a common biochemical function as E3 ligase within the ubiquitination cascade. The TRIM proteins represent one of the largest E3 ligase families counting in human more than 70 members. These proteins are implicated in a plethora of cellular processes such as apoptosis, cell cycle regulation, muscular physiology, and innate immune response. Consistently, their alteration results in several pathological conditions emphasizing their medical relevance. Here, the genetic and pathogenetic mechanisms of rare disorders directly caused by mutations in TRIM genes will be reviewed. These diseases fall into different pathological areas, from malformation birth defects due to developmental abnormalities, to neurological disorders and progressive teenage neuromuscular disorders. In many instances, TRIM E3 ligases act on several substrates thus exerting pleiotropic activities: the need of unraveling diseasespecific TRIM pathways for a precise targeting G. Meroni (*) Department of Life Sciences, University of Trieste, Trieste, Italy e-mail: [email protected]

therapy avoiding dramatic side effects will be discussed. Keywords

Tripartite motif, TRIM · RING domain · Ubiquitination · E3 ubiquitin ligases · Rare genetic diseases

14.1

Introduction

The Tripartite Motif (TRIM) family constitutes the largest subfamily of RING domain-containing proteins [1]. In addition to an N-terminal RING domain, these proteins share the presence of one or two additional Zn-binding domains named B-box (B-box 1 and B-box 2) and a coiled-coil region, hence the term Tripartite Motif (TRIM) or RBCC as acronym to indicate this family (Fig. 14.1a). C-terminal to the Tripartite Motif, the TRIM family members display different domains or domain compositions, which permit an extra subclassification of the family into at least nine groups (Fig. 14.1a) [2]. Possessing a RING domain, the majority of TRIM family members act as E3 ubiquitin ligases within the ubiquitination cascade [3, 4]. Ubiquitination is a posttranslational modification that consists in the covalent bond of Ubiquitin moiety(ies) usually on Lysine residues of the specific targets. This process is regulated through the intervention of a cascade of enzymes for: (1) the activation of the ubiquitin peptide

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_14

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(E1 ubiquitin activating enzyme); (2) the conjugation of the activated ubiquitin (E2 ubiquitin conjugating enzyme); and (3) the specific transfer of the properly oriented ubiquitin peptide(s) on the substrate (E3 ubiquitin ligase enzyme) [5]. The topologies and distribution of ubiquitin or ubiquitin chains on the targets via the action of this cascade of enzymes determine the fate of the target itself [6] (Fig. 14.1b). Exerting this function, TRIM family members facilitate/catalyze the proper and correct transfer of the ubiquitin moiety to specific targets. In many instances TRIM-mediated ubiquitination of the substrate is meant for proteasome- or lysosome-mediated degradation but signals for the regulation of substrate activity or distribution can also be conveyed (Fig. 14.1c) [7]. Additionally, E3 ligaseindependent biological roles of TRIM proteins have been proposed, e.g., RNA-binding [8]. As ubiquitination regulates the stability and activity of many, if not all, proteins, it is not surprising that TRIM family members are implicated in a variety of cellular processes, from transcription to apoptosis, from cell cycle regulation to signal transduction [4, 9]. This involvement, often associated with spatial and temporal specific expression, implicates TRIM proteins in many physiological processes and, following their alteration, in several pathological conditions. The pathologies in which TRIM proteins are implicated are manifold and frequently each TRIM family member can have pleiotropic functions. By regulating the stability of oncogenes and tumor suppressors, several TRIM proteins participate in neoplastic processes [10, 11]. Further, the majority of TRIM proteins are implicated in innate immunity pathways either as positive or negative regulators of the cellular response to invading pathogens, whether viruses or bacteria [12–14] and in autoimmune disorders [15]. In addition, Genome-Wide Association Studies (GWAS) highlighted the contribution of polymorphic variants of TRIM genes to the genetic susceptibility to multifactorial disorders. The involvement in the pathologies above is addressed elsewhere; this chapter will focus on the direct implication of TRIM genes in Mendelian rare genetic diseases. Indeed, the possibility

G. Meroni

to exploit genomic massive sequencing of affected and non-affected individuals in families with inherited diseases boosted the identification of mutations in TRIM genes in several forms of genetic disorders. Here below, the state-of-the-art in this field will be summarized.

14.2

Genetic Diseases Caused by Mutations in Class I TRIM Genes

Subclass I TRIM family members are characterized by the presence of both B-box 1 and 2 within the tripartite motif and by a complex C-terminal portion that includes: a COS domain, employed by these proteins to associate with the microtubules [2], followed by a fibronectin type III repeat (FN3) and a PRY-SPRY domain (Fig. 14.1a). The members of this subgroup are mainly expressed during embryonic development and are involved in the definition of the midline in several developing structures. Accordingly, mutations in some of these genes are associated with pediatric pathologies caused by developmental malformations.

14.2.1

TRIM18/MID1 and Opitz G/BBB Syndrome

The first TRIM gene to be associated with a genetic disease was TRIM18, most commonly dubbed MID1 (from here onward, MID1), which is the gene responsible for the X-linked form of Opitz G/BBB Syndrome (XLOS; OMIM 300000). XLOS is a disorder characterized by defects in the development of embryonic midline structures and caused by loss-of-function (LOF) mutations in the MID1 gene [16–18]. The most characteristic clinical features of XLOS are facial anomalies, which include ocular hypertelorism, broad nasal bridge, frontal bossing and cleft lip and/or palate, as well as laryngo-tracheo-esophageal abnormalities and hypospadias. Imperforate anus and congenital heart defects are also present [17]. Neurologically, XLOS patients may also present with developmental delay and intellectual

14

TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders

R

B1 B2

CC

313

C-ter

TRIPARTITE MOTIF RBCC

" Fig. 14.1 TRIM E3 ubiquitin ligases. (a) Domain composition of the tripartite motif proteins and subclassification (class I to IX) of TRIM proteins based on C-terminal domains (right-hand side). Dashed brackets indicate that some of the TRIM members might not display the

included domain. R RING domain, B1 B-box 1 domain, B2 B-box 2 domain, CC Coiled-coil region. (b) Ubiquitination cascade. Ub ubiquitin. (c) Scheme of TRIM E3 ligase activity

disabilities and brain abnormalities, such as cerebellar hypoplasia and agenesis of the corpus callosum. Cerebellar development defects are observed also in the murine Mid1 knockout mouse model [19]. XLOS is a disorder presenting with highly variable expressivity. More than 80 different mutations have been detected in MID1 to date; they are represented by either complete/partial deletions of the gene or

point mutations (missense, nonsense, splice site mutations), the latter especially within the C-terminal region [18]. In cells, MID1 is associated with the microtubular apparatus and missense and truncating mutations, at least those tested experimentally, result in decreased affinity of the protein for the microtubules suggesting that the LOF mechanism apply to the activity exerted on this cytoskeletal structure (Fig. 14.2a)

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G. Meroni

R

B1 B2

CC

R

B1 B2

CC

R

B1 B2

CC

Fig. 14.2 Class I TRIM proteins in genetic diseases. (a) Microtubular localization of GFP-TRIM18/MID1 in Cos-7 cells (left-hand panel); decreased affinity for the microtubule of GFP-TRIM18/MID1 carrying a Cys266Arg XLOS mutation (right-hand panel). Bottom, schematic representation of TRIM18/MID1 domain composition; dashed line indicates the presence of XLOS deletions encompassing the entire gene while full line

indicates the region where point mutations have been detected. (b) Schematic representation of TRIM1/MID2 domain composition and the nature and position of the COS domain missense mutation found in MRX101 patients. (c) Schematic representation of TRIM36 domain composition and the nature and position of the PRY-SPRY missense mutation found in anencephaly patients

[20, 21]. Interestingly, point mutations are distributed along the entire length of the protein with the exception of the RING domain, which is involved only when complete or large deletions/ duplications are concerned. This may suggest that RING mutations, if occurring, can exert a different role with possible implication in different pathological conditions (Fig. 14.2a). Since the identification of MID1 as the gene implicated in XLOS, several biochemical and biological findings have been reported. Despite this, the pathogenesis of the disease is still enigmatic. Biochemically, in vitro ubiquitin E3 ligase activity of MID1 in cooperation with several E2

conjugating enzymes has been described [22, 23]. In biological context, the first reported target of MID1 E3 activity was the catalytic subunit of serine/threonine protein phosphatase 2A (PPP2CA) [24]. MID1 directly interacts, through the B-box 1 domain, with Alpha4 (α4) that is one of the atypical regulatory subunits of PPP2CA driving the latter to ubiquitin-mediated proteasomal degradation [24–27]. Later on, α4 was also reported to be a MID1 substrate [28, 29] and the mechanism of self-regulation of the MID1/α4/PP2Ac complex involves a series of ubiquitination and dephosphorylation events that have been long studied but still remain to be

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TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders

completely unraveled. Whatever the mechanism, the MID1/α4/PP2Ac complex influences PP2Ac stability [30]. Altered PP2Ac activity affects mTORC1 complex formation and signaling [31] and, further, MID1 assembles with factors involved in mRNA transport and protein translation [30, 32]. This pathway can play a significant role in the pathogenesis of XLOS although the underlying mechanisms are still unraveled. Another signaling pathway in which MID1 was reported to be involved is the Sonic Hedgehog (Shh) pathway. MID1 leads, in a direct or indirect way, to the proteolytic cleavage of the kinase Fu [33] and controls the nucleus–cytoplasmic shuttling of the Shh target GLI3 [34]. Although the mechanism of action of MID1 in this pathway is still poorly investigated, the involvement of midline structure definition in embryonic development can be more easily interpreted through alteration of this pathway. Indeed, as demonstrated during very early stage chicken development, Mid1 and Shh have crossrepressive relationship [35] and the two genes in human, MID1 and SHH, when mutated result in opposite phenotypes: an enlargement (XLOS) versus a narrowing (Holoprocencephaly, OMIM 142945) of the midline, respectively. Another mechanism in which MID1 is likely involved is the control of cytokinesis. One of the assessed substrates, BRAF35, has a role during neuronal differentiation [36] and cytokinesis [37, 38] and MID1 is associated with the midbody at the conclusion of the mitosis [20, 39]. MID1dependent BRAF35 ubiquitination might regulate its subcellular localization [40]. MID1 and BRAF35 are both expressed in proliferative compartments during embryonic development and their interaction, either implicated in the repression of neuronal genes or in the regulation of cell division/cell cycle, or both, can be relevant for the pathogenesis of the Opitz syndrome. Recently, MID1 has been found in association with the microtubule-organizing and midbody protein Astrin, consistently MID1 silencing leads to cytokinetic defects [39, 41]. It is possible that MID1 implication in mTORC1 and Shh pathways and in the process of cytokinesis might be different sides of the same

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coin, which warrants further investigation. Interestingly, the implication of MID1 in cytokinesis through the Astrin protein involves principally, the closest TRIM18/MID1 paralogue, TRIM1/ MID2, addressed in the following section.

14.2.2

TRIM1/MID2 and X-Linked Intellectual Disability

TRIM1, also known as MID2 (from here onward, MID2), is the closest TRIM18/MID1 paralogue and is located as MID1 on the X chromosome in human and mouse [42, 43]. The two gene products interact forming a microtubularassociated complex but the precise stoichiometry and dynamics of the heteromeric complex is presently unknown [27]. Despite being in complex with MID1, MID2 has never been found mutated in XLOS patients. However, it is implicated in a form of X-linked recessive intellectual disability (Mental Retardation, X-Linked 101—MRX101, OMIM 300928). Out of 11 affected males of a large Indian family, 6 were genetically evaluated and a missense mutation in MID2 was found to segregate with the pathological condition. The mutation was found in the heterozygous state in carrier females who are unaffected [44]. The onset of the signs occurs at birth and this intellectual pathology is characterized by global developmental delay, impaired cognition, poor or lack of speech, and in some patients the occurrence of seizures. Some affected individuals present with long face and large ears, squint and strabismus, underscoring a broader developmental involvement [44]. The missense mutation segregating in this family, Arg347Gln, involves a very conserved amino acid in the COS domain of MID2 (Fig. 14.2b). This domain is implicated in the association of the protein with the microtubules and, consistently, a mimic of the mutated protein does not present the classical filamentous distribution but is found in cytoplasmic bodies and aggregates [44]. MID2 controls the abovementioned Astrin through direct E3 ligase activity regulating its protein levels on exit from the cell cycle [39]. The Arg347Gln MID2 is still

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able to localize at the midbody, bind and ubiquitinate Astrin, thus depriving of pathogenetic hints in MRX101 [39]. In the pathological conditions described so far, the same cellular consequence of the mutant protein is thus observed for both MID2 and MID1. Opitz syndrome is also characterized by the presence of intellectual disability but whether the onset of this sign in the two conditions is linked to the formation of MID1-MID2 complex function is still to be determined.

14.2.3

TRIM36 and Anencephaly

Another member of the TRIM subgroup I implicated in a birth defect is TRIM36, which maps on chromosome 5q22.3 and is responsible for anencephaly, an extreme form of neural tube defect (OMIM 206500). Anencephaly is characterized by the absence of cranial vault and brain tissues in the fetus and is incompatible with life. Alteration of TRIM36 was detected in a 20-week-old fetus, born to consanguineous parents: a homozygous Pro508Thr missense mutation identified in the PRY-SPRY domain (Fig. 14.2c). The mutation, detected by whole-exome sequencing, segregated with the disorder in the family [45]. Exogenous expression of the mutated form of TRIM36 leads to disrupted microtubules, disorganized spindles, loosely arranged chromosomes, abnormal cytokinesis, decreased cell proliferation, and increased apoptosis compared to the wild-type protein in different cell lines [45]. Similar results were obtained by cellular knockdown of TRIM36 using siRNA suggesting a complex mechanism, involving precise dosage of the wild-type product instead of a classic loss-of-function usually presented by recessive disorders, inducing proliferation defects during neurulation underlying this condition [45]. The function of TRIM36 during development was studied in Xenopus, where the frog orthologue is implicated in cortical rotation and consequently in dorsal axis formation [46]. How this function is related to neural tube closure is it not known; however, the microtubular apparatus is crucial for the morphogenetic movements necessary for neural tube closure and the xMid1/xMid2

complex was also found implicated in neural tube closure in the same species [47, 48]. So far, no data on TRIM36 E3 ubiquitin ligase activity and possible substrates are known. Although the mechanisms underpinning the pathogenesis of the three disorders described above are not defined yet, it is fascinating that members of subclass I are all implicated in developmental disorders, often related to central nervous system and midline structures. The remaining 3 members (TRIM9, 46, and 67) have not been so far associated to any genetic disorders; however, they are all microtubular proteins some of which have roles in midline and CNS development in mouse models and have been associated with cerebellar disorders [49–52]. It may be interesting to evaluate their possible cooperation, likely through direct interaction, in some of these processes.

14.3

Genetic Diseases Caused by Mutations in Class VII TRIM Genes

Class VII TRIM family members are defined by the presence of a C-terminal 6-bladed β-propellerlike structure composed of 6 NHL (NCL-1, HT2A, LIN41) repeats, in some members preceded by a Filamin-type immunoglobulin domain (IGFLMN) (Fig. 14.1a) [1, 43]. Within their tripartite motif, some members present both B-box 1 and B-box 2 whereas some others have retained only B-box 2. Members of this group have been implicated in genetic diseases that are illustrated here below.

14.3.1

TRIM2 and Charcot–Marie– Tooth

Charcot–Marie–Tooth (CMT) disease represents a subgroup of inherited peripheral neuropathies characterized by symptoms typically starting from the feet and progressing in a distal to proximal pattern. CMT patients present with muscle weakness and atrophy in the lower limbs resulting in gait difficulties and can develop skeletal abnormalities (e.g., pes cavus, scoliosis). The

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onset of the clinical signs occurs in the first 2 decades of life with the tendency to start early in childhood and to show a more severe course and complex phenotype in the autosomal recessive forms. Type 2 forms, also known as axonal CMT, contrary to type 1, are characterized by normal or moderately slow nerve conduction velocity [53]. To date, more than 50 genes are implicated in CMT forms. Mutations in TRIM2 on chromosome 4q31.3 have been found in the axonal type CMT type 2R (OMIM 615490), a recessive condition characterized by onset in early childhood. One of the patients, born to consanguineous parents, carries a homozygous Asp667Ala missense variant localized just before the fifth NHL repeat and leading to a change of a highly conserved residue in evolution [54] (Fig. 14.3a). In the other reported

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patient, a compound heterozygous mutation was identified: Glu227Val, within the tripartite coiledcoil domain, and a 1 bp deletion leading to a frameshift and a premature truncation of the TRIM2 protein, Lys567Argfs7 at the NHL domain level [55] (Fig. 14.3a). In the case of the variants detected in the compound heterozygote, the patient’s fibroblasts showed that TRIM2 is highly unstable indicating loss-of-function as pathogenetic mechanisms. The patients with TRIM2 mutations present peripheral axonal neuropathy, muscle weakness and atrophy, slow motor nerve conduction velocities, loss of myelinated fibers, accumulation of neurofilaments within axons, and swollen myelinated fibers. In the more severely affected patient, deceased before 3 years of age, respiratory insufficiency, tracheomalacia, and vocal cord paralysis were also present [54].

CC

Fig. 14.3 Class VII TRIM proteins in genetic diseases. (a) Schematic representation of TRIM2 domain composition with the homozygous mutation found in one CMT2R patient (top) and the compound heterozygous mutations found in another patient (bottom). (b) TRIM32 domain structure and position of some of the point mutations found in LGMD8R patients (top); dashed line indicates

the presence of LGMD8R-associated deletions encompassing the entire gene. The single mutation found in BBS11 patients is shown (bottom). (c) Schematic representation of TRIM71 domain structure and the nature and position of the NHL missense mutation found in Congenital Hydrocephalus patients

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The Trim2 gene trapped (null) knockout mouse line shows gait ataxia and neurodegeneration accompanied by axonal swelling and development of neurofilament aggregates, a phenotype that recapitulates CMT although, differently from human, implicates not only the peripheral but also the central nervous system [56]. This axonopathy is characterized by disorganized intermediate neurofilaments and accumulation of NF-L in axons followed by progressive neurodegeneration [56]. Indeed, NF-L was found to be a substrate of TRIM2 ubiquitin ligase activity explaining its accumulation in Trim2 knockout mouse axons and possibly elucidating the axonal defects in TRIM2-mutated CMT patients [56].

14.3.2

TRIM32 and Limb Girdle Muscular Dystrophy

TRIM32 is an NHL-containing family member mutated in patients with an autosomal recessive form of Limb Girdle Muscular Dystrophy (LGMD Type 8R, formerly known as 2H, OMIM 254110) [57]. The clinical manifestations in LGMD8R range from mild muscular impairment to severe muscle weakness in wheelchair-bound patients. This disorder is heterogeneous; indeed some of the same TRIM32 LGMD8R mutations can be associated with a more severe form of muscular disease, Sarcotubular Myopathy (STM) [57]. Histological analyses of LGMD8R patients’ muscles revealed the presence of rounded fibers with internal nuclei, presence of atrophic and hypertrophic fibers, degenerated myofibrils and Z-lines, presence of enlarged vacuoles [58]. All mutations associated with LGMD8R and STM are either point mutations within the NHL region, leading to missense or premature truncation of the protein, or deletions of large portions of the gene [59] (Fig. 14.3b). Reproduction of either complete LOF (null Trim32) or of an NHL-contained missense mutation (Asp489Asn, corresponding to the Asp487Asn human mutation) in murine models recapitulate the progression of the human disease as described above [60, 61]. Further, these models revealed the presence of disorganized sarcomeres and autophagic double-membrane vacuoles. In

addition, a neurological component with altered neurofilaments was identified. The murine models, assessing the instability of the missense mutation, indicate LOF as a mechanism underlying the LGMD8R. While this is intuitive with large gene deletions that completely abolish TRIM32 activity, it is likely that the NHL-contained point mutations will alter the binding to the target substrates. As for the latter, several TRIM32 E3 ligase activity substrates have been reported to date. Some of them are clearly related to the muscular fiber structure and physiology (actin, desmin, dysbindin) whereas others are important cell cycle regulators (c-Myc, p53) [11, 59]. The two different groups of targets indicate that TRIM32 might be implicated in both muscle atrophy and regeneration, with implication in the biology of satellite cells [62, 63]. Interestingly, a missense mutation in TRIM32 B-box 2 domain (Pro130Ser) has been associated with a form of Bardet–Biedl Syndrome (BBS11; OMIM 209900), a ciliopathy characterized by multi-organ abnormalities clinically diverse from muscular dystrophies [64] (Fig. 14.3b). The role of TRIM32 at the primary cilium is currently unclear but this finding further highlights the pleiotropic role of TRIM32 and of its specific mutations.

14.3.3

TRIM71 and Congenital Hydrocephalus

Congenital Hydrocephalus (CH) is a condition characterized by enlarged brain ventricles, progressive distension of the cerebral ventricular system arising from failed cerebrospinal fluid passage and homeostasis [65]. The genetics underlying this condition is difficult and still unclear. Recently, a large cohort of CH probands was screened for de novo mutations and among the genes mutated in these patients, TRIM71 was found [66]. The patients with TRIM71 mutations exhibited neurodevelopmental delay and epilepsy, and in one case open schizencephalic clefts, in addition to CH. Two heterozygous missense mutations were found in three CH patients: p.Arg608His and p. Arg796His [66]. Interestingly, these variants are

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at homologous positions in different NHL repeats where arginine residues are evolutionary conserved and histidine substitution are predicted to alter folding of the blade of the propeller domain (Fig. 14.3c). The recurrent mutation in two patients and the analogy between the two missense variants detected in heterozygous state suggest that a gain-of-function mechanism underlies the congenital defect in these patients. Indeed, Trim71 null mouse line exhibits a different and worse phenotype, exencephaly and embryonic lethality [67]. Besides the E3 ligase activity [68], TRIM71 also mediates posttranscriptional silencing of mRNAs through direct interactions with UTRs of target genes via the NHL domain and the latter might be hampered in these patients [69]. Interestingly, Trim71 is expressed in the developing neuroepithelium and ventricular zone and in particular in the ciliated neuroepithelium as are the other genes found mutated in CH. This suggests a common physiological/embryological function, the alteration of which leads to Congenital Hydorcephalus. The descriptions above highlighted a common theme among class VII TRIM members: the disease-causing mutations affect mainly the NHL domain. In several instances, this domain is necessary to bind the target substrates of TRIM-NHL E3 ligase activity. However, the NHL domain mediates both protein–protein and protein–RNA interactions. The Drosophila TRIM-NHL member BRAT binds singlestranded RNA and the mammalian members of this group are involved in miRNA binding through the top surface of the NHL β-propeller structure [8]. It will be interesting to further investigate these properties and the relationship between TRIM-NHL-mediated RNA regulation and ubiquitin E3 ligase activity.

14.4

Genetic Diseases Caused by Mutations in Other Classes of TRIM Genes

Class I and VII list several genes associated with genetic disorders, mainly with pediatric and teenage onset, as these subclasses includes genes with

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strong and often specific temporo-spatial expression during development [1]. Additional rare genetic diseases are caused by mutations in TRIM genes from other subgroups and they are briefly described here below.

14.4.1

TRIM8 and Epileptic Encephalopathy

Early-Onset Epileptic Encephalopathy (EOEE) is a heterogeneous group of neurodevelopmental disorders characterized by intractable pharmacoresistant seizures and unfavorable developmental outcome. Many forms are described and variants in several genes have been reported [70]. Up to now, chromosome 10q24.32 located TRIM8 was reported mutated in 6 EOEE diagnosed patients. In addition to epileptic episodes, the patients present with growth delay, dysmorphic facial appearance, and skeletal malformations. They often present with difficulties or complete lack of meaningful words and social smiling. All patients carry de novo truncating mutation within exon 6 of TRIM8. The variants are represented either by insertion or deletion of 1 bp causing frameshifts or by nonsense substitution (Fig. 14.4a) [70–72]. All the variants leave an intact tripartite motif, the stability of which is not investigated, and lack the C-terminal portion that in TRIM8, belonging to class V, does not present any homology with other domains and whose function is not known (Fig. 14.1a) [2, 43]. If stable the truncated TRIM8 product might function in a dominant negative manner thus impacting on the function of wild-type TRIM8. TRIM8 plays divergent roles in many biological processes and signaling pathways. TRIM8 is a nuclear protein that exerts either a tumor suppressor action, playing a prominent role in regulating p53 tumor suppressor activity, or an oncogene function, through the positive regulation of the NF-κB pathway. Moreover, TRIM8 is aberrantly expressed in glioblastoma and its expression suppresses cell growth and induces a significant reduction of clonogenic potential in glioblastoma cell lines [73, 74]. How these

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Fig. 14.4 Other classes of TRIM proteins in genetic diseases. (a) Schematic representation of TRIM8 domain composition and position of some of the mutations found in EOEE patients. (b) TRIM37 domain structure; dashed line indicates the presence of MULIBREY-associated deletions encompassing the entire gene while full line indicates the presence of point mutations along the entire length of the product. (c) Schematic representation of TRIM20 domain structure and the positions of the missense mutations found in FMF patients (continuous lines). The most common mutation is also indicated. (d) TRIM44 domain structure and position and nature of the missense mutation detected in aniridia patients

findings can help in understanding the pathogenesis of EOEE is still unknown but, in this respect, involvement of TRIM8 in the regulation of stemness in neural tissues is intriguing.

14.4.2

TRIM37 and MULIBREY Nanism

MULIBREY (MUscle, LIver, BRain, and EYes) nanism (OMIM 253250) is caused by homozygous or compound heterozygous mutation in the TRIM37 gene, which encodes a peroxisomal and nuclear protein [75, 76]. Consistently with a ubiquitous expression of the TRIM37 gene, this disorder affects several tissues in particular of mesodermal origin. MULIBREY nanism patients present with severe pre- and postnatal growth impairment including occasional progressive cardiomyopathy, characteristic facial features, failure of sexual maturation, insulin resistance with

type 2 diabetes, and an increased risk for tumors. Patients often display short stature and muscular hypotonia, and dysmorphic facial features. The neurological involvement concerns large cerebral ventricles and cisternae but normal intelligence is observed. Hepatomegaly and metabolic alterations are also associated with the disease as well as both male and female infertility [77]. To date, more than 20 TRIM37 mutations have been found associated with this disorder. These mutations are mainly represented by frameshift, splice site alteration and stop codon, all leading to premature truncation of the protein product along its entire length, even proximal to the N-terminus. Intragenic rearrangements and complete gene deletion are also reported, likely promoted by repetitive Alu elements numerous in this genomic region [77]. Most patients are from Finland where a major mutation due to founder effect is the most represented [75]. A Trim37 knockout mouse line

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recapitulates several features of the human disease suggesting a loss-of-function as pathogenetic mechanism [78]. The product of the TRIM37 gene is unique within the TRIM family as it presents the MATH domain C-terminal to the tripartite motif (Fig. 14.4b). This 133-residue-domain is involved in the formation of oligomeric structures and is shared with the TRAF E3 ubiquitin ligases [79]. Interestingly, like TRAF proteins also TRIM37 is involved in innate immunity and antiviral defense. TRIM37 ubiquitin ligase activity was demonstrated against PEX5 and K646 for the regulation of peroxisomal import suggesting MULIBREY as a peroxisomal biogenesis disorder [80]. Recently, TRIM37 has been found implicated in centriole formation through its E3 ligase activity and this can also open novel avenues to understand MULIBREY nanism pathogenesis [81].

and inflammation in the peritoneum, synovium, or pleura. This disorder is prevalent in Arabic, Turkish, Armenian, and Sephardic Jewish populations, hence the name, and the ancestral M694 V mutation account for the majority of the mutated alleles. The pathogenetic variants fall into two clusters, one at the N-terminus and one within the PRY-SPRY domain and depending on whether monoallelic or biallelic mutations are detected they are involved in the dominant or recessive form, respectively [84]. They are mainly missense mutations suggesting that the periodic nature of inflammatory attacks in FMF is consistent with a protein that functions adequately at steady state but decompensates under stress. TRIM20 product, like several innate immunity TRIM family members, senses perturbation in intracellular homeostasis leading to the activation of the inflammasome complex and downstream activation of pro-inflammatory pathways [85, 86].

14.4.3

14.4.3.2 TRIM44 and Aniridia TRIM44 does not present a domain N-terminal to the B-box 1 and its incomplete tripartite motif is composed of B-box 1 and B-box 2, unusually separated by a long stretch of acidic residues, and coiled-coil region (Fig. 14.4d). Heterozygous mutations in the TRIM44 gene have been detected in the autosomal dominant form of Aniridia, AN3 (OMIM 617142). In a 4-generation-family with affected individuals showing decreased progressive visual acuity, bilateral defects of the iris, cataract, and, in some patients, glaucoma, a missense mutation in the TRIM44 gene leading to Gly155Arg substitution segregated with the disease [87]. This mutation has been shown to affect PAX6 expression, a master gene for eye field development and whose mutations are considered the major cause of aniridia [87]. TRIM44 has been shown involved in tumorigenesis through the activation of the AKT/mTOR pathway [88] and molecularly it has been shown to stabilize another TRIM family member, TRIM17, possibly through a USP-like function [89]. How these processes underlie the control of PAX6 in aniridia is at present not known.

RING-less TRIM Family Members and Genetic Diseases

Two rare genetic disorders are caused by mutations in non-orthodox members of the TRIM family in that they do not possess a RING domain, although evolutionarily they are closely related to the classical TRIM proteins [43]

14.4.3.1 TRIM20 and Familial Mediterranean Fever In the case of TRIM20, better known as MEVF or Pyrin, the position of the RING domain is taken by the PAAD or PYRIN domain (Fig. 14.4c). Proteins containing a Pyrin domain are frequently involved in inflammation and innate immunity processes and include intracellular pathogen receptors. Therefore, it is not surprising that mutations in this gene, mainly expressed in blood cells, cause Familial Mediterranean Fever (OMIM 249100, AR; 134,610, AD) [82, 83]. Familial Mediterranean fever (FMF) is an autosomal disorder with onset in childhood or adolescence and characterized by recurrent attacks of fever lasting 24–48 h, which may occur several times per week to once per year,

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Conclusions and Future Perspectives

The number of TRIM family members implicated in rare genetic diseases is growing but unfortunately our knowledge of the pathogenesis is not increasing at the same pace. Even when findings are reported, the mechanisms are not dissected at a level needed to clearly understand the molecular pathogenetic processes involved and to propose a therapeutic approach. Gene therapy, sometimes offered for LOF genetic diseases would not be advisable in many of the cases addressed above due to the lack of the necessary therapeutic window in disorders very often arising during embryonic development. The design of targeting approaches requires more information on the pathogenetic role of genetic disease-TRIM protein and many limitations should be overcome. First, the E3 ligase activity should be correlated more strictly to the pathogenetic process. In many instances, we lack information on the natural substrates and even when targets are known, the topology of the ubiquitin modification and hence the fate of the target and the ubiquitination machinery involved are not assessed. Further complicating matters, for some TRIM proteins, several substrates are reported and in addition to their role in the genetic disorder, they are shown to be involved in other physiological (immunity, miRNA processing) and pathological (cancer, HIV infection) processes. These pleiotropic roles are currently representing an obstacle to the design of specific therapeutic approaches targeting these molecules but avoiding dramatic undesirable effects. In addition, other biochemical functions, associated or not with the E3 ligase activity, might play a role in the disease pathogenesis. In the particular case of TRIM proteins, an issue still to be addressed properly is their homoand hetero-interaction dynamics [90]. Through the formation of different homo- and heterocomplexes, the details of which are completely lacking, these proteins can diversify their cellular and physiological roles. Besides, also the pathogenetic variants can play a role in this dynamics thus impacting differently on the diseases.

In addition, lack of solved TRIM protein structures is a hurdle toward therapy. Despite the importance of TRIM E3 proteins in many diseases, the published structures include a handful of single or tandem domains of TRIM proteins. Structural biology studies, tightly coupled with biochemical analyses, will provide additional information to further dissect TRIM complex stoichiometry, the E3–E2 interactions, and the structural basis for substrate selection in physiological and pathological conditions.

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13. Ozato K, Shin DM, Chang TH, Morse HC III (2008) TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol 8(11):849–860 14. Nisole S, Stoye JP, Saib A (2005) TRIM family proteins: retroviral restriction and antiviral defence. Nat Rev Microbiol 3(10):799–808 15. Jefferies C, Wynne C, Higgs R (2011) Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease? Nat Rev Immunol 11 (9):617–625 16. Quaderi NA, Schweiger S, Gaudenz K, Franco B, Rugarli EI, Berger W et al (1997) Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22. Nat Genet 17:285–291 17. Fontanella B, Russolillo G, Meroni G (2008) MID1 mutations in patients with X-linked Opitz G/BBB syndrome. Hum Mutat 29(5):584–594 18. Li B, Zhou T, Zou Y (2016) Mid1/Mid2 expression in craniofacial development and a literature review of X-linked opitz syndrome. Mol Genet Genomic Med 4(1):95–105 19. Lancioni A, Pizzo M, Fontanella B, Ferrentino R, Napolitano LM, De Leonibus E et al (2010) Lack of Mid1, the mouse ortholog of the Opitz syndrome gene, causes abnormal development of the anterior cerebellar vermis. J Neurosci 30(8):2880–2887 20. Cainarca S, Messali S, Ballabio A, Meroni G (1999) Functional characterization of the Opitz syndrome gene product (midin): evidence for homodimerization and association with microtubules throughout the cell cycle. Hum Mol Genet 8(8):1387–1396 21. Schweiger S, Foerster J, Lehmann T, Suckow V, Muller YA, Walter G et al (1999) The Opitz syndrome gene product, MID1, associates with microtubules. Proc Natl Acad Sci U S A 96(6):2794–2799 22. Han X, Du H, Massiah MA (2011) Detection and characterization of the in vitro E3 ligase activity of the human MID1 protein. J Mol Biol 407(4):505–520 23. Napolitano LM, Jaffray EG, Hay RT, Meroni G (2011) Functional interactions between ubiquitin E2 enzymes and TRIM proteins. Biochem J 434(2):309–319 24. Trockenbacher A, Suckow V, Foerster J, Winter J, Krauss S, Ropers HH et al (2001) MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat Genet 29 (3):287–294 25. LeNoue-Newton M, Watkins GR, Zou P, Germane KL, McCorvey LR, Wadzinski BE et al (2011) The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation. J Biol Chem 286(20):17665–17671 26. Liu J, Prickett TD, Elliott E, Meroni G, Brautigan DL (2001) Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit alpha 4. Proc Natl Acad Sci U S A 98 (12):6650–6655

323 27. Short KM, Hopwood B, Yi Z, Cox TC (2002) MID1 and MID2 homo- and heterodimerise to tether the rapamycin- sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders. BMC Cell Biol 3(1):1 28. Du H, Huang Y, Zaghlula M, Walters E, Cox TC, Massiah MA (2013) The MID1 E3 ligase catalyzes the polyubiquitination of Alpha4 (alpha4), a regulatory subunit of protein phosphatase 2A (PP2A): novel insights into MID1-mediated regulation of PP2A. J Biol Chem 288(29):21341–21350 29. Watkins GR, Wang N, Mazalouskas MD, Gomez RJ, Guthrie CR, Kraemer BC et al (2012) Monoubiquitination promotes calpain cleavage of the protein phosphatase 2A (PP2A) regulatory subunit alpha4, altering PP2A stability and microtubule-associated protein phosphorylation. J Biol Chem 287(29):24207–24215 30. Winter J, Basilicata MF, Stemmler MP, Krauss S (2016) The MID1 protein is a central player during development and in disease. Front Biosci 21:664–682 31. Liu E, Knutzen CA, Krauss S, Schweiger S, Chiang GG (2011) Control of mTORC1 signaling by the Opitz syndrome protein MID1. Proc Natl Acad Sci U S A 108(21):8680–8685 32. Aranda-Orgilles B, Trockenbacher A, Winter J, Aigner J, Kohler A, Jastrzebska E et al (2008) The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex. Hum Genet 123(2):163–176 33. Schweiger S, Dorn S, Fuchs M, Kohler A, Matthes F, Muller EC et al (2014) The E3 ubiquitin ligase MID1 catalyzes ubiquitination and cleavage of Fu. J Biol Chem 289(46):31805–31817 34. Krauss S, Foerster J, Schneider R, Schweiger S (2008) Protein phosphatase 2A and rapamycin regulate the nuclear localization and activity of the transcription factor GLI3. Cancer Res 68(12):4658–4665 35. Granata A, Quaderi NA (2003) The Opitz syndrome gene MID1 is essential for establishing asymmetric gene expression in Hensen’s node. Dev Biol 258 (2):397–405 36. Hakimi MA, Bochar DA, Chenoweth J, Lane WS, Mandel G, Shiekhattar R (2002) A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Proc Natl Acad Sci U S A 99(11):7420–7425 37. Lee M, Daniels MJ, Garnett MJ, Venkitaraman AR (2011) A mitotic function for the high-mobility group protein HMG20b regulated by its interaction with the BRC repeats of the BRCA2 tumor suppressor. Oncogene 30(30):3360–3369 38. Lee M, Venkitaraman AR (2014) A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis. Cell Cycle 13(16):2554–2563 39. Gholkar AA, Senese S, Lo YC, Vides E, Contreras E, Hodara E et al (2016) The X-linked-intellectual-disability-associated ubiquitin ligase Mid2 interacts with

324 astrin and regulates astrin levels to promote cell division. Cell Rep 14(2):180–188 40. Zanchetta ME, Napolitano LMR, Maddalo D, Meroni G (2017) The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2associated factor 35, BRAF35. Biochim Biophys Acta Mol Cell Res 1864(10):1844–1854 41. Zanchetta ME, Meroni G (2019) Emerging roles of the TRIM E3 ubiquitin ligases MID1 and MID2 in cytokinesis. Front Physiol 10:274 42. Buchner G, Montini E, Andolfi G, Quaderi N, Cainarca S, Messali S et al (1999) MID2, a homologue of the Opitz syndrome gene MID1: similarities in subcellular localization and differences in expression during development. Hum Mol Genet 8(8):1397–1407 43. Sardiello M, Cairo S, Fontanella B, Ballabio A, Meroni G (2008) Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties. BMC Evol Biol 8:225 44. Geetha TS, Michealraj KA, Kabra M, Kaur G, Juyal RC, Thelma BK (2014) Targeted deep resequencing identifies MID2 mutation for X-linked intellectual disability with varied disease severity in a large kindred from India. Hum Mutat 35(1):41–44 45. Singh N, Kumble Bhat V, Tiwari A, Kodaganur SG, Tontanahal SJ, Sarda A et al (2017) A homozygous mutation in TRIM36 causes autosomal recessive anencephaly in an Indian family. Hum Mol Genet 26 (6):1104–1114 46. Cuykendall TN, Houston DW (2009) Vegetally localized Xenopus trim36 regulates cortical rotation and dorsal axis formation. Development 136 (18):3057–3065 47. Suzuki M, Hara Y, Takagi C, Yamamoto TS, Ueno N (2010) MID1 and MID2 are required for Xenopus neural tube closure through the regulation of microtubule organization. Development 137(14):2329–2339 48. Suzuki M, Morita H, Ueno N (2012) Molecular mechanisms of cell shape changes that contribute to vertebrate neural tube closure. Develop Growth Differ 54(3):266–276 49. van Beuningen SF, Will L, Harterink M, Chazeau A, van Battum EY, Frias CP et al (2015) TRIM46 controls neuronal polarity and axon specification by driving the formation of parallel microtubule arrays. Neuron 88(6):1208–1226 50. Do LD, Gupton SL, Tanji K, Bastien J, Brugiere S, Coute Y et al (2019) TRIM9 and TRIM67 are new targets in paraneoplastic cerebellar degeneration. Cerebellum 18(2):245–254 51. Boyer NP, Monkiewicz C, Menon S, Moy SS, Gupton SL (2018) Mammalian TRIM67 functions in brain development and behavior. eNeuro 5(3) 52. Menon S, Boyer NP, Winkle CC, McClain LM, Hanlin CC, Pandey D et al (2015) The E3 ubiquitin ligase TRIM9 is a filopodia off switch required for netrin-dependent axon guidance. Dev Cell 35 (6):698–712

G. Meroni 53. Baets J, De Jonghe P, Timmerman V (2014) Recent advances in Charcot-Marie-Tooth disease. Curr Opin Neurol 27(5):532–540 54. Pehlivan D, Coban Akdemir Z, Karaca E, Bayram Y, Jhangiani S, Yildiz EP et al (2015) Exome sequencing reveals homozygous TRIM2 mutation in a patient with early onset CMT and bilateral vocal cord paralysis. Hum Genet 134(6):671–673 55. Ylikallio E, Poyhonen R, Zimon M, De Vriendt E, Hilander T, Paetau A et al (2013) Deficiency of the E3 ubiquitin ligase TRIM2 in early-onset axonal neuropathy. Hum Mol Genet 22(15):2975–2983 56. Balastik M, Ferraguti F, Pires-da Silva A, Lee TH, Alvarez-Bolado G, Lu KP et al (2008) Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration. Proc Natl Acad Sci U S A 105(33):12016–12021 57. Frosk P, Weiler T, Nylen E, Sudha T, Greenberg CR, Morgan K et al (2002) Limb-girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3-ubiquitin-ligase gene. Am J Hum Genet 70(3):663–672 58. Schoser BG, Frosk P, Engel AG, Klutzny U, Lochmuller H, Wrogemann K (2005) Commonality of TRIM32 mutation in causing sarcotubular myopathy and LGMD2H. Ann Neurol 57(4):591–595 59. Lazzari E, Meroni G (2016) TRIM32 ubiquitin E3 ligase, one enzyme for several pathologies: from muscular dystrophy to tumours. Int J Biochem Cell Biol 79:469–477 60. Kudryashova E, Wu J, Havton LA, Spencer MJ (2009) Deficiency of the E3 ubiquitin ligase TRIM32 in mice leads to a myopathy with a neurogenic component. Hum Mol Genet 18(7):1353–1367 61. Kudryashova E, Struyk A, Mokhonova E, Cannon SC, Spencer MJ (2011) The common missense mutation D489N in TRIM32 causing limb girdle muscular dystrophy 2H leads to loss of the mutated protein in knock-in mice resulting in a Trim32-null phenotype. Hum Mol Genet 20(20):3925–3932 62. Cohen S, Zhai B, Gygi SP, Goldberg AL (2012) Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy. J Cell Biol 198(4):575–589 63. Nicklas S, Otto A, Wu X, Miller P, Stelzer S, Wen Y et al (2012) TRIM32 regulates skeletal muscle stem cell differentiation and is necessary for normal adult muscle regeneration. PLoS One 7(1):e30445 64. Chiang AP, Beck JS, Yen HJ, Tayeh MK, Scheetz TE, Swiderski RE et al (2006) Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11). Proc Natl Acad Sci U S A 103(16):6287–6292 65. Tully HM, Dobyns WB (2014) Infantile hydrocephalus: a review of epidemiology, classification and causes. Eur J Med Genet 57(8):359–368 66. Furey CG, Choi J, Jin SC, Zeng X, Timberlake AT, Nelson-Williams C et al (2018) De Novo mutation in

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genes regulating neural stem cell fate in human congenital hydrocephalus. Neuron 99(2):302–314. e4 67. Maller Schulman BR, Liang X, Stahlhut C, DelConte C, Stefani G, Slack FJ (2008) The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure. Cell Cycle 7(24):3935–3942 68. Nguyen DTT, Richter D, Michel G, Mitschka S, Kolanus W, Cuevas E et al (2017) The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation. Cell Death Differ 24(6):1063–1078 69. Aeschimann F, Kumari P, Bartake H, Gaidatzis D, Xu L, Ciosk R et al (2017) LIN41 posttranscriptionally silences mRNAs by two distinct and position-dependent mechanisms. Mol Cell 65 (3):476–489. e4 70. Euro E-RESC, Epilepsy phenome/genome P, Epi KC (2014) De novo mutations in synaptic transmission genes including DNM1 cause epileptic encephalopathies. Am J Hum Genet 95(4):360–370 71. Assoum M, Lines MA, Elpeleg O, Darmency V, Whiting S, Edvardson S et al (2018) Further delineation of the clinical spectrum of de novo TRIM8 truncating mutations. Am J Med Genet A 176 (11):2470–2478 72. Sakai Y, Fukai R, Matsushita Y, Miyake N, Saitsu H, Akamine S et al (2016) De novo truncating mutation of TRIM8 causes early-onset epileptic encephalopathy. Ann Hum Genet 80(4):235–240 73. Venuto S, Castellana S, Monti M, Appolloni I, Fusilli C, Fusco C et al (2019) TRIM8-driven transcriptomic profile of neural stem cells identified glioma-related nodal genes and pathways. Biochim Biophys Acta, Gen Subj 1863(2):491–501 74. Caratozzolo MF, Micale L, Turturo MG, Cornacchia S, Fusco C, Marzano F et al (2012) TRIM8 modulates p53 activity to dictate cell cycle arrest. Cell Cycle 11(3):511–523 75. Avela K, Lipsanen-Nyman M, Idanheimo N, Seemanova E, Rosengren S, Makela TP et al (2000) Gene encoding a new RING-B-box-coiled-coil protein is mutated in mulibrey nanism [In process citation]. Nat Genet 25(3):298–301 76. Kallijarvi J, Avela K, Lipsanen-Nyman M, Ulmanen I, Lehesjoki AE (2002) The TRIM37 gene encodes a peroxisomal RING-B-box-coiled-coil protein: classification of mulibrey nanism as a new peroxisomal disorder. Am J Hum Genet 70(5):1215–1228 77. Brigant B, Metzinger-Le Meuth V, Rochette J, Metzinger L (2018) TRIMming down to TRIM37:

325 relevance to inflammation, cardiovascular disorders, and cancer in MULIBREY nanism. Int J Mol Sci 20(1) 78. Kettunen KM, Karikoski R, Hamalainen RH, Toivonen TT, Antonenkov VD, Kulesskaya N et al (2016) Trim37-deficient mice recapitulate several features of the multi-organ disorder Mulibrey nanism. Biol Open 5(5):584–595 79. Gupta I, Varshney NK, Khan S (2018) Emergence of members of TRAF and DUB of ubiquitin proteasome system in the regulation of hypertrophic cardiomyopathy. Front Genet 9:336 80. Wang W, Xia ZJ, Farre JC, Subramani S (2017) TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import. J Cell Biol 216 (9):2843–2858 81. Balestra FR, Strnad P, Fluckiger I, Gonczy P (2013) Discovering regulators of centriole biogenesis through siRNA-based functional genomics in human cells. Dev Cell 25(6):555–571 82. Consortium TFF (1997) A candidate gene for familial Mediterranean fever. Nat Genet 17(1):25–31 83. Consortium TFI (1997) Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. Cell 90 (4):797–807 84. Westwell-Roper C, Niemietz I, Tucker LB, Brown KL (2019) Periodic fever syndromes: beyond the single gene paradigm. Pediatr Rheumatol Online J 17(1):22 85. Manukyan G, Aminov R (2016) Update on pyrin functions and mechanisms of familial mediterranean fever. Front Microbiol 7:456 86. Sharma D, Malik A, Guy C, Vogel P, Kanneganti TD (2019) TNF/TNFR axis promotes pyrin inflammasome activation and distinctly modulates pyrin inflammasomopathy. J Clin Invest 129(1):150–162 87. Zhang X, Qin G, Chen G, Li T, Gao L, Huang L et al (2015) Variants in TRIM44 cause aniridia by impairing PAX6 expression. Hum Mutat 36 (12):1164–1167 88. Wei CY, Wang L, Zhu MX, Deng XY, Wang DH, Zhang SM et al (2019) TRIM44 activates the AKT/mTOR signal pathway to induce melanoma progression by stabilizing TLR4. J Exp Clin Cancer Res 38(1):137 89. Urano T, Usui T, Takeda S, Ikeda K, Okada A, Ishida Y et al (2009) TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase. Biochem Biophys Res Commun 383(2):263–268 90. Napolitano LM, Meroni G (2012) TRIM family: Pleiotropy and diversification through homomultimer and heteromultimer formation. IUBMB Life 64(1):64–71

Part IV Diet

We Are What We Eat: Ubiquitin– Proteasome System (UPS) Modulation Through Dietary Products

15

Eleni Panagiotidou and Niki Chondrogianni

Abstract

During lifetime, the molecular mechanisms that are responsible for cellular defense against adverse conditions such as oxidative and heat stress tend to be less efficient, thus gradually leading to the natural phenomenon of aging. Aging is linked to increased oxidative stress and is characterized by the accumulation of damaged macromolecules. The accumulation of oxidized and misfolded proteins is also accusable for various neurodegenerative pathologies that are linked to aging. Among self-defense mechanisms of cells, proteostasis network is responsible for the proper biogenesis/folding/ trafficking of proteins and their elimination through proteolysis. The ubiquitin-proteasome system (UPS) is the major proteolytic mechanism that has attracted the interest of many researchers as an antiaging target. Interestingly, many natural compounds have been identified as potent UPS activators. Given that diet is a manageable environmental factor that affects aging, consumption of natural dietary products that may E. Panagiotidou Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece e-mail: [email protected] N. Chondrogianni (*) Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece e-mail: [email protected]

potentially enhance the UPS function, would contribute to increased health span and delayed onset or progression of age-related disorders. Herein, we summarize natural compounds and extracts derived from edible products that have exhibited antiaging and anti-aggregation properties and the beneficial properties have been linked to the UPS modulation. Keywords

Ubiquitin–proteasome system (UPS) · Proteasome modulation · Aging · Age-related diseases · Dietary compounds

Abbreviations Aβ AD ALP APP C-L CP CT-L DUB D3T EVOO GHE HD HGPS

Amyloid β Alzheimer’s disease Autophagy-lysosome pathway Amyloid precursor protein Caspase-like Core proteasome Chymotrypsin-like Deubiquitinating enzymes/ deubiquitinases 3H-1,2dithiole-3-thione Extra virgin olive oil Guarana hydroalcoholic extract Huntington’s disease Hutchinson-Gilford progeria syndrome

# Springer Nature Switzerland AG 2020 R. Barrio et al. (eds.), Proteostasis and Disease, Advances in Experimental Medicine and Biology 1233, https://doi.org/10.1007/978-3-030-38266-7_15

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HSP HTT NHDF Nrf2 PD PDMs PKA PolyQ RP UPS T-L 6-OHDA 18α-GA

15.1

Heat shock protein Huntingtin Normal human dermal fibroblasts Nuclear factor (erythroid-derived 2)-like 2 Parkinson’s disease Polyphenol digested metabolites Protein kinase A Polyglutamine Regulated particle Ubiquitin-proteasome system Trypsin-like 6-hydroxydopamine 18α-glycyrrhetinic acid

Introduction

Protein integrity is vital for the normal function of all cell types. To maintain a healthy proteome, cells have developed a protein quality control system which is crucial for cell metabolism and stress adaptation. The proteostasis network is responsible for: (a) protein synthesis, stabilization, folding mostly by chaperons of the heat shock protein (HSP) family and trafficking, and (b) protein degradation mainly through the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway (ALP) [1, 2]. Molecular chaperons recognize misfolded protein species and if the repair is impossible, they cooperate with the proteolytic systems favoring the degradation of these proteins [3, 4]. Attenuation of proteostasis mechanisms is linked to aging and is considered as one of the hallmarks of aging [5, 6]. Moreover, impaired function of proteostasis has also been reported in various age-related disorders such as Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s (HD) diseases which are characterized by chronic expression of misfolded or aggregated proteins [7]. The average life expectancy has progressively elevated in the last decades. Subsequently, the development of age-related disorders has ensued this rapid increase. Aging and age-related

neurodegeneration have been associated with increased oxidative stress but also with nutrients and their metabolites that may alleviate these toxic processes, playing a pivotal role in the battle against organismal time decline [8]. For example, a plethora of studies gives prominence to the antiaging properties of polyphenolic compounds (phenolic acids, flavonoids, stilbenes, lignans) [9]. Given that these substances mainly contribute to plant protection from adverse environmental conditions through their antioxidant activities, it is possible to be effective as antiaging factors in mammalian cells as well [10]. These compounds can be easily isolated from plants and are included in daily nutritional products like fruits, vegetables, and cereals. Among the proteostasis mechanisms, the UPS system has been extensively investigated for its involvement in aging and age-related disorders [11]. Due to its capacity to degrade prone-to-aggregation proteins and oxidatively damaged proteins, UPS activation by natural or synthetic substances consists a desirable strategy against aging and aggregation-related disorders [12–14]. Herein, we review natural compounds that have been shown to positively affect the UPS and to exert beneficial properties against aging and age-related disorders. The reported substances can be found in edible natural products that we daily consume in the context of a normal nutrition or in dietary supplements.

15.2

The UPS System

The UPS is a key player for the degradation of short-lived, misfolded, and damaged proteins and under normal conditions it is involved in cell cycle progression, apoptosis, and cell proliferation, among others [15]. The proteasome is a large multi-subunit enzymatic complex that is the core of the UPS system catalyzing the ATP/ubiquitindependent and independent proteolysis (Fig. 15.1) [16, 17]. Ubiquitin is a conserved protein that modifies and tags proteins for degradation through the

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We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products

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Fig. 15.1 The UPS system. Misfolded, oxidatively damaged or normally folded proteins are degraded in an ATP/ ubiquitin-dependent or independent manner by the 26S and 20S proteasomes, respectively

ubiquitination process which includes three steps. (i) The E1 ubiquitin-activating enzyme activates the ubiquitin which (ii) is transferred to E2 ubiquitin-conjugating enzyme. Subsequently, the ubiquitin-charged E2 binds to (iii) the E3 ubiquitin-ligase enzyme which catalyzes the final step of ubiquitination. E3 ligase carries the substrate for degradation and thus ubiquitin is transferred to the substrate. This cycle is repeated and results in the poly-ubiquitination of the target protein which in turn (iv) is recognized and degraded by the 26S proteasome followed by the release of the ubiquitin molecules (through the action of deubiquitinases) to be reused [17]. The 26S/30S (referred to 26S proteasome hereafter) proteasome is formed following the capping of the 20S core proteasome (CP) by one (26S) or two (30S) 19S regulated particles (RP) and is mostly responsible for the degradation of

ubiquitinated proteins. The 20S proteasome is composed of 28 subunits (14-α type and 14-β type). The 20S subunits form four rings with seven subunits of the same type per two rings in a barrel-like structure. The two inner rings are formed by β subunits (β1-7) and exert proteolysis through β1 (caspase-like/C-L), β2 (trypsin-like/TL), and β5 (chymotrypsin-like/CT-L) subunits. The two outer rings are formed by α-type subunits and create a gated channel through which the protein substrate reaches the three catalytic centers [18]. The 19S RP consists of two sub-complexes: the lid that consists of nine subunits (Rpn3, Rpn 5-Rpn9, Rpn11, Rpn12, and Rpn15) and the base, which interacts directly with the 20S core and is composed of hexameric AAA-ATPases (Rpt1-6) and tetrameric non-ATP-ases namely Rpn1, Rpn2, Rpn10, and Rpn13. Rpn11 subunit possesses deubiquitination activity while Rpn3 and Rpn10

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Table 15.1 Human genes that encode the 26S/30S and 20S proteasome subunits 20S CP α subunits α6 α2 α7 α3 α5 α1 α4

Gene PSMA1 PSMA2 PSMA3 PSMA4 PSMA5 PSMA6 PSMA7

β subunits β1 β2 β3 β4 β5 β6 β7

Gene PSMB6 PSMB7 PSMB3 PSMB2 PSMB5 PSMB1 PSMB4

serve as ubiquitin receptors [19, 20] (Table 15.1). In the 26S proteasome ubiquitin-ATP-dependent proteolysis, proteins tagged with ubiquitin are recognized by Rpn3 and Rpn10 receptors and deubiquitinating enzymes (DUB) like Rpn11, USP14, and UCH37 remove the ubiquitin tag [21]. Subsequently, the base of the 19S complex unfolds the protein and promotes it to the catalytic centers for degradation [22]. On the other hand, the 20S proteasome has been suggested to promote the degradation of unfolded and oxidized proteins in an ATP-independent manner and does not require poly-ubiquitination of target proteins and 19S-mediated protein unfolding [16] (Fig. 15.1).

15.3

Proteasome Activation

Proteasome activation is feasible through genetic manipulation, post-translational modifications of specific proteasome subunits, or conformational alterations of the 20S core structure [14]. Treatment with specific compounds (including some that are part of our diet) can also modulate the expression and/or the function of the proteasome and they will be further presented below (Fig. 15.2). The transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is the key response factor against oxidative stress that regulates the induction of the expression of various antioxidant enzymes [23]. A cross talk between Nrf2 and the proteasome has been reported and several studies have shown that Nrf2 is able to modulate the 20S and 19S gene expression following oxidative

19S RP Base subunits Rpt1 Rpt2 Rpt3 Rpt4 Rpt5 Rpt6 Rpn1 Rpn2 Rpn10 Rpn13

Gene PSMC2 PSMC1 PSMC4 PSMC6 PSMC3 PSMC5 PSMD2 PSMD1 PSMD4 ADRM1

Lid subunits Rpn3 Rpn5 Rpn6 Rpn7 Rpn8 Rpn9 Rpn11 Rpn12 Rpn15

Gene PSMD3 PSMD12 PSMD11 PSMD6 PSMD7 PSMD13 PSMD14 PSMD8 SEM1

stress or dietary Nrf2 modulation [24, 25]. Apart from Nrf2, Rpn4 is another transcription factor in yeast that has been shown to regulate proteasome biogenesis. Under normal conditions, Rpn4 is short lived and rapidly degraded by the proteasome. However, upon proteasome inhibition following proteotoxic stress, Rpn4 is stabilized to upregulate the expression of proteasome genes. With regard to genetic manipulation of the proteasome, we have previously demonstrated that stable overexpression of the β5 catalytic subunit or the hUPM1/POMP chaperone in human fibroblasts may induce all three proteasomal activities [26, 27], while PSMD11 (RPN-6 in C. elegans) has also been shown to be correlated with increased proteasome assembly and activity [123, 124]. Proteasome activation has also been succeeded through post-translational modifications such as phosphorylation. 26S upregulation has been achieved through cAMP-dependent protein kinase A (PKA) phosphorylation of the Rpt6 proteasome subunit [28]. Moreover, PKA-mediated phosphorylation of Rpn6 has been shown to enhance the degradation of ubiquitinated proteins [29]. Additionally, inhibition of the proteasome deubiquitinases such as USP14 and UCH37 resulted in elevated proteasome activities [30]. Finally, various peptides and compounds have been shown to favor proteasome function through conformational alterations that mainly affect the α-gated channel of the 20S core. Molecules that modulate 20S and/or 26S proteasome function are known as gate-openers or stimulators [14].

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Fig. 15.2 Proteasome activation. Increased proteasome activity or content may occur through genetic manipulation, post-translational modifications, and conformational alterations. Natural or synthetic compounds can modulate

15.4

UPS Modulation in Aging and Neurodegenerative Diseases

Aging is a natural decline of the organism and its function during lifetime affects, in several rhythms, almost whole animal and plant kingdoms. This natural process is characterized by the attenuation of self-defense mechanisms and the decline of homeostatic mechanisms. Impairment of proteostasis has been associated with aging and results in reduced capacity of cells to repair misfolded or damaged proteins or to remove them through degradation pathways [31]. Proteasome activation has been shown to decelerate aging in vivo and in vitro [32]. The accumulation of misfolded proteins also characterizes many neurodegenerative disorders

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proteasome activity and function either directly as stimulators or through modulation of molecules responsible for proteasome activation

that are linked to aging such as AD, HD, PD, and Hutchinson–Gilford progeria syndrome (HGPS). AD is the most common cause of dementia and is characterized by loss of synaptic connections and brain atrophy. The accumulation of amyloid β (Aβ) plaques and hyper-phosphorylated Tau protein in neurofibrillary tangles are distinctive AD features [33]. Aβ is formed by the Amyloid Precursor Protein (APP) when the latter is cleaved by β- and γ-secretases. The proteasome is one of the major agents for Αβ metabolism, albeit the Aβ aggregates lead to proteasome impairment resulting in accumulation of aggregates in a vicious cycle [34]. Additionally, the E3 ubiquitin ligase HRD1 and the deubiquitinase UCHL-1 are downregulated during AD further highlighting the importance of UPS restoration in this disorder [35, 36].

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Huntingtin (HTT) is the most involved protein in HD pathogenesis. Mutations in HTT gene are related to expansion of the CAG triplet resulting in a polyglutamine (polyQ) tract in the N-terminus of the HTT protein [37]. Like in other proteinopathies, there is evidence of the UPS implication in HD. Mutations in ubiquitin UBB+1, PARKIN, NHLRC1 (an E3 ubiquitin ligase), and reduction of E1 and E2 ubiquitin enzyme activities along with 26S proteasome inhibition by HTT are characteristic alterations that lead to the UPS impairment in HD [38, 39]. PD is characterized by the accumulation of Lewy bodies (eosinophilic intracytoplasmic inclusions); α-synuclein is the main component [40]. α-synuclein has been shown to impede both the 20S and 26S proteasome function [41]. Moreover PARKIN, an E3 ligase, is the second most involved protein in PD pathology. PARKIN has been shown to enhance the interaction between 19S proteasome subunits underlining the importance of this ligase on the 26S proteasome activation [42]. PARKIN is affected by Lewy bodies that along with PARKIN mutations result in the degradation impairment observed in PD [43, 44]. Finally, progeroid syndromes are genetic conditions characterized by premature aging. Given that aging is accompanied by impairment of proteostatic mechanisms subsequently the UPS may also affect this type of syndromes. HGPS is a genetic condition in which a point mutation in the LMNA gene is observed. There are not many studies referring to UPS implication in HGPS; however, the downregulation of β5 and β7 proteasome subunits and reduction of CT-L proteasome activity have been reported [45]. Therefore, the UPS also appears as an attractive target against HGPS.

15.5

UPS and Diet

Several compounds have been shown to induce the UPS expression and/or function and thus to affect the progression of aging and age-related disorders. These compounds are summarized in the following sections and in Table 15.2.

15.5.1

Aggregation-Related Disorders

Well known for its stimulant activity on the central nervous system, guarana (Paullinia cupana) is consumed widely in Brazil in high-energy drinks and dietary supplements. Guarana compounds such as polyphenols, caffeine, and other methylxanthines have been associated with its beneficial biological properties which include antioxidant, antimicrobial, and chemophylactic activities in carcinogenesis [75]. Moreover, guarana powder has been shown to prevent Aβ aggregation in human neuronal-like cells while guarana water extract reduced the formation of polyQ aggregates expressed in C. elegans, suggesting a beneficial effect in AD and HD, respectively [76, 77]. According to a recent study, treatment with guarana hydroalcoholic extract (GHE) increased proteasomal and lysosomal activities and reduced intracellular ROS in C. elegans. It was also demonstrated that GHE eliminated the toxic phenotypes associated with protein misfolding and accumulation in nematode models of HD and AD. All the data above suggest the contribution of protein degradation mechanisms in GHE-mediated protection against polyQ protein aggregation and Aβ-induced toxicity [46]. Numerous studies have extensively investigated the pivotal role of the Mediterranean Diet in the prevention of a wide range of age-related pathological conditions [78]. The extra virgin olive oil (EVOO) is one of the most characteristic components of the Mediterranean Diet and it has attracted the interest of many researchers due to its antioxidant and antiaging properties while many of EVOO’s ingredients have been proposed as nutraceuticals against AD disease [79, 80]. Oleocanthal is one of the EVOO’s phenols responsible for its bitter and pungent taste. Many studies demonstrated the potential neuroprotective and anti-aggregation effects of oleocanthal [81–83]. According to a recent study, oleocanthal upregulated the expression of PSMD1 and PSMB4 subunits in neuron-like SH-SY5Y cells under stress and normal conditions, suggesting the involvement of oleocanthal in the degradation of misfolded proteins rather than oxidized ones. Moreover, oleocanthal increased

PSMD1, PSMB4

26S proteasome

26S proteasome

Aβ proteasome-dependent reduction

Extra virgin olive oil (EVOO)

Leaves of Psidium guajava (guava tea), white mulberry and red wine

Mangifera indica (mango), leaves of Annona squamosa and Camellia sinensis

Blueberries, raspberries, peanuts, the skin of grapes, red wine, cocoa and various other herbs

Oleocanthal

Morin

Isoquercitrin

Resveratrol

T-L activity

CT-L activity, UBA-1

UPS target CT-L activity

Source Paullinia cupana

APP695transfected HEK293 cells C. elegans (CL2006) Mouse (3xTgAD) (cerebral cortex)

APP695transfected SH-SY5Y cells

APP695transfected SH-SY5Y cells

AD

AD

AD

AD

(continued)

[49–51]

[48]

[48]

[47]

References [46]

Natural compound/ extract Guarana hydroalcoholic extract (GHE)a Disorder AD/HD

Table 15.2 Natural compounds/extracts that trigger UPS activation and affect aging and/or age-related disorders Model C. elegans (CL2006, CL4176, AM141) SH-SY5Y cells

We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products

Structure

15 335

Variety of widely consumed products including green tea, honey, red wine, and onions

Variety of plants including citrus fruits

Quercetin

Rutin

CT-L activity

CT-L activity CT-L activity

CT-L activity

uba-1, ubq-1, CT-L activity

PARKIN, UCH-L1, CT-L activity

Rhodiola rosea

Salidroside

CT-L activity

UPS target CT-L activity

CT-L activity, Psmd1

Source Honey, mushrooms, lobsters, shrimps, wine

Extract of many plants including Zanthoxylum chiloperone, Eurycoma longifolia and Aerva lanata

Structure

Canthin-6-one

Natural compound/ extract Trehalose

Table 15.2 (continued)

C. elegans (CL2006) APP695transfected SH-SY5Y cells HFL-1 cells mHTTtransfected Neuro2a cells APP695transfected SH-SY5Y cells

A30P α-synucleintransfected SH-SYS5 cells

PC12 cells

Model Glial cultures from R6/1 mouse model of HD HD human fibroblasts

AD

Aging HD

AD

PD

PD

Disorder HD

[57]

[58] [59]

[57]

[56]

[55]

[54]

[53]

References [52]

336 E. Panagiotidou and N. Chondrogianni

Pueraria lobata

Glycyrrhiza radix

18α-glycyrrhetinic acid (18α-GA)

PSMB6, PSMB6, PSMB7

Ginkgo biloba

Puerarin

HRD-1, proteasome function

Arbutus unedo (strawberry tree)

Polyphenol digested metabolites (PDMs) from leaves and fruits of Arbutus uneda Ginkgo biloba extract EGb761

CT-L activity, T-L activity, PBS-5, PAS-1-7, RPT-6 CT-L activity, T-L activity, C-L activity

CT-L

CT-L activity

CT-L activity

CT-L activity

CT-L activity, PSMC2

CT-L activity, T-L activity, C-L activity

Cruciferous vegetables

Sulforaphane

HFL-1 cells

C. elegans (CL2006, CL4176, CL2331) SH-SY5Y cells exposed to 7PA2conditioned medium C. elegans

mHTTtransfected HEK293 cells MPP+-treated SH-SY5Y cells

mHTTtransfected HEK293 cells Fibroblasts from patients with HGPS hESCs (human embryonic stem cells) Yeast cells transformed with α-synuclein

Aging

AD

PD

HD

PD

Aging

HGPS

HD

(continued)

[66, 67]

[66]

[65]

[64]

[63]

[62]

[61]

[60]

15 We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products 337

Proteasome function

Rubia cordifolia L.

Cruciferous vegetables

Many plants including Betula pubescens, Ziziphus mauritiana and Prunella vulgaris

Polysaccharide 5

3H-1,2dithiole-3-thione (D3T)a

Betulinic acida

CT-L activity

CT-L activity, T-L activity, C-L activity, PSMA1, PSMA4, PSMB3, PSMB5, PSMB6

UPS target Proteasome function

Structure

Source Ganoderma mushrooms

Natural compound/ extract Ganoderic acid DM

Table 15.2 (continued)

Purified proteasome

Mouse

T-REx293 expressing Aβ42-EGFP

Model T-REx293 expressing Aβ42-EGFP

AD

AD

AD

Disorder AD

[70]

[69]

[68]

References [68]

338 E. Panagiotidou and N. Chondrogianni

CT-L activity, PSMB5

Broccoli, spinach, tomatoes and brussels sprouts

Bee pollen Plethora of natural edible products

Lipoic acid

Bee pollen extract Zinc

HFL-1 cells Humans (population study)

NHDF cells

IMR90, W138 cells

Aging Aging

Aging

Aging

[73] [74]

[72]

[71]

a Compounds that have demonstrated anti-aggregation properties, but the association, if there is any, between their potency to act as proteasome activators and their contribution in protection against age-related disorders has not been studied yet

CT-L activity CT-L activity

CT-L activity, T-L activity, C-L activity

Green olives, olive leaves and EVOO

Oleuropein

15 We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products 339

340

HSP90 levels, a chaperone that is essential for proteasome integrity and function [47, 84, 85]. Morin is a flavonol present in guava leaf tea (Psidium guajava), white mulberry (Morus Alba L.), and red wine. It is known for its antioxidant activity as well for its antiapoptotic and neuroprotective properties [86, 87]. Regarding its neuroprotective effects in AD, morin restricted β- and γ-secretase activities to impede Aβ aggregation as well as to disaggregate preformed fibrils and to act against H2O2-induced stress in APP695-transfected SH-SY5Y cells. In the same cell line, morin positively affected the 26S proteasome activity under H2O2 stress conditions. The same results were also produced by another flavonoid namely isoquercitrin [48]. The potential therapeutic properties of resveratrol including life span extension, neuroprotection, and cytoprotection, foreshadow the potential contribution of this polyphenol in age-related disorders [88, 89]. Resveratrol’s effects on neurodegenerative disorders have been studied both in cellular and organismal level in models of AD; the effects on UPS among their positive outcomes have also been investigated [90]. For instance, in APP695transfected HEK293 cell line, resveratrol reduced Aβ levels through proteasomal degradation [49]. The same phenotype was reported in C. elegans; treatment with resveratrol reduced Aβ aggregates in CL2006 strain which expresses the human Aβ1-42 and paralyzes as Aβ aggregates accumulate [91]. Reduced Aβ aggregates have also been linked to proteostasis factors including UBA-1 (the only ubiquitinactivating enzyme in C. elegans) and enhanced proteasome activity [50]. Resveratrol was also shown to activate the brain proteasome function in the 3xTg-AD mouse model. Moreover, resveratrol administration enhanced the levels of neprilysin, an amyloid-degrading enzyme and diminished the levels of the amyloidogenic secretase BACE-1 [51]. On top of these effects, the already known effect of resveratrol on deacetylase SIRT1 [92] seems to dictate a preferential degradation of phosphorylated-Tau through enhanced deacetylation of Tau [51]. The abovementioned biochemical alterations led to

E. Panagiotidou and N. Chondrogianni

ameliorated cognitive behavior and function. Natural sources of resveratrol are blueberries, raspberries, peanuts, the skin of grapes, red wine, cocoa and various other herbs [93]. Trehalose is a disaccharide that is characterized as an autophagy enhancer [94]. During the last decades, many studies have reported the longevity effects of trehalose and its neuroprotective properties against HD [95]. In vivo experiments with mouse models of HD showed that trehalose administration reversed many aspects of pathological phenotype caused by polyQ aggregates [96]. In HD human fibroblasts, trehalose increased proteasome activities and decreased HTT protein levels [53]. Similar results were observed in glial cultures from R6/1 mice [52]. Trehalose is found naturally in honey, mushrooms, lobsters, shrimps, wine, and other edible products. Canthin-6-one is a natural derived alkaloid that can be isolated by many herbs including the edible plant Aerva lanata. In PD cellular models (PC12 cells), canthin-6-one promoted α-synuclein degradation through the UPS. Specifically, this alkaloid promoted the expression of Psmd1 gene by activating PKA, subsequently favoring the UPS activation [54]. Salidroside is a glycoside with antiaging, antioxidant, and neuroprotective properties. In SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), salidroside alleviated pSer129-α-synuclein burden. Additionally, in the same PD model salidroside elevated the protein levels of PARKIN and UCH-L1 which are two of the most involved constituents of UPS in PD. Free ubiquitin protein levels were also increased due to salidroside treatment along with the 20S proteasome activity (CT-L activity). Moreover, treatment with salidroside enhanced 20S proteasome activity in SH-SY5Y cells transfected with A30P α-synuclein [55]. The herb Rhodiola rosea is the main source of salidroside. Rhodiola rosea is consumed in the form of tea, made from the roots of the plant and it has been used as an antiaging herb in traditional Chinese medicine. Quercetin is a flavonoid found in many edible products like green tea, onions, and red wine.

15

We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products

Many studies reveal the positive outcome of quercetin in AD in vivo and in vitro [56]. In CL2006 transgenic strain of C. elegans, quercetin was able to reduce Aβ aggregates and the downstream paralysis phenotype. Among the defense mechanisms, quercetin promoted the UPS activation. Restriction of uba-1 and ubq-1 expression hindered the protective effects of quercetin against paralysis. The same research group observed enhanced proteasome activity in CL2006 animals treated with quercetin [56]. Proteasome activation by quercetin was also observed in APP695-transfected SH-SY5Y cells [57]. The same action was also revealed for rutin, a flavonoid mostly derived from citrus fruits that has the potential to prevent Aβ aggregation [97]. Both quercetin and rutin target agents related to proteasome activation, like Nrf2 [98]. Finally, in an HD cell line model, quercetin eliminated mHTT aggregates through proteasome activity enhancement [59]. Sulforaphane is an organic compound mainly found in many dietary products like onions, broccoli, cherries, and red grapes as well as in edible products like green tea and red wine. Through its strong antioxidant activity, sulforaphane exerts protective effects against neurodegenerative diseases [99, 100]. In murine neuroblastoma cells, sulforaphane enhanced proteasome activity via the Nrf2 transcription factor leading to elevated expression of proteasome subunits and thus to resistance against oxidative stress [25]. In an HD cellular model (mHTT-transfected HEK293 cells) treatment with sulforaphane enhanced mHΤΤ degradation by the proteasome [60]. Additionally, sulforaphane was also able to induce proteasome activation in fibroblasts derived from patients with HGPS and improved the growth rate of these cells. In the same study, increased levels of HSP27 protein were also reported; sulforaphane has been shown to enhance proteasome activity through upregulation of HSP27 [61, 101]. As mentioned above polyphenols exhibit antioxidant and anti-aggregation activities. Interestingly, a recent study revealed the implication of polyphenol digested metabolites (PDMs) in PD [63]. PDMs from leaves and fruits of Arbutus

341

unedo limited α-synuclein aggregates in a yeast model of PD through proteostasis network modulation, including proteasome elevated activity. Moreover, PDMs influenced the transcription levels of HRD-1, an ubiquitin ligase which is induced by unfolded protein response (UPR). HRD-1 recognizes and ubiquitinates misfolded proteins in ER favoring their degradation through the proteasome indicating a cross talk between the UPR and the UPS in the clearance of α-synuclein by PDMs. Many phenolic acids and flavonoid compounds such as myricetin, quercetin, and kaempferol along with terpenes attribute to Ginkgo biloba extract strong antioxidant properties [102]. Treatment of mHTT-transfected HEK293 cells with Ginkgo biloba extract EGb761 was able to decrease the amount of poly-Q proteins. Accordingly, it was reported that EGb761 promotes proteasome activation and elevated transcript levels of PSMB5, PSMB6, and PSMB7 subunits [64]. Puerarin is mainly isolated from Pueraria lobata and belongs to the isoflavone family. Beverages made by roots of Pueraria lobata contain a small number of isoflavones including puerarin. Many studies reveal the positive effects of puerarin in neuronal survival and cytoprotection in PD organismal and cellular models [103, 104]. In 1-methyl-4phenylpyridinium (MMP+)-treated SH-SY5Y cells, treatment with puerarin increased CT-L activity and lowered the amount of α-synuclein and ubiquitin-conjugated proteins, suggesting the implication of the UPS in puerarin positive results in PD [65]. 18α-glycyrrhetinic acid (18α-GA) is a triterpenoid isolated from Glycyrrhiza radix. We have shown that 18α-GA enhanced proteasome activity and function through elevated protein expression of PBS-5, PAS-1-7 and RPT-6 subunits that are orthologs of PSMB5, α-subunits and Rpt-6 proteasome subunits, respectively in C. elegans. In the same study, 18α-GA was able to promote deceleration of the paralysis phenotype in a proteasome-dependent manner in nematode models of AD [66]. 18α-GA has been shown to enhance

342

E. Panagiotidou and N. Chondrogianni

proteasome activity through the induction of Nrf2 transcription factor in human primary cells or its ortholog, namely SKN-1, in C. elegans [66, 67]. Finally, in the same study 18α-GA reduced Aβ toxicity in SH-SY5Y cells while parallel proteasome inhibition abolished this effect. Ganoderic acid and polysaccharide 5 isolated from Ganoderma mushrooms and the plant Rubia cordifolia L., respectively, were shown to induce Aβ clearance through the proteasome in T-REx293 embryonic kidney cell line expressing Αβ42-EGFP [68]. Finally, there are two natural compounds that were shown to possess anti-aggregation properties and have also been shown to modulate the UPS, but the association, if there is any, between their potency to act as proteasome activators and their contribution in protection against aggregate accumulation has not been studied yet. 3H-1,2dithiole-3-thione (D3T) can be found in cruciferous vegetables. In a recent study, treatment of a mouse model of AD (Tg2576) with D3T markedly decreased the levels of insoluble Aβ40-42 and enhanced the expression of Nrf2 [105]. Interestingly, D3T has been previously revealed to elevate the 26S proteasome activation and increase the expression of multiple proteasomal subunits in an Nrf2dependent manner [69]. Whether the Nrf2mediated proteasome activation is responsible for the protection against Aβ-toxicity remains to be elucidated. Last but not least, betulinic acid is a triterpenoid present in a plethora of plants and has been studied for its neuroprotective effects [106]. Betulinic acid has been reported as stimulator that enhances 20S proteasome activity [70]; the link between betulinic acid action as proteasome stimulator and the neuroprotection that it exerts is still not investigated.

15.5.2

Aging

Oleuropein is a phenolic compound found in high amounts in green olives, olive leaves, and EVOO. Oleuropein effectively exerted antiaging activity and enhanced proteasome function suggesting for the first time that proteasome

activators are interesting targets for the development of antiaging agents. More specifically oleuropein extended the life span of IMR90 and W138 human primary embryonic fibroblasts and increased CT-L, C-L, and T-L activities. Potential conformational changes of the proteasome structure by oleuropein were suggested to be responsible for the observed proteasome enhancement [71]. Quercetin is not only efficient against age-related disorders, but it positively affects the process of aging. Quercetin has been shown to prolong the life span of S. cerevisiae and C. elegans [107]. Constant treatment of HFL-1 cells with quercetin increased CT-L proteasome activity and proteasome quantity and delayed the senescence phenotype [58]. Lipoic acid is an organosulfur compound that is naturally synthesized by animals and is an essential cofactor in oxidative metabolism [108]. Nevertheless, lipoic acid can also be found in common ingredients of our diet like broccoli, spinach, tomatoes, and brussels sprouts. Lipoic acid is a strong antioxidant acting as a radical scavenger. With regard to its antiaging properties, α-lipoic acid has been shown to increase life span in C. elegans and D. melanogaster [109–111]. In normal human dermal fibroblasts (NHDF), treatment with lipoic acid positively affected the 20S proteasome activity and upregulated the protein levels of PSMB5 subunit leading to attenuation of oxidative damage and enhanced cell proliferation [72]. Sulforaphane is an isothiocyanate and its protective effects against neurodegeneration and aging have attracted the interest of many researchers [112]. Sulforaphane exerts its protective effects through interaction with the Nrf2 pathway [113–115]. Sulforaphane treatment maintained self-renewal and polypotency and delayed differentiation of human embryonic stem cells through Nrf2 activation and elevated proteasome activity [62]. Bee pollen is widely used and consumed by humans since ancient times [116]. Due to its composition (mixture of polyphenolic compounds) bee pollen has been shown to protect cells through antioxidant and anti-inflammatory

15

We Are What We Eat: Ubiquitin–Proteasome System (UPS) Modulation Through Dietary Products

mechanisms [117, 118]. Treatment of HFL-1 cells with bee pollen was shown to trigger the proteasome activity and to elevate protein expression levels of β2 and β5 subunits [73]. 18α-GA has been shown to extend the life span of treated human fibroblasts and to positively affect proteasome activity in an Nrf2dependent process [67]. Furthermore, increased life span and enhanced CT-L and T-L activities were also observed in wild-type C. elegans upon 18α-GA administration [66]. Finally, it is known that zinc is an essential trace element involved in the maintenance of many homeostatic mechanisms and its deficiency has been associated with aging [119, 120]. In a population study, zinc administration to the elderly increased CT-L proteasome activity and reduced proteotoxicity [74].

343

vated, along with the incidence of age-related disorders, consumption of dietary products with potent UPS activation properties may represent a promising strategy for health span extension and improvement. Acknowledgments We would like to thank Mrs. Nikoletta Papaevgeniou and Mary A. Vasilopoulou for comments on the manuscript. Part of the research presented here from our lab has been cofinanced by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation under the call RESEARCH—CREATE— INNOVATE (project codes: T1EDK-00353 and T1EDK01610). NC lab is also cofinanced under the Action “Action for the Strategic Development on the Research and Technological Sector” (project STHENOS-b, MIS 5002398).

References 15.6

Conclusions

Aging is a natural, unavoidable process that affects almost all organisms. UPS dysfunction is considered as one of the hallmarks of aging and is reported in many age-related pathologies [121]. An important number of investigations reveals the contribution of diet on aging. Diets that are high in fish, fresh fruit and vegetable consumption, like the Mediterranean-type Diet, have been suggested to favor healthy aging and to lower the risk of age-related pathologies. The abovementioned dietary products are rich in phytochemicals which are known for their antioxidant properties and their positive effects on aging and many pathologies including neurodegeneration [122]. Nevertheless, clinical trials and populational studies are missing for most of these compounds. Another pitfall is the fact that all experimental procedures are performed using isolated compounds usually in the μM range. Nevertheless, these concentrations are often unreachable through diet. On top of that, other constituents of dietary extracts may also have adverse effects that can dampen the beneficial properties of a single compound. Therefore, more detailed studies are needed. Given that the life span and the population of older adults have been ele-

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