Ubiquitin and Disease (Molecular Biology Intelligence Unit) 1-57059-545-3

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M OLECULAR BIOLOGY I N T ELLI G EN C E U N I T

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4

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Josep M. Argilés • Francisco J. López-Soriano Javier Pallarés-Trujillo ○

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Ubiquitin and Disease ○

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R.G. LANDES ○

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C O M P A N Y ○

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MOLECULAR BIOLOGY INTELLIGENCE UNIT 4

Ubiquitin and Disease Josep M. Argilés, Ph.D. Francisco J. López-Soriano, Ph.D. Javier Pallarés-Trujillo, Ph.D. Department of Biochemistry and Molecular Biology University of Barcelona Barcelona, Spain

R.G. LANDES COMPANY AUSTIN , TEXAS U.S.A.

MOLECULAR BIOLOGY INTELLIGENCE U NIT 4 Ubiquitin and Disease R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1998 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-545-3

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

CIP applied for, but not received at time of publication.

MOLECULAR BIOLOGY INTELLIGENCE UNIT 4 PUBLISHER’S N OTE R. G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, TissueE ngineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics.

Ubiquitin and Disease

Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are published within 90 to 120 days of receipt of the manuscript. We would like to thank our readers for their continuing interest and welcome any comments or suggestions they may have for future books.

Josep M. Argilés, Ph.D.

Francisco J. López-Soriano, Ph.D. Javier Pallarés-Trujillo, Ph.D.

Kemper Department of Biochemistry and MolecularJudith Biology University of BarcelonaProduction Manager Barcelona, Spain R.G. Landes Company

R.G. LANDES COMPANY AUSTIN , TEXAS U.S.A.

CONTENTS 1. Proteolysis: A Pleyade of Systems ............................................................ 1 Lysosomal Protein Degradation ............................................................. 3 Nonlysosomal Proteolytic Systems ......................................................... 5 2. The Ubiquitin System and Proteolysis ................................................... Structural and Functional Relationships of the System Components ............................................................... The Yeast Ubiquitin System Components and Their Eukaryotic Homologs ...................................................... The 26S Proteasome .............................................................................. The 20S Proteasome Subunits .............................................................. The 19S Cap Complex Regulatory Subunits ........................................ Ubiquitin-Fusion Proteins and Ubiquitin-like Proteins ..................... Drugs and Inhibitors .............................................................................

13

3. Alzheimer’s and Other Neurodegenerative Diseases ............................ Alzheimer’s, A Disease Related to Regeneration ................................. BAP, A Key Molecule in Understanding Alzheimer’s Disease ............ BAP Accumulation ................................................................................ BAP Aggregation ................................................................................... The Tau Protein ..................................................................................... The Link Between Tau Phosphorylation and APP .............................. Ubiquitin and Alzheimer’s Disease ...................................................... Ubiquitin Participates in Neural Regeneration ................................... Ubiquitin and NF- κB Generation ........................................................ Other Neurodegenerative Disorders ....................................................

61 61 61 63 68 69 73 75 78 79 79

4. Cancer ...................................................................................................... Cyclins and Cyclin-dependent Kinases ................................................ The Suppressor p53 Protein .................................................................. Ubiquitin-controlled Oncogenic Proteins ........................................... DNA Repair ........................................................................................... Ubiquitin-related Oncogenes ............................................................... A Possible Role for Ubiquitin System in Metastasis ............................

91 91 94 94 95 96 96

5. Muscle Wasting and Dystrophies ......................................................... Cancer Cachexia .................................................................................. Muscle Denervation and Atrophy ...................................................... Starvation and Malnutrition ............................................................... Other Pathological States .................................................................... Regulation of the System in Skeletal Muscle: TNF as an Activator .. Anabolic Hormones and the Ubiquitin System ................................. Ubiquitin System and Mechanotransduction in Muscle Growth ..... Neuromuscular Activity and the Maintenance of Muscle Mass ....... Wasting and Muscle Regeneration ..................................................... Stress Response in Muscle Wasting .................................................... The Ubiquitin System and Muscle Wasting Inducers ....................... Muscle Dystrophies .............................................................................

13 33 44 44 45 47 47

101 101 101 102 102 103 103 105 106 106 108 109 109

6. Other Pathological States ...................................................................... 117 Immune Response-related Diseases ................................................... 117 Cataract Formation and Blindness ..................................................... 120 Hypertension and Ischemia ................................................................ 120 Liver Diseases ....................................................................................... 121 AIDS ..................................................................................................... 121 The Angelman Syndrome ................................................................... 122 7. Ageing .................................................................................................... 127 8. The Role of Other Proteolytic Systems in Disease .............................. 137 Lysosomal: Cathepsins ........................................................................ 137 Nonlysosomal: Calpains ...................................................................... 140 Interaction Between the Ubiquitin System and Others..................... 141 9. Cell Injury and Cell Death: Apoptosis ................................................. Apoptosis and Disease ......................................................................... The Role of Ubiquitin in Apoptosis ................................................... Manipulation of Apoptosis as a Therapeutic Strategy.......................

147 147 153 154

10. The Regulation of the Ubiquitin System ............................................. 161 Mechanisms Controlling the Ubiquitin System ................................ 161 Proposed Rate-Limiting Steps in the Regulation of the Ubiquitin System .................................................................. 165 11. The Design of Therapeutic Strategies .................................................. 169 Index ................................................................................................................ 173

PREFACE

O

nly a few years ago, protein degradation was a poorly understood field, especially from a regulatory point of view. Although the molecular basis of protein synthesis (its constituents, enzymatic reactions and regulatory mechanisms) has been extensively studied and is very well understood, the subject of protein degradation has not made progress at the same rate. Indeed, the biology of proteolysis is only now coming to light. Protein degradation plays an essential role in animal cells. In addition to its involvement in enzyme regulation and cell remodelling, it provides an important source of amino acid carbon for gluconeogenesis and other biosynthetic and oxidative pathways when an exogenous substrate is not available. Ubiquitin and Disease deals with the involvement of the ubiquitin-dependent proteolytic system in protein degradation in a variety of pathological conditions. The book analyzes most of the conditions in which defects in ubiquitin gene expression and/or synthesis are involved in the understanding of the molecu lar basis of th e disease. Am on g th ese are Alzh eim er’s an d oth er neurodegenerative diseases, muscle dystrophies, cancer, muscle wasting and autoimmune diseases. The importance of ubiquitin-dependent proteolysis is also studied in ageing and special emphasis is placed on understanding the regulation of this ATP-dependent proteolytic system in order to design future therapeutic strategies. The book has three different parts. The first is introductory while the second constitutes the core of the book and analyzes the involvement of ubiquitin in different pathologies. Finally, the last part concerns the regulation of the system and the design of therapeutic strategies. The first chapter considers the different proteolytic systems. At present, there are two main cellular systems for the degradation of cellular proteins: the vacuolar system (in a membrane-surrounded acidic compartment), which includes lysosomes, endosomes and endoplasmic reticulum, and the cytosolic nonlysosomal pathways. The latter are believed to be regulated completely independently from the former because in addition to the differences in subcellular localization, there are differences in substrate specificity, optimum pH, sensitivity to inhibitors and physiological functions. The second chapter extensively describes the structural and functional relationships of the system com ponents, ubiquitin-activating enzym es, ubiquitin-transferring enzym es, the deubiquitinating enzym es and the proteasome or multicatalytic protease system. It also takes into consideration the yeast ubiquitin system components and their eukaryotic homologs. The next chapter concerns the role of the ubiquitin system in Alzheimer’s disease, the most common form of senile dementia, affecting more than 15 million people worldwide. The disease is characterized, in advanced cases, by a marked deterioration in memory and all cognitive functions. Ubiquitin is always associated with the typical alterations found in the disease, with an increased amount of soluble or immunoreactive ubiquitin in the cerebral cortex

of brains from patients with Alzheimers’s disease, compared with the levels found in control brains. Moreover, the increase is correlated with the degree of neurofibrillar changes. The ubiquitin system is also involved in DNA repair mechanisms and a misfunction of these systems may not only lead to gene alterations related to an impaired cellular stress response or a neurotissue repair mechanism, but also to other possible etiologies of the pathological state, such as impaired circulatory function. In addition to Alzheimer’s disease, the ubiquitin system also seems to be involved in a variety of neurodegenerative diseases including Parkinson’s disease and Diffuse Lewy bodies disease, amyotrophic lateral sclerosis, the prion or spongiform encephalopathies, cerebral amyloid angiopathies and, finally, a miscellaneous group of different types of dementias, all of which can either have a hereditary basis or developsp oradically. Chapter four is devoted to the role of the ubiquitin system in cancer, a disease based on uncontrolled cell proliferation. The ubiquitin system is clearly involved in the control of the cell cycle. It participates in the control of cyclin dependent-kinases (CDKs) through the regulation of cyclin degradation. Cyclins are small proteins that bind to the CDKs, activating them in a cyclin-CDK complex. The successive activation and deactivation of specific cyclin-CDK complexes permits the transition from one cell cycle state to another. In addition, there are other ubiquitin-controlled proteins whose functions are also relevant for cell proliferation. Among them, the tumor suppressor protein p53 not only plays an important role in cell cycle regulation, but also in cell survival. The next chapter is devoted to the role of the ubiquitin system in muscle wasting. This pathological state is generally thought to be caused by an increase in protein breakdown. Indeed, protein degradation is activated in numerous pathological conditions and may represent an important factor in loss of body weight since skeletal muscle represents over 40% of total body mass in humans. The apparent selectivity of the ATP-ubiquitin-dependent pathway makes it an attractive mechanism which could account for the specificity of protein degradation within skeletal muscle. Thus recent studies have focused on the role of ubiquitin in muscle. Muscle dystrophies are degenerative neuromuscular diseases, commonly characterized by progressive muscle fiber necrosis. Such diseases lead to muscle wasting and weakness, and eventually to death, due to cardiac or respiratory failure. Chapter six considers the involvement of the ubiquitin system in a variety of immune-related diseases. Indeed, the ubiquitin system may potentially be involved in any type of immune related disease, since it is most likely be related with the expression of MHC-I molecules and the function of the antigen-specific T cell receptors. In addition, in relation to the antigen-specific T cell receptor, the ubiquitin system has been shown to ubiquitinate multiple intracellular lysines in response to receptor engagement. Such ubiquitination has been shown to depend on receptor phosphorylation through the tyrosine kinase activity of its own receptor; taking all this into consideration, the ubiquitin system might well have a role in signal transduction after T cell activation.

The involvement of the ubiquitin system in ageing is analyzed in chapter seven. Ageing can be considered as a nonpathological degenerative process that causes a progressive deterioration and loss of physiological functions and, finally, death. The importance of the ubiquitin system in ageing is related to the role of the system in repair mechanisms, both DNA and stress response. Chapter eight considers the involvement of other proteolytic systems in disease. Indeed, it is by no means the aim of this book to suggest that the ubiquitin system is the only proteolytic system which is involved in disease. Other proteolytic systems may have key roles in some diseases and even more than one system may be involved in a particular pathological state. The aim of this chapter is to describe the possible involvement of both the lysosomal and nonlysosomal proteolytic systems and to relate them to the ubiquitin-dependent proteolytic system. Chapter nine deals with the involvement of ubiquitin in the mechanism of apoptosis. Little is known about the role of protease(s) in apoptotic death, although preliminary work has suggested the involvement of calpains (calciumdependent proteases). The nature of their involvement is very controversial since, in some reports, inhibitors of calpain blocked apoptosis, showing a positive function of calpain in the induction of apoptosis, while other work showed that calpain inhibitors accelerated apoptosis, thus suggesting a negative role. Ubiquitin certainly seems involved in apoptosis. Chapter ten is devoted to the mechanisms controlling the ubiquitin system: control at the transcriptional level, direc

CHAPTER 1

Proteolysis: A Pleyade of Systems A

lthough the molecular basis of protein synthesis (its constituents, enzymatic reactions and regulatory mechanisms) has been extensively studied and is very well understood, the subject of protein degradation has not made progress at the same rate. Indeed, the biology of protein degradation is only now coming to light. Protein degradation plays an essential role in animal cells. In addition to its involvement in enzyme regulation and cell remodeling, it provides an important source of amino acid carbon for gluconeogenesis and other biosynthetic and oxidative pathways when an exogenous substrate is not available. Because the amino acids are largely generated at the expense of proteins (which are rapidly regained with food intake), such degradation acts as a form of fuel reserve. However, it differs from carbohydrate and fat storage in that normal intracellular proteins, rather than specialized molecules such as glycogen or triacylglycerides, are used as the amino acid source. Protein degradation is required for a large number of cell events (Fig. 1.1). For instance, in the regulation of metabolic pathways, it is involved in the degradation of key metabolic enzymes with short half-lives and in the degradation of post-transcriptional activators of mRNA and of regulatory proteins such as oncogene products, heat-shock proteins and receptors. It also controls processing and activation of enzymes to their mature forms (such as lysosomal enzymes) or activation of proteolytic enzymes (such as trypsin). It also has a very important role in the removal of abnormal proteins, which may be either conformationally defective or denatured by oxidative stress. Protein degradation is involved in the regulation of cell shape and motility through the degradation of cytoskeletal and contractile proteins and in cell mitosis and signal transduction (by proteolytic activation of kinases; e.g. protein kinase C (PKC)). Proteolysis is also involved in tissue repair and regeneration, contributing to the clearing of necrotic tissue and participating in the activation of growth factors. It is also involved in apoptosis and turnover of cells with short life-spans such as erythrocytes, leukocytes and platelets, and in the alterations of cell phenotype associated with embryogenesis and organogenesis or cellular atrophy or hypertrophy. Protein degradation has a crucial role in host defense, participating in antigen presentation and the killing of microbes. The targeting and assembly of cellular proteins is also controlled proteolytically. Thus the processing of leader and propeptides during biogenesis and the degradation of excess subunits of “multimeric” proteins is controlled by proteind egradation. Concerning the different systems involved in protein degradation, the lysosomes, with their substantial complement of broad-specificity proteases, were once considered the locus of cellular protein degradation. Early investigations into protein degradation in mammalian cells concentrated on the lysosomes because lysosomal proteases were found to be highly active against a wide variety of peptide and protein substrates. However, the use of inhibitors of lysosomal function soon revealed that intracellular proteolysis is by no means restricted to the lysosomes.1 At present, there are two main cellular systems for thed egradation Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

2

Ubiquitin and Disease

Fig. 1.1. The different proteolytic systems in the cell: main functions.

of cellular proteins: the vacuolar system (in a membrane-surrounded acidic compartment), which includes lysosomes, endosomes and endoplasmic reticulum, and the cytosolic nonlysosomal pathways. The latter are believed to be regulated completely independently from the former because in addition to the differences in subcellular localization, there are differences in substrate specificity, optimum pH, sensitivity to inhibitors and physiological functions.

Proteolysis: A Pleyade of Systems

3

Lysosomal Protein Degradation The lysosome is a heterogeneous organelle and constitutes one of the main sites of intracellular degradation. More than 50 lysosomal enzymes have been identified and shown to degrade almost all biological molecules: protein, lipid, carbohydrate and nucleic acids.2 Among these enzymes, lysosomal proteases, also called cathepsins, are present in all mammalian cell types with the exception of enucleated red blood cells. The name cathepsin is derived from a Greek term meaning “to digest”. These enzymes, often glycoproteins, are relatively small (as compared with other nonlysosomal proteases) with molecular mass in the 20-40 kDa range. They are optimally active at acidic pH values and are unstable in either neutral or alkaline conditions. The properties of the lysosomal cathepsins are very similar in different species and cell types. The number of lysosomes (and therefore the concentration of lysosomal proteinases in the cell) varies in different cells and is particularly high in the liver, spleen, kidney and macrophages. The degradation of proteins by lysosomes involves exopeptidases, that cleave bonds only near the ends of molecular chains, and endopeptidases, that hydrolize peptide bonds in the middle of the protein sequence (Table 1.1). The two types of enzyme work synergistically to achieve complete protein degradation. Usually, the breakdown of proteins begins with the action of endopeptidases, followed by trimming of the resulting oligopeptides by exopeptidases. The exopeptidases include aminopeptidases and carboxypeptidases, whilst the endopeptidases can be classified, according to the identity of the catalytic group at the active site, into cysteine or aspartic proteinases (Table 1.1). Proteins can be taken up and degraded by lysosomes by multiple pathways involving endocytosis, autophagy or direct transport. Extracellular proteins can be degraded by lysosomes either intracellularly after endocytic uptake3,4 or extracellularly by secretion of lysosomal enzymes.5 Many plasma membrane proteins and certain other proteins within the vacuolar apparatus are also degraded by lysosomes via endocytosis.6 In autophagy (also

Table 1.1 Lysosomal proteases Protease Endopeptidases: Cathepsin B Cathepsin L Cathepsin H Cathepsin M Cathepsin N Cathepsin S Cathepsin T Cathepsin D Cathepsin E Exopeptidases: Cathepsin C Dipeptidyl peptidase Cathepsin III Cathepsin I (A) Cathepsin IV (B2) Lysosomal carboxypeptidase C

Optimum pH

MW (kDa)

Type of protease

5 5 5 5-7 3.5 3.5 6 3.5 2.5

25 24 28 30 20 25 35 42 100

Cys Cys Cys Cys Cys Cys Cys Asp Asp

5-6 4.5-5.5 6 5.5 5-6 5.5

20-40 130 N.D. 100-650 50 25

Aminopeptidase Aminopeptidase Aminopeptidase Carboxypeptidase Carboxypeptidase Carboxypeptidase

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Ubiquitin and Disease

termed autophagocytosis) there is a sequestration of cytoplasmic components in doublemembrane autophagic vesicles. These vesicles originate from the endoplasmic reticulum 7,8 or from a pre-existing organelle known as the phagophore.9 There are two different types of autophagic lysosomal degradation, macro- and microautophagy. Owing to the extreme heterogeneity of autophagic vacuoles, De Duve and Wattiaux10 coined the term microautophagy to express the idea that the sequestered volume could extend below the accepted limit for (macro)autophagy. In microautophagy, the lysosomal membrane invaginates at multiple locations to form single-membrane intralysosomal vesicles which later, following disintegration, release their contents which are then exposed to the action of lysosomal hydrolases.11 Both micro- and macroautophagy appear to be rather nonselective in that many proteins and other substances are taken up at similar rates.12 They form spontaneously in the normal, untreated cell, macroautophagy being controlled by amino acids (and, in some cases, by serum or other growth-promoting factors in cell media) and are induced by deprivation,13 while microautophagy predominates under basal conditions.14 Although the degradation of proteins in lysosomes is largely nonselective, lysosomes are also able to degrade cytosolic proteins in a highly selective manner, which is dependent upon a specific amino acid sequence within the substrate proteins. This is the case for the direct transport of specific proteins into the lysosome.15 This proteolytic pathway is restricted to cytosolic proteins containing peptide sequences that are biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). The mechanism by which this kind of protein is targeted to lysosomes is similar in many aspects to the import of newly synthesized proteins into the mitochondrion, endoplasmic reticulum, nucleus or peroxisome.15 The direct transport mechanism is particularly active in response to stress or starvation and involves the participation of members of the heat-shock 70 kDa family. As a result, the transport of both RNase A by fibroblast lysosomes15 and glyceraldehyde-3-phosphate dehydrogenase16 is highly selective and stimulated by ATP and hsc73, a heat shock cognate protein of 73 kDa.17 This heat-shock protein may participate in the lysosomal degradation of proteins containing KFERQ-like peptide regions, either by facilitating the binding of the substrate proteins to the receptor or protein translocation channel in the lysosomal membrane, or by unfolding proteins prior to lysosomal import.15 The spectrum of proteins that are degraded by lysosomal proteolysis is very wide, and consequently the functions and roles of lysosomes are numerous. They are responsible for the degradation of plasma proteins and lipoproteins, and necrotic cells or cell fragments that are internalized by receptor-mediated endocytosis and phagocytosis. Examples of some of these substrates are albumin,18 low density lipoproteins19 and hemoglobin.20 Apart from their involvement in the degradation of cytosolic proteins, they also participate in the processing of several secretory proteins such as thyroglobulin 21 and renin.22 Lysosomal proteases are also involved in bone resorption and the degradation of collagen in the bone matrix.23 Cathepsins also seem to participate in cellular differentiation as indicated by changes in their gene expression or activity. This is the case of cathepsin B; the activity of this proteolytic enzyme increases during the differentiation of myoblasts to myotubes.24 Lysosomal proteases also have a role in antigen-presenting cells by degrading endocytosed protein antigens. Antigen processing forms the basis for recognition of antigenic epitopes by T lymphocytes, a central feature of which is the proteolysis of antigens to produce the immunogenic peptides that are recognized by T cells. Whereas B lymphocytes express an antigen receptor (immunoglobulin) that recognizes antigens in their native conformation, T cells do not recognize conformational aspects of antigen structure. Instead, T cells recognize antigens as short linear peptides associated with major histocompatibility complex (MHC) proteins: these peptide-MHC complexes are “presented” by antigen-presenting cells. Therefore, antigen processing may be defined as the conversion of native protein antigens to pep-

Proteolysis: A Pleyade of Systems

5

tide-MHC complexes, which are displayed on the plasma membrane, where they are available for T cell recognition. There are two types of MHC molecules, classes I and II, which differ not only in structure but also in function. MHC-II molecules are transported to endocytic compartments where they bind peptides that are catabolically-derived from exogenous (extracellular) antigens internalized by endocytosis or phagocytosis. Following peptide loading of class II molecules, the peptide-class II complex is then translocated to the cell surface for presentation to the T cells.25,26 The regulation of lysosomal protease activity involves both local pH changes and the presence of specific inhibitors. As already pointed out, the optimum pH values for lysosomal proteolytic enzymes are in the acidic range (Table 1.1) and therefore changes in intralysosomal pH play an important role in protein turnover in these organelles. In relation to this, the presence of an H + -ATPase (proton pump) in the lysosomal membrane, which generates an acidic environment, is essential for the activation of lysosomal enzymes.27 There are three classes of cellular H + -ATPases. They are located in the mitochondria (F1F0ATPase), plasma membrane (E1E2-ATPase) or intracellular vacuolar membranes, such as the lysosome (H + -ATPase). The lysosomal H + -ATPase is characterized by a requirement for ATP and Mg2+ for maximal activation, a high sensitivity to proteolysis and a lack of response to inhibitors of mitochondrial and plasma membrane H + -ATPase.28 In addition to the activation of lysosomal proteinases, the proton gradient may also contribute to the transport of products of lysosomal hydrolysis to the cytosol.29 Concerning inhibitors of lysosomal protein degradation, two types have been identified, the cystatins (present in cells) and the plasma proteins α2-macroglobulin and α-cysteine proteinase.30 The cystatins are a group of low molecular weight inhibitors which, in addition to being involved in the regulation of the activity of lysosomal enzymes, may have a critical role in the protection of cell injury caused by the intracellular release of proteinases.30 Cystatins are classified according to the cell type in which they are found. Cystatin A is present in epithelial cells and leukocytes, cystatin B is found in lymphocytes and monocytes, cystatin C is present in neuroendocrine cells, and cystatin S is found in salivary glands.

Nonlysosomal Proteolytic Systems As we have previously stated, cells are in a dynamic state of synthesis and degradation, and although lysosomal protein degradation systems have a pivotal role in the maintenance of cellular homeostasis, the role of the nonvacuolar systems also seems to be crucial and can explain many pathological states.

Calpains and Calcium-Dependent Proteolysis Calcium ions act as cellular messengers, and various calcium-binding proteins are activated when intracellular calcium concentrations increase upon stimulation of cell-surface receptors. The calcium-dependent proteases, or calpains, are cytosolic cysteine proteases which require calcium for proteolytic activity. Consequently, these proteolytic enzymes play an important role in various physiologically important calcium mobilization. The family of calpains (only found among animals) can be divided into two groups on the basis of distribution (ubiquitous and tissue specific). The best known are the ubiquitous enzymes µ- and m-calpain (or calpain I and calpain II respectively). Their Ca2+ requirements for protease activity differ, at least in vitro (ca. µM and mM for µ- and m-calpains, respectively). Because the intracellular calcium concentration fluctuates normally at submicromolar levels, the former is more likely to operate in cells under physiological conditions, although there may be an unknown in vivo mechanism involving, for example, nuclear materials for the activation of the m-calpain.31 Although a relatively large amount of information concerning the structural and enzymological properties of these enzymes is known, little information is available about their physiological roles.

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Calpains are heterodimers consisting of two subunits: an 80 kDa (catalytic) subunit and a 30 kDa subunit.32 The large subunit, responsible for protease activity, is divided into four domains (I to IV from the N-terminus) (Fig. 1.2). Domain II is a cysteine-protease domain, similar to the plant cysteine protease papain, while the fourth domain (IV) is a Ca2+ -binding protein, similar to calmodulin. This explains the name calpain (“cal” as in calmodulin and “pain” as in papain). Presumably binding of calcium to domain IV induces conformational changes in the molecule that result in the activation of the protease domain. While different family members of the large subunit have been found, the smaller subunit (which also contains a calcium-binding domain and a hydrophobic membranebinding domain at its N-terminal) is common to all isoenzymes. Cong et al33 demonstrated that the small subunit is involved in the regulation of calcium sensitivity to calpain activity. The structure of the tissue-specific calpains bears great similarity to this domain pattern. n-calpain-1 (nCL-1) (also known as p94) contains three distinct segments (NS, IS-1 and IS-2) that are not found in other calpain species, and it is expressed only in skeletal muscle.34 The protein, however, is only just detectable in muscle due to rapid autolytic degradation following translation.35 Although the possibility that the enzyme directly degrades muscle proteins has been ruled out, the most likely possibility is that nCL-1 acts as a biomodulator in the activation of an enzyme or protein essential for muscle function. n-calpains-2 and 2' (nCL-2 and nCL-2') are generated by alternative splicing from the same gene and are predominantly expressed in the stomach.36 Interestingly, nCL-2' has no calcium-binding domain (Figure 1.2) and seems to be active without calcium. The levels of expression of these two tissue specific calpain forms is roughly the same, implying that nCL-2' is not just a minor splicing product but plays an important role in the stomach along with µ- and m-calpains and nCL-2. Although the physiological function of the calpain system remains unclear, four groups of in vitro substrates have been identified:37 (1) cytoskeletal proteins such as α-actinin or talin, and microtubule-associated proteins, (2) membrane proteins such as growth factor receptors (epidermal growth factor receptors), adhesion molecules (integrin) and ion transporters (Ca2+ -ATPase), (3) kinases (such as PKC) and phosphatases (such as calcineurin), and (4) cytokines (interleukin-1- α) and transcription factors (Fos, Jun). The mechanism of calpain proteolysis is not simply digestive but results in alteration, this being known as limited proteolysis. From this point of view, calpain activity is basically involved in regulatory processes. However, since limited proteolysis by calpains can also lead to a certain degree of destabilization of the protein structure, it may influence subsequent attacks by other cellular proteases, thus facilitating destruction. It is for this reason that calpain involvement in protein turnover should not be overlooked. Concerning the regulation of the calpain system, Goll et al38 demonstrated that the catalytic site of the calpains is normally blocked and that addition of calcium concentrations high enough to induce proteolytic activity unblocks the active sites. Therefore calcium concentrations that are high enough to induce proteolytic activity cause a significant conformational change in the calpain molecule, thus making its active site available to the susbstrate. However, the calcium concentration required to induce proteolytic activity (2-20 µM or 200-700 µM for µ- and m-calpains, respectively) is much higher than the free calcium concentrations that are normally found inside living cells (0.2-0.8 µM free calcium). Therefore, cells must contain some kind of mechanism to lower the calcium concentration required to induce calpain activity. Coolican and Hathaway39 reported that phospholipids (phosphatidylserine and phosphatidylinositol) lower the calcium concentration required for calpain activation. This may indicate that the enzymes are activated at the plasma membrane where these types of phospholipid are found. Indeed, various pieces of evidence

Proteolysis: A Pleyade of Systems

7

Fig. 1.2. Domain structures of calpain isoenzymes. Domain structures of calpain isoenzymes are schematically presented. Domain II corresponds to the cysteine protease segment; domain IV corresponds to the calmodulin-like domain. n-calpain-1 (nCL-1), also known as p94, contains three distinct segments (NS, IS1 and IS2) that are not found in other calpain species, and it is expressed only in skeletal muscle.

indicate that calpains are activated at the biological membrane in the presence of calcium and phospholipids (phosphatidylinositol-bisphosphate, (PIP2) in particular) when intracellular calcium concentrations increase by stimulation of cell surface receptors. Indeed, the inactive form (a procalpain) is found mainly in the cytosol. When the calcium concentration increases, the hydrophobic regions of the enzyme become exposed after calcium binding, and the enzyme translocates to the cytoplasmic surface of the plasma membrane. This binding markedly lessens the calcium requirement for enzymatic activation and it is then, in the presence of calcium and phospholipids, that autoproteolytic activation starts with proteolysis of the 80 kDa subunit and subsequent enzyme activation occurs.40,41 Further proteolysis of the 30 kDa subunit leads to complete

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Ubiquitin and Disease

activation. Finally, the active protease is released from the membrane back into the cytosol, where its activity may be inhibited either by prolonged exposure to high levels of calcium (which causes excessive autoproteolysis) or by conjugation to cytosolic inhibitors. In addition, calpains interact with a specific endogenous protein called calpastatin.42 It exerts the opposite effects to those of calcium and phospholipids, acting as a competitive inhibitor with high affinity and strict specificity of the protease activity.43 Calpastatin is ubiquitously expressed and acts at levels comparable to those of total calpain activity. Interestingly, the calcium concentration required for calpastatin to bind to and inactivate the calpains is approximately the same ( µ-calpain) or significantly less (m-calpain) than the concentration required for the proteolytic activity of calpains themselves.44 Consequently, rising calcium concentrations would induce calpastatin binding before the proteolytic activity is turned on. It is therefore essential to take into consideration phospholipids as the molecules that are able to overcome calpastatin inhibition. In this way, calpastatin inhibitory effects can be counterbalanced by calcium and phospholipids, both acting as accelerators of the protease system. Other proteins which also inhibit calpain activity are α2-macroglobulin and kininogens.45 Unlike calpastatin, these proteins are present extracellularly and suppress the activity of calpains released by injured cells.46 The activity of the calpain system is therefore tightly regulated. This tight regulation (the mechanisms of which are to some extent still unknown) together with the requirement of high, physiologically abnormal calcium concentrations, has strongly hindered the understanding of its physiological function. However, since one of the main substrates of the calpain system are cytoskeletal proteins and since these proteins control cell shape and various functional processes, calpain-mediated alteration of cytoskeletal proteins may play pivotal roles in these phenomena. Calpains have been implicated in long-term potentiation of memory47 and neuronal cell death after transient ischemia.48,49 Furthermore, calpains have been postulated to play roles in cell division,50 apoptosis,51 cell differentiation and proliferation,52,53 embryonic development 54 and stomach hydrogen chloride secretion.55 Calpain participates in the partial proteolysis and activation of some membrane proteins and therefore has a potential role in signal transduction. The activation of PKC and other well-known kinases by calpains has drawn considerable attention because of the role of these enzymes in transmembrane signal transduction.

ATP and Ubiquitin-Dependent Proteolysis In the late 1970s, Hershko et al56 observed that abnormal cytoplasmic proteins were degraded at a neutral pH in a process that required ATP. Additional studies revealed that multiple factors were required to support this activity. The first such factor was a small polypeptide called ubiquitin. Ubiquitin-dependent proteolysis is governed by an elaborate pathway in which covalent attachment of this small (76 residues), highly conserved protein to other cellular proteins targets them for degradation by cytoplasmic, ATP-dependent proteolysis. It is an essential degradative pathway in eukaryotic cells and accounts for more than 90% of short-lived protein turnover. The multi-ubiquitinization of proteins and their destruction by cytosolic proteolysis is associated with a large multisubunit complex known as the proteasome. Eukaryotic proteasomes manifest an unusually robust spectrum of proteolytic activities and consist of a heterogeneous population whose members differ in subunit composition and vary in response to differentiation and other changes in cell state. Among the substrates of the ubiquitin-dependent proteolytic pathway are damaged and abnormal proteins, as well as naturally short-lived proteins such as transcriptional regulators, oncoproteins and regulators of cell cycle progression. In addition to its role in the turnover of proteins, the binding of ubiquitin to a variety of proteins may regulate numerous other basic cellular functions, for example, regulation

Proteolysis: A Pleyade of Systems

9

of gene expression, progression of mitotic cycle, modification of receptors, biogenesis of ribosomes, DNA repair, and early cell growth and development. However, further work is needed to establish a direct link between ubiquitin conjugation and these functions, as well as to elucidate the regulatory mechanisms involved. In spite of this, our understanding of ubiquitin-dependent proteolysis has benefited from the complementary perspectives afforded by biochemical and genetic approaches. Biochemical studies have permitted the identification and characterization of the enzymatic activities involved in ubiquitin-protein conjugation and the degradation of ubiquitinated proteins, while genetic studies have provided insight into the generality of ubiquitin-dependent proteolysis and its function in vivo. In the next chapter, we will deal extensively with the structure and function relationships of the system components together with the genes and enzymes involved.

References 1. Seglen PO. Inhibitors of lysosomal function. Meth Enzymol 1983; 96:737-764. 2. De Duve C. Lysosomes revisited. Eur J. Biochem 1983; 137:391-397. 3. Del Rosso M, Fibbi G, Pucci M et al. Modulation of surface-associated urokinase. Binding, interiorization, delivery to lysosomes, and degradation in human keratinocytes. Exp Cell Res 1991; 193:346-355. 4. Schmidt SL. The mechanism of receptor-mediated endocytosis: More questions than answers. Bioessays 1992; 14:589-596. 5. Ishii Y, Hashizume Y, Watanabe T et al. Cysteine proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung. J Histochem Cytochem 1991; 39:461-468. 6. H are JF. Mechan ism s of m em bran e protein turn over. Biochim Biophys Acta 1990; 1031:71-90. 7. Dunn WA. Studies on the mechanisms of autophagy: Formation of the autophagic vacuole. J Cell Biol 1990; 110:1935-1945. 8. Ueno T, Muno D, Kominami E. Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes from leupeptin-administered rat liver. J Biol Chem 1991; 266:18995-18999. 9. Seglen PO, Gordon PB, Holen I. Nonselective autophagy. Semin Cell Biol 1990; 1:441-448. 10. De Duve C, Wattiaux R. Functions of lysosomes. Annu Rev Physiol 1966; 28:435-492. 11. Dice JF. Molecular determinants of protein half-lives in eukaryotic cells. FASEB J 1987; 1:349-357. 12. Kominami E, Hashida S, Khairallah EA et al. Sequestration of cytoplasmic enzymes in an autophagic vacuole. Lysosomal system induced by injection of leupeptin. J Biol Chem 1983; 258:6093-6100. 13. Schworer CM, Shiffer HA, Mortimore GE. Quantitative relationship between autophagy and proteolysis during graded amino acid deprivation in perfused rat liver. J Biol Chem 1981; 256:7652-7658. 14. Mortimore GE, Lardeux BR, Adams CE. Regulation of microautophagy and basal protein turnover in rat liver: Effects of short-term starvation. J Biol Chem 1988; 263:2506-2512. 15. Dice JF, Terlecky SR. Selective degradation of cytosolic protein s by lysosom es. In : Ciechanover AJ, Schwartz AL, eds: Cellular Proteolytic Systems. New York: Wiley-Liss, 1994:55-64. 16. Aniento F, Roche E, Cuervo AM et al. Uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase by rat liver lysosomes. J Biol Chem 1993; 268:10463-10470. 17. Chiang HL, Terlecky SR, Plant CP et al. A role for a 70-kilodalton heat shock protein in lysosomal proteolysis of intracellular proteins. Science 1989; 246:282-285. 18. Baricos WH, Zhou Y, Fuerst RS et al. The role of aspartic acid and cysteine proteinases in album in degradation by rat kidney cortical lysosom es. Arch Biochem Biophys 1987; 256:687-691. 19. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986:232:34-47.

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20. Diment S, Stahl P. Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J Biol Chem 1985; 260:15311-15317. 21. Dunn AD, Crutchfield HE, Dunn JT. Proteolytic processing of thyroglobulin by extracts of thyroid lysosomes. Endocrinology 1991; 128:3073-3080. 22. Wang PH, Do YS, Macaulay L et al. Identification of renal cathepsin B as a human proreninprocessing enzyme. J Biol Chem 1991; 266:12633-12638. 23. Delaissé JM, Ledent P, Vaes G. Collagenolytic cysteine proteinases of bone tissue. Cathepsin B, (pro)cathepsin L and a cathepsin L-like 70 kDa proteinase Biochem J 1991; 279:167-174. 24. Béchet DM, Ferrara MJ, Mordier SB et al. Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B. J Biol Chem 1991; 266:14104-14112. 25. Gradehandt G, Ruede E. The endo-lysosomal protease cathepsin B is able to process conalbumin fragments for presentation to T cells. Immunology 1991; 74:393-398. 26. Harding CV, Collins DS, Slot JW et al. Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell 1991; 64:393-401. 27. Klionsky DL, Herman PK, Emr SD. The fungal vacuole. Composition, function, and biogenesis. Microbiol Rev 1990; 54:266-292. 28. Mellman I, Fuchs R, Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem 1986; 55:663-700. 29. Ohkuma S. In: Glaumann H, Ballard FJ, eds: Lysosomes: Their Role in Protein Breakdown. London: Academic Press, 1987:115-148. 30. Barrett AJ. The cystatins. A new class of peptidase inhibitors. Trends Biochem Sci 1987; 12:193-196. 31. Mellren RL, Song K, Mericle MT. m-Calpain requires DNA for activity on nuclear proteins at low calcium concentrations. J Biol Chem 1993; 268:653-657. 32. Suzuki K, Sorimachi H, Yoshizawa T et al. Calpain: novel family members, activation, and physiological function. Biol Chem Hoppe-Seyler 1995; 376:523-529. 33. Cong J, Thompson VF, Goll DE. Effect of monoclonal antibodies specific for 28-kDa subunit on catalytic properties of the calpains. J Biol Chem 1993; 268:25740-25747. 34. Sorimachi H, Imajoh-Ohmi S, Emori Y et al. Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and µ-types. Specific expression of the mRNA in skeletal muscle. J Biol Chem 1989; 264:20106-20111. 35. Sorimachi H, Toyama-Sorimachi N, Saido TC et al. Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem 1993; 268:10593-10605. 36. Sorimachi H, Ishihura S, Suzuki K. A novel tissue-specific calpain species expressed predominantly in the stomach comprises two alternative splicing products with and without Ca2+ -binding domain. J Biol Chem 1993; 268:19476-19482. 37. Saido TC, Sorimachi H, Suzuki K. Calpain: New perspectives in molecular diversity and physiological-pathological involvement. FASEB J 1994; 8:814-822. 38. Goll DE, Thompson VF, Taylor RG et al. Role of the calpain system in muscle growth. Biochimie 1992; 74:225-237. 39. Coolican SA, Hathaway DR. Effect of L- α-phosphatidylinositol on a vascular smooth muscle Ca2+ -dependent protease. J Biol Chem 1984; 259:11627-11630. 40. Cottin P, Poussard S, Desmazes JP et al. Free calcium and calpain I activity. Biochim Biophys Acta 1991; 1079:139-145. 41. Melloni E, Pontremoli S. The calpain-calpastatin system. Structural and functional properties. J Nutr Biochem 1991; 2:467-476. 42. Suzuki K, Imajoh S, Emori Y et al. Calcium-activated neutral protease and its endogenous inhibitor. Activation at the cell membrane and biological function. FEBS Lett 1987; 220:271-277. 43. Maki M, Takano E, Osawa T et al. Analysis of structure-function relationships of pig calpastatin by expression of m utated cDNAs in Escherichia coli. J Biol Chem 1988; 263:10254-10261.

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44. Kapprell HP, Goll DE. Effect of Ca2+ on binding of the calpains to calpastatin. J Biol Chem 1989; 264:17888-17896. 45. Crawford C. Protein and peptide inhibitors of calpains. In Mellgren RL, Murachi T, eds. Intracellular Calcium-dependent Proteolysis. Boca Raton: CRC Press, 1990:75-89. 46. Crawford C, Willis AC, Gagnon J. The effects of autolysis on the structure of chicken calpain II. Biochem J 1987; 248:579-588. 47. Del Cerro S, Arai A, Kessler M et al. Stimulation of NMDA receptors activates calpain in cultured hippocampal slices. Neurosci Lett 1994; 167:149-152. 48. Hong SC, Goto Y, Lanzino G et al. Neuroprotection with a calpain inhibitor in a model of focal cerebral ischemia. Stroke 1994; 25:663-669. 49. Robert-Lewis JM, Savage MJ, Marcy VR et al. Immunolocalization of calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain. J Neurosci 1994; 14:3934-3944. 50. Yamaguchi R, Maki M, Hatanaka M et al. Unphosphorylated and tyrosine-phosphorylated forms of a focal adhesion protein, paxillin, are substrates for calpain II in vitro: implications for the possible involvement of calpain II in mitosis-specific degradation of pallixin. FEBS Lett 1994; 356:114-116. 51. Squier MKT, Miller AKC, Malkinson AM et al. Calpain activation in apoptosis. J Cell Physiol 1994; 159:229-237. 52. Song DK, Malmstrom T, Kater SB et al. Calpain inhibitors block Ca2+-induced suppression of neurite outgrowth in isolated hippocampal pyramidal neurons. J Neurosci Res 1994; 39:474-481. 53. Saito Y, Saido TC, Sano K et al. The calpain-calpastatin system is regulated differently during human neuroblastoma cell differentiation of Schwannian and neuronal cells. FEBS Lett 1994; 353:327-331. 54. Em ory Y, Saigo K. Calpain localization chan ges in coordin ation with actin -related cytoskeletal changes during early embryonic development of Drosophila. J Biol Chem 1994; 269:25137-25142. 55. Yao X, Thibodeau A, Forte JC. Ezrin-calpain I interactions in gastric parietal cells. Am J Physiol 1993; 265:C36-C46. 56. Hershko A, Ciechanover A, Rose IA. Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP. Proc Natl Acad Sci USA 1979; 76:3107-3110.

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CHAPTER 2

The Ubiquitin System and Proteolysis Structural and Functional Relationships of the System Components Ubiquitin

U

biquitin is a small (76 residues) and abundant peptide, which is found in all eukaryotic cells. Its primary sequence is remarkably conserved, with only three amino acid differences between the yeast and the human sequences. The secondary structure includes five strands of beta-pleated sheets, three and a-half turns of alpha-helix and seven reverse turns. Ubiquitin appears as a tightly packed globular protein with four C-terminal residues that protrude from the main globular domain to form the site of ubiquitin protein conjugation (Fig. 2.1). The fact that ubiquitin is heat-stable and tolerates a wide range of pH can be explained by the fact that approximately 87% of its polypeptide chain is involved in hydrogen bonds. The functional sites of ubiquitin are the C-terminus, through which the acceptor proteins are ligated, and Lys-48, which serves as acceptor site for the own ubiquitin in the formation of polyubiquitin chains.1,2

Ubiquitin-activating Enzymes Ubiquitin activation is catalyzed by the ubiquitin-activating enzyme and is a prerequisite for any type of ubiquitin transfer. Although a few variants of ubiquitin-activating enzymes have been described and their functional involvement is still not known (Table 2.1), ubiquitin activation can be considered as the most common step in all the ubiquitin-related pathways. The mechanism of activation is summarized in Figure 2.2. First, the Mg2+ -dependent hydrolysis of ATP between the alpha- and beta-phosphoryl groups drives the formation of ubiquitin adenylate on Gly-76, the complex being tightly bound to the E1 enzyme. Second, the adenylate transfers the ubiquitin to an active sulfhydryl group of E1 in a reaction that occurs rapidly in the absence of Mg2+ . Third, the E1-ubiquitin thiol ester undergoes another round of Mg2+ -dependent AMP-ubiquitin formation, yielding a ternary complex in which E1 carries two molecules of C-terminally-activated ubiquitin.3 Once ubiquitin is activated, the E1-ubiquitin thiol ester of the ternary complex undergoes transthiolation with the active sulfhydryl of a ubiquitin-conjugating enzyme E2. The formation of the ternary complex is quite important since the occupancy of the nucleotide/ adenylate site of E1, either by Mg2+ -ATP or by AMP-ubiquitin, strongly stimulates the ubiquitin transthiolation between the E1 and E2 enzymes (Fig. 2.3).3 The primary structure of the E1 enzymes has 43-53% of amino acid sequence identity between yeast and human enzymes.4 They are characterized by sequence homologies which are consistent with the described mechanism of ubiquitin activation. Firstly, there are the nucleotide binding domains. Such domains are characterized mainly by the Gly-X-Gly-XX-Gly motif, where X is any type of amino acid.5,6 Such a motif is followed in some cases by a residue of lysine and an additional typical Asp-X-X-Gly sequence.7,8 Secondly, there are Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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C-terminal

Lys-48

Fig. 2.1. Tridimensional structure of ubiquitin. The C-terminal (red) (residue 76) binds covalently to the proteins that are going to be degraded. The e-amino group of Lys-48 (blue) may bind another ubiquitin ubiquitin molecule to from polyubiquitin chains. The residues in green correspond to the ones that interact with the ubiquitin-conjugating enzyme E2. Reprinted with permission from: Stryer L. Biochemistry, WH Freeman and Co., New York.

Table 2.1. E1 enzymes S. cerevisiae

wheat

UBA1

A. thaliana

mouse

human

UBA1

ube1/A1S9X X-linked

UBE1 X-linked (orUBE1L)

UBA2 UBA3

ube2 Y-linked Sby/A1S9Y-1 Y-linked

UBA2 UBA3 AxR1

APP-BP1

The Ubiquitin System and Proteolysis

15

Fig. 2.2. The ubiquitin-mediated proteolysis. The ubiquitin-activating enzyme (E1), together with ATP, generates a high energy link (thioester bond) with ubiquitin (Ub), that is transfered to the ubiquitin-conjugating enzyme (E2). This permits substrate recognition by the multicatalytic protease complex (26S proteasome) and consequently its proteolytic degradation in an ATPdependent process. In some cases, the substrate recognition by the E2 enzyme is mediated by the formation of an ubiquitin-protein ligase (E3)-substrate complex. Following ATP-dependent proteasome action, ubiquitin is released from the proteasome by means of a peptidase or isopeptidase activity either soluble or linked to the proteasome. The specificity of the degradation pathway is mediated by both E2 and E3 enzymes.

five conserved cysteines of which Cys-600 is believed to act as the acceptor site for ubiquitinE1 thiolester formation (Fig. 2.4).5 This is suggested by its inclusion in a region with relative homology to the regions surrounding the active cysteine of 19 different E2 enzymes. Other regions of the E1 enzyme that will probably be studied in the near future are those involved in its intracellular localization and in its binding with the E2 enzymes during the ubiquitin transfer. In the first case, a putative targeting sequence for its nuclear localization has already been suggested.5

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Fig. 2.3. Activation of the ubiquitin system. Ubiquitin activation is catalyzed by the ubiquitinactivating enzyme and is a prerequisite for any type of ubiquitin transfer. The mechanism of activation involves the Mg2+ -dependent hydrolysis of ATP between the alpha- and beta-phosphoryl groups that drives the formation of ubiquitin adenylate on Gly-76, the complex being tightly bound to the E1 enzyme. The adenylate transfers the ubiquitin to an active sulfhydryl group of E1, in a reaction that occurs rapidly in the absence of Mg2+ . Finally, the E1-ubiquitin thiol ester undergoes another round of Mg2+ -dependent AMP-ubiquitin formation, yielding a ternary complex in which E1 carries two molecules of C-terminally-activated ubiquitin. Once ubiquitin is activated, the E1-ubiquitin thiol ester of the ternary complex undergoes transthiolation with the active sulfhydryl of a ubiquitin-conjugating enzyme E2.

Ubiquitin-Transferring Enzymes Common Features After activation, the activity of the E2 and E3 enzymes is involved in transferring ubiquitin to the protein which has to be degraded. Some aspects of the mechanism of ubiquitin transfer from the E1 to the E2 enzymes (and probably from the E2 to the E3 enzymes) seem to be common to all the ubiquitin system enzymes involved in ubiquitin conjugation, regardless of their substrate specificities. This has been suggested by the common structural motifs found in all the E2 enzymes studied 1 and in the expanding group of E3 enzymes with the hect domain.9 These structural motifs in the E2 enzymes were first described by comparison of their primary sequence. It was revealed that all have a conserved domain of roughly 16 kDa, called the UBC domain, with 35% sequence homology.1 In this domain, two highly conserved sequences can be seen. One is centrally located in the UBC domain with a highly conserved cysteine in the middle of the sequence. This cysteine is responsible for the ubiquitin binding to the E2 enzyme through a thiolester link, since its

The Ubiquitin System and Proteolysis

17

Fig. 2.4. Conjugation with ubiquitin. Cys-600 is believed to act as the acceptor site for ubiquitinE1 thiolester formation. This is suggested by its inclusion in a region with relative homology to the regions surrounding the active cysteine of 19 different E2 enzymes.

substitution by other amino acids leads to a complete loss of enzymatic activity.10–12 Other amino acids of the same sequence have also been shown to contribute to the correct binding activity of the cysteine residue.13 The other highly conserved sequence is rich in basic residues and is located at the N-terminal end of the UBC domain and is thought to be involved in the binding of the E1 to the E2 enzymes since small deletions in the sequence have led to a reduced ubiquitin-binding activity.10 In addition, the presence of a common E1 binding site, present in all the E2 enzymes (Table 2.2), is also suggested by the fact that a single E1 enzyme is able to act on different E2 enzymes of the same or different species.14,15 Interestingly, a direct influence of this region on the ubiquitin-binding activity of the critical cysteine residue by the distant position in the tertiary structure of two E2 enzymes could be discarded. Binding between E1 and E2 enzymes has been reported.1 Thus, studies of directed mutagenesis and binding between these enzymes may be very helpful in finding out which residues are involved in the interaction. In the same way, the studies performed by Cook et al16,17 on the three-dimensional structure of two different E2 enzymes almost consisting in the UBC domain have also been very useful for understanding the common and specific features of the mechanism of ubiquitin transfer. They reported the tertiary structure of the Arabidopsis thaliana UBC1 enzyme and the Saccharomyces cerevisiae UBC4 enzyme, both with different functions and only 42% sequence identity. Interestingly, in spite of their differences, different E2 enzymes (consisting mainly in the UBC domain) have both a highly conserved secondary structure and a conserved overall folding (Table 2.3). The surface topology of both enzymes reveals common and different areas. These common areas have been related to the ubiquitin-accepting cysteine residue and probably to the E1 binding region, while divergent surface regions have been proposed to be involved in the interaction with their respective substrates or E3 enzymes. Concerning the E3 enzymes, special attention has to be given to their hect domain. This was first described in the mammalian E6-AP ubiquitin ligase and has been found in

ubcH3

UBC3/cdc34

ubcH6

ubcH5A/B/C

r-homolog

ubcD1

AtUBC8 AtUBC9 AtUBC10 AtUBC11 AtUBC12 ubc2

UBC4/UBC5 UBC6

AtUBC4 AtUBC5 AtUBC6

UBC8 UBC9

UBE2I

UBE2G ubcH2 ubcH9

ubc7

UBC7

ubcH10

UBC10

ubcH8 UBE2L1 UBE2L2 UBE2L3

E2F1 ubcH7

E2-C benUBC ubcM4

ubcH4

ubcM1

Other eukaryotic UBCs

UBE2G is muscle specific. Other eukaryotic UBCs are different Class III enzymes cloned from Drosophila; an E2-E3 hybrid of 230 kDa cloned from rabbit reticulocytes and the Arabidopsis thaliana UBC7/13/14 enzymes.

hHR6A hHR6B

r-homolog ubcH1

Dhr6 m-homolog

ubc1

C. elegans Clam Drosophila Mouse Rat Rabbit Human

UBC2/RAD6

rhp6+ AtUBC1 AtUBC2 AtUBC3

UBC1

S. pombe A. thaliana

S. cerevisiae

Table 2.2 Nomenclature of E2 enzymes

18 Ubiquitin and Disease

DNA replication metabolic adaptation

cell cycle control (G1-S transition)

DNA repair repression of retrotransposition peptide transport metabolic adaptation sporulation

cellular growth metabolic adaptation stress response endocytosis of membrane proteins sporulation G0 induction

Functions

cleave of ubiquitin fusion proteins

involvement in the secretory pathway metabolic adaptation endocytosis of membrane proteins

S.C.UBC4/UBC5-like S.C.UBC4/UBC5 cellular growth stress response turnover of transcriptional factors

S.C. UBC3-like S.C UBC3

S.C. UBC2-like S.C UBC2/RAD6

S.C. UBC1-like S.C.UBC1

E2 ENZYMES

Table 2.3. Main characteristics of E2 enzymes

fructose-1,6-bisphosphatase ste2 receptor ste 6 receptor

Clb cyclin abnormall proteins MAT-alpha2

Gcn4

cln2 cln3 sic

Gcn4 (transcriptional factor)

histones

fructose-1,6-bisphosphatase abnormal proteins ste 2 receptor

Substrates

degradation box (Deg1 and Deg2)

destruction box

PEST sequence

PEST sequence PEST sequence

N-end rule

UBR1(E3)

continued

ubiquitin system components

signal

RAD18 (E3-like)

Interaction with other

Degradation

The Ubiquitin System and Proteolysis 19

S.C.UBC10-like S.C.UBC10 peroxisome biogenesis

cell cycle control (G2-M progression)

cell cycle control (G2-M progression)

S.C.UBC9-like S.C.UBC9

ubcH9 (human homolog) UBE2I (human homolog)

No phenotypic defects

Stress response induced by cadmium turnover of transcriptional factors

S.C.UBC8-like S.C.UBC8

UBE2G (human homolog muscle-specific)

S.C.UBC7-like S.C.UBC7

p53

Clb5 (S phase cyclin) Clb2 (mitotic cyclin)

MAT-alpha2

MAT-alpha2

p53

UbcH6 (human homolog) involvement in the secretory pathway turnover of transcriptional factors

p53 c-fos p105 precursor of NF-kB

UbcH5 (human homolog)

S.C.UBC6-like S.C.UBC6

p53

AtUBC8 (A. thaliana homolog)

Table 2.3. Continued

destruction box destruction box

degradation box (Deg1 and Deg2)

degradation box (Deg1 and Deg2)

RAD52 (ubiquitin-like protein) UBL1 (ubiquitin-like protein)

suggested E3 suggested E3

UBC6

UBC7

ubiquitin p105 ligase Rsp5 E6-AP

E6-AP(E3)

E6-AP(E3)

20 Ubiquitin and Disease

involved in neuronal conectivity

completion and exit from mitosis

linked to the Golgi membrane completion and exit from mitosis mitotic cyclins A and B

mitotic cyclins A and B

p53

p53 p53

p53 c-fos p105 precursor of NF-κB actine, troponine T, MyoD lysozime

(1) UBE2L1 (= FAD3) has been suggested to be the gene involved in Alzheimer’s disease.

ubcH10 (human homolog of EC-2) ben UBC (Drosophila)

ubcH4 (human) UbcM1 (mouse) E2-C (clam oocytes)

Other UBCs:

UbcM4 (mouse) ubcH7 (human homolog of E2-F1) ubcH8 (human homolog of E2-F1) UBE2L1 (1) UBE2L2 UBE2L3

UBCs close related with s.c. UBC4/UBC5 E2-F1 (rabbit)

Other eukaryotic UBCs

E3C (belongs to the cyclosome) E3C

E6-AP

E6-AP E6-AP

ubiquitin p105 ligase E3L E3L

E6-AP

The Ubiquitin System and Proteolysis 21

22

Ubiquitin and Disease

nearly all members of the E3 family.9 The homolog domain is located at the C-terminal region of the E6-AP ligase and includes a completely conserved cysteine residue. Very interestingly, Scheffner et al18 have reported that the E3 proteins involved in substrate recognition also have ubiquitin-binding catalytic activity through a thiolester link on the highly conserved cysteine of the hect domain. Thus, the E3 proteins would participate in a thiolester cascade involving the E1-E2-E3 enzymes, finally transferring ubiquitin to the substrate. In relation to the common mechanism of ubiquitin transfer through the E1-E2-E3 enzymes, it should be noted that some tight complexes between E2 and E3 enzymes have been detected.19,20 The sequences associated with such types of binding have not been identified yet. However, their presence may facilitate the ubiquitin transfer from the E1 initial enzyme to the final substrate. Features Providing Specificity Although the UBC and hect domains (in the E2 and E3 enzymes respectively) suggest common features in the mechanism of ubiquitin transfer, the main role of these enzymes is to provide specificity in the ubiquitin-related pathways, such specificity being necessarily related to their structural differences. On this basis, the structural diversity of the ubiquitinconjugating enzymes has been classified by taking into account their additional regions. Thus, apart from class I enzymes, which are the smallest (consisting mainly of the UBC domain), there are enzymes with either extra C-terminal or extra N-terminal extensions from the core domain, which are called class II and class III enzymes, respectively. Class IV enzymes contain both N- and C-terminal extensions.1 Although all the additional parts of the E2 enzymes may indeed be involved in substrate specificity, at present only the highly acidic C-terminal extensions of the S. cerevisiae UBC2 and UBC3 enzymes (class II) have been shown to interact in in vitro assays with histones.21–23 This interaction has been shown to be functionally important for the ubiquitin transfer activity of the UBC2 and UBC3 enzymes, since the removal of the acidic tails results in reduced ubiquitin conjugation.21–23 Very interestingly, in the case of the S. cerevisiae UBC2 enzyme, the interaction between its acidic tail and histones is related to histonepolyubiquitination and is essential for sporulation.22,23 Furthermore, at sporulation the UBC2 enzyme has pleiotropic functions, one of them being DNA repair. The acidic tail of the S. cerevisiae UBC2 enzyme is not involved in DNA repair, this being reinforced by the lack of the tail in other UBC2 homologs from different species.24–26 Thus, specific sequences other than the acidic C-terminal extension of the UBC2 enzyme may be involved in the substrate specificity related to DNA repair and other functions. Some may also be involved in the interaction with ubiquitin-protein ligases (E3 enzymes). The E3 enzymes, together with specific sequences in the E2 enzymes, are probably the most important factors involved in the proteolytic diversity of the ubiquitin system, and thus in substrate specificity (Table 2.4). They act as substrate-recognizing proteins for the E2 enzymes, providing them with more specificity and a wider range of action. In the case of class I enzymes, which do not have any type of C- or N-terminal extensions, their need for the E3 enzymes seems more important. This has been suggested since it is almost impossible to transfer ubiquitin from the E1 enzyme to test proteins in in vitro conjugation assays.27 Maybe the most widely reported example of collaboration between a class I E2 enzyme and an E3 enzyme is the action of E2-F1 (a rabbit homolog of ubcH7) and E6-AP in the proteolytic degradation of p53. Other ligases have also been described to act with E2-F1 in the degradation of different substrates. In addition to class I enzymes, class II enzymes have also been shown to collaborate with E3 enzymes. This is the case for the UBC2 enzymes, which have been shown to increase their field of action thanks to two of the first known E3 enzymes.15,19,28,29 These are the

N end rule N end rule N end rule N end rule

Degradation signal

fur4p (uracil permease) Gap1 (amino acid transporter)

Substrates

targets possibly related with RNA metabolism

targets possibly related with RNA metabolism

ubiquitin fusion proteins cell cycle control (G2-M transition)

brain development

stress response sporulation endocitosis of membrane proteins

peptide transport

Functions

Other eukaryotic hect domain proteins Drosophila HYD imaginal disc growth and differentiation Mouse Nedd4 Rat p100

murine homolog of Rsp5 human homolog of Rsp5 (hRPF1) S.C.ufd4 S.C.Tom1 S.C.Hct4 S.C.Hct5

S.C.-like hect domain proteins S.C.Npi/Rsp5

UBR1-like S.C.UBR1 E3α (rabbit homolog) E3β (rabbit/close related) S.C.UBR2

E3 ENZYMES

Table 2.4. Main characteristics of E3 enzymes

AtUBC8 ubcH5

AtUBC8 ubcH5

UBC2 UBC2

continued

(1)

Interactions with other ubiquitin system components

The Ubiquitin System and Proteolysis 23

partial proteolytic processing of the p105 precursor of NF-κB

Ubiquitin p105 ligase

(1) Similarity to Rsp5 (2) Mutation lead to Angelman syndrome (3) In Liddle’s syndrome deletion of a binding subunit of the transporter to the Nedd4 protein leads to hypertension (4) It is part of the cyclosome

DNA repair

lysozime mitotic cyclins A and B

exit from mitosis

E3-C (cyclin specific from clam)

E3-like factors RAD18 (yeast)

actine, troponine T, MyoD

Human Nedd4

Other eukaryotic E3s E3-L (muscle specific from rabbit)

amiloride-sensitive Na+ channel

Human E6AP (interacting withE6)

regulation of blood pressure

proposed as DNA binding transcriptional regulator p53

Rat UreB1

Table 2.4. Continued

UBC2

E2F1 ubcH5 E2F1 E2-C ubcH10 E2F1 ubcH5

(4)

AtUBC8 (2) E2F1 ubcH5/ubcH6 ubcH7/ubcH8 ubcH4 (3)

24 Ubiquitin and Disease

The Ubiquitin System and Proteolysis

25

E3-alpha (whose yeast homolog is UBR1) and the E3-beta, from rabbit reticulocytes.30 Whereas the former binds to substrates with N-terminal amino acid residues that are basic or have bulky hydrophobic side chains, the latter recognizes only small uncharged residues at the N-terminus of the substrates. Such recognition of destabilizing N-terminal residues is the basis for protein degradation by the “N-end rule pathway”.31 It has been reported that the E3-alpha enzyme has two separated “head” sites for the binding of the two types of N-terminal residues.32 Thus, multiple substrate binding sites on an E3 enzyme also provide pathways for the proteolytic diversity. This, together with the fact that the number of hect domain proteins is increasing in a progressive manner,9,33 reinforces the very important role of the E3 enzymes in substrate specificity. There are, however, other strategies that confer specificity on the ubiquitin system. A good example is the combined action of heterodimer complexes between different E2 enzymes, as is the case of the UBC6-UBC7 complex involved in the metabolism of the yeast MAT-alpha 2 transcriptional repressor.34 As mentioned above, the C- or N-terminal extensions of the E2 enzymes of classes II, III or IV may also be involved in substrate specificity. They can also function in cellular localization or in the regulation of E2 enzymatic activity. The former has been clearly shown in the UBC6 enzyme which has a hydrophobic (type II) signal-anchor sequence at the end of its C-terminal extension that localizes the protein to the endoplasmatic reticulum, with the active domain of the enzyme facing the cytoplasmic side of the membrane.1 The latter has been implicated in the regulation of the C-terminal extension of the UBC3 enzyme during the cell cycle.14 Concerning its cellular localization, an N-terminal extension of a 500 kDa murine E2 (called UbcM1) of class IV has been shown to have putative transmembrane spans.1

The 26S Proteasome The 26S proteasome is the ATP-dependent multicatalytic protease complex involved in the ubiquitin-related pathways for protein degradation, its estimated molecular mass being between 1,700-2,000 kDa. Like ubiquitin, the 26S proteasome has ubiquitous distribution in all eukaryotes, being present in all tissues.35,36 Also, as a logical consequence of its role in ubiquitin-dependent protein degradation, the 26S proteasome is involved in the same cellular functions as ubiquitin when they are mediating protein degradation. Concerning its cellular localization, this is mainly cytosolic and nuclear.37 The structure/function of the 26S proteasome complex has been analyzed by considering its two main parts. The 20S proteasome has a hollow cylindrical structure with the proteolytic core inside and is only able to hydrolize peptides in an ATP-independent process (see Fig. 2.5). The other part involves the two end/on 19S cap complexes which provide the 20S proteasome with the ability to degrade proteins in an ATP-dependent process.35,36

The 20S Proteasome The 20S proteasome has been named the “multicatalytic protease complex”, owing to its unusual ability to cleave peptide bonds on the carboxyl side of basic, hydrophobic and acidic amino acid residues, such activities being referred to as trypsin-like, chymotrypsinlike and peptidylglutamyl-peptide hydrolase activities.38 Recently two more enzymatic activities have been found to be associated with the 20S proteasome. These are the “branchedchain amino acid preferring” (BrAAP) that cleaves preferentially bonds on the carboxyl side of branched-chain amino acids, and “small neutral amino acid preferring” (SNAPP) that cleaves peptide bonds between the small neutral amino acids Ala-Gly or Gly-Gly.39 In addition, site-directed mutagenesis, inhibitor and structural studies have demonstrated that the active centers consist of N-terminal threonine amino acids on 20S proteasome subunits.40

26

Ubiquitin and Disease

Fig. 2.5. Activation of the proteasome.The structure/function of the 26S proteasome complex has been analyzed by considering its two main parts. The 20S proteasome has a hollow cylindrical structure with the proteolytic core inside, and is only able to hydrolize peptides in an ATPindependent process. The other part involves the two end/on 19S cap complexes which provide the 20S proteasome with the ability to degrade proteins in an ATP-dependent process. The assembly of the 26S proteasome requires Ca2+ and ATP.

The proteasome therefore represents the first described threonine protease. Interestingly, there are three active centers on the yeast 20S proteasome.41 Structural studies on 20S proteasomes have shown that they consist of a cylindrical complex of approximately 700 kDa where numerous low molecular mass subunits ranging from 20 to 35 kDa are arranged in four stacked rings, each containing seven subunits.36 The study of cloned cDNAs from these subunits revealed that the Thermoplasma acidophilum 20S proteasome had the simplest subunit composition with a single type of alpha and beta subunits.42,43 Further comparative studies of the described sequences of the eukaryotic 20S proteasome subunits have classified them as alpha- or beta-type subunits, in order of their sequence homology to the Thermoplasma subunits.36,44 The structure of the Thermoplasma 20S proteasome has been shown to be similar to the eukaryotic 20S proteasomes.45 On these lines, whereas the alpha subunits make up the rings at each end of the cylinder, the beta subunits are placed in the two inner rings, thus forming an α7β7β7α7 assembled complex.45 Based on X-ray crystallography data, the three-dimensional structure of the Thermoplasma proteasome complex is a hollow cylinder of 148 Å in length, 113 Å in diameter and contains a central channel with three large cavities (Fig. 2.6).46 The two outer cavities are placed between the alpha and beta rings. The third cavity is placed at the center of the complex and is formed by the beta rings. Interestingly, the binding of a peptide aldehyde

The Ubiquitin System and Proteolysis

27 Fig. 2.6. Th ree dim en sion al X-ray structure of the Thermo– plasma proteasome. (a) Top view of the 20S proteasome, showing the entrance to the channel. (b) View of the proteasome cut op en alon g th e 7- fold axis. ( c) Sc h em at ic d r awin g of a proteasome cut open showing the three cavities, the gates and the protealytically active sites. Rep r in ted with p er m ission from: Löwe J, Stock D, Jap B et al. Science 1995; 268: 533-539.

28

Ubiquitin and Disease

inhibitor marks the active site in the central cavity at the N-terminus of a beta subunit. Access to the central cavity is restrained by the outer gates. These are formed by the alpha subunits which leave an opening of 13 Å in diameter. Interestingly, phylogenetic studies on many proteasome subunits from different organisms, subdivide both the alpha- and beta-type subunits into seven branches, where only one member from any given organism is found in each branch.36 The only exceptions are the LMP2, LMP7 and MECL1 subunits which are known to replace their constitutive homologs after cellular activation.36,41 With additional immunoelectron microscopic data47 such results suggest that the 20S proteasome is a dimer consisting of two identical subcomplexes, each containing seven different alpha and seven different beta subunits.36 The highly conserved sequences of the alpha subunits are located mainly at the N-terminal regions, the C-terminal regions being more variable.44 In the case of the beta subunits, their most variable regions have been found at the N-terminus.44 Such structural features are related to functional considerations. Probably the conserved regions of alpha and beta subunits could be involved in the assembly of the proteasome, while the variable regions could be involved in the regulation and/or the catalytic processes.48 Concerning the beta subunits, five of the seven beta subtypes are synthesized as proproteins from which peptides have to be cleaved to free the active centers on the N-terminal Thr-1.40,49,50 In relation to the catalytic mechanism, Seemüller et al40 have identified by site-directed mutagenesis that the active center is located in the N-terminal threonine of beta subunits. Furthermore the authors have suggested that proteolysis occurs through the nucleophilic attack of the threonine on the substrate. Such nucleophilic attack may be activated by the amino group of the Thr-1 or by the Lys-33, both of which have been proposed to act as possible proton acceptor-donors. Thus, stripping the proton from the active center would initiate the attack, followed by the return of the proton to the departing amino-terminal group of the cleaved substrate. The functional role of Lys-33 in the catalytic mechanism has been shown to be essential, since mutation of it leads to complete inactivation. In the case of the beta subunit autolysis, the catalytic mechanism may have a certain parallelism.41 Lys-33 is also essential for the autocatalytic cleavage which occurs between Thr-1 and Gly-1. Other highly conserved residues such as Ser-129, Ser-169 and Asp-166, although not essential for catalysis, have been proposed to be important for the beta subunit structure, and may have a substitute role in the mechanism of proteolysis.41 Apart from the catalytic mechanism which may have been highly conserved during evolution, some consideration has to be given to substrate specificity. On these lines, each enzymatic activity seems to be related to specific “binding pockets”. They interact with an amino acid residue near the cleavage site, making the interaction with the N-terminal threonine easier. In addition, another unspecific “binding pocket” may occur around the inner beta ring annulus and may be involved in nonspecific acid or basic hydrolysis.41 This would act together with the former as a second hydrolytic point, generating the eight amino acid fragments result of the proteasome activity.

The 19S Cap Complexes The 26S proteasome has been shown to hydrolize proteins in the presence of ATP, thanks to the addition of two 19S cap complexes, both equal to the 20S proteasome.35,36 At least in cellular extracts, such complexes have been shown to be free or assembled with the 20S proteasome in an ATP-dependent process.51,52 They have also been referred to as ATPase or PA700 complexes, since they have ATPase activities36 or act as activators for the proteasomedependent protein proteolysis.51,52 The name “19S cap complex” comes from its location in the ultrastructure of the 26S proteasome.35

The Ubiquitin System and Proteolysis

29

Electron microscopy and digital image analysis studies carried out on negatively stained 26S proteasomes have shown that their structures consist of a central 20S proteasome cylinder to which two additional cap-shaped substructures are attached at both ends.35 Such substructures, which correspond to the 19S cap complexes, have been reported to have a dumb-bell shaped-like appearance. In addition, they seem to be highly asymmetrical and appear roughly V shaped, with some distinct protein mass intruding into the cleft of the V. They are bound to the 20S proteasome in opposite orientations (trans configuration), reflecting the dimeric organization and the two-fold symmetry of the 20S proteasome. Concerning the composition of the 19S cap complexes, they have been shown to be made up of at least 15 different subunits with molecular masses ranging from 25-110 kDa.36 Many of them have been cloned and sequenced and their enzymatic activities have been characterized. Subunits involved in the recognition of ubiquitinated proteins have also been identified, together with a subunit involved in the release of ubiquitin from the protein remnants after proteolysis. The former are important because they bind the proteasome substrate and may introduce certain specificity into the degradation process. The latter is important since it may have a role in the control of the proteolytic rate, as will be discussed later. In addition, among the 13 identified subunits in the S. cerevisiae 26S proteasome, six have been shown to be ATPase subunits, based on their sequence similarity with a novel family of ATPases, called AAA-ATPases.36 Their role seems to be associated with the ATPdependent association of the 19S cap complexes with the 20S proteasome. However, they are also believed to act by unfolding and transporting the protein substrates into the 20S proteolytic core. This hypothesis is based on several points. Firstly, in vitro degradation studies with the Thermoplasma 20S proteasome have clearly demonstrated that a protein must be unfolded and transported through the outer narrow gate to the inner compartment, in order to be degraded by the complex.53 Secondly, a parallelism clearly exists between the ATPases of the 19S cap complex and the ATP-dependent chaperones ClpA and ClpX involved in the activation of the ATP-dependent ClpP protease of Escherichia coli.54 The molecular mechanism of unfolding and transport is not yet understood, but it has been suggested that the high number of ATPases found in the 19S cap complex may provide alternative binding sites for particular proteins, thus making the unfolding and translocation of different proteins easier.55

The Deubiquitinating Enzymes The deubiquitinating enzymes, also known as ubiquitin C-terminal hydrolases (see Table 2.5), are involved in the hydrolysis of the linkage between the C-terminal glycine of ubiquitin and various adducts.30 These enzymatic activities are mainly involved in the release of ubiquitin from byosynthetic precursors, such as the polyubiquitin linear chains and the ubiquitin fusion proteins, or in the recycling of ubiquitin from conjugated proteins (isopeptidase activity) before or after proteolysis.30 In addition, they have been suggested to act on incorrectly ubiquitinated proteins or abnormal polyubiquitin chains in order to prevent their interference in protein degradation.56 Another important role of these enzymes seems to be the maintenance of the pool of free ubiquitin in front of intracellular nucleophiles such as glutathione or polyamines, which would rapidly deplete it by reacting with the E1-ubiquitin or the E2-ubiquitin thiol esters.56 Finally, it has to be remembered that ubiquitination does not always have a degradative role. Ubiquitin conjugation is also believed to modulate protein functions through changes in protein structure.30 Thus, the H2A and H 2B histones have been shown to be norm ally ubiquitinized and to becom e deubiquitinated during mitosis.57 The deubiquitinating enzymes have been arranged into two distinct classes: one consisting of a set of relatively small molecules which cleave ubiquitin from small peptides and

null mutants phenotypically normal

Functions

Tre17 (human homolog) Unp (murine homolog) S.C.Ubp5

S.C.Ubp2 S.C.Ubp3 S.C.Ubp4/Doa4

The UBP family S.C.Ubp1(93kDa)

null mutants phenotypically normal

null mutants phenotypically normal null mutants phenotypically normal DNA repair cellular growth stress response sporulation vacuolar biogenesis

null mutants phenotypically normal

30 kDa enzyme from calf thymus purified by Wilkinson

Yuz related S.C.Yuz (26kDa) Rat PGP9.5 Bovine PGP9.5 Human PGP9.5 (26 kDa) Bovine enzyme 54% identical to PGP9.5 (30 kDa)

ENZYME

ubiquitin fusion proteins

ubiquitin fusion proteins isopeptide linked ubiquitin conjugates MAT-alpha2

part of the 26S proteasome

ubiquitin fusion proteins: like polyubiquitin or fused to ribosomal proteins ubiquitin fusion proteins ubiquitin fusion proteins

(2) (2)

(1)

Interactions with other ubiquitin system components

ubiquitin fused to small compounds, possibly to metallothionein, the 52 amino acid human ribosomal extension protein and histone2A

cleaves ubiquitin fused to small proteins

Substrates

Table 2.5. Main characteristics of the ubiquitin-C-terminal hydrolases

30 Ubiquitin and Disease

unanchored ubiquitin chains

unanchored ubiquitin chains

ubiquitin fusion proteins

ubiquitin fusion proteins

required for normal eye development early growth response gene induced by IL-3 ubiquitin fusion proteins early growth response gene induced by IL-2

stress response sporulation turnover MAT-alpha2 and other proteins stress response sporulation turnover of MAT-alpha2 and other proteins null mutants phenotypically normal

mutants with slight growth defect null mutants phenotypically normal mutants with defect in chromatinmediated gene silencing

(1) All PGP9.5 are neuronal-specific. (2) Protooncogen. (3) Promotes proteolysis. (4) Inhibits proteolysis. (5) Its gene (located in X chromosome) has been suggested to be involved in a type of blindness associated to defects in protein turnover.

Other eukaryotic hydrolases Hydrolase highly expressed in retina UCH1 to UCH10 from chick muscle

Other eukaryotic Ubps Faf (Drosophila) DUB1 (human) DUB2 (human)

S.C.Ubp15 S.C.Ubp16

Isopeptidase T (human homolog)

S.C.Ubp11 S.C.Ubp12 S.C.Ubp13 S.C.Ubp14

S.C.Ubp6 S.C.Ubp7 S.C.Ubp8 S.C.Ubp9 S.C.Ubp10

(5)

(4)

(3)

(3)

The Ubiquitin System and Proteolysis 31

32

Ubiquitin and Disease

intracellular nucleophiles (e.g. Yuh1 in yeast) and the other by a group of larger proteins which cleave ubiquitin from a range of protein substrates.55 This latter group of enzymes is the so-called Ubp (ubiquitin-specific processing protease) family which is characterized by several short highly conserved sequences probably involved in the deubiquitinating active center of the enzymes. Among these sequences the most common are the Cys and His boxes. Other short sequences are also relatively conserved. Ever since the beginning of ubiquitin system-related research deubiquitinating enzymes have been studied and several possible functions have been suggested. It is only recently, however, that some investigations have demonstrated a key role for the deubiquitinating enzymes in the regulation of the ubiquitin system. The search for Ubp-like sequences in different protein sequence databases has revealed a large family of Ubp-like proteins. In yeast, Hochstrasser 55 has identified up to 16 Ubp proteins, this exceeding the number of the identified E2 enzymes in the same organism. Such a high number of Ubps suggests a high degree of protein specificity, this probably being related to the control of specific protein turnover. In addition, this high number of Ubps may be related to the diverse functions proposed for the deubiquitinating enzymes. The study of some of them has suggested that they may control the activity of the ubiquitin system in two different ways, either by having an inhibitory effect on proteolysis or by having a promoting effect.55 The negative regulation of the ubiquitin system has been suggested by the studies carried out with the Drosophila fat facets ( faf) gene, which codes for Ubp enzyme.58 This gene is required for normal eye development, and mutations in the Cys and His boxes of the gene behave as n u ll m utation s in tr an sgen ic flies, su ggestin g the cr itical role for the deubiquitinating activity. In addition, different mutants in a 20S proteasome subunit have been shown to suppress the defect in eye development seen in faf flies. Thus, these data have suggested that faf may function by reversing a specific ubiquitination and consequently preventing or slowing its degradation by the proteasome. Considering that the ubiquitin conjugation step is highly dynamic, with the rapid addition and removal of ubiquitin subunits, it could be expected that changes in the activity of certain deubiquitinating enzymes would modulate the dynamic balance between ubiquitination and deubiquitination.55,59 The positive regulation has mainly been suggested by the studies carried out with the mammalian isopeptidase T60–62 or the Doa4 deubiquitinating enzyme.63 The promoting activity of proteolysis is based on the removal of molecules with inhibitory potential against the proteasome. The mammalian isopeptidase T and its yeast functional homolog Ubp14 are both Ubp enzymes that act preferentially on unanchored ubiquitin chains. Such chains can be directly synthesized by a number of E2 enzymes or generated in the course of substrate proteolysis. Interestingly, a yeast mutant lacking the Ubp14 enzyme has been shown to have defects in protein degradation and to accumulate unanchored ubiquitin chains. Based on this, such unanchored chains may act as a negative control for proteasome action. In contrast, an increase in the activity of the isopeptidase T or the Ubp14 enzyme would be expected to promote proteolysis by preventing proteasome inhibition. The other known deubiquitinating enzyme that has a promoter role in proteolysis is the yeast Doa4 enzyme.63 This enzyme is part of the 26S proteasome and it is believed to act by deubiquitinating the peptides that remain after proteolysis. This has been suggested since small ubiquitinated species that are slightly larger than unanchored ubiquitin chains accumulate in Doa4 mutants. As in the case of the unanchored ubiquitin chains, the ones linked to the proteolytic remnants are also believed to inhibit the proteasome activity by a negative feedback mechanism. In contrast, Doa4 is believed to promote proteolysis by removing such inhibitor molecules. In fact, this promoting activity has been clearly demonstrated by the elimination of Doa4 from yeast cells, which stabilizes the degradation of different proteins to different degrees. The importance of Doa4 in the control of the proteolytic activity

The Ubiquitin System and Proteolysis

33

of the proteasome has also been demonstrated, with changes in its cellular levels having been shown to be partially rate-limiting in the degradation of the MAT-alpha 2 repressor. In addition to the above data, the involvement of deubiquitinating enzymes in pathological states reinforces the suggestion that deubiquitination has a key role in the control of the ubiquitin system. A high degree of similarity between the deubiquitinating enzyme Doa4 and the human oncogene tre2 has been found.63 This oncogen is a sequence from Ewing sarcomas that can cause tumors in a nude mouse assay.64 Furthermore, sequences homologous to Doa4 have been shown to be derived from the human Tre17 gene located in chromosome 17.65,66 This gene is transcribed into at least two alternatively spliced mRNAs. One of them has no Ubp activity and its over expression induces transformation in nude mice.65 Thus both the tre oncogene product and the Tre17-transforming protein may act as dominant negative variants that would interfere with the normal function of Tre17.67 Interestingly, the involvement of Doa4-like activities in cancer development has been reinforced by the characterization of other homologous genes, such as the murine unp gene68,69 and its human homolog hunp.70 This latter gene is over expressed in small cell lung tumors and is located within a chromosomal region 3p21.3 which is known to be rearranged in these tumor cells.

The Yeast Ubiquitin System Components and Their Eukaryotic Homologs Ubiquitin-activating Enzymes (E1) UBA1: The most studied ubiquitin activating enzyme is a homodimer formed by two UBA1 proteins in S.cerevisiae.71 This UBA1 gene has been cloned and shown to encode a 114 kDa protein with the typical n ucleotide bin din g con sen sus sequen ce Gly-X-Gly-X-X-Gly of the E1 enzymes.5 In addition, the UBA1 protein has been purified.71 The importance of the E1 formed by UBA1 proteins is reflected by the fact that its deletion in yeast is lethal.5 In addition, its activating activity is believed to be needed for almost all known ubiquitin transfers. In fact, the UBA1 protein or its rabbit homolog has been used in many in vitro ubiquitin-transfer assays. Furthermore, the use of temperature-sensitive mutants obtained from possible mammalian homologs of UBA1 has shown that ubiquitin activation is linked to most of the known ubiquitin mediated functions. For example, with these mutants at nonpermissive temperatures a lack of ubiquitin activation results in the arrest of the cell cycle72-74 and failure to degrade short-lived proteins or to degrade abnormal proteins.75,76 In addition to the UBA1 gene, other possible eukaryotic homologs have been cloned from wheat,77 mouse73,78 and humans.79-81 Three genes named UBA1, UBA2 and UBA3 have been isolated from wheat.77,82,83 Whereas the amino acid sequence of wheat UBA2 is nearly identical to UBA1, the sequence of UBA3 is significantly different. Concerning the mouse homologs, mapping data have indicated that the mouse ube/AIS9x is located on the X chromosome,84 while sby/AIS9Y-I is linked to the Y chromosome, identified as a candidate spermatogenic gene.85,86 A human UBE1 ( UBE1L) gene has been shown to have a high degree of identity to UBA1 and to be located in the 3p21 chromosomal region (X-linked).79,87 This gene is well expressed in normal lung tissue, but shows little or not expression in lung cancer-derived cells.88 UBA2: The UBA2 gene has been characterized and shown to encode a 71 kDa protein which shares with the UBA1 gene the nucleotide-binding consensus sequence and the conserved Cys residue believed to form a thiolester with ubiquitin.89 The UBA2 protein is located in the nucleus whereas the UBA1 is located both in the nucleus and the

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cytoplasm. UBA2 has also been shown to be essential for life since it is required for cell viability.89 However, UBA1 and UBA2 cannot complement each others essential functions even if their protein subcellular localizations are altered by mutagenesis.89 Thus, they may have different functions. UBA3: The UBA3 gene encodes a 33 kDa protein and shares the highly conserved sequences involved in ubiquitin activation. Its function is still unknown. Two genes which may represent a new family of ubiquitin activating enzymes have been cloned from humans90 and A. thaliana.91 They are located in the plasma membrane and are involved in the response to first messengers such as growth factors or hormones. In humans the protein has a molecular mass of 59 kDa and has been named APP-BP1 (from amyloid precursor protein-binding protein).90 This protein is closely related to the metabolism of the amyloid precursor protein (APP) and consequently with Alzheimer's disease (see chapter 3). This protein, which has E1 features, is 61% similar to the protein encoded by the A. thaliana AXR1 gene required for normal response to the hormone auxin.90 Although ubiquitin activation has been supposed to be the least specific step in the ubiquitin system pathway, UBA1-like proteins are constantly being described, suggesting some specificity in the activation process. In addition, Hochstrasser 55 has suggested that an increasing number of E1 enzymes may also be related to the activation of ubiquitin-like proteins since they are believed to need ubiquitin activation which is poorly sustained by the UBA1 related E1 enzyme. The best studied ubiquitin-like protein is the mammalian ubiquitin cross-reactive protein UCRP.92

Ubiquitin-conjugating Enzymes (E2) UBC1: This is a class II enzyme which is known to have a role in cellular growth, in the stress response,93 and in the endocytosis of membrane proteins such as receptors and transporters.55 Interestingly, the UBC1 enzyme acts together with the UBC4 and UBC5 enzymes in these cellular functions,93,94 since a single gene is dispensable, and only the deletion of all three is inviable.93 On the basis of this functional overlap, it would not be strange to find substrates ubiquitinated by the three enzymes. In fact, this has been reported for the degradation of abnormal proteins in the stress response and for the ubiquitination involved in the endocytosis of yeast membrane proteins such as the mating-pheromone ste2 receptor.95 Something similar has been suggested for the uracil permease,33 the general amino acid transporter Gap1,33 the ste6 protein 96 and the multidrug transporter Pdr5,97 in the activation of their endocytic pathway. In addition, the UBC1 enzyme has a specific role in the cell cycle after germination of ascospores of S. cerevisiae.93 UBC2: This is a class II enzyme involved in DNA repair, sporulation, repression of retrotransposition, and peptide transport through the “N-end rule” pathway.55 The involvement of the UBC2 enzyme in DNA repair was suggested after the identification of its gene sequence, which is identical to yeast DNA repair RAD6 gene.98 It has a central role in one of the three gene groups involved in the yeast DNA repair (see ref. 99 for a review). Mutants in the UBC2/RAD6 gene produce diverse phenotypes related to impaired DNA repair.1 The role of UBC2 in DNA repair is also suggested by the fact that there is an increased expression of the UBC2 gene in response to DNA-damaging agents.100,101 Functional homologs of UBC2 for DNA repair have been cloned from Schizosaccharomyces pombe,24 Drosophila25 and humans.26,102 However, they are class I enzymes without the acidic C-terminal extension of UBC2. Thus, this extension is not necessary for DNA repair.24–26 Conversely, the interaction between UBC2 and RAD18, a protein involved in the recognition of DNA damaged regions, has been reported to be necessary for activation of DNA repair.103

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Concerning the function of UBC2 in sporulation, it has been demonstrated that the enzyme undertakes its function through histone polyubiquitination.22 This is mediated by the highly acidic tail of the UBC2 enzyme, as indicated by the occurrence of RAD6 mutants which lack the highly acidic tail and are defective in sporulation and also in histone polyubiquitination. Further evidence of this is that the addition of the first four residues of the acidic tail in the RAD6 mutants restores both histone polyubiquitination and sporulation.23 However, the involvement of the acidic tail in sporulation is not a ubiquitous mechanism since the S. pombe UBC2 homolog is effective in sporulation but lacks the C-terminal extension.24 Another functional feature of the UBC2 enzyme is the repression of retrotransposition, this being related to transcriptional events. Eukaryotic retrotransposons are defective retroviruses in the envelope sequence which translate from one genome position to another. They are mediated through RNA intermediate which is believed to use reverse transcriptase to m ake a DNA copy to be in tegrated in to an other poin t. In addition , retrotransposons affect the transcription of certain genes. For example, when the delta elements (equivalent to the retrovirus LTR sequence) of the yeast Ty transposon are inserted into the promoter region of the LYS2 gene, a negative effect on its transcription is observed.104 Interestingly, this negative effect is reversed by over- or underexpression of histones,104 probably ubiquitinated, since RAD6 mutants have been shown to avoid this reversion.105 Although it is still a controversial field, these results suggest a role for UBC2 in determining the specificity of transcriptional initiation through changes in the chromatin structure, thanks to histone ubiquitination. Several indirect observations have also suggested that ubiquitinated histones may be preferentially enriched in transcriptionally active regions of the chromatin.106 Given that the UBC2 enzyme is the major ubiquitin-conjugating enzyme involved in mono- and polyubiquitination of histones,22,98 the involvement of these activities in UBC2 functions should not be regarded as strange. Finally, the UBC2 enzyme is also able to conjugate polyubiquitin with different substrates, depending on their N-terminal unstabilizing amino acid (“N-end rule” pathway).19,31 In such cases, the ubiquitin transfer by the UBC2 enzyme needs the cooperation of the yeast ubiquitin ligase UBR1.19,31 Although yeast mutants lacking the UBR1 gene have no significantly different phenotypes,107 it is known that the UBC2-UBR1 activity has an important role in peptide transport.55 This and other yet unknown functions of the UBC2-UBR1 activity may overlap with the activities of other ubiquitin-conjugating enzymes. Homologs for the DNA repair function of the U BC2 gene have been cloned from S.p ombe,24 Drosophila25 and humans.26,102 A family of two human homologs for DNA repair have been named as hHR6A and hHR6B.108 Another DNA repair gene, the UbcH1 shows a 69% identity with RAD6.109 A mouse homolog of hHR6B has been shown to be involved in fertility.110 A RAD6 homolog of 14 kDa has been characterized in rabbit muscle.111 In A.thaliana the UBC2 homologs constitute a family of three genes named AtUBC1, AtUBC2 and ATUBC3.112 AtUBC1 is a 16 kDa enzyme.10 UBC3: This is a class II enzyme involved in the control of the cell cycle and DNA replication.1,55 It is characterized by a highly acidic C-terminal extension, which is even larger than the UBC2 extension. As with the UBC2 enzyme, the acidic tail mediates ubiquitin conjugation to histones in vitro.21 These ubiquitin conjugations may be closely related to the known UBC3 functions, but their role is still not completely understood. They may possibly be related to chromatin changes. The identification of the sequence of the UBC3 gene has shown that it is identical to the cdc34 gene,113 therefore involving it in the control of the cell cycle. It is known that the UBC3 enzyme takes part in its control through the degradation of Cln cyclins and suppressor proteins such as p40, which allow the transition from G1 to S phase.21,113 In addition,

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the enzyme has been reported to mediate the spindle pole body separation. Its involvement in the degradation of the transcriptional factor Gcn4 (involved in the synthesis of amino acids and nucleotides) also may be related to changes in the cell cycle during fasting situations. A functional homolog of UBC3 has been localized in the far telomeric region of the human 19p13.3 chromosome.114 UBC4 and UBC5: These are almost identical class I enzymes.1 Based on their observed mutant phenotypes94 and some of their reported substrates,1,36,55 they are involved in cellular growth, stress response and sporulation. Concerning their involvement in cellular growth, the UBC4 enzyme is able to degrade Clb cyclins in vitro. Cyclins are related to the control of cell cycle progression. Concerning the stress response, both the UBC4 and UBC5 genes have heat-shock regulatory elements in their promoter regions and are induced by heat-shock and cadmium.94 In addition, both enzymes have been shown to participate in the degradation of abnormal proteins generated in cellular stress situations.94 In addition to the substrates related to cellular growth or the stress response, UBC4 and UBC5 enzymes participate in the degradation of the yeast MAT-alpha 2 repressor.34 They also have a role in the endocytosis of different types of membrane proteins. In such cases, simply the ubiquitination of the proteins (without any kind of protein degradation), enhances their endocytosis, thus leading them to the endolysosomal pathway. In yeast, both UBC4 and UBC5 mediate the endocytosis of the mating-pheromone ste2 receptor,95 the ste6 protein 96 and possibly the uracil permease,33 the general amino acid transporter Gap1,33 and the multidrug transporter Pdr5.97 Concerning functional homologs in A. thaliana, a family of five genes (known as AtUBC8 to AtUBC12) has been characterized as being homologous to UBC4. These genes are not induced by heat shock.115 In addition, the product of the AtUBC8 gene has been shown to participate in the degradation of the p53 protein, collaborating with the E6-AP ubiquitin ligase.116 This protein is known to halt the cell cycle and induce DNA repair when DNA damage is detected. In Caenorhabditis elegans the Ubc2 gene has a high degree of amino acid identity and complements the function of UBC4 and UBC5.117,118 In Drosophila the UbcD1 gene is functionally equivalent to UBC4 and UBC5.119 Furthermore, in the rat, an UBC4 homologous gene has been characterized and one isoform is only expressed in testicles.120,121 Two human homologs of the UBC4- UBC5 family have been cloned. The UbcH5 and UbcH6 enzymes have been characterized and have been shown to participate in the degradation of p53 in collaboration with E6-AP.116,122 Given that stress situations may produce DNA damage, it would seem likely that p53 turnover is controlled by the family of UBC4 and UBC5 homologs, either during the stress response or simply in situations of DNA damage. Three types of ubcH5 (A, B and C) have been identified.123 The Ubc H5 has been implicated in the degradation of c-jun and c-fos,124 which are known to form a heterodimer transcriptional activator involved in the early-growth response.125 Very probably, the same homolog also participates in the proteolytic processing of the p105 precursor of the mammalian NF- κB p50 subunit.126 Considering the very wide field of action of both the UBC4 and UBC5 enzymes, future research will, no doubt, concentrate on finding different types of signals involved in specificity. On these lines, different isoforms for these enzymes have been detected in A. thaliana115 and humans.116,123,127 In addition, they have been shown to act with the E6-AP ubiquitin ligase in the degradation of the p53 protein 116,123,127 and with a new ligase involved in the processing of p105 precursor of NF- κB.126 They are also involved in ubiquitination related to specific phosphorylations (for membrane receptors or transcriptional factors) and to specific protein sequences (such as the destruction boxes in clb cyclins). Therefore, the un-

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37

derstanding of the signals involved in specificity is at an early stage and is a promising field of research. UBC6: This is a class II enzyme with a C-terminal extension bearing a signal anchor sequence which inserts the protein into the membrane.1,128 The enzyme has been localized in the endoplasmic reticulum membrane and possibly in the nuclear envelope, with the catalytic UBC domain facing the cytoplasmic compartment.1,128 It belongs to a family of proteins involved in the secretory pathway. Its over expression (and to a lesser degree UBC4 over expression) restores the phenotype of the yeast secretion mutant sec61.1 The Sec61 protein belongs to a multisubunit complex required for translocation of proteins across the endoplasmicr eticulum me mbrane.129-131 Thus, it has been hypothesized that UBC6 (and UBC4) may be involved in the degradation of unprocessed protein precursors that are not translocated through the rough membrane and which may be toxic for the cell.1 It has also been suggested that UBC6-like proteins may be involved in the turnover of membrane proteins such as the cytochrome P450 2E1.132 In addition to being involved in the metabolism of membrane proteins, the UBC6 enzyme may also participate in the control of transcriptional factors as is the case for the UBC4/UBC5 enzymes. It has been reported that a UBC6-UBC7 complex is involved in the degradation of the yeast MAT-alpha 2 repressor.34 At least two different signals for its degradation have been identified.1 Interestingly, the UBC4/UBC5 enzymes have been shown to participate in the degradation of this transcriptional repressor. This functional involvement may be extended to other transcriptional factors and nuclear proteins in their transit through the nuclear pore, as suggested by Jentsch.1 UBC7: This is a class I enzyme whose gene sequence predicts a protein of 18 kDa. Its functions have been shown to be closely related to the stress response and the control of transcriptional factors.1,55 In the first case, studies carried out with UBC7 mutants have shown that the enzyme is involved in the stress response induced by cadmium but not by heat shock.1 Considering possible links with disease, the stress induced by cadmium may represent any type of heavy metal or chemical stress produced by physiological imbalances with toxic consequences. Interestingly, cadmium induces the expression of the UBC4, UBC5 and UBC7 enzymes,133 and therefore one should bear all of them in mind in connection with this type of toxicity. The UBC7 enzyme is also involved in the control of transcriptional factors. For example, the UBC6UBC7 complex participates in the degradation of the yeast MAT-alpha 2 repressor.34 The human UBE2G homolog with a high degree of amino acid identity with UBC7 of both S. cerevisiae and C. elegans.134 Interestingly, it is highly expressed in human skeletal muscle. UBC8: This is a class II enzyme whose gene has been cloned and the protein predicted to have a molecular mass of 25 kDa. No phenotypic defects have been found in null mutants. Thus, its functions may overlap with other ubiquitin conjugating enzymes.1,55 In A. thaliana a family of three UBC8 homologs (named AtUBC4, AtUBC5 and AtUBC6) has been identified.112 In humans a UBC8 homolog, called UbcH2, has been cloned, characterized and located on chromosome 7.135 UBC9: This is a class I enzyme whose gene predicts a molecular mass of 18 kDa. Its function seems to be related to coordinating the transition from G2 to mitosis in the cell cycle, through the degradation of Clb cyclins and with the collaboration of certain still uncharacterized ubiquitin ligases (see chapter 4).1,55 Thus, deletion of the UBC9 gene leads to G2/M phase arrest in S. cerevisae.1,55 Interestingly, a human homolog of UBC9 called UbcH9 has been cloned and characterized. It codes a 17 kDa protein and is located in the 16p13.3 chromosomal region.136,138 Another human homolog

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named UBE2I has been identified and shown to interact with RAD52 and UBL1 (ubiquitin-like proteins) and also with p53.139 UBC10: This gene has been cloned and indicates a 21 kDa protein. It has also been identified as the PAS2 gene, which is involved in peroxisome biogenesis by a still unknown mechanism. UBC11, 12 and 13: These genes have been cloned. Predicted protein sizes are 17, 21 and 18 kDa respectively. Their functions have not been determined. Other Eukaryotic E2 Enzymes not Homologous with S. cerevisiae In rabbit reticulocytes an E2 purified from a new fraction called E2-F1 has been shown to participate in the degradation of p53 in collaboration with the E6-AP ligase.140,141 Its human homolog ( UbcH7) has been cloned and characterized.122 It has also been shown to degrade the p53 protein in collaboration with the E6-AP ligase.122 Taking into account that the p53 protein is a transcriptional factor involved in the arrest of the cell cycle and activation of DNA repair, its degradation by these enzymes not only suggests a possible role of the enzyme in transcriptional events but also in DNA repair. UbcH8 has also been cloned and shown to belong to the same family.142 Finally, the human genes known as UBE2L1, UBE2L2 and UBE2L3, which are localized on chromosomes 14, 12 and 22 respectively, are believed to be homologous to the same family and probably involved in the degradation of p53, c-fos, and the maturation of the NF- κB,143 like E2-F1. Interestingly, the UBE2L1 has been proposed to be the FAD3 gene involved in the development of Alzheimer’s disease.144 E2-C was purified and cloned from clam oocytes as a cyclin selective ubiquitin-conjugating enzyme involved in the degradation of mitotic cyclins A and B and it is required for the completion of mitosis.145 The E3-C ubiquitin ligase, which is a part of the cyclosome, also participates in this process. Its human homolog has recently been cloned and named UbcH10.146 Concerning other eukaryotic E2s, the Drosophila bendless gene encodes a new E2 enzyme involved in the synaptic connectivity between a subset of central nervous system neurons.147 Other reported eukaryotic E2 enzymes are three different class III enzymes (with N-terminal extension, but no C-terminal extension), which have been cloned from mouse and Drosophila.1 In addition, an E2 of 230 kDa which seems to be an E2-E3 hybrid has been identified in rabbit reticulocytes.148 The UbcM1 murine ubiquitin-conjugating enzyme has been characterized as a membrane protein. Its gene sequence seems to encode a 500 kDa enzyme with several putative alpha helical transmembrane spans and potential N-linked glycosylation sites. Structural predictions also suggest that the UBC domain may face the cytosol as in the case of the UBC6 enzyme. The enzyme has been localized in the Golgi network and possibly the endoplasmic reticulum.1 Finally, and very recently, by means of differential mRNA display, Thomson and collaborators149 have isolated a family of inactive variants of the E2 enzyme (UEV: ubiquitinconjugating E2 enzyme variant proteins). These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeast to mammals. These proteins may be involved in the control of differentiation and could exert its effects by altering cell cyle distribution.

Ubiquitin-protein Ligases (E3) Ubr1: The gene has been cloned from S. cerevisae and encodes a 225 kDa protein.55,107 It forms a tight complex with the UBC2 enzyme19 and is able to recognize substrates through specific N-terminal residues, which are basic (Arg, His, Lys) or bulky hydrophobic (Leu, Phe, Trp, Tyr) amino acids.107 The Ubr1 protein has also been shown

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39

to form a thiolester link with ubiquitin in the process of ubiquitin transfer.59 Yeast mutants lacking the Ubr1 gene do not have a significant phenotype.107 Recently, however, a functional involvement of Ubr1 in peptide transport has been reported.150 The E3-alpha ubiquitin-protein ligase from rabbit reticulocytes is the functional homolog of the Ubr1 protein. It was initially partially purified by affinity chromatography and estimated to have an apparent molecular mass of 350 kDa composed of two 180 kDa subunits.151,152 The E3-alpha enzyme was originally described as recognizing proteins through their basic (Arg, His, Lys) or bulky hydrophobic (Leu, Phe, Trp, Tyr) N-terminal amino acids. These two types of protein substrates were called type I and type II and were shown to interact with E3-alpha at different sites. A third type of protein substrate which was neither basic nor hydrophobic was also shown to interact with the enzyme.30 In addition to the E3-alpha enzyme, another ubiquitin-protein ligase called E3-beta has been purified from rabbit reticulocyte extracts where the former was removed.153 This enzyme recognizes subtrates through their N-terminal residue, but through Thr, Ser and Ala (type III substrates) in this case. In spite of their different substrate specificities, the E3-alpha and E3-beta enzymes seem to be close related. Ubr2: This protein of 217 kDa has only been identified in S. cerevisiae with a 22% identity to Ubr1. Its functions have not been determined. Hect Domain Proteins The family of hect domain ( H omologous to the E6-AP Carboxy Terminus) proteins are a group of ubiquitin ligases characterized by the presence of a domain originally present in the human E6-AP protein ligase. This domain is essential for ubiquitin-thiolester formation. Interestingly, near 40 different genes have been identified as having this domain.9 Npi/Rsp5: The gene sequence predicts a 92 kDa protein which may have a Ca2+ -dependent phospholipid interaction motif in its N-terminal region.33,55 This ubiquitin-protein ligase is essential for viability, stress response and sporulation.154 Some of these functions are probably carried out together with some of the enzymes of the UBC4UBC5 family since it has been shown to interact with AtUBC8 and ubcH5.9 Interestingly, the Npi1/Rsp5 protein has a homolog both in humans and in mice, the latter being involved in brain development.33 Ufd4: The gene encodes a 168 kDa protein which degrades ubiquitin fusion proteins. Tom1: The gene encodes a 374 kDa protein which is essential for viability. This protein is involved in the transition from the G2 to the M phase of the cell cycle. Hct4 and Hct5: The genes encode 103 kDa and 168 kDa proteins respectively. Other Eukaryotic Hect Domain Proteins: E6-AP: The gene has been cloned and the purified protein has an apparent molecular mass of 100 kDa.9,155 The E6-AP protein is a human ubiquitin-protein ligase known to be involved in the degradation of the p53 protein. The p53 protein inhibits the cell cycle and induces DNA repair, thus preventing the perpetuation of DNA alterations that may lead to cancer development.156 The importance of the p53 protein turnover in cancer development was first suggested by the studies carried out with the oncogenic human papillomavirus.157 In these studies, cancer development was found to be closely related to increased p53 protein degradation, which was mediated by the human papillomavirus E6 protein. We now know that the complex formed by the E6 protein with the E6-AP ubiquitin-protein ligase binds to the p53 protein, triggering its ubiquitin-dependent protein degradation.157 From a mechanistic point of view, the E6-AP ubiquitin-protein ligase forms a thiolester link with ubiquitin in the process of ubiquitin transfer to the substrate.13 In contrast with Ubr1, its binding

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to the E2s enzymes is weak. Interestingly, the E6-AP ligase induces the degradation of p53, acting either with the UBC4/UBC5 family of ubiquitin-conjugating enzymes or with the E2F1 family E2 enzymes. The E6-AP ligase may be involved in cancer development and also in other DNA repair and stress-response related diseases. Matsuura et al158 reported that the Angelman syndrome gene associated with deletions in the human chromosome 15q11-q13, corresponds to the human E6-AP gene called UBE3A . In addition to the relevance of E6-AP in disease, the discovery of the E6-AP ubiquitin-protein ligase has been the starting point in the expansion of the family of ubiquitin-protein ligases,102 this reinforcing their hypothetical role in providing specificity to the ubiquitin-mediated proteolysis. In addition to E6-AP ligase, many other ubiquitin ligases are present in a wide spectrum of animals ranging from Drosophila to humans. Other Eukaryotic Ubiquitin-protein Ligases E3L: This has been purified and is a homodimer of an apparent molecular mass of 550 kDa.159 It is different from other known E3s, such as E3-alpha/Ubr1, E3-beta and E6-AP. Interestingly, this ligase seems to be involved in the degradation of muscle proteins since it shows substrate specificity for actin, troponin T and MyoD.159 It acts through a non-“N-end rule” pathway and in concert with the E2-F1 enzyme. The E3L ligase is also involved in the degradation of typical “N-end rule” substrates (such as lysozyme) through different signals than the N-terminal residue. Although this enzyme has been found in different mammalian tissues, it may be of interest in studies on muscle disorders and/or physiology related research. E3-C: This protein is involved in cell dependent conjugation and degradation of mitotic cyclins in conjunction with E2-C (from clam oocytes) or the human ubcH10.146 The ligase is a component of a large particle (approx. 1,500 kDa), the cyclosome160 or anaphase-promoting complex. Ubiquitin p105 ligase: This 320 kDa ligase isolated from rabbit reticulocytes is implicated in the partial processing of p105, the precursor of the p50 subunit of the transcription factor NF- κB.126 This molecule is a cytosolic protein heterodimer, composed of 50 and 65 kDa subunits, normally inactivated through their binding to inhibitory proteins (I- κB). Activation of NF- κB involves dissociation from the I- κB proteins. Then it is translocated to the nucleus and binds to specific κB-specific DNA sequences activating gene transcription. Either E2F1 or ubcH5 participate in conjunction with this ligase in the processing of the p105 precursor protein. RAD18: This is another protein that binds (it constitutes an E3-like factor) to DNA damaged regions and to the UBC2/ RAD6 enzyme,161 probably acting as an E3-like factor in DNA repair. Certain heat-shock proteins which are believed to facilitate the degradation of misfolded proteins have also been suggested to act as E3-like factors.162

Ubiquitin C-terminal Hydrolases Yuz Related Ubiquitin C-terminal Hydrolases: This class of ubiquitin C-terminal hydrolases has apparently been highly conserved during evolution, Yuz being the yeast form of a large family of small eukaryotic proteins of around 30 kDa.30 Two types of activity have been reported in this family of enzymes. One type of activity is able to cleave ubiquitin from small peptides or proteins that may either be biosynthetic precursors of ubiquitin or of small proteins, while the other type cleave ubiquitin from small intracellular nucleophiles, such as amines or glutathion, that would rapidly consume the free ubiquitin pools.

The Ubiquitin System and Proteolysis

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Yuz:

The gene has been cloned and shown to encode a 26 kDa protein. The Yuz enzyme cleaves the linkage between ubiquitin and the alpha amino group of small proteins fused to it.163,164 Null mutants are phenotypically normal,55,165 therefore suggesting the occurrence of other ubiquitin C-terminal hydrolases with overlapping activities. Other eukaryotic ubiquitin C-terminal hydrolases related to Yuz are: The PGP9.5 protein is a hydrolase that is highly expressed in neuronal cells (1-5% of total protein), to a lesser degree in the testis, and is undetectable in many peripheral tissues.166,167 Its human gene has been cloned and shown to encode a 26, kDa protein.166 The bovine and rat homologs have also been cloned and are almost identical.166,167 Mayer and Wilkinson 168 have shown that PGP9.5 is probably the minor hydrolase resolved from a mixture of three 30 kDa hydrolases obtained from calf thymus. PGP9.5 has also been purified from bovine retina by affinity chromatography on a ubiquitin-Sepharose column, and its catalytic properties have been characterized.169 A major 30 kDa C-terminal hydrolase has been obtained from calf thymus and has been shown to be 54% identical to the PGP9.5 hydrolase.166 This hydrolase is believed to be the same as that characterized by Rose and collaborators from reticulocytes and erythrocytes and is an enzyme which acts on a variety of C-terminal amide derivatives of ubiquitin and small compounds.170,171 Probably the same 30 kDa hydrolase also coincides with the enzyme that acts on ubiquitin protein fusions such as ubiquitin metallothionein or the natural fusion protein of ubiquitin to the 52 amino acid human ribosomal extension protein. The enzyme purified from erythrocytes by Moskovitz172 and shown to act on branched ubiquitin-epsilon amino-histone 2A conjugates is most likely the same 30 kDa hydrolase. Finally this last protein is thought to be similar to the not well characterized ubiquitin isopeptidase first described from rat liver nucleoli by Andersen et al.173,174 A third 30 kDa ubiquitin C-terminal hydrolase has also been obtained from calf thymus by Mayer and Wilkinson.166

The Yeast Ubp Family and Their Eukaryotic Functional Homologs The Ubp enzymes (ubiquitin-specific processing proteases) are characterized by the highly conserved elements known as Cys and His boxes, which are believed to be involved in the active site. These enzymes are larger than the Yuz-related ubiquitin C-terminal hydrolases and cleave ubiquitin from a range of protein substrates rather than from peptides or small compounds. Many yeast Ubp mutants do not have striking phenotypic abnormalities.63,175 Thus, it has been considered that there may be a considerable overlap in the Ubp functions, although other possibilities have also been taken into account.55 Ubp1: The gene has been cloned and is known to encode a 93 kDa protein. The enzyme has been shown to process natural ubiquitin-protein fusions, such as ubiquitin linked to ribosomal proteins or linear polyubiquitin, when it is coexpressed with Ubp1. Null mutants are phenotypically normal.165 Ubp2: The gene has been cloned and encodes a 102 kDa protein. The enzyme cleaves ubiquitin-protein fusions. Ubp2 mutants are phenotypically normal. Ubp3: The gene has been cloned and encodes a 102 kDa protein. The enzyme cleaves ubiquitin-protein fusions. No phenotypic defects have been reported in Ubp3 mutants. Ubp4/Doa4: The gene has been cloned and encodes a 105 kDa protein. Interestingly, this deubiquitinating enzyme is thought to be part of the 26S proteasome, since purified preparations of the yeast 26S proteasome contain the Doa4 protein. The enzyme is believed to work in conjunction with the 26S proteasome, probably in the latter stages of proteolysis. This is suggested by the fact that Doa4 mutants accumulate small ubiquitinated species only slightly larger than unanchored ubiquitin chains,

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which are thought to be ubiquitinated proteolytic remnants of 26S proteasome activity.63 The enzymatic activity of Doa4 has been shown to cleave ubiquitin-protein fusions and isopeptide-linked ubiquitin conjugates (isopeptidase activity).55,63 The Doa4 mutant was first isolated as a result of its inability to degrade the MAT-alpha 2 repressor protein. The characterization of the Doa4 mutant also revealed its involvement in the degradation of many other substrates.63 In addition, the function of the Ubp4/Doa4 enzyme seems to be very broad. For example, a role in DNA repair, cellular growth, the stress response, sporulation and vacuolar biogenesis has been reported.55,63 Still related to Doa4, Eytan et al176 reported a ubiquitin C-terminal hydrolase from rabbit reticulocytes which is also an integral part of the 26S proteasome. In addition to its deubiquitinating activity it is tightly coupled to the proteolytic action of the complex. This hydrolase releases ubiquitin from conjugates that are good substrates for proteolysis, and also acts on adducts in which a single ubiquitin is attached to the protein. Thus it has been suggested that the hydrolase may have a role in the release of ubiquitin from linkage to amino groups of the protein substrate at the final stages of the ubiquitin proteolytic pathway. Its relationship with Doa4 remains to be understood. Eukaryotic Homologs of Ubp4 are: Human Tre17: the human tre17 gene encodes an Ubp enzyme with high homology to Doa4.65,69 However, this gene is transcribed into at least two alternatively spliced mRNAs, one of them being involved in the synthesis of a protein product which lacks the Ubp activity.65,66 Interestingly, this tre17 variant is oncogenic when it is overexpressed in nude mice.65,66 Based on these observations it is believed that the transforming Tre17 protein acts as a dominant negative variant, interfering with the normal function of tre17.67 Obviously, the transforming activity of the Tre17 protein reinforces the role of Doa4 and its functional homologs in the control of the cell cycle. Human tre2 oncogene: This is an oncogene whose sequence was first identified by Papa and Hochstrasser 63 as having the highest degree of similarity with Doa4. Molecular mapping revealed that the tre2 oncogene is a hybrid sequence derived from several human genetic loci, the homologous sequences to Doa4 having arisen from the tre17 gene.65,66 The function of the tre2 oncogene, if there is one, may be similar to the negative variant of the Tre17 protein. Murine Unp: The gene has been cloned and shown to encode a Ubp enzyme close related to tre17.68 The Unp gene has proto-oncogene properties.69 Human Unp: The human homolog of the murine Unp is located within the chromosomal region 3p21.3 and is rearranged in small cell lung tumors.70 Ubp5: The gene has been cloned and shown to encode a 92 kDa protein. The enzyme cleaves ubiquitin-protein fusions. No phenotypic defects have been reported in Ubp5 mutants. Ubp6: The gene has been cloned and shown to encode a 57 kDa protein. Ubp7: The gene has been cloned and shown to encode a 123 kDa protein. The enzyme cleaves ubiquitin-protein fusions. Ubp8: The gene has been cloned and shown to encode a 54 kDa protein. Ubp8 mutants have a slight growth defect. Ubp9: The gene has been cloned and shown to encode a 86 kDa protein. The enzyme cleaves ubiquitin-protein fusions. Ubp9 mutants are viable. Ubp10/Str4: The gene has been cloned and shown to encode a 90 kDa protein. Ubp10 mutants have a defect in chromatin-mediated gene silencing.

The Ubiquitin System and Proteolysis

43

Ubp11: The gene has been cloned and shown to encode a 83 kDa protein. Ubp12: The gene has been cloned and shown to encode a 143 kDa protein. Ubp13: The gene has been cloned and shown to encode a 84 kDa protein. Ubp14: The gene has been cloned and shown to encode a 91 kDa protein. The enzyme acts mainly on unanchored ubiquitin chains, most of which are generated in the course of proteolysis. Interestingly, this activity is believed to be important in preventing the 26S proteasome inhibition by the released ubiquitin chains in a negative feedback mechanism. Consequently, the Ubp14 enzyme has been suggested to have an important regulatory role in the degradation of a number of proteins, where increased Ubp14 activity would have a promoting proteolytic role and Ubp14 downregulation an inhibitory effect. The importance of the Ubp14 activity in the control of certain proteolytic processes has been suggested by the following observations. First, a yeast mutant lacking the Ubp14 enzyme has been reported to have defects in the degradation of distinct proteins, this being associated with a striking accumulation of unanchored ubiquitin chains. Second, an overabundance of unanchored ubiquitin chains generated otherwise in wild-type yeast cells is associated with defects in protein degradation. As to the functional involvement of the Ubp14 enzyme, it has been reported to induce resistance to amino acid analogs (a form of stress response) and to be involved in sporulation and the degradation of the MAT-alpha 2 repressor and other proteins.55

The Eukaryotic Homologs of Ubp14 are: Isopeptidase T: The enzyme has been purified and characterized from erythrocytes by Hadari et al61 and has an apparent molecular mass of 100 kDa. It acts on branched polyubiquitin chains through the ubiquitin-Lys-48-ubiquitin linkage, but not on the isopeptide linkage between the polyubiquitin chain and the protein substrate.60-62,177 A Ubp gene corresponding to a 100 kDa protein has also been cloned from erythrocytes and may correspond to the isopeptidase T gene.178 Interestingly, human isopeptidase T is the functional homolog of the yeast Ubp14 enzyme. The two proteins are 31% identical and share similar enzymological properties. In addition, isopeptidase T has been shown to reverse the defects observed in the yeast Ubp14 mutant.55 Ubp15: The gene has been cloned and shown to encode a 143 kDa protein. Ubp15 mutants have a slight growth defect. Ubp16: The gene has been cloned and shown to encode a 57 kDa protein. Other Eukaryotic Ubiquitin Hydrolases Faf: The product of the Drosophila faf gene required for normal eye development and embryogenesis is a Ubp-type deubiquitinating enzyme.179 In contrast to isopeptidase T, there is some evidence suggesting that this enzyme may have an inhibitory role in protein degradation by reversing the ubiquitination of certain proteins and therefore preventing or slowing their degradation by the 26S proteasome.55,179 DUB1: The gene has been cloned from mouse erythrocytes and shown to encode an Ubptype enzyme.180 The enzyme product cleaves ubiquitin-protein fusions. In addition, the DUB1 gene is induced by the cytokine interleukin-3 (IL-3) as an early growth response gene. In normal conditions, the expression of this gene is rapidly down-regulated. However, if continuous expression is induced with a steroid-inducible promoter, the cell cycle is arrested at the G1 phase.

44

Ubiquitin and Disease

DUB2: The gene has been cloned from T cells and shown to encode an Ubp-type enzyme. Interleukin-2 (IL-2) has been shown to induce its expression as an early-response gene which is rapidly down-regulated in T cells.181 Others: Swanson et al182 reported the cloning of a novel ubiquitin C-terminal hydrolase expressed in all tissues but at levels 5- to 10-fold higher in the retina than elsewhere. The gene was localized in the short arm of the X chromosome. Several pieces of evidence suggest that this gene is an excellent candidate for several X-linked retinal disorders mapped within the same chromosomal region. In relation to muscle disorders and/or physiology is the reported identification of 10 ubiquitin C-terminal hydrolases from chick muscle.183 They were named UCH1 to UCH10 and their apparent molecular masses range from 27 to 810 kDa. In addition, they seem to have different substrate specificities. All are capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids. Five of the enzymes, UCHs 1 through to 5, are also capable of generating free ubiquitin from poly-His-tagged diubiquitin. UCH1 and UCH7 can remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin (RGA)-alpha NH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin-binding domain of myosin light-chain kinase. Multiple species of ubiquitin-alpha NH-protein hydrolase activities have been identified in reticulocyte extracts.184 In addition to the three 30 kDa C-terminal hydrolases found in calf thymus by Mayer and Wilkinson,168 the characterization of a 100-200 kDa ubiquitin C-terminal hydrolase was also reported.

The 26S Proteasome It consists of the 20S proteasome subunits together with the 19S cap complex regulatory subunits.

The 20S Proteasome Subunits The 20S proteasome of T. acidophilum consists of a single type of alpha and beta subunit. These have been the basis for arranging all the eukaryotic subunits by sequence similarity.36,41,44 Among the eukaryotic proteasomes, the S. cerevisiae and the human 20S proteasomes are the most extensively studied. They consist of seven different alpha and seven different beta type subunits, all of which have been cloned (see Table 2.6 for nomenclature). In addition, the structural studies carried out with this yeast 20S proteasome complex have shown that all 14 subunits are present in unique localizations.41 Site-directed mutagenesis and inhibitor studies carried out by Seemüller et al40 on the Thermoplasma 20S proteasome have clearly shown that nucleophilic attack on the protein substrate is mediated by the N-terminal threonine of the beta subunit. Based on the presence of this N-terminal threonine and on the characterization of inhibitor binding sites, Groll et al41 have reported that the S. cerevisiae 20S proteasome has three different beta subunits with catalytic activity. The b5/PRE2 subunit has both chymotrypsin-like and trypsin-like specificity, and the b1/PRE3 subunit has peptidylglutamyl peptide hydrolytic (PGPH) specificity. The third subunit b2/PUP1 acts on large residues regardless of whether they are neutral, basic or acidic. Apart from b5/PRE2, b1/PRE3 and b2/PUP1, other beta-type subunits have been reported to affect proteolytic activities. For example, mutations in the b4/C11 and b7/PRE4 subunits affect the chymotrypsin-like and the PGPH activity respectively. However, the 20S proteasome structure reported by Groll et al41 has shown that these subunits are adjacent to b5/PRE2 and b1/PRE3 respectively. These authors have argued that the reported mutations in the b4/C11 and b7/PRE4 subunits would disrupt their contact with the b5/PRE2 and b1/PRE3 subunits, which would result in distortion of the active N-terminal threonine.

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Table 2.6. Nomenclature of the 20S proteasome subunits New systematic S. cerevisiae name α 1 s.c. α 2 s.c. α 3 s.c. α 4 s.c. α 5 s.c. α 6 s.c. α 7 s.c. β 1 s.c. β 1i β 2 s.c. β 2i β 3 s.c. β 4 s.c. β 5 s.c. β 5i β 6 s.c. β 7 s.c.

C7/PRS2 Y7 Y13 PRE6 PUP2 PRE5 C1/PRS1 PRE3

A. thaliana

Drosophila

mice

rat

human

MC3

iota C3 C9

TAS-g64

PROS25 PROS29 PROS28.1

PSM-30

PRO35

iota C3 C9 C6 zeta C2 C8 Y/delta LMP2 Z MECL1 C10 C7 X/MB1 LMP7/LMP7a C5 N3/beta BPRO526

delta

zeta C2 C8 delta

PUP1 PUP3 C11/PRE1 PRE2 C5PRS3 PRE4

C10II C7I C5

LMP7 C5

LMP7 C5 RN3

For additional nomenclature, see also: Sc11, PRE8 and PRE9 as a-type subunits of S. cerevisiae, and PRE7 as a b-type subunit. Different isoforms of RC6 and MC3 have been identified.

The 19S Cap Complex Regulatory Subunits The ATPase Subunits Seven ATPase subunits have been cloned and subsequently characterized as members of the 19S cap complex (Table 2.7).36,55,185 At least six of them have been shown to be members of a novel family of ATPases called AAA-ATPases.186 The S. cerevisiae subunits and their functional human homologs are shown in Table 2.6. All the studied ATPases are essential for viability, and point mutations in several of them cause defects in protein degradation.187,188 As suggested by Hochstrasser,55 this high number of different ATPases may provide alternative binding sites that would favor the unfolding of particular proteins as well as their translocation into the proteasome interior. Interestingly, some of these subunits are similar or identical to proteins involved in transcriptional activation. For example, the human S4, S6 and S7 subunits have been shown to be closely related to proteins which interact or modulate the TAT protein transcriptional activator of the human immunodeficiency virus (HIV).189 In addition, the S. cerevisiae Cim3/sug1 subunit participates in transcriptional activation and is a component of the RNApII complex.190 YAT1, YAT2 and YAT5 are yeast AAA-ATPase genes which, based on sequence similarity, may encode additional 19S cap complex ATPase subunits.191 Some of the data concerning the conformational changes and binding activities of these ATPases in transcriptional events may be useful in understanding the mechanism of substrate unfolding and translocation to the proteasome interior. Concerning the function of these ATPase subunits, the Cim3/Sug1 and Cim5/YAT3

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Table 2.7. Regulatory subunits of the 26S proteasome Saccharomyces cerevisiae

Human

ATPase subunits S4 Cim5/YTA3 Cim3/Sug1 Crl13/Sug2 YAT1 YAT2 YAT5

kDa 49 52 45 49 48 48 49

Non-ATPase subunits Nin1 Sen3 Nas1/Hrd2 Sun1 Sun2 Nas2 Nas3 Doa4

kDa 32 104 109 30 60 25 38

ATPase subunits S4 S7 p45 Sug2

Non-ATPase subunits p31 S1 or p112

kDa

kDa

p28 or S5 p58 tre17

subunits have been shown to be required for G2-M cell cycle progression.55,188 An interesting question would be what is the degree of specific involvement of each subunit in different cellular functions? The Non-ATPase Subunits Several non-ATPase subunits have also been identified as components of the 19S cap complex (Table 2.7).36,55 Among them, subunits involved in polyubiquitin binding are believed to be of utmost importance for substrate recognition and consequently for cell viability.55 The first to be identified was the human S5a subunit.192 Other functional homologs have been cloned from Drosophila and A. thaliana.193,194 The yeast functional homolog of S5a is called SUN1. Surprisingly, its deletion has no consequences for cell growth or the stress response. The presence of more polyubiquitin binding subunits has been suggested by Hochstrasser.55 Given that substrate recognition may have a role in the control of proteolysis, and that the Drosophila S5a functional homolog is only loosely associated with the 19S cap complex,193 many other substrate recognition subunits may be found as free subunits with short half-lives. Other non-ATPase subunits associated with the 19S cap complex are those with deubiquitinating activities. Among them, the Doa4 subunit has a key role in the regulation of 26S proteasome-mediated proteolysis, an increased Doa4 activity having a promoting proteolytic effect.55,63 Its human homolog may be the tre17 protein with Ubp activity. In addition to Doa4, the enzyme reported by Eytan et al176 may be taken into account when considering possible deubiquitinating enzymes associated with the 26S proteasome. Four additional genes encoding non-ATPase subunits of the 19S cap complex have also been cloned.195 The yeast SEN3 gene, which encodes the largest subunit (104 kDa), has been shown to influence the tRNA splicing endonuclease system.36 Concerning its cellular func-

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tions, this gene is essential for viability, resistance to stress conditions, sporulation and degradation of ubiquitinated proteins.2 Its human homolog has been identified and called p112 (S1).36 The Nin1 gene has been cloned and encodes a 32 kDa protein essential for viability and the G2-M cell cycle progression.55 It is involved in the degradation of ubiquitinated proteins. Its human homolog is the p31 subunit.36 The SUN2 gene has been cloned and encodes a 60 kDa protein that is essential for viability. Like the SUN1 gene, it is a high copy suppressor of the temperature-sensitive nin1-1 mutant.36,55 Its human homolog is the p58 subunit.36 The Nas1/Hrd2 gene has been cloned and encodes a 109 kDa protein essential for viability. It has a low level of similarity to SEN3. Other proteins associated with the 19S cap complex are Nas2 and Nas3.55

Ubiquitin-Fusion Proteins and Ubiquitin-like Proteins Ubiquitin is invariably encoded as a fusion protein that must undergo a post-translational cleavage to produce the mature ubiquitin. Processing of the ubiquitin fusion proteins by ubiquitin occurs virtually cotranslationally and does not seem to be used to regulate the cellular levels of free, mature ubiquitin.196,197 Although this was first described and characterized in yeast,198 it has also been found in eukaryotes. Ubiquitin is expressed from four loci in yeast. UBi1 and UBi2 encode identical polypeptides consisting of a single ubiquitin moiety fused to the N-terminus of a ribosomal protein of the large subunit, UBi3 encodes a protein consisting of single ubiquitin moiety fused to the N-terminus of a ribosomal protein of the small subunit.197 The expression of these ribosomal proteins with N-terminal fusions of ubiquitin appears to facilitate the efficiency with which they are incorporated into ribosomes. Expression in both yeast and bacteria of other proteins with engineered N-terminal ubiquitin fusions often increases the efficiency of their expression.199,200 The evolution of ubiquitin-ribosomal fusion proteins may therefore represent a naturally occurring use of ubiquitin to enhance expression and synthesis of the associated ribosomal proteins. It is also possible that the fusion of ubiquitin to these ribosomal proteins provides a connection between the protein synthetic and protein degradative apparatus that facilitates their coordinate regulation. The function of UBi4 seems to be related to the maintenance of the cellular ubiquitin pools during stress conditions, when ubiquitin conjugation is a particularly active process. Indeed, UBi4 consists of five head-to-tail repeats of ubiquitin and is highly expressed in response to a variety of environmental and metabolic stresses including, heat shock, starvation and UV radiation exposure.201 Although as we have previously stated, ubiquitin is present in a highly conserved form, several investigations have identified ubiquitin-like coding sequences that share from 40 to 80% or more amino acid sequence homology with that of ubiquitin.202–206 Although some of these ubiquitin-like proteins have C-terminal sequences that might allow them to conjugate with other cellular proteins, they do not. Only the conserved, canonical, “true” ubiquitin is able to conjugate. Some of these ubiquitin-like proteins are often associated with viral function, being either expressed by viruses (such as baculovirus or togavirus) or produced by mammalian cells in response to viral infection. An attractive speculation is that these ubiquitin-like proteins are one aspect of a broader strategy to modify host ubiquitin-dependent pathways in response to viral infection, either for the benefit of the virus in the case of the virally-encoded proteins or for the benefit of the host in the case of host-encoded proteins.207

Drugs and Inhibitors The identification of inhibitors that can selectively block proteasome function in intact cells has already led to dramatic progress in understanding the physiological roles of the proteasome (see Table 2.8). In addition to proving useful as investigative tools, these

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Table 2.8. Inhibitors of the proteasome Compound

References

Lactacystin

208

Arsenoxides

224,225

Peptidyl aldehydes MG115 MG132 PSI ZLLF ZLLL ALLN

210-223

Antitumorals cisplatin aclarubicin mitomycin c

226-228

compounds also have therapeutic potential. Perhaps one of the most interesting studies concerning proteolytic inhibitors is that of Fenteany et al208 describing a specific natural inhibitor of the 20S proteasome, the Streptomyces metabolite lactacystin. This compound was first noted for its ability to inhibit cell cycle progression and promote neurite outgrowths in a neuroblastoma cell line.209 3H-labeled lactacystin specifically and covalently reacts with the N-terminal threonine of one particular subunit of the mammalian proteasome (XMB1) thereby inhibiting its activity. The mechanism by which lactacystin inhibits the proteasome involves spontaneous formation of the active lactacystin analog clasto-lactacystin beta-lactone (lactonization). This is the chemical form that interferes with the proteasome. Lactacystin provides a pharmacological tool that can be used to study the specific roles of the proteasome in cellular regulation in vivo. For example, the inhibition of the proteasome may prevent the degradation of growth inhibitory proteins or it may block the proteolytic activation of growth permissive proteins in the cell, whilst lactacystin induces expression of the cell cycle inhibitor p21WAF1/CIP1 in human cancer cells. A number of membrane-permeable peptide inhibitors with C-terminal reactive aldehyde groups (peptide aldehydes) have been widely used to inhibit proteasome activities.210-212 Among them, MG115 (carbobenzoxyl-Leu-Leu-norvalinal) 213 and MG132 (carbobenzoxylLeu-Leu-leucinal) 214 reduce degradation of ubiquitinized proteins by the 26S complex without affecting its ATPase or isopeptidase activity. Interestingly, their potency against the activated 20S particle is greater than against the 26S proteasome.210 Their effects are reversible, and after removal, normal rates of protein degradation are restored; in addition, their efficacy in reducing proteolysis in intact cells appears greater than in crude extracts or with purified 26S proteasomes.210 Cells are fully viable for at least 10-20 hours in their presence and during this time rates of protein synthesis and ATP content are unaltered. Other peptidyl aldehydes that have proved to have potent inhibitory effects on the proteasom e are PSI (N-benzyloxycarbonyl-Ile-Glu-Ala-leucinal),215 ZLLF (benzyloxycarbonyl-Leu-Leu-phenylalaninal), ZLLL (benzyloxycarbonyl-Leu-Leu-leucinal) and ALLN (N-acetyl-Leu-Leu-norleucinal).216 The latter induces cell cycle arrest in platelet-

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derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome.217 Inhibition of the proteasome results in accumulation of the tumor suppressor protein p53, which is followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. The use of this inhibitor has therefore served to support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cyclep rogression. The use of this type of inhibitor has also led to suggestions that a component of the proteasome (or multicatalytic proteinase complex) that preferentially cleaves bonds after branched-chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the proteasome.218 Indeed, at least five distinct proteolytic components of the 20S proteasome have been described:219 trypsin-like, chymotrypsin-like, peptidyl-glutamyl peptide hydrolyzing, small neutral amino acid-preferring and BrAAP. Vinitsky et al218 reported that the most potent inhibition of this proteolytic activity is obtained with the peptidyl aldehyde benzyloxycarbonyl-Gly-Pro-Phe-leucinal (ZGPFL). They demonstrated that both the Pro residue in the P3 position and the Leu in the P1 are very important in proteolytic inhibition. Several other proteasome peptidyl aldehydes have been reported but have not been studied extensively. Traenckner et al introduced the peptide aldehyde carbobenzoxyl-IleGlu(O-t-Bu)-Ala-leucinal,220 which can also enter cells and causes accumulation of ubiquitin conjugates.211 Iqbal et al221 synthesized a number of potent dipeptides that inhibit proteolytic activity and can stabilize short-lived mutant proteins. A novel group of potent peptide aldehydes has been synthesized that contain large hydrophobic groups at the P4 position which cause a dramatic increase in potency.222 Finally, Spaltenstein et al223 have designed and synthesized a novel group of protease inhibitors (tripeptide alpha',beta'-epoxyketones) that inactivate the proteasome at nanomolar concentrations. In spite of the efficiency of these peptidyl aldehydes, some of them are not specific for the proteasome but also act on other proteases. They are effective towards both serine and cysteine proteinases by forming, respectively, hemiacetal and thioacetal adducts with these enzymes. Consequently, inhibition of intracellular degradation of ubiquitin-protein conjugates by peptidyl aldehydes cannot be credited exclusively to the action of the proteasome. Therefore in studies involving these compounds it is important to show that selective inhibitors of lysosomal proteolysis or of calpains do not have similar effects and/or that the sensitivity of a response to different aldehyde inhibitors correlates with their potency against the proteasome. Another group of compounds capable of inhibiting ubiquitin-dependent proteolysis is that of trivalent arsenoxides. Klemperer and Pickart 224 demonstrated that the simplest trivalent arsenoxide inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate. The same group later demonstrated that phenylarsenoxides are considerably more powerful inhibitors that seem to act on the ubiquitin-protein ligase E3.225 This is suggested by the fact that a complex of E3-alpha and the 14 kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin with the E3 component of the complex mediating binding. In addition, p-aminophenylarsenoxide inhibits isolated E3, the inhibition being rapidly reversed by the addition of dithiothreitol (which contains a vicinal thiol group) but not by β-mercaptoethylamine (a monothiol). One has to take into consideration that, in certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of this enzym e. Thus, a bifunctional phenylarsenoxide (brom oacetylam ino– phenylarsenoxide) rapidly and irreversibly inactivates E3 but bromoacetyl aniline (lacking an arsenoxide moiety) does not inhibit E3. It thus seems clear that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site.

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Ubiquitin and Disease

Finally, a group of anti-tumoral compounds have also been suggested to be inhibitors of the proteasome. Isoe et al demonstrated that both cysplastin and aclarubicin (and also mitomycin C), which are chemotherapeutic agents used in cancer research, are able to block ubiquitin-dependent proteolysis.226 While the former seems to inhibit the conjugation of ubiquitin to proteins, the latter inhibits the proteolytic process per se.226 Another study by the same group found that the antitumor drug aclacinomycin A (known as a DNAintercalative agent) inhibits the degradation of ubiquitinized proteins in rabbit reticulocytes lysates.227 Later studies revealed that, of all the catalytic activities of the bovine pituitary 20S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug.228

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19. Dohmen J, Madura K, Bartel B et al. The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme. Proc Natl Acad Sci USA 1991; 88:7351-7355. 20. Reiss Y, Heller H, Hershko A. Binding sites of ubiquitin-protein ligase. Binding to ubiquitinprotein conjugates and of ubiquitin-carrier protein. J Biol Chem 1989; 264:10378-10383. 21. Goebl MG, Yochem J, Jentsch S et al. The yeast cell cycle gene CDC34 encodes a ubiquitinconjugating enzyme. Science 1988; 241:1331-1335. 22. Sung P, Prakash S, Prakash L. The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity. Gene Dev 1988; 2:1476-1485. 23. Morrison A, Miller EJ, Prakash L. Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae. Mol Cell Biol 1988; 8:1179-1185. 24. Reynolds P, Koken MHM, Hoeijmakers JHJ et al. The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae. EMBO J 1990; 9:1423-1430. 25. Koken MHM, Reynolds P, Bootsma D et al. Dhr6, a Drosophila homolog of the yeast DNA-repair gene RAD6. Proc Natl Acad Sci USA 1991; 88:3832-3836. 26. Koken MHM, Reynolds P, Jasper-Dekker I et al. Structural and functional conservation of two human homologs of the yeast DNA-repair gene RAD6. Proc Natl Acad Sci USA 1991; 88:8865-8869. 27. Jentsch S, Seufert W, Sommer T et al. Ubiquitin-conjugating enzymes: novel regulators of eukaryotic cells. Trends Biochem Sci 1990; 15:195-198. 28. Ciechanover A, Schwartz AL. How are substrates recognized by the ubiquitin-mediated proteolytic system? Trends Biochem Sci 1989; 14:483-488. 29. Hershko A. The ubiquitin pathway for protein degradation. Trends Biochem Sci 1991; 16:265-268. 30. Hershko A, Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem 1992; 61:761-807. 31. Varshavsky A. The N-end rule. Cell 1992; 69:725-735. 32. Reiss Y, Hershko A. Affinity purification of ubiquitin protein ligase on immobilized protein substrates. Evidence for the existence of separate NH 2-terminal binding sites on a single enzyme. J Biol Chem 1990; 265:3685-3690. 33. Hein C, Springael JY, Volland C et al. NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur1 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol 1995; 18:77-87. 34. Chen P, Johnson P, Sommer T et al. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor. Cell 1993; 74:357-369. 35. Peters JM. Proteasomes:protein degradation machines of the cell. Trends Biochem Sci 1994; 19:377-382. 36. Hilt W, Wolf DH. Proteasomes: Destruction as a programme. Trends Biochem Sci 1996; 21:96-102. 37. Peters JM, Franke WW, Kleinschmidt JA. Distinct 19S and 20S subcomplexes of the 26S proteasome and their distribution in the nucleus and the cytoplasm. J Biol Chem 1994; 269:7709-7718. 38. Wilk S, Orlowski M. Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex. J Neurochem 1983; 40:842-849. 39. Orlowski M, Cardozo C, Michaud C. Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl site of branched chain and small neutral amino acids. Biochemistry 1993; 32:1563-1572. 40. Seemüller E, Lupas A, Stock D et al. Proteasome from Thermoplasma acidophilum: a threonine protease. Science 1995; 268:579-582.

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199. Butt TR, Jonnalagadda S, Monia BP, et al. Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proc Natl Acad Sci USA 1989:86:2540-2544. 200. Ecker, DJ, Stadel JM, Butt TR et al. Increasing gene expression in yeast by fusion to ubiquitin. J Biol Chem 1989; 264:7715-7719. 201. Finley D, Ozkaynak E, Varshavsky A. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 1987; 48:1035-1046. 202. Haas AL, Ahrens P, Bright PM et al. Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin. J Biol Chem 1987; 262:11315-11323. 203. Banerji J, Sands J, Strominger JL et al. A gene pair from the human major histocompatibility complex encodes large proline-rich proteins with multiple repeated motifs and a single ubiquitin-like domain. Proc Natl Acad Sci USA 1990; 87:2374-2378. 204. Kas K, Michiels L, Merregaert J. Genomic structure and expression of the human fau gene: encoding the ribosomal protein S30 fused to a ubiquitin-like protein. Biochem Biophys Res Commun 1992; 187:927-933. 205. Michiels L, Van der Rauwelaert E, Van HF et al. fau cDNA encodes a ubiquitin-like-S30 fusion protein and is expressed as an antisense sequence in the Finkel-Biskis-Reilly murine sarcoma virus. Oncogene 1993; 8:2537-2546. 206. Olvera J, Wool J. The carboxyl extension of a ubiquitin-like protein is rat ribosomal protein S30. J Biol Chem 1993; 268:17967-17974. 207. Driscoll J, Finley D. A controlled breakdown: Antigen processing and the turnover of viral proteins. Cell 1992; 68:823-825. 208. Fenteany A, Standaert RF, Lane WS et al. Inhibition of proteasome activities and subunitspecific amino-terminal threonine modification by lactacystin. Science 1995; 268:726-731. 209. Omura S, Fujimoto T, Otoguro K. Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells. J Antibiot 1991; 44:113-116. 210. Rock KL, Gramm C, Rothstein L et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on the MHC class I molecules. Cell 1994; 78:761-771. 211. Figueiredo-Pereira ME, Berg KA, Wilk S. A new inhibitor of the chymotrypsin-like activity of the m ulticatalytic proteinase com plex (20S proteasom e) induces accum ulation of ubiquitin protein conjugates in a neuronal cell. J. Neurochem 1994; 63:1578-1581. 212. Vinitsky A, Michaud C, Powers JC et al. Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex. Biochemistry 1992; 31:9421-9428. 213. Lee DH, Goldberg AL. Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharom yces cerevisiae. J Biol Chem 1996; 271: 27280-27284. 214. Read MA, Neish AS, Luscinskas FW et al. The proteasome pathway is required for cytokineinduced endothelial-leukocyte adhesion molecule expression. Immunity 1995; 2:493-506. 215. Traenckner EBM, Pahl HL, Henkel T et al. Phosphorylation of human IκB- α on serine 32 and 36 controls IκB- α proteolysis and NF-kB activation in response to diverse stimuli. EMBO J 1995; 14:2876-2883. 216. Alkalay I, Yaron A, Hatzubai A et al. Stimulation-dependent IkBa phosphorylation marks the NF-kB inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 1995; 92:10599-10603. 217. Dietrich C, Bartsch T, Schanz F et al. p53-dependent cell cycle arrest induced by N-acetylL-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts. Proc Natl Acad Sci USA 1996; 93:10815-10819. 218. Vinitsky A, Cardozo C, Sepp-Lorenzino L et al. Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes. J Biol Chem 1994; 269:29860-29866. 219. Orlowski M, Cardozo C, Michaud C. Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids. Biochemistry 1993; 32:1563-1572.

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220. Traenckner EB, Wilk S, Baeuerle PA. A proteasome inhibitor prevents activation of NF-kB and stabilizes a newly phosphorylated form of IκB- α that is still bound to NF-kB. EMBO J 1994; 13:5433-5441. 221. Iqbal M, Chatterjee S, Kauer JC et al. Potent inhibitors of proteasome. J Med Chem 1995; 38:2276-2277. 222. Akopian TN, Kisselev AF, Goldberg AL. Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum. J Biol Chem 1997; 272:1791-1798. 223. Spaltenstein A, Leban JJ, Huang JJ et al. Design and synthesis of novel protease inhibitors: tripeptide alpha’,beta’-epoxyketones as nanomolar inactivators of the proteasome. Tetrahedron Lett 1996; 37:1343-1346. 224. Klemperer NS, Pickart CM. Arsenite inhibits two steps in the ubiquitin-dependent proteolytic pathway. J Biol Chem 1989; 264:19245-19252. 225. Berleth ES, Kasperek EM, Grill SP et al. Inhibition of ubiquitin-protein ligase (E3) by monoand bifunctional phenylarsenoxides: evidence for essential vicinal thiols and a proximal nucleophile. J Biol Chem 1992; 267:16403-16411. 226. Isoe T, Naito M, Hirai R et al. Inhibition of ubiquitin-ATP-dependent proteolysis and ubiquitination by cisplatin. Anticancer Res 1991; 11:1905-1910. 227. Isoe T, Naito M, Shirai A et al. Inhibition of different setps of the ubiquitin pathway system by cisplatin and aclarubicin. Biochim Biophys Acta 1992; 1117:131-135. 228. Figueiredo-Pereira ME, Chen WE, Li J, et al. The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsinlike activity of the bovine pituitary 20S proteasome. J Biol Chem 1996; 271:16455-16459.

CHAPTER 3

Alzheimer’s and Other Neurodegenerative Diseases Alzheimer’s, A Disease Related to Regeneration

A

lzheimer’s disease (AD) is the most common form of senile dementia. It affects more than 15 million people worldwide and will probably affect many more in the future because of increased life expectancy. The disease is characterized, in advanced cases, by a marked deterioration in memory and all cognitive functions as a result of a progressive degeneration and loss of cortical and limbic neurons.1 Cholinergic transmission seems to be deeply involved in this process,2 especially in terms of cognitive deterioration and loss of memory. Typical histological lesions include numerous extracellular senile plaques, composed of dystrophic neurites and axons with a central deposit of beta-amyloid protein (BAP) and surrounded by activated microglia and astrocytes. Beta-amyloid deposits are also found in the walls of meningeal and cerebral blood vessels.3 They also include cellular neurofibrillary tangles composed mainly of paired helical filaments of aberrantly hyperphosphorylated tau protein (which binds to microtubules).4 The main component of the amyloid structures found in senile plaques is the BAP, which aggregates in insoluble fibrillar complexes with an antiparallel beta-pleated sheet structure.3 BAP is also present in neurofibrillary tangles. In this case, as in senile plaques, it is found associated with components such as ubiquitin, alpha-1-antichymotrypsin (ACT), apolipoprotein E, heparan sulfate proteoglycans and aluminum salts.5 The etiology of AD may be linked to a defect in the mechanism of regeneration of nervous tissue. Nerve cells are often subject to regenerative processes. Alterations in the regeneration mechanism can either happen early in life, in people affected with genetic defects (such as in Down’s syndrome, where AD appears as early as 30-40 years of age) or in older people, sporadically, as a consequence of a progressive process associated with ageing and influenced by toxic agents or trauma.6 Thus, when the mechanism involved in the regeneration of old cells (a normal consequence of ageing) partially loses its capabilities, it triggers the appearance of the disease. Interestingly, cases of a genetic (familial) component (autosomal dominant) are linked to defects in chromosomes 21, 19 and 14 which involve amyloid precursor protein (APP), apolipoprotein E and ACT.3,7,8

BAP, A Key Molecule in Understanding Alzheimer’s Disease Beta-amyloid protein (BAP) is a peptide formed by 40-43 amino acids. It is derived from the APP which is a larger protein (a transmembrane glycoprotein) that is ubiquitously expressed and involved in both wound and neural tissue repair (Fig. 3.1). Although it is bound to have a physiological role, BAP may be a very toxic compound (leading to the appearance of AD) if it is subject to polymerization and subsequent fibril formation. Fibrils are formed by an antiparallel beta-pleated sheet of BAP,3 thus constituting an amyloid deposit. Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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Fig. 3.1. A model of the human amyloid precursor protein. This schematic representation of the human amyloid precursor protein (APP) shows the most important domains and the sites of cleavage (a, b and g) leading to the generation of BAP.

The term “amyloid” is generic for a class of otherwise unrelated peptides which under appropriate conditions aggregate to form insoluble filaments of about 7-9 nm in diameter.3 Similar types of amyloid deposits have been found in other pathological states such as type II diabetes where they accumulate in the form of amyelin 9 and also in neurodegenerative diseases other than AD.10 It has been shown that BAP amyloids are highly toxic and can promote the development of AD. However, BAP has been found under normal physiological conditions, for example, in the extracellular medium of cell cultures, in the human cerebrospinal fluid and also in immunoreactive species in human serum.11 So, BAP may be a functional protein that is overexpressed in particular conditions, but is not an abnormal protein. The presence of diffuse plaques with a preamyloid status (where BAP is accumulated but not aggregated) has been described.12 The presence of small numbers of amyloids with a restricted distribution in aged brains represents the step prior to uncontrolled BAP activity found in AD.13 Bearing all this in mind, the mechanisms involved in BAP accumulation and BAP aggregation are essential in the understanding of AD development. Based on studies to date, it could be argued that such BAP deposition may be the consequence of a mistake in the control of a neurotissue repair mechanism. The presence of dead neurons or dysfunctional neurons in aged brains activates a mechanism to restore the lost synaptic connections. Control of this mechanism may fail for an unknown reason. Such a mechanism would operate, for instance, after traumatic or toxically-induced neuronal lesions. Combining the above information suggests a particular neuronal tissue repair mechanism in AD. Death of neurons as a result of toxic, ischemic or traumatic agents in aged brains, or by the action of cytokines or apoptotic mechanisms, may induce the disruption of cellular anchorage interactions in the neighboring cells. This process is involved in cellular growth activation 14–16 and is reflected in an increase in mitogenic APP forms.17–19 Increased expression would result in an increased secretion of protease nexin 2 (PN2), which acts through the gamma subunit of the nerve growth factor (NGF) (with protease activity) promoting neurite outgrowth.20,21 Once the neurite reached the proximity of other cells, some of its membrane receptors would be involved in intercellular interactions. In this way, NGF is also involved in the expression of the nonmitogenic APP695 isoform, which has cellular adhesion properties.22 Microglia may either kill nonfunctional cells or degrade already dead cells through a tumor necrosis factor- α (TNF)-mediated cytotoxic mechanism.23–26 TNF secretion is also

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involved in the activation of cathepsin G secretion from astrocytes. In addition to TNF secretion, activated microglia secrete interleukin-1 (IL-1) and NGF,23–26 both of which are involved in the induction of the expression of APP isoforms.20 IL-1 induces ACT secretion in hepatocytes, and a similar action in astrocytes has been proposed.27 Since ACT is involved in the control of cathepsin G,28,29 special consideration must be given to this serine protease inhibitor which may be involved in BAP internalization.30 A defect in such internalization may lead to overexpression of BAP, contributing to development of AD.

BAP Accumulation cDNA cloning studies suggest that BAP is part of a much larger precursor protein, named APP. The gene for APP is located on chromosome 21. APP is a 90-130 kDa membrane glycoprotein with a beta peptide (4 kDa) spanning the border between the extracellular domain and the transmembrane region. There are several isoforms of APP, three of them containing BAP31,32 (two of them being mitogenic while the other is a cell-adhesion molecule) derived from a single gene by alternative mRNA splicing. These putative BAP precursors contain 770 (APP770), 751 (APP751) and 695 (APP695) amino acids, the first two differing from the third by the presence of a Kunitz-type protease inhibitor insert (KPI),31,33 which confers the mitogenic capacity.

The Secretory Pathway of PN2 and Other APP Related Forms In the best characterized secretory pathway, APP is cleaved in the middle of the BAP sequence generating a soluble form of APP. In those APP isoforms with the KPI insert (found in proteases inhibitors), the secreted APP (100 kDa), which contains almost all of the APP N-terminal extracellular domain, is identical to PN2, which is a serine protease inhibitor.34 Proteolysis is carried out by a relatively unknown and uncharacterized protease, originally named as alpha-secretase and cleaves APP at the Gln-15 of the BAP sequence in its extracellular domain near the membrane. The rest of the APP molecule, from the Leu-17 of BAP to the intracellular C-terminus of APP, remains linked to the membrane as an 11 kDa peptide (Fig. 3.2). In addition to its role in neurite outgrowth and wound repair, PN2 could also be involved in the control of protease activities related to BAP generation as will be discussed later. So its metabolism and functions may be closely related to BAP accumulation. However, a pathological role for PN2 has been proposed, based on a possible misfunction in its production or clearance and on the fact that it is an amyloidogenic protein since it contains a fragment of BAP.31 In fact PN2 also accumulates in senile plaques. Although its BAP sequence has the lowest aggregation activity, a possible role in aggregation as a nucleation species cannot be excluded, especially given the heterogeneity of BAP fragments present in senile plaques.11 Nevertheless, in normal conditions the exposed secretory pathway seems to preclude amyloidogenesis, given that the alpha-secretase cleaves the APP protein in the middle of the BAP sequence.31 Some recent reports concerning regulatory aspects of this mechanism point out the same precluding amyloidogenic feature. For example, it has been shown that either the direct activation of protein kinase C (PKC), through phorbol esters, or indirect activation through the muscarinic receptor m1, increases soluble APP secretion and decreases BAP production.35,36 This mechanism, which is involved in soluble APP secretion and in negative control of BAP production, does not involve the direct phosphorylation of APP by PKC. Another protein, possibly the alpha-secretase or a protein involved in trafficking of vesicles, is phosphorylated.11 Although the secretion of PN2 has been well characterized, other pathways related to the secretion of APP-related forms must exist. The secretion of soluble forms of APP with

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Fig. 3.2. Formation of protease nexin 2 and amyloidogenic fragments. Both protein nexin 2 (PN2), formed by the secretory pathway, and the amyloidogenic fragments (AF), generated by the lysosomal pathway, could have a role in BAP aggregation.

O-linked sugars that are tyrosine-sulphated and able to bind heparin, has been characterized.32,37 These forms lack the C-terminus of APP. In addition, the extracellular core domain of APP has been shown to be part of heparan sulphate proteoglycans.38 However, further characterization of these proteins is needed in order to obtain their APP sequence. Interestingly, the secretion or extracellular generation of soluble APP forms corresponding exactly to the cleavage in the N-terminus of BAP, carried out until now by a beta-secretase.11 In fact the extensive secretion and association of APP related forms with the extracellular matrix has been shown in cell cultures of the R14 line, a nerve-like cell related to the rat eye.39

The Extracellular Metabolism of APP To date, two proteases that act in the extracellular matrix have been identified by Abraham et al as being able to cleave APP in the N-terminus of BAP, thus producing a BAP molecule associated with the rest of the APP C-terminus domain, either associated or not with the membrane (Fig. 3.3).12,29 These proteases have been characterized from homogenates of human or monkey AD brains as a cathepsin G-like protease and as a metalloprotease (and seem to be released mainly by astrocytes). The cathepsin G-like protease is a serine

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65 Fig. 3.3. Molecular mechanisms leading to the formation of BAP. BAP can either be formed extracellularly via the action of proteases or intracellularly in acidic compartments via cathepsin D.

protease, activated by calcium with the same proteolytic pattern and the same immunoreactivity as cathepsin G. Two proteins with such activity have been purified: one of 30 kDa and another of 68 kDa, the first probably being a fragment of the second. Both are inhibited by ACT and PN2, which are serine protease inhibitors that accumulate in the senile plaques. The other extracellular protease involved in the generation of the N-terminus of BAP (betasecretase) is a cysteine-metalloprotease (according to its response to metalloprotease inhibitors), which is active between pH 5.5 and 7.5. Cathepsin G and the metalloproteases both act by remodeling the extracellular matrix in different physiological ways,40,41 coordinating their activity with oxidative extracellular enzymes.42 In general terms, the action of the different extracellular proteases is controlled by their specific protease inhibitors.43 Such control drives the correct proteolytic processing and avoids tissue damage. As part of this mechanism one type of protease inhibitor can be degraded by other types of protease. However, it has been shown that a malfunction in the metabolism of one type of complex protease-protease inhibitor can lead to a malfunction in the coordinated extracellular action of different proteases.42 Since the N-terminus of the BAP has sequence homology with serine proteases and binds ACT,44 it may be suggested

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that such proteolysis may be important for the regulation of the uptake of the proteaseprotease inhibitor complex by the cell that is necessary for the synthesis of more inhibitor and so for the control of protease activity.45 A similar role could also be mediated by the soluble BAP peptide. In fact Joslin et al30 have shown that BAP competes for the binding ACT-cathepsin G cellular receptor. Finally these extracellular proteases may generate BAP fragments from the different soluble forms of APP; for example, HSPG forms, the glycosaminoglycan forms of APP without the C-terminus, or PN2. Such fragments may have a role in BAP aggregation.

The Endolysosomal Pathway of APP Leads to Potential BAP Precursors Once APP has been processed in the secretory pathway involved in PN2 generation, both the full-length membrane-bound APP and the remaining 10 kDa fragment of APP are internalized and degraded in an endosomal-lysosomal pathway. Both the NPTY sequence (near the cytoplasmic C-terminus) and the phosphorylation by PKC in the cytosolic domain have been shown to direct internalization to clathrin-coated pits.46 The APP molecule and the 10 kDa fragment accumulate in lysosomes of leupeptintreated cells, along with different BAP C-terminal proteolytic fragments (from 8 to 22kDa). 11,47–49 In view of this, it is clear that the lysosomal metabolism of APP and the 10kDa fr agment can generate amyloidogenic peptides that, in uncontrolled proteolysis, exocytosis or cell breakdown could be important in inducing aggregation and also BAP accumulation after the action of extracellular proteases.

Intracellular BAP Generation Several authors have shown that BAP can normally be secreted into the extracellular medium of different cell cultures (see ref. 11 for a review). For example, 4 kDa and 3 kDa peptides that immunoreact with BAP antibodies have been identified. Whereas the 4 kDa peptide is exactly BAP, the 3 kDa peptide originates from the point of alpha-secretase excision and so it has to be derived from the 10 kDa fragment. Moreover, a heterogenous mixture of BAP related-peptides (in lower amounts) has also been detected. In addition, and in favor of the hypothesis that BAP is normally secreted, the BAP peptide has been found in the cerebrospinal fluid and immunoreactive related BAP peptides in the serum.11 The finding that drugs which interfere with pH gradients in vesicular compartments (such as ammonium chloride, chloroquine or monensin) markedly inhibit BAP production in most cell types, suggests that an acidic compartment is necessary for BAP generation. The lysosomal compartment does not seem to be involved, so endosomes and late Golgi or secretory vesicles have been proposed to be involved in BAP generation.11 The exclusion of the lysosomal compartment in BAP generation has been suggested because lysosomotropic inhibitors such as leupeptin, a serine and cysteinylproteinase inhibitor that inhibits 70% of proteinase activities, failed to inhibit BAP secretion.11,49 Also BAP production was not decreased in I-cells in which some lysosomal functions are deficient due to a genetic defect in mannose phosphorylation. Finally, BAP has not been found in isolated lysosomes. At this point three questions have to be considered. The secretion of APPs seems to decrease BAP production and, in addition, the lysosomal metabolism of APP does not seem to be involved in BAP generation. So, why, when and through what kind of APP processing is BAP generated? The recent characterization of intracellular proteases involved in BAP generation will probably help in such understanding. For example, Ladror et al have characterized a cathepsin D (an aspartate protease) which is able to cleave at the N-terminus (betasecretase) and C-terminus (gamma-secretase) of the BAP peptide, thus generating BAP from APP.49 When using a double mutant substrate (in residues 687 and 688) a 100-fold

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increase in the production of BAP was observed. Whereas hydrolysis at the N-terminus has been shown to take place only at the Asp-1 of BAP, hydrolisis at the C-terminus involves both Ala-42 and Thr-43 in 32% and 68% respectively. According to this, cathepsin D is able to produce BAP 1-42 and BAP 1-43. Interestingly, once BAP is produced, BAP 1-43 can be degraded into six pieces by cathepsin D. However BAP 1-42 is resistant to proteolysis. Given that transfecting cells with the mutated APP leads to the accumulation of amyloidogenic fragments, the authors suggest that the role of cathepsin D may be to produce and secrete the APP cleaved by the N-terminus of BAP. The C-terminal hydrolysis, and so the generation of soluble BAP, would occur later and at a lower rate, being the final step in the total hydrolysis of BAP. In addition to being present in lysosomes, cathepsin D is found in clathrin-coated vesicles, endosomes, trans-Golgi and secretory vesicles.49 The protease has a role in the secretion of galβ1-4glc NAc-alpha-2-6-sialyl transferase, an enzyme released in the acute phase. Thus, cathepsin D may have a similar role in the processing and secretion of the N-terminus of BAP (contained in APP), which is also an acute-phase protein. APP may either be directly processed or internalized into clathrin-coated vesicles and later processed in endosomes and secretory vesicles.49 Although cathepsin D is normally found as an acidic intracellular protease, it has been shown that both cathepsin D and B, in a free form, can accumulate in relatively large amounts in senile plaques and are not exclusively confined to lysosomes or bound to the fibrils.50 Both cathepsins are considered as early markers in AD, being highly accumulated in the cortex and hippocampus of AD brains. So an increased, uncontrolled secretion of such cathepsins, together with increased BAP secretion, may participate in the generation of additional extracellular BAP.

Neurons, Astrocytes and Microglia in Alzheimer’s Disease Protease inhibitors might also be involved in the control of BAP generation. These compounds have several origins, some of them related to either neurons, astrocytes or the microglia. Astrocytes and microglia are involved in normal brain repair.24,26,51 PN2 and ACT are both serine-protease inhibitors which accumulate in senile plaques. PN2 has been shown to promote neurite outgrowth 21,22 while ACT has been shown to bind to BAP in the amyloid fibrils.44 Given that cathepsin G produces the N-terminal cleavage of BAP in the extracellular medium 12,29 and that both PN2 and ACT inhibit cathepsin G activity,28,29 the study of the balance between inhibitors and extracellular proteases has to be seriously considered in order to understand AD etiologies. Cathepsin G (a serine protease), elastase (a metalloprotease) and myeloperoxidase (an extracellular enzyme involved in the generation of oxidizing molecules) are involved in the action of neutrophils against infection, alien materials and removal of either dead or damaged tissue.42 On top of their degradative action, these proteolytic enzymes participate in the remodelling of the extracellular matrix during tissue repair. Obviously, the action of such types of hydrolytic and oxidative enzymes has to be highly regulated in order to avoid irreversible tissue damage. Protease inhibitors are usually found in higher amounts than proteases and inhibit protease activity by binding to the enzyme.52 The expression of some inhibitors is under hormonal or cytokine control. Once the protease has been inhibited, the cellular binding and uptake of the inhibitor-protease complexes (as in the case of ACT-cathepsin G) induces the synthesis of acute-phase proteins which may also be involved in the same tissue regeneration mechanism.45 Given that BAP binds ACT and that has been shown to compete for the ACT-cathepsin G cellular receptor,30 its accumulation may be a consequence of a lack of cellular internalization. Another striking aspect related to protease inhibitors is that their

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activity is cross-related, showing interdependence between their control mechanisms. For example, an uncontrolled myeloperoxidase from neutrophils has been shown to act upon the inhibitor of elastase (alpha-1-proteinase inhibitor), leading to an increase in cathepsinG. This seems to be the case, for instance, in the course of events in acute rheumatoidar thritis.42 ACT also controls the production of reactive oxygen species (ROS) by neutrophils.53 Considering this aspect, any defect in the metabolism and control of the protease inhibitors involved in brain repair may well be responsible for BAP amyloid generation. In fact, ROS are produced by microglia in brain repair situations and are overexpressed in AD where they seem to promote BAP aggregation.23 Astrocytes are glial cells with important physiological functions which respond vigorously with cellular hypertrophy to diverse types of neurological injury. Their response occurs rapidly and can be detected within one hour of a mechanical trauma. However, reactive astrocytosis is also detected in some other situations of brain damage such as in infections, ischemic, inflammatory or degenerative diseases, as is the case of AD. In these cases, astrocytes have been shown to secrete cathepsin G and ACT, among other proteases and protease inhibitors. This molecular profile is typically produced by hematogenous cells during wound repair in peripheral tissues. So astrocytes are believed to act as rapidly responding resident cells of the central nervous system (CNS) in different neuronal tissue repair situations.28,51,54 Microglia are brain macrophages and are its only immunological barrier. They destroy all types of foreign or dead material and release both degradative agents such as proteases, ROS, or nitrogen reactive species, and regenerating molecules such as NGF or IL-1.23–26 Moreover microglia secrete TNF,55 a cytokine that can be cytotoxic to damaged cells, that has been shown to activate astrocytes and cathepsin G secretion.23–26 The role of both microglia and astrocytes in brain repair is very clear. However, uncontrolled activation of microglia may be involved in AD acceleration rather than protecting against the disease.56 Given that brain repair is mediated by different APP isoforms and BAP and that microglia induces (through NGF or IL-1) the expression of APP isoforms, overexpression of APP related forms, together with a defect in their normal cellular processing, could indeed promote BAP accumulation and consequently AD development. In agreement with this, anti-inflammatory drugs slow development of AD.57

BAP Aggregation The aggregation of BAP does not always involve the full amino acid sequence. Thus the hydrophobic domain of BAP (residues 29-42) is more likely to be involved in aggregation that the hydrophilic domain (residues 1-28). In addition, polymerization requires the involvement of a nucleation factor which acts as a prime.3 Cell pH is among the factors that may influence the aggregation of BAP;3,58 a decrease in pH, possibly due to lysosomal intervention, increases aggregation. The pH may influence the protonation state of some amino acids and may thus decrease the solubility of BAP. This is especially important around the pI of the peptide. Furthermore high salt concentrations of metals such as zinc and aluminum also seem to play a very important role in BAP aggregation.3,59 Thus the amyloid deposit includes these metals. Aluminium is present as aluminosilicates in AD plaques and tangles and thus diets containing this metal should be considered as being of special risk for the appearance of the disease. There is, however, a significant lack of epidemiological studies. Another factor contributing to enhanced BAP aggregation is dependent on possible mutations affecting this molecule. To illustrate this point, one has to take into consideration that about 5% of the AD cases have a genetic component and that in those cases there are point mutations in the sequence of the BAP such as the appearance of a mutation Glu 22Gln which results, not in a enhanced accumulation but in increased aggregation and thus con-

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tributes to the higher incidence of the disease.3,60 Finally different proteins can also influence aggregation (apolipoprotein E4, apolipoprotein E2, serum amyloid P, heparan-sulphate proteoglycans and ACT).3

The Tau Protein The tau protein, which can exist in six isoforms with molecular masses ranging from 55 to 68 kDa, is thought to stabilize microtubules in axons, thus providing the basis for axonal transport.61,62 The function of microtubules in the brain, although not yet fully established, seems to be associated with the movement of cytoplasmic constituents, especially in axoplasmic flow. Tau is also responsible for one of the main characteristics of AD: the neurofibrillary tangles of which the pair-helical filaments (PHF) are one of the main AD components.62,63 PHF appear as double-stranded structures, showing periodic variations in width between about 8 and 20 nm with a spacing between cross-overs of about 80 nm. The whole PHF can be considered as a twisted ribbon. Another form of abnormal filament is the so-called straight filament (SF), also found in AD tangles. These filaments are termed straight because they seem to have a uniform width of 15 nm. Some observations indicate that they represent alternative forms of assembly of the same subunit (tau protein).63 Each tangle contains paired helical filaments as major components and single straight filaments as minor components.64 PHF is assembled inside the cell at an early stage as an exclusive hyperphosphorylated form which is the full-length form.65,66 Once PHF has accumulated it is gradually cleaved off at its N-terminus which is followed by ubiquitination.67 The C-terminal fragment is later removed outside the cell (as a consequence of cell death) constituting the extracellular tangles68,69 which will later have a key role in the aggregation of BAP.70 Microtubules serve as tracks for intracellular transport. Tau is thought to stabilize microtubules in axons and thus provide the basis for axonal transport. The cellular function of tau is to promote the nucleation and elongation of microtubules and to protect them against disassembly. These functions can be traced back to different domains of tau. Assembly not only requires the internal repeat domain but also regions from either side which act as jaws; the flanking region in turn tends to promote the formation of bundles (parallel arrays) which may be important in neurite outgrowth.62 The core of PHF is made up of the three or four tandem repeat regions which normally function as microtubule-binding domains.64 Goedert et al have reported that assembly of PHFs results from three repeat-tau and that of SFs from four repeat-tau.71 There are at least six different isoforms of the human tau protein which differ either in having three or four repeats in the microtubule-binding domain or by the presence or absence of additional inserts near the N-terminus of the protein. The different isoforms arise from a single gene by alternative mRNA splicing. It seems likely that all isoforms of the tau protein are incorporated into PHF, given that antibodies against different parts of the longest tau isoform react with neurofibrillary tangles (NFT).63 No differences have been detected in the expression of the mRNAs corresponding to the different isoforms of the tau protein in AD brains versus aged normal controls. Thus if an abnormality in the tau protein contributes to the assembly of PHF in AD, it is likely to be some form of post-translational modification rather than the level of expression. In fact it appears that abnormal forms of the tau protein can be detected in AD and that the modification may reside mainly in the phosphorylation state of the protein.63 Other alterations in the tau protein (not linked to phosphorylation) are its binding to ubiquitin and its glycation.72 The importance of phosphorylation of the tau protein is related to its main role in tubulin assembly. Phosphorylation is a negative factor that interferes with the binding of tubulin. Conversely, dephosphorylation (by means of phosphatase activity) is a key factor in maintaining the phosphorylation balance of the tau protein.64,73

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Interestingly, Goedert et al have shown that glycosaminoglycanes (GAGs) such as heparin, heparan sulphate, chondroitin sulphate or dermatan sulphate promote PHF assembly.71 Phosphorylation of tau is not required and does not inhibit or promote PHF formation. In fact tau has several sites of heparan binding in the N-terminal fragment comprising the acidic, basic, and proline-rich regions, and in the C-terminal half which includes the repeat region. They have also shown that, like phosphorylation, heparin inhibits tau binding to microtubules, leading to microtubule disassembly. Reinforcing the role of GAGs in PHF formation, the same authors reported that an accumulation of heparan sulphate precedes tangle formation whereas GAGs stimulate tau phosphorylation and coexist in nerve cells of AD brains. Also heparan sulphate GAGs have been found in amyloid plaques and neurofibrillary tangles where they are bound to BAP and APP. APP isoforms contain chondroitin sulphate which may directly promote tau polymerization if the two molecules happen to be in the same compartment as a consequence of alterations in the cell’s physiology such as oxidative stress. Such an interaction would require a membrane abnormality since glycosylation in normal cells takes place in the lumen of the rough endoplasmic reticulum and the Golgi apparatus. Such an abnormality may be caused by BAP-induced oxidative stress.64 NFT may exert additional oxidative stress on the tangle-bearing neuron. This may be mediated by advanced glycosylation end-products (formed by nonenzymatic glycosylation and oxidation) that are capable of generating reactive oxygen intermediates and are found in NFT and senile plaques of AD brains. Consistent with this fact is the colocalization of oxidizing stress markers in such structures.75 In addition, free radicals have been shown to induce crosslinking of tau in vitro, thus contributing to PHF formation.75 Although phosphorylation of certain residues of the tau protein is involved in some of the physiological proper ties of the protein su ch as its cellu lar degradation , 76,77 hyperphosphorylation (involving both residues which are not normally phosphorylated and a higher phosphate stoichiometry of certain residues) leads to the formation of PHF-tau (Table 3.1). Interestingly, the presence of a relatively highly phosphorylated form of tau (which does not form PHF) has been shown in the foetus.78 Morishima-Kawashima et al78 and Hasegawa et al79 have made major contributions to mapping the phosphorylation sites of the AD abnormal tau protein. Phosphorylation can occur in either a proline- or nonproline-directed fashion (Fig. 3.4). Proline-directed phosphorylation features a proline contiguous to the phosphoacceptor serine or threonine at the carboxy terminal side.80 Phosphorylation sites of PHF-tau and fetal tau (which is also highly

Table 3.1 Kinase-phosphatase systems involved in tau phosphorylation/ dephosphorylation Enzyme

Pro-directed

Binding to microtubules

Direct phosphorylation

cdc2 MAPK GSK3B 35/41 K PKA PKC CamK Casein K

yes yes yes no no no no no

yes yes yes no yes no no no

yes yes no yes yes ? ? ?

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Fig. 3.4. Phosphorylation sites of PHF-tau. Phosphorylation can occur in either a proline- or nonproline-directed fashion. Proline-directed phosphorylation features a proline contiguous to the phosphoacceptor serine or threonine at the C-terminal side. There are 19 sites of phosphorylation in pathological PHF-tau, all of them (except for Ser-262) are localized in the N- and C-terminal flanking regions of the microtubule binding domain (see text for more details).

phosphorylated) provide four important findings: (1) there are 19 sites of phosphorylation in pathological PHF-tau, all of them (except for Ser-262) are localized in the N- and C-terminal flanking regions of the microtubule-binding domain, half of them being shared by fetal tau; (2) PHF-tau is phosphorylated at almost twice as many sites as fetal tau, and in this latter form most of them are proline-directed; (3) 7 of 10 additional phosphorylation sites in PHF-tau are nonprotein kinase proline-directed sites; and (4) the extent of phosphorylation at given sites in fetal tau is less than in PHF-tau. Concerning the kinase systems involved in these phosphorylation patterns, neuronal cdc2-like kinase, MAP kinases and GSK3B kinases are proline-directed, all of them being physically associated with microtubules and thus detected in neurofibrillary tangles. Whereas the first two can directly phosphorylate the tau protein, GSK3B kinases can only phosphorylate it when it has already been phosphorylated by other kinases. Nonproline directed kinases involve protein kinases A and C and calmodulin kinase, casein kinase and 35/41 kinase.74,78 It is very interesting to point out that a 35/41 kinase phosphorylates Ser-262 (which is located within the first microtubule binding repeat), inhibiting microtubule-binding several-fold more than the incorporation of 10 phosphates by MAP kinase.81 In conclusion, all the above mentioned kinases are involved in the redistribution and reorganization of microtubules, all these enzymes being additionally interphosphorylated, rendering the situation still more complicated. The proline-directed kinases are involved in a very complex kinase network that participates in signal transduction related to neural regeneration. It has been proposed that AD may result from accumulated defects in the kinase network that governs the proline-directed kinases such that their inappropriate activation is sustained in the affected neurons.80 The transduction pathway for NGF (involved in both neuroregeneration and neuroprotection) first involves binding to the appropriate extracellular ligand, dimerization of the receptor and cross-tyrosine phosphorylation.80 After this event, either activation of the MEK-MAP cascade or activation (via phospholipase C- γ) of Raf occurs, the final

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Fig. 3.5. Signal transduction pathways associated with tau hyperphosphorylation. There are basically two kinase systems involved in the transduction pathway for NGF. One of the is associated with Ras-GAP, while the other is based on PLCg activity. Both pathways finally lead to MAP kinase activation which then drives tau phosphorylation.

result being phosphorylation of the tau protein and subsequent microtubule rearrangement (Fig. 3.5). In this signal transduction pathway a very important role is also undertaken by phosphatases as we will discuss shortly. Several studies have revealed similarities between the phosphorylation state of biopsyderived normal adult human tau, postmortem human fetal tau and postmortem PHF-tau, thus suggesting that hyperphosphorylation may be the consequence of a defect in phosphatase activity rather than the result of hyperphosphorylation per se. In this way, PHF tau may be generated as a result of defective phosphatases in the AD brain rather than as a consequence of hyperactive kinases.70 Recently it has been demonstrated that normal tau is phosphorylated in many of the fetal sites but is rapidly dephosphorylated after tissue resection by active protein phosphatases. This suggests that phosphorylated tau forms must have a rapid turnover in vivo, probably regulated by a dynamic balance between multiple protein kinases and protein phosphatases.

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Fig. 3.6. Interaction between PHF-tau and BAP. Due to the action of toxic agents or during ageing, there is an enhanced hyperphosphorylation of tau that leads to an increased BAP deposition. In addition, these agents can also induce directly the accumulation of BAP. Microglia can also contribute to this BAP formation from APP. BAP accumulation is finally responsible for neural toxicity.

So hyperphosphorylation of tau can be a consequence of upregulation of protein kinases or downregulation of protein phosphatases. Three protein phosphatases are known to dephosphorylate tau on proline-directed sites in vitro: protein phosphatase I, 2A and 2B. The activity of these enzymes are decreases in the brain of AD patients.82

The Link Between Tau Phosphorylation and APP An important functional and biochemical link exists between tau hyperphosphorylation and APP. First, tau participates in neuronal regeneration and synaptic plasticity.83–85 Secreted APPs and tau hyperphosphorylation seem to be involved both in neuronal plasticity and in neuroprotective mechanisms against different types of insults involved in AD development (Fig. 3.6).75 On the other hand, the tau-induced hyperphosphorylation by secreted APPs through MAP kinase activation has recently established a biochemical link. Greenberg and Kosik86 have demonstrated that APP can activate MAP kinase in cortical neurons, this fact having been related to tau phosphorylation, thus providing a potential link between two of the central elements of AD: APP and hyperphosphorylated tau. The accumulation of secreted APP could include a sustained MAP kinase activation and consequently PHF-tau formation and an impairment in synaptic plasticity.

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The involvement of APP in neuronal plasticity has already been mentioned above. The involvement of tau hyperphosphorylation has been suggested recently by different observations. The term “neuronal plasticity” is widely used to express the adaptability of neurons to different situations. Neurons try to restore lost synaptic connections as a consequence of nonfunctional or dead neurons. Interestingly, loss of synapses87,88 and a failure in neuronal plasticity has been related to dementia in AD.83 Supporting this argument, aged brains of healthy humans show an age-related increase in the dendritic extent of single neurons in most of the brain regions studied to date. Such age-related increases have not been found in AD.83 Callahan et al83 have suggested that tau hyperphosphorylation is involved in neuronal plasticity, having shown that exclusively tangle-bearing neurons in AD brains show a reduction in neuronal plasticity, this being measured by expression of GAP43, a protein which is known to be associated with this function. Other observations have also suggested a role for tau hyperphosphorylation in synaptic plasticity. For example, the expression of tau hyperphosphorylated epitopes (AT8 and PHF1 epitopes) has been associated with situations of extensive neurogenesis where there is rapid neurite outgrowth.84,85,89 Another link between APP and tau hyperphosphorylation is their involvement in neuroprotective mechanisms. Such mechanisms act against different types of insults such as glucose deprivation, ischemia, calcium imbalance, or free radical accumulation, which have been shown to induce PHF-tau-like hyperphosphorylation and to be a risk for AD development. Interestingly, the secreted forms of APP have neuroprotective effects against some of these insults75 whilst activation of neuroprotective signaling pathways involves MAP kinase activity and tau phosphorylation in a PHF-like manner.90,91 Recently, Greenberg and Kosik86 reported a direct biochemical link between APP and tau hyperphosphorylation. They showed that the secreted forms of APP activate MAP kinases in PC 12 pheochromocytoma cells or cultured cortical neurons, inducing increased amounts of hyperphosphorylated tau in the AT8 epitope. NGF also provides a biochemical link between APP and tau hyperphosphorylation. NGF is probably the neurotrophic factor most involved both in neuroprotective mechanisms and neuronal plasticity. The link between APP and tau hyperphosphorylation is suggested by the fact that the secreted APP (PN2) acts through the NGF receptor, potentiating the NGF activity, and the fact that NGF action in neuronal plasticity and neuroprotection is mediated through MAP kinase activation and tau hyperphosphorylation.75 Given that both APP and tau hyperphosphorylation seem to have a role in neuronal tissue repair and neuroprotective mechanisms, it may be suggested that environmentally-induced or genetic defects in the signal transduction pathways of these mechanisms may well be involved in AD development. A continuing controversy concerns which molecule is ultimately responsible for AD development. Is it BAP or PHF? Given that there is enough evidence to suggest that APP and tau hyperphosphorylation are involved in the same mechanisms, this controversy does not seem very relevant. This is because a genetic defect could induce either BAP or PHF, and either of them could induce the other, as a consequence of an altered mechanism. Both direct and toxicological evidence has led to the same conclusion. BAP has been shown to induce tau hyperphosphorylation and cell death.92,93 Thus, BAP action could induce PHFlike-tau and PHF through different types of imbalance situations, where free radical accumulation and oxidative stress could promote PHF aggregation, as suggested above. On the other hand, Trojanowski et al70 reported that injection of purified PHF-tau into the rodent brain (but not other plaque and tangle components), induces codeposits of BAP. This may be related to the released PHF from degenerating neurons which may promote the aggregation of the normally-secreted soluble BAP. Finally, amyloid deposition occurs in the absence of NFT in AD.94 However, this type of amyloid deposition is related to age and to nonpathologically related diffuse senile plaques. In view of the above it is possible that BAP

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or PHF could be generated as a consequence of a genetic defect, and that either could promote the aggregation of the other. Thus, the glial reaction involved in neuronal protection or regeneration would exponentially enhance such a positive feed-back mechanism, promoting neuritic senile plaques and AD development. Such promoting activity would be a consequence of the IL-1 overproduction between others trying to activate a mechanism which is blocked first by the genetic defect and later by BAP and PHF toxicity. More dead neurons would further activate the glial reaction and thus neuronal degeneration.

Ubiquitin and Alzheimer’s Disease The events that generate the aberrant tau and beta amyloid proteins seem to be the key to understanding the etiology of AD. The study of the other components associated with these abnormal proteins may provide clues to these events. But is ubiquitin always associated with the typical alterations of AD? Taddei et al95 and Wang et al96 reported an increased amount of soluble or immunoreactive ubiquitin respectively in the cerebral cortex of brains from patients with AD as compared with the levels found in control brains. Moreover, the increase reported by Wang et al96 was strongly correlated with the degree of neurofibrillar changes. These changes were nonexistent in the cerebellum where the ubiquitin levels were normal and much less remarkable in the white than in the grey matter of the cortex. Although these results suggest a permanent association of ubiquitin with the neurofibrillary tangles, this has become more evident from the study carried out by Morishima and Ihara97 on the composition of paired helical filaments. These authors purified and analyzed the components of such paired helical filaments and showed that they are composed of abnormally full-length phosphorylated tau proteins and ubiquitin, many of them associated with their microtubule-binding domain. Shin et al5 have found additional evidence concerning the permanent association of ubiquitin with the typical alterations of AD. They demonstrated that intracerebral injection of abnormally full-length phosphorylated tau protein into rats induces codeposits of ubiquitin and the other typical components of AD such as BAP, ACP, apolipoprotein E and heparan sulphate proteoglycans, both in the senile plaques and in the neurofibrillary tangles. But why does ubiquitin accumulate in the neurofibrillary tangles and senile plaques? What is its function in such places? Taking into account that the ubiquitin system degrades mainly abnormal proteins generated in cellular stress situations, some authors have proposed a similar action in the neurons affected by AD.98,99 Under stress situations caused, for example, by temperature, heavy metals, oxidants or free radicals, perturbations of protein structure or an impaired protein synthesis leads to cell damage and death. Most cells avoid this by means of both the synthesis of heat-shock proteins which prevent protein denaturation and the synthesis of components of the ubiquitin system which degrade the abnormal proteins.100 According to the parallelism proposed between cellular stress situations and the situation of the neurons affected by AD, Cisse et al have shown the presence of heat-shock proteins in the brains of AD patients.101 Cellular accumulation of free and conjugated ubiquitin may be interpreted as a certain blockage of degradative action protecting against stress conditions. In accordance, an accumulation of ubiquitin and ubiquitin conjugates during deficits of proteasome activity has been described.102,103 On the other hand, ubiquitin accumulation could represent hyperactivity of the proteolytic system, unable to protect the cell. Such hyperactivity could lead to cellular degradation and degeneration in an unregulated way. But it has also been suggested that this could be related to regulated activity of the ubiquitin system in cellular degradation.104 This proposal takes into consideration that ubiquitin has a role in programmed cell death.105,106 In this case, the presence of damaged neurons may activate an apoptotic mechanism mediated by the ubiquitin system.

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The action of the ubiquitin system does not necessarily have to lead to cellular degradation. As in cellular protection against stress conditions, ubiquitin could be involved in neuronal regeneration and reparative rather than destructive metabolic events. This seems probable because Takada et al107 and Savedia and Kiernan 108 have implicated the ubiquitin system in neurite outgrowth and axonal regeneration respectively. In the first case, neuronal differentiation and neurite outgrowth in PC12 cells, promoted by NGF, were associated with the increase of high molecular weight ubiquitin-protein conjugates, suggesting that the ubiquitin system has a role in this process.107 In the second case, the axonal regeneration that occurs after transection of motor neuron axons in the rat was associated with a twofold increase in ubiquitin mRNA content, while there was no significant change when axonal regeneration was prevented by nerve ligation.108 The ubiquitin-dependent proteolytic pathway is also involved in axonal regeneration through the degradation of tRNA-mediated arginylated proteins. Such arginylation, which renders proteins with N-terminal acidic amino acids sensitive to the ubiquitin pathway, has been shown to be rapid after crushing of sciatic nerves and is related to axonal regeneration.109 More studies are needed to determine the exact role. In addition to the activity of the ubiquitin system in neurite outgrowth or axonal regeneration, ubiquitin has a role both in the synaptic connectivity of Drosophila neurons and in other cellular adhesion events.110–112 It thus seems probable that the ubiquitin system could act in a neurotissue repair mechanism. This idea is reinforced by the fact that the ubiquitin system is able to degrade (at least in vitro) the extracellular domains of APP,113 a protein also involved in AD which has a role in neuronal growth and neuronal adhesion events. In addition, Chow et al have cloned and sequenced a 59 kDa APP-binding protein called APP-BP1114 which interacts with the C-terminal region of APP in vitro and has sequence similarity with the ubiquitin activating enzyme E1. Although these studies have included the APP-BP1 protein in a new family of E1-related proteins (with structural variations), it is also believed to have a role in a cellular ubiquitination pathway.114 The implication of APP and its proteolytic fragments in tissue repair mechanisms such as injury healing or neurotissue repair has been widely supported. For example, the serine protease and the inhibitor of serine proteases (nexin II), identified in the APP structure,34,115 have previously been implicated in healing and in neurite outgrowth.21,116 In addition, the mitogenic effect on fibroblasts of the secreted fragments of the APP751 isoform 33 and the APP requirement for their normal growth in cell cultures117 is consistent with the APP role in tissue repair. Finally, in a similar fashion, structural features of APP that are related to cellular adhesion proteins have been described.4,118 In relation to the neuronal tissue, it is noteworthy that APP is likely to be a heparan sulphate proteoglycan (HSPG) 38,119 since these molecules are typically located in the synaptic space and act as receptors of intercellular adhesion promoting molecules.120 Thus it may be suggested that ubiquitin and APP could cooperate in a mechanism that would repair neuronal tissue damage, promoting neurite and/or axonal growth and the restoration of the lost synaptic connections. This idea is supported by the fact that APP is an acute-phase protein expressed and/or secreted in response to physiological injury. Moreover, both ubiquitin and APP are expressed in neuron injury situations that may be subject to a repair event. For example, Bacci et al99 have shown the presence of ubiquitin and APP in dystrophic axons of different origin, including p-bromophenylacetylurea-induced dystrophic axons in rats. Also, Nakamura et al121 have detected the presence of ubiquitin and APP in a highly atrophic rat hippocampus with extensive neuronal loss induced by ibotenic acid. In addition, axonal injury situations have been described, for instance, axonal transection or brain axonal injury after head trauma, in which the ubiquitin mRNA or APP are expressed respectively.108,122

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At this point, several questions arise. Is ubiquitin really involved in the control of APP functions in vivo? Could impaired degradation of APP by the ubiquitin system lead to an APP tissue repair dysfunction and to the development of AD? Although it is known that the extracellular domains of the APP isoforms are cleaved into its beta-amyloid sequence31 and that the APP secreted fragments are critical to mediate its function,22,117 more research is needed to determine the different APP proteolytic transformations and their physiological relevance. APP conjugation to ubiquitin could also have an important role in internalization (this is, for example, the case for the platelet-derived growth factor (PDGF) receptor) 123 and further processing such as lysosomal degradation or BAP generation. A possible APP-ubiquitin related dysfunction may be related to mutations in the ubiquitin system-related genes such as ubiquitin-peptidases or proteasome genes. But it may also be related to alterations in signal transduction mechanisms involved in ubiquitin system activation. An intriguing question is whether TNF or other cytokines can regulate ubiquitin and APP function. This possibility is strongly suggested by the fact that TNF activates the ubiquitin system has a role as a cellular growth and differentiation factor and also in the regulation of cellular adhesion proteins.124 Gregori et al have shown that either BAP or some of its fragments selectively inhibit the ubiquitin-dependent protein degradation in vitro.125 They operate through the chymotrypsin-like activity of the 20S proteasome, having no effect on either conjugate formation or conjugate deubiquitinitation. Apparently, there are at least two regions of BAP that are important for inhibition: the N-terminal region and a portion of the transmembrane domain, which have a cooperative effect when they are together in the BAP molecule. The same authors have shown that while BAP inhibits the chymotrypsin-like activity of the proteasome 20S, it has no effect on the proteolytic activity of the protease chymotrypsin, suggesting that BAP does not interact with the active site of the proteasome subunit. These and other observations have led to a model in which BAP allosteric effects induce conformational changes in the proteasome, thus preventing the substrate from interacting with the subunit active site.125 Taking into account the close relationship between APP and the ubiquitin system, such BAP inhibition of ubiquitin-dependent proteolytic activity could be related to a negative control in the APP metabolism mediated by the ubiquitin system. PN2 is a serine protease inhibitor derived from the APP mitogenic isoforms (APP771 and APP750) by the activity of alpha-secretase, which cleaves the APP molecule at residue 16 of the BAP sequence near the cell membrane. Thus, PN2 is secreted into the extracellular medium to activate (together with other molecules) neurite outgrowth. Gregori et al have shown that the secreted forms of APP770, APP751, and APP695 are degraded in vitro by the ubiquitin-dependent proteolytic system, so it seems very likely that PN2 would correspond to the secreted forms of APP770 and APP751.113 In agreement with this, APP (in the PN2 sequence) has been characterized as a protein rich in PEST sequences.126 The structural motif known as a PEST sequence is flanked by basic residues and is enriched in proline (P), glutamic acid (E), aspartic acid (D), serine (S) and threonine (T). PEST sequences target substrates for ubiquitin system recognition.127 PN2 accumulation in the dendritic plaques and in capillary walls may result in the constant stimulation of MAP kinases (and thus generation of PHF-tau and PHF). On the other hand, an alteration in the normal PN2 metabolism may block or disrupt the activity of other protease inhibitors or proteases involved in neuronal regeneration. Additionally, accumulation of PN2 in capillary walls may be associated with ischemic and hypoglycaemic stimuli which would further activate both PHF-tau and PHF formation. Finally, the ubiquitin system is also involved in DNA repair mechanisms.128 At this level, a DNA repair dysfunction may not only lead to gene alterations related to an impaired cellular stress response or a neurotissue repair mechanism, but also to other possiblee tiologies

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of AD, such as impaired circulatory function, as will be discussed later. If it is accepted that the involvement of the ubiquitin system in different repair events could be used in the prevention of AD, the study of its enzymes and their physiological control should be encouraged in the future both in neuronal tissue and other related tissues. Although the primary structure of ubiquitin is normal in neurons affected by AD,95 further studies could be directed towards the different enzymes of the system; for example, conjugase, ligase, peptidase, isopeptidase or proteasome activities. Are their genes and their genetic expressions normal? What different factors related to the disease, like for example the PHF-tau-ubiquitin complex, could modify or inhibit their activity? These and other intriguing questions may lead to a better understanding of the disease. Nowadays, a ubiquitin-conjugating gene ( UBE2L1) has been detected which has been suggested to correspond to the FAD3 gene involved in AD development.129

Ubiquitin Participates in Neural Regeneration Until now we have considered that the ubiquitin system may have a role in three repair mechanisms, namely the cellular stress response, DNA repair and the neurotissue repair mechanism where it would share a role with APP. An impairment of either of them could lead to the development of the disease. Among the possible causes of such impairment, one has to consider the interaction between the PHF-tau protein and the ubiquitin system. With regard to this interaction, abnormally full-length phosphorylated tau protein and ubiquitin are the components of paired helical filaments, which are one of the hallmarks of AD.97 Some authors think that the cellular events that lead to the aberrant tau protein (PHF-tau) and to the paired helical filaments are the clue to the development of AD. It has been reported that glucose deprivation in cortical or hippocampus neuron cultures raises the intracellular calcium levels, and this induces changes in both PHF-tau and ubiquitin, and neuronal degeneration.130–132 In addition, the presence of PHF-tau-like and ubiquitin immunoreactivity in response to a cerebral ischemic insult has been shown in cats.133 So it seems that an impaired cerebral circulatory function could be one of the first factors involved in PHF-tau generation. Once generated by abnormal phosphorylation, the PHF-tau protein could be an important factor leading to other typical changes in AD. In support of this hypothesis, Shin et al5 have demonstrated that intracerebral injection of PHF-tau protein in the rat induces codeposits of ubiquitin, BAP and the other typical components of the disease. However, other possibilities cannot be discarded, for instance, the initial generation of BAP. Takashima et al92 have reported that beta amyloid protein raises protein kinase activity in cultured neurons, this leading to a highly phosphorylated tau protein and cell death as a consequence of this phosphorylation. The tau protein is a microtubule-associated protein that is associated with ubiquitin, in normal growth conditions, in a human neural cell line.134 Thus, the tau protein could be degraded normally by the ubiquitin system.134 On the other hand, it has been shown that the ubiquitin-associated PHF-tau protein is relatively resistant to proteolysis.135 So it may be relevant to ask: is the tau protein really degraded by the ubiquitin system under normal conditions? Does the PHF-tau-ubiquitin complex block the normal activity of the ubiquitin system? Perhaps through the inhibition of peptidase, isopeptidase or proteasome activities? In fact, the inhibition of the ubiquitin system by the PHF-tau-ubiquitin complex may be one of the determinant steps for the development of AD. Such inhibition may lead to an impaired proteolytic degradation of APP and, consequently, to the generation of the BAP, as has been suggested by Vazquez et al20 Not only would this lead to a malfunction of the neuronal reparative effects of APP but also to the neurotoxic effect of BAP. Moreover, the inhibition of the ubiquitin system could lead to a lack of abnormal protein degradation in the ubiquitin-mediated stress response.

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Another important role of the ubiquitin system is to prevent the aggregation of hyperphosphorylated tau. A defective response of the ubiquitin system to antioxidative enzymes could result in an accumulation of reactive oxygen species that could induce membrane damage and alter compartmentation, resulting in PHF-tau generation and subsequent aggregation due to the contact with GAGs, as we have previously seen.

Ubiquitin and NF- κB Generation The ubiquitin-dependent proteolytic system may also have a role in AD development through activation of the nuclear transcription factor κB (NF- κB). NF- κB is a heterodimeric complex consisting of a p50 and a p65 subunit, which is tightly regulated through the inhibitor I- κB proteins. Such proteins bind to NF- κB, forming an inactive cytoplasmic ternary complex. Release from the I-κB proteins allows NF- κB to become active and translocate to the nucleus. Recently, Palombella et al136 have shown that NF- κB can be activated by the ubiquitin dependent proteolytic pathway through processing of the 105 kDa precursor protein (p105) of the p50 subunit or through degradation of the I- κB proteins after phosphorylation. NF- κB is involved in immune and inflammatory responses so it may have different implications at the level of the microglia.136,137 NF- κB is involved in the transcription of the class I major histocompatibility complex (MHC) gene, the interleukin-6 (IL-6) gene and cell adhesion genes like E-selectin, ICAM, and VCAM-1.135 Given that MHC-I molecules increase in number in the AD microglia and that normally they act in macrophage-mediated activation processes, it would not be out of place to suggest that they may have a role related to microglia. It also has to be remembered that the ubiquitin-dependent pathway mediates the generation of antigenic peptides from cytoplasmic proteins for presentation on MHC-I molecules.138,139 IL-6 expression (which is induced by the ACT-cathepsin G complex) has been shown to have a role in inducing the synthesis of other acute-phase proteins, probably implicated in the neuronal regeneration process.140 Finally, the expression of cell adhesion molecules is involved in axon growth and guidance and thus in the process of neuronal regeneration.141 The Ig superfamily includes, among others, the neural cell adhesion molecule (NCAM) and the neuron-glia cell adhesion molecule (Ng-CAM). The action of CAMs seems to be mediated through complex interactions. For example, the axonin-1 CAM interacts with Ng-CAM and with integrins to which APP seems to be related. At this point it could be asked: is expression of APP controlled by the ubiquitin system through NF- κB activation?

Other Neurodegenerative Disorders The involvement of the ubiquitin system in neurodegenerative disorders other than AD has been suggested following the finding of ubiquitin-related intraneuronal inclusions by means of immunohistochemical characterization. Future studies will probably reveal further involvement of the ubiquitin system in the generation of such diseases. Among the neurodegenerative diseases related to ubiquitin, several groups may be considered. Lewy body diseases, including Parkinson’s disease and diffuse Lewy bodies disease, amyotrophic lateral sclerosis, the prion or spongiform encephalopathies, cerebral amyloid angiopathies and, finally, a miscellaneous group of different types of dementias, all of which can either have a hereditary basis or develop sporadically. The main features of these diseases can be found in Table 3.2. Lewy body diseases are characterized by the presence of intracytoplasmic neuronal inclusions named Lewy bodies (LBs). They include Parkinson’s disease and diffuse Lewy body disease.142 Patients suffering from Parkinson’s disease display severe and progressive deficits in motor behavior predominantly as a consequence of the degeneration of dopaminergic neurons located in the mesencephalon and projecting to striatal regions.143 In some cases, they develop dementia. Diffuse Lewy body disease

Alzheimer’s disease Lewy body diseases Parkinson’s disease Diffuse Lewy’s body disease Amyotrophic lateral sclerosis (ALS) Common ALS ALS (DSO) Other dementias Progressive supranuclear palsy Picks disease Huntington’s disease Spongiform encephalopaties Ovine scrapie Mad cow disease Creutzfeld-Jacob disease Gerstmann-Straussler-Scheinker disease Cerebral amyloid angiophaties (CAA) Down syndrome

DISEASE

Table 3.2 Neurodegenerative diseases

BAP NFT PHF-tau BAP

NFT PHF-tau BAP

(-) (-) (-) NFT PHF-tau

NF

NF

NF

NF NF

BAP

(-) (-) (-) (-)

NF

NFT PHF-tau BAP

Presence of

prion prion prion prion

Lewy bodies Lewy bodies

CA2/3 neurites

CA2/3 neurites CA2/3 neurites

UB UB UB UB UB UB

UB UB UB

UB UB

UB UB

UB proteasome proteasome

Immunoreactivity for

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is characterized by a widespread distribution of LBs in the cortex, in addition to the usual subcortical sites. The disease is usually accompanied by a neurobehavioral syndrome that may include hallucinations, delusions and psychosis, all patients eventually becoming demented.142 It is known that the severity of dementia in patients with diffuse Lewy body disease is related to cortical LBs density. Interestingly, the presence of large numbers of ubiquitinated dystrophic neurites called CA2/3 in the hippocampus region (where LBs are most abundant) has been detected.144,145 Such abnormal neurites are thought to be related to abnormal neuronal connectivity. The close relationship between LBs and the CA2/3 dystrophic neurites has also been suggested by their immunocytochemical characterization. In addition, LBs have not only been shown to have ubiquitin immunoreactivity like the CA2/3 dystrophic neurites but also to have high molecular weight polyubiquitinated proteins146 and immunoreactivity against the proteasome.147 Both LBs and CA2/3 dystrophic neurites are also positive for immunoreactivity with antibodies against a neurofilament protein and the neuronal ubiquitin hydrolase PGP 9.5, whereas they do not stay with antibodies against PHF-tau protein or paired helical filaments.145,148 Interestingly the cyclin-dependent kinase 5 is also present in LBs.148 So it has been proposed that cdk5 could be involved in the phosphorylation of neurofilament proteins. The ubiquitin system could be involved in the control of the cdk5 through the degradation of its cyclin. Finally, in spite of the differences between LBs and the intracellular alterations seen in AD, it is interesting to consider that the density of neocortical LBs from patients with Parkinson’s disease correlates with the density of neocortical senile plaques.149 Amyotrophic lateral sclerosis (ALS) is another neurodegenerative disorder which generates a motor clinical syndrome and/or a frontal lobe dementia and is characterized by the presence of different types of ubiquitin-immunoreactive intraneuronal inclusions.150 Initially the disease is associated with selective vulnerability of the corticomotoneuronal system. Thisind uces excessive excitatory neurotransmitter release at the corticomotoneuronalspinal and spinal-motoneuronal synapses, leading finally to the loss of neurons.151,152 However, nonmotor systems and many subcortical structures are also affected in dementia.153 The presence of ubiquitinated and neurofilament rich inclusions in the lower motor neurons has been considered to be the hallmark of ALS.154,155 However some cases of dementia have also been associated with ubiquitinated inclusions in the amygdala. Such inclusions do not react with tau antibodies and are different from AD or LB inclusions.154 Interestingly patients with familial amyotrophic lateral sclerosis (associated with a point mutation in the Cu,Zn- superoxide dismutase gene SOD1) have unusual pathological features which include neurofibrillary tangles in neurons of the globus pallidus, substantia nigra, locus coeruleus and inferior olivary nuclei, and the absence of ubiquitin immunoreactive inclusions in motor neurons.156 In addition to AD and Lewy body disease dementias, which are the most frequent, there are other less common dementias which have been shown to be associated with ubiquitin related inclusions. They include progressive supranuclear palsy (PSP) and Pick’s disease (PD) which are frontotemporal dementias, and Huntington’s disease (HD). PSP is characterized by alterations in specific cortical and subcortical brain areas which are different from AD.157,158 Interestingly, several subcortical nuclei and to a lesser extent, the cerebral cor tex areas have been shown to have n eurofibrillar y tan gles, although they are immunonegative against ubiquitin and consist of straight filaments of tau protein.159 Senile plaques are rare or absent in these areas.158 Pick’s disease is characterized by tau and ubiquitin immunoreactive spherical cortical intraneuronal inclusions.150 Pick’s disease type C also has neurofibrillary tangles that react strongly with antibodies against tau protein and ubiquitin in some cases. Such tangles have been shown to consist, by electron microscopy, of paired helical filaments identical to those

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of AD. No BAP amyloid deposition has been described in Pick’s disease.160 In the case of Huntington’s disease, patients have dystrophic neurites that immunohistochemically resemble the CA 2/3 neurites seen in Lewy body disease. They are immunoreactive with ubiquitin and APP in cortical areas.161 The prion or spongiform encephalopathies have also been shown to have ubiquitin related inclusions. Prion diseases of animals include the ovine scrapie and “mad cow” disease (bovine spongiform encephalopathy), whereas prion diseases of humans include the Creutzfeld-Jakob disease, the Gerstmann-Straussler-Scheinker disease and kuru. Their etiology is based on cellular alterations arising from the presence of an abnormal prion protein. This protein, which is normally associated with neurons, can change its conformation followin g certain m utation s, aggregatin g in to amyloid fibrils, in ducin g in turn a neurodegenerative disorder with dementia. The striking feature of this disease is that it is sometimes an infectious disease where the abnormal prion protein is the transmissible agent via food intake. In addition, the disease can be inherited or sporadic. It has been demonstrated that infectious transmission occurs because the pathological prion protein induces conformational changes in the normal ones, thus promoting fibril aggregation.162–165 Ubiquitin-protein conjugates between others proteins have been identified in late endosome-like organelles where the abnormal prion protein seems to be accumulated and where it is believed to induce the conformational change.166 In addition, in later stages of the disease, an increase in the expression of the polyubiquitin C and heat-shock protein 70 genes has been shown.167 Cerebral amyloid angiopathies comprise a heterogenous group of hereditary or sporadic disorders that are characterized clinically by ischemic and/or hemorrhagic strokes and histologically by BAP amyloid deposition in the wall of leptomeningeal and cerebral cortical blood vessels. Such BAP deposition occurs without the presence of neurofibrillary tangles and has been shown to be the consequence of mutations in the APP molecule.168,169 Finally, Down’s syndrome (which is caused by the trisomy of the chromosome 21) also has to be considered as being closely related to AD because of the presence of neurofibrillary tangles and BAP amyloid deposition.6,170 Oxidative stress, immune response and apoptotic mechanisms have been proposed to be involved in most neurodegenerative disorders. So the involvement of the ubiquitin system in such mechanisms (as we have previously emphasized) reinforces the idea (together with the experimental findings of ubiquitin presence in those diseases) that the system may have a key role in the etiology of these pathological states. In addition, defects in neuroprotective and neuroreparative mechanisms have been proposed as possible causes. Thus AD and other neurodegenerative disorders may be good models with which to study the involvement of the ubiquitin system in such mechanisms. Interestingly, all the neurodegenerative disorders briefly described have features in common. Such overlap may be helpful for obtaining biochemical correlations in the study of the etiology of suchdiseases.

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153. Jackson M, Lowe J. The new neuropathology of degenerative frontotemporal dementias. Acta Neuropathol Berl 1996; 91:127-134. 154. Anderson VE, Cairns NJ, Leigh PN. Involvement of the amygdala, dentate and hippocampus in motor neuron disease. J Neurol Sci 1995; 129:75-78. 155. Migheli A, Attanasio A, Schiffer D. Ubiquitin and neurofilament expression in anterior horn cells in amyotrophic lateral sclerosis: Possible clues to the pathogenesis. Neuropathol Appl Neurobiol 1994; 20:282-289. 156. Orrell RW, King AW, Hilton DA et al. Familial amyotrophic lateral sclerosis with a point mutation of SOD-1: Intrafamilial heterogeneity of disease duration associated with neurofibrillary tangles. J Neurol Neurosurg Psychiatry 1995; 59:266-270. 157. Cruz-Sánchez FF. Antigenic determinant properties of neurofibrillary tangles. Relevance to progressive supranuclear palsy. J Neural Transm 1994; 42:165-178. 158. Verny M, Duyckaerts C, Delaere P et al. Cortical tangles in progressive supranuclear palsy. J Neural Transm 1994; 42:179-188. 159. Taraszewska A, Barcikowska M, Pasierbski W. A case of progressive supranuclear palsy with widespread appearance of neurofibrillary changes and associated senile and vascular brain lesions. Folia Neuropathol 1995; 33:93-99. 160. Love S, Bridges LR, Case CP. Neurofibrillary tangles in Niemann-Pick disease type C. Brain 1995; 118:119-129. 161. Jackson M, Gentleman S, Lennox G et al. The cortical neuritic pathology of Huntington’s disease. Neuropathol Appl Neurobiol 1995; 21:18-26. 162. Weissmann C. Molecular biology of transmissible spongiform encephalopathies. FEBS Lett 1996; 389:3-11. 163. Wells GAH, Wilesmith JW. The neuropathology and epidemiology of the bovine spongiform encephalopathy. Brain Pathol 1995; 5:91-103. 164. Dearmond SJ, Prusiner SB. Prion protein transgenes and the neuropathology in prion diseases. Brain Pathol 1995; 5:77-89. 165. Ghetti B, Dlouhy SR, Giaccone G et al. Gerstmann-Straussler-Scheinker disease and the Indian kindred. Brain Pathol 1995; 5:61-75. 166. Arnold JE, Tipler C, Laszlo L et al. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. J Pathol 1995; 176:403-411. 167. Kenward N, Hope J, Landon M et al. Expression of polyubiquitin and heat-shock protein 70 genes increases in the later stages of disease progression in scrapie-infected mouse brain. J Neurochem 1994; 62:1870-1877. 168. Coria F, Rubio I. Cerebral amyloid angiopathies. Neuropathol Appl Neurobiol 1996; 22:216-227. 169. Hendryks L, Van Duijn CM, Cras P et al. Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the beta-amyloid precursor protein gene. Nature Genet 1992; 1:218-221. 170. Selkoe DJ. Biochemistry of altered brain proteins in Alzheimer’s disease. Annu Rev Neurosci 1989; 12:463-469.

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CHAPTER 4

Cancer I

n the last ten years, one of the main aims in cancer research has been the elucidation of the mechanism that controls the cell cycle. It is also known that the ubiquitin system takes part in this mechanism and that its activity permits and promotes cellular proliferation. Handley-Gearhart et al1 described a mutation in ubiquitin-activating enzyme (E1) that is the primary defect responsible for cell-cycle arrest in a temperature-sensitive mutant cell line (ts20) derived from Chinese hamster ovary E36 cells. These authors demonstrated the relevance of the activating enzyme through the transfection and expression of human E1 cDNA into these cells, which completely rescued the ts20 phenotype at the nonpermissive temperature. In addition, other authors have reported that subunit mutations in the ubiquitin-related proteasome activity cause yeast mitosis arrest.2,3 Other evidence involving the ubiquitin system in the control of the cell cycle has been reported by Mori et al4 They showed a clear relationship between the ubiquitin conjugation of H2A histone and DNA synthesis in a temperature-sensitive mutant cell line defective in DNA replication (tsFT5) isolated from the mouse mammary carcinoma cell line FM3A. But how does the ubiquitin system act in the control of the cell cycle?

Cyclins and Cyclin-dependent Kinases The ubiquitin system is involved in the control of cyclin dependent-kinases (CDKs) through the regulation of cyclin degradation.5 Cyclins are small proteins that bind to the CDKs, activating them in a cyclin-CDK complex. The successive activation and deactivation of specific cyclin-CDK complexes permits the transition from one cell cycle state to another (for example from G1 phase to DNA replication). G1 cyclins (Cln1,2,3 in yeast, and D,E,C in mammalian cells) and B-type cyclins in yeast (S-phase: Clb5,6; or mitotic: Clb1-4) or mitotic cyclins in mammalian cells (A and B cyclins) have been described; they are arranged according to the cell cycle state where they act. Additionally, a Cdc28 (CDK) in budding yeast, a Cdc2 (CDK) in fission yeast, and 1,4,5,6 CDKs (G1) and cdc2 (mitotic CDK) in mammalian cells have been described.6 The activity of each CDK complex is controlled at several levels: (1) binding or degradation of the cyclins, (2) phosphorylation of the catalytic subunit, and (3) binding of inhibitory proteins like the sic protein in yeast or the CKIs in mammalian cells.7 Genetic studies in yeast showed that some of the cyclin-Cdc28 complexes are regulated by the ubiquitin system. Yaglom et al8 reported that once the Cln3-Cdc28 complex is activated, Cln3 cyclin is degraded by the ubiquitin system (Fig. 4.1). They showed that the ubiquitin conjugating enzyme involved in such control is the UBC3 enzyme, which is encoded by the gene described as cdc34. They also demonstrated, using a temperature-sensitive yeast mutant defective in Cdc28 kinase, that the Cln3-Cdc28 complex phosphorylates its own cyclin. This is necessary for cyclin recognition by the UBC3 enzyme and the ubiquitin transfer that leads to cyclin degradation and complex inactivation. Some aspects of cellular

Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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Fig. 4.1. The ubiquitin system in the cell cycle progression. Ubiquitin-conjugating enzymes (UBC) play a very important role in the cell cycle progression allowing cyclin degradation by the proteasome. UBC3 is a ubiquitin-conjugating enzyme codified by the cdc34 gene, that mediates the Cln3, Cln1, Cln2 and sic supressor protein degradation. UBC9 mediates conjugation to Clb5 and Clb2 cyclins.

functions of the Cln3 cyclin and the Cln3-Cdc28 complex may help us to understand the cyclin-related context in which the ubiquitin system acts. The Cln3 cyclin is a sensor for cellular size that commits yeast cells to division. Cln3 then binds to Cdc28 forming an activated complex which triggers the START signal for cell cycle progression. This occurs as a result of the phosphorylation and activation of the SBF transcription factor, which promotes the transcription of the Cln1 and Cln2 cyclin genes and other genes related to the START point. The expression of these cyclins leads to the formation of the corresponding complexes (Cln1-Cdc28 and Cln2-Cdc28), which are involved in the activation of the MBF transcription factor that promotes the transcription of S-phase cyclins (Clb5 and Clb6) and other genes associated with DNA synthesis.6 Results reported recently by several authors suggest that ubiquitin activity is extended to the control of other cyclins and is not limited to the Cln3 cyclin (Fig. 4.1). For example, Deshaies et al9 proposed that the Cln2-Cdc28 complex could be regulated by the ubiquitin system. They described Cdc34 temperature-sensitive mutant cells which are defective for

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Cln2 cyclin degradation. This suggests that the ubiquitin-conjugating enzyme UBC3 (Cdc34) can also transfer ubiquitin to the Cln2 cyclin, inducing its proteolytic degradation and inactivation of the Cln2-Cdc28 complex. In addition, the same authors have shown that phosphorylation of the Cln2 cyclin in the activated complex by its partner kinase is necessary for ubiquitin transfer.9 Very recently, another protein, Cdc53 (which is believed to be a ubiquitinprotein ligase) has also been involved in Cln2 degradation.10 The activity of S-phase Clb5-Cdc28 kinase is also controlled by the ubiquitin system, which takes part in both the activation and deactivation of the kinase.11,12 Activation is mediated through the proteolytic degradation of the sic suppressor protein. This protein prevents uncontrolled Clb5-cdc28 kinase activity.6 Activation of the Clb5-Cdc28 kinase by the ubiquitin system was suggested by Schwob et al12 based on the fact that temperaturesensitive Cdc34 mutant yeast cells are unable to destroy the sic protein and cannot enter S-phase of the cell cycle. So, it seems that the activation of the Clb5-Cdc28 kinase could involve the UBC3 ubiquitin conjugating enzyme, whose action would permit the G1 to S-phase transition. When the Clb5-Cdc28 kinase has already acted, the ubiquitin system also seems to be involved in its deactivation. For example, Seufert et al11 have reported that the ubiquitin-conjugating enzyme UBC9 is involved in Clb5 degradation. Interestingly, Seufert et al11 have also demonstrated the involvement of the UBC9 ubiquitin-conjugating enzyme in the degradation of mitotic cyclins. This is the case for the Clb2 cyclin, whose action permits transition into the M-phase of the cell cycle. It has been proposed that the specificity of the UBC9 enzyme for S-phase or mitotic cyclins could be given by different ubiquitin ligases E3. The mitotic cyclin-CDKs complexes downregulate normal cellular processes such as vesicle transport or transcription, and reorganize the cytoskeleton elements (such as the microtubule network), the actin microfilaments and the nuclear lamina, thus enabling cell division.6 The study of CDK control by the ubiquitin system in the yeast cell cycle has lead to greater understanding of this type of process. However, this knowledge must be extended to mammalian cells in order to know more about the proteins that could be involved in cancer development. The CDK4/cyclin D and the CDK2/cyclin E are both complexes from the G1 phase that are inhibited by the p27 CKI, whose activation permits and promotes the transition to DNA synthesis. Recently, Pagano et al13 reported that the human UBC2 and UBC3 ubiquitin-conjugating enzymes are involved in p27 degradation and so in CDK4/cyclin D and CDK2/cyclin E activation. This leads to retinoblastoma protein phosphorylation which is involved in the activation of DNA synthesis-related transcription factors.14 In addition, as in yeast, phosphorylation by CDK2 and subsequent ubiquitinization lead to the degradation of cyclin E, the difference with yeast being that separation of the CDK2 of the cyclin preceeds degradation.15 The ubiquitin system also has an important role in the transition through mitosis of the eukaryotic cells. Glozer et al16 have shown that cdc2/cyclin A and the cdc2/cyclin B deactivation by the ubiquitin system through cyclin degradation allows the decondensation of chromosomes and the reformation of the nuclear membrane. Moreover, the ubiquitinproteasome pathway has also been shown to degrade sister chromatid cohesion proteins that are essential for binding sister chromatids to each other.17 Although the UBC4 conjugating enzyme is involved in the degradation of these proteins, the cellular signal for degradation could come from a specific ubiquitin-protein ligase E3 associated with an anaphasepromoting complex of 20S named cyclosome.18,19 In fact, Aristarkhov et al20 have cloned a new conjugating enzyme from clam oocytes (which bears no homology with UBC4) that seems to be involved in the degradation of the mitotic cyclins A and B.

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The Suppressor p53 Protein Although the control exerted by the ubiquitin system on cyclins/CDKs seems to be essential for correct cell cycle progression, there are other ubiquitin-controlled proteins whose functions are also relevant for such a mechanism. Among them, the tumor suppressorp rotein p53 not only plays an important role in cell cycle regulation, but also in cell survival.21-23 Initially, it was reported that p53 is a target for the “high risk” human papilloma virus (HPV) E6 oncoproteins and that these oncoproteins stimulate the ubiquitin-dependent degradation of p53 in crude reticulocyte lysates. Such degradation was related to the low levels of p53 observed in HPV-transformed cervical carcinoma cell lines, emphasizing the suppressor activity of p53 in the cell cycle and a possible mechanism for the tumorigenicity of these oncogenic proteins.23 The importance of p53 in the control of the cell cycle seems quite probable if we take into consideration that mutations that inactivate or alter its suppressor activity are frequently associated with changes in its stability and seem to be a genetic change that occurs in human cancer.24,25 In fact, it is known that the suppressor activity of p53 on the cell cycle is linked to DNA repair events. It has been shown that p53, which shows a transient accumulation in response to DNA damage, is a transcription factor with a very short half-life in normal cells. Thus p53 induces the transcription of target genes like p21, which is involved in the cell cycle arrest, and GADD 45, which is involved in DNA repair.22,25 The role of the ubiquitin system in the proteolytic degradation of p53 is very clear since most of the enzymes related to the system, including ubiquitin-activating (E1) 26 and conjugating enzymes (E2),27,28 ubiquitin-protein ligases (E3),29 and proteasome 26S24 have been shown to be involved. The proteolytic degradation of p53 seems to be activated by phosphorylation.30 The E6 oncoprotein of the human papilloma virus binds to an E3 ubiquitin protein ligase of 100 kDa, known as E6-AP, and targets p53 for degradation.29 E6-AP accepts ubiquitin from an E2 ubiquitin-conjugating enzyme and transfers it to the targeted substrate.29 The conjugation step involves a new conjugating enzyme known as E2-F1, described in rabbits,27,28 that also participates in NF- κB maturation and c-fos degradation. Very recently, a human homolog of the rabbit enzyme has been found and named as UBE2L3.31 In addition, human homologs of the yeast UBC4-UBC5 enzymes, named ubcH5 and ubcH6,32,33 and human homolog of E2F1, named ubcH7 and ubcH8,34,35 have been described to degrade p53 protein. DNA damage is recognized by specific proteins that induce the cellular accumulation of p53 and activate (through p53) the DNA repair complexes directly or indirectly.25 Moreover, the ubiquitin-conjugating enzyme UBC2 has been shown by Bailly et al36 to be involved in DNA repair through the interaction with a DNA damage-recognizing protein. All this leads to the possibility that ubiquitin-dependent protein degradation could also be involved in signal transduction from DNA damage to the inhibition of p53 degradation.

Ubiquitin-controlled Oncogenic Proteins Apart from cyclins and p53, there are other proteins whose presence is necessary for normal cell cycle progression. At present, we know more about these proteins and their functions, thanks to the study of the known oncogenes. As is the case of cyclins and p53, some oncogenic products that may be controlled by the ubiquitin system have been described. One of them is a cytostatic factor (CSF-1) which is the product of c-mos. In Xenopus oocytes, CSF-1 functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation. Ishida et al37 and Nishizawa et al38 have both demonstrated that the rapid turnover of CSF-1 in the early stage is mediated by the ubiquitin system. At the late stage, CSF-1 becomes stable by full phosphorylation; this seems to prevent ubiquitin system recognition.38 The CSF-1 cytostatic activity prevents oocyte prolif-

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eration, arresting the cell cycle at the second meiotic metaphase through the inhibition of the ubiquitin-dependent degradation of mitotic cyclins. It is after fertilization that eggs are released from their metaphase arrest. In addition, it has been demonstrated by Nishizawa et al39 that after fertilization the ubiquitin system is also involved in CSF-1 degradation and inactivation. They have also shown that CSF-1 dephosphorylation is a key signal for the ubiquitin system recognition. Other oncogenes whose products seem to be degraded by the ubiquitin system are c-jun and c-fos.40–42 The c-jun and c-fos genes are early growth-response genes whose products form an heterodimer transcriptional factor.43 Interestingly, Stancovski et al44 have shown that the ubiquitin-conjugating enzyme E2-F1 involved in c-fos degradation is the same one that targets the p53 suppressor protein, although it acts with a novel ubiquitin-protein ligase E3. Such degradation seems to be enhanced by phosphorylation and binding to c-jun.42 Hermida-Matsumoto et al45 have also implicated the ubiquitin-conjugating enzyme E214K in this process. The products of c-fos and c-jun are unstable and quickly degraded by the ubiquitin system but not the v-fos and v-jun products.40,42 This could explain the oncogenic potential of v-fos and v-jun .27 Thus, a lack of ubiquitin system recognition of oncogenic products like v-fos and v-jun could be a mechanism of oncogenesis. Another oncogene controlled by the ubiquitin system is bcl-1/PRAD1.6 Its product is a D1 cyclin that is overexpressed in some human cancers whose reported control by the ubiquitin system is through the degradation of the CKI p27, as previously mentioned. Increased degradation of suppressor proteins such as p53 or p27 by the ubiquitin system could be oncogenic. A lack of degradation of cell cycle-promoting proteins like the v-fos and v-jun products could also be oncogenic. Stability changes in these oncogenic proteins due to mutations could be a mechanism of oncogenesis. However, mutations in the ubiquitin system genes could also produce the same oncogenic effects. This means that it would not be strange to find ubiquitin system-related oncogenes.

DNA Repair Although the ubiquitin-mediated control of oncogenic proteins may be important, it seems clear that some of them become oncogenic as a result of mutations or alterations in their own oncogenes. Thus the DNA repair involved in such mutations could be very important for avoiding cancer development. Following from this, the implication of the ubiquitin system in DNA repair mechanisms also seems to be relevant for avoiding cancer development. The involvement of the ubiquitin-conjugating enzyme UBC2 in DNA repair has been reviewed by Jentsch et al.21,46 The UBC2 gene is identical to the RAD6 gene, which has been extensively studied as a DNA-repair gene in yeasts. Some evidence of the role of UBC2 has come from the characterization of yeasts UBC2/RAD6 mutants. These mutants show a high sensitivity to DNA-damaging agents, defects in postreplication repair of UVdamaged DNA, and enhanced G.C-T.A transversions. Koken et al47 have identified two human ubiquitin-conjugating DNA repair genes ( HHR6A and HHR6B) which are homologs of the yeast RAD6 gene. Other experiments suggesting a role for the ubiquitin system in DNA repair are those where the UBC2 gene and the yeast polyubiquitin gene UBi4 were transcriptionally activated in response to DNA-damaging agents. Recently, an approximation to the molecular mechanism involved in the reparative action of UBC2 has been reported 36 in which the UBC2/RAD6 enzyme forms a specific complex with the RAD18 protein which is also the product of a DNA repair gene. The study demonstrated that the interaction is important for the reparative effect of both proteins. However, it was also observed that RAD18 binds a single strand, whereas RAD6 has no affinity for DNA. So it was proposed that RAD18 could target damaged DNA regions for RAD6 recognition while ubiquitin-conjugating activity would modulate the activity of stalled DNA repairma chinery.

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In fact, there is no reason to question the involvement of the ubiquitin system in DNA repair. But is there any direct evidence of impaired DNA repair in cancer development? Complementation group C of xeroderma pigmentosum is a skin cancer caused by impaired DNA repair. This impairment affects the nucleotide excision repair mechanism that removes DNA damage as part of an oligonucleotide fragment followed by replacement with new DNA.48 In this context, Masutani et al49 have characterized a possible nucleotide excision repair complex which may be important for the prevention of skin cancer. The repair complex would be composed of the human homolog of the yeast RAD4 repair gene and the yeast RAD23 repair gene. Interestingly, all the RAD23 derivatives share a ubiquitin-like N-terminus. Taking this into account, the ubiquitin system has an important role in DNA repair, and alterations in this cellular mechanism could certainly lead to cancer development; consequently, it would not be surprising if changes in specific ubiquitin-related genes could lead to the development of this disease.

Ubiquitin-related Oncogenes Ubiquitin-related oncogenes do indeed exist. Papa and Hochstrasser 50 described a yeast Doa4 gene that is homologous to the human tre-2 oncogene. Both genes encode a ubiquitinreleasing enzyme that cleaves ubiquitin from substrate remnants still bound to the proteasome. Deletion of the Doa4 gene induces defects in cellular growth and DNA repair. We have considered that ubiquitin-related alterations in the control of both cellular functions could lead to cancer development. Other authors have also cloned and sequenced a ubiquitin releasing enzyme from yeast, named UBP5, which is closely related to the human tre-2 oncogene and the mouse unp oncogene.51 Further evidence supporting the existence of ubiquitin-related oncogenes is the overexpression of the murine unp oncogene which also encodes a ubiquitin-releasing enzyme and which leads to oncogenic transformation of NIH3T3 cells. Gray et al52 have cloned cDNA from the human homolog of the unp gene ( unph), and have reported the overexpression of unph RNAm in small cell tumors and adenocarcinomas of the lung. Although the role of ubiquitin-releasing enzymes in the induction of tumor growth is an interesting field in cancer research, another ubiquitin-related malfunction has been described which could also be involved in tumor growth. It has been reported that the human UBE1L gene, whose product has a high degree of identity to the ubiquitin-activating enzyme, is well expressed in normal lung tissue but hardly or not at all in lung cancer-derived cell lines. Kok et al53 have shown that there are no cancer-specific mutations in this gene, but its lack of expression is correlated with a highly decreased sensitivity towards DNAse I of the promoter region. So, either an altered promoter or the absence of a yet unknown transcription factor could be the cause of the UBE1L nonexpression.

A Possible Role for Ubiquitin System in Metastasis The malignancy of a tumor is closely related to the capacity for generating secondary tumor sites or metastases. This phenomenon involves the generation of secondary tumors at sites distant from the primary ones, and is mediated through different invasive processes. Metastatic malignant cells invade normal adjacent tissues, penetrate into the capillary lumen and into the systemic circulation. Some malignant cells will then arrest at sites distant from the primary tumor and will invade through the vessel walls into the surrounding tissue, thus forming a secondary tumor. Although at present there is no direct evidence involving the ubiquitin system in the metastatic process, several factors indicate a possible link. The first is that invasive processes occur not only in pathological conditions but also in some normal conditions. The involve-

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ment of the ubiquitin system in metastasis could be suggested by its role in normal invasive processes such as wound repair or nerve regeneration, as mentioned in chapter 3. A common feature of invasive processes is the degradation of the extracellular matrix (ECM) which allows cells to migrate into adjacent tissues. In the case of tumor growth, malignant cells have been shown to degrade the ECM through secretion of extracellular proteolytic factors, mainly plasminogen activators or metalloproteinases, or through decreased secretion of their respective inhibitors. Given that the ubiquitin system is involved in the metabolism of an extracellular serine protease inhibitor like protease nexin 2, its possible involvement in metastasis through the metabolism of other extracellular proteinase inhibitors would be an interesting field of research. Another possible link between metastatic events and ubiquitin comes from the fact that the ubiquitin system may be involved in the expression of collagenase, one of the main metalloproteinases involved in ECM degradation by malignant cells. This is suggested because the ubiquitin system is involved in the control of c-jun and c-fos, which form the active heterodimeric transcriptional activator AP-1,44 also involved in the expression of collagenase.54 Finally, the reported involvement of the ubiquitin system in the metabolism of the amyloid precursor protein (APP) could of utmost importance for the cell-ECM relationship, and in the control of cellular growth and cell interactions. APP has not only been shown to be a cell adhesion and mitogenic molecule and also an extracellular matrix protein (heparan-sulphate proteoglycan), but also to have high extracellular homology with beta-integrins.55 These compounds are cell surface receptors with binding domains to laminin and fibronectin, two of the main ECM proteins involved in the control of the cell-ECM relationships.56 They are also involved in cellular growth and migration processes.

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36. Bailly V, Lamb J, Sung P et al. Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Develop 1994; 8:811-820. 37. Ishida N, Tanaka K, Tamura T et al. Mos is degraded by the 26S proteasome in a ubiquitin dependent fashion. FEBS Lett 1993; 324:345-348. 38. Nishizawa N, Okazaki K, Furuno N et al. The second codon rule and autophosphorylation govern the stability and activity of mos during the meiotic cell cycle in Xenopus oocytes. EMBO J 1992; 11:2433-2446. 39. Nishizawa N, Furuno N, Okazaki K et al. Degradation of Mos by the N-terminal proline (Pro-2) dependent ubiquitin pathway on fertilization of Xenopus eggs:possible significance of natural selection for Pro-2 in Mos. EMBO J 1993; 12:4021-4027. 40. Treier M, Staszewski LM, Bohmann D. Ubiquitin-dependent c-jun degradation in vivo is mediated by the g domain. Cell 1994; 78:787-798. 41. Jariel-Encontre I, Pariat M, Martin F et al. Ubiquitinylation is not an absolute requirem en t for degradation of c-jun protein by the 26S proteasom e. J Biol Chem 1995; 270:11623-11627. 42. Tsurumi C, Ishida N, Tamura T et al. Degradation of c-fos by the 26S proteosome is accelerated by c-jun and multiples protein kinases. Mol Cell Biol 1995; 15:5682-5687. 43. Wong JM, Mafune KI, Yow H et al. Ubiquitin-ribosomal protein S27 a gene overexpressed in hum an colorectal carcinom a is an early growth response gene. Cancer Res 1993; 53:1916-1920. 44. Stancovski I, Gonen H, Orian A et al. Degradation of the proto-oncogen product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: Identification and characterization of the conjugating enzymes. Mol Cell Biol 1995; 15:7106-7116. 45. Hermida-Matsumoto ML, Chock PB, Curran T et al. Ubiquitinylation of transcription factors c-Jun and c-Fos using reconstituted ubiquitinylating enzymes. J Biol Chem 1996; 271:4930-4936. 46. Jentsch S, Seufert W, Hauser HP. Genetic analysis of the ubiquitin system. Biochim Biophys Acta 1991; 1089:127-139. 47. Koken MH, Hoogerbrugge JW, Jasper-Dekker I et al. Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification. Dev Biol 1996; 173:119-132. 48. Lehmann AR. Nucleotide excision repair and the link with transcription. Trends Biochem Sci 1995; 20:402-404. 49. Masutani C, Sugasawa K, Yanagisawa J et al. Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. EMBO J 1994; 13:1831-1843. 50. Papa FR, Hochstrasser M. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature 1993; 366:313-324. 51. Xiao W, Fontanie T, Tang M. UBP5 encodes a putative yeast ubiquitin specific protease that is related to the human Tre-2 oncogene product. Yeast 1994; 10:1497-1502. 52. Gray DA, Inazawa J, Gupta K et al. Elevated expression of Unph, a proto-oncogene at 3p21.3, in human lung tumors. Oncogene 1995; 10:2179-2183. 53. Kok K, Berg AVD, Veldhuis PMJF et al. The genomic structure of the human UBE1L gene. Gene Expres 1995; 4:163-175. 54. Brenner DA, O’Hara M, Angel P et al. Prolonged activation of jun and collagenase genes by tumor necrosis factor- α. Nature 1989; 337:661-663. 55. Crowther RA. Structural aspects of pathology in Alzheimer’s disease. Biochim Biophys Acta 1991; 1096:1-9. 56. Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol 1995; 11:549-599.

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CHAPTER 5

Muscle Wasting and Dystrophies uscle wasting is generally thought to be caused by an increase in protein breakdown. Indeed, protein degradation is activated in numerous pathological conditions and may represent an important factor in loss of body weight since skeletal muscle represents over 40% of total body mass in humans.1 Bearing this in mind, a growing interest has developed concerning the proteolytic systems that are activated in skeletal muscle and that are responsible for muscle wasting in pathological states. The apparent selectivity of the ATP-ubiquitindependent pathway makes it an attractive mechanism which could account for the specificity of protein degradation within skeletal muscle. Thus recent studies have focused on the role of ubiquitin in muscle.

M

Cancer Cachexia Cachexia is a poorly understood syndrome characterized by anorexia, weight loss, profound metabolic abnormalities and progressive host wasting which may result in death.2 Several studies have indicated that cachexia can explain about 30% of all cancer deaths in humans.3 Tissue wasting involves mainly adipose tissue and skeletal muscle. Muscle wasting during cancer cachexia is generally accepted to be caused by an increase in protein breakdown with little or no change in protein synthesis.4 Recently Llovera et al5 reported that both the presence of ubiquitin conjugates and the expression of different ubiquitin genes is highly elevated in the skeletal muscle of cachectic tumor-bearing rats. In addition, incubations of skeletal muscles from tumor-bearing rats revealed little or no involvement of either lysosomal cathepsin and cytosolic Ca2+ -dependent calpains in the increased muscle proteolysis encountered in the skeletal muscle of AH130 Yoshida-bearing rats.6 Activation of the ubiquitin system in skeletal muscle during cancer cachexia seems to be unrelated to the high circulating levels of glucocorticoids found in these animals,7 and can be reverted by clenbuterol (an adrenergic β2-agonist) treatment.8 Later work on the involvement of the ubiquitin-dependent pathway in muscle wasting during cancer cachexia has confirmed the initial observations.9,10 Preliminary observations made in skeletal human muscle from cachetic pancreatic patients also points towards the ubiquitin system being responsible for the activation of proteolysis in skeletal muscle.

Muscle Denervation and Atrophy Muscle atrophy is a common phenomenon observed during ageing11 and can be influenced by environmental and other external influences such as physical activity, disuse and immobilization, nutrition and food restriction, and injuries, drugs and diseases.12 It involves molecular and cellular changes in muscle tissue which lead to atrophy and loss of fibers and finally results in a major decrease in muscle mass.12 Riley et al13 made the first observations concerning the involvement of the ubiquitin-dependent proteolytic system in muscle atrophy. They used muscles which became atrophic after one week of microgravity conditions aboard Spacelab 3. Microgravity produced atrophy and concomitant increases Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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in conjugation of proteins to ubiquitin, as determined by a quantitative immunoassay in both soleus and extensor digitorum longus (EDL) muscles.13 Denervation in the rat (caused by cutting the sciatic nerve) results in rapid atrophy of the soleus muscle causing a 30% reduction in muscle weight in only a few days. Atrophy is accompanied by enhanced protein degradation with no changes in synthesis. Medina et al showed that denervation-induced proteolysis was markedly inhibited by blocking ATP production but was not affected by other treatments preventing lysosomal and Ca2+ -dependent proteolysis.14 They also showed that there were increases in ubiquitin conjugates and in the expression of the ubiquitin genes in the atrophic muscle versus the contralateral intact muscle. These changes mirror almost exactly the changes in overall protein degradation and breakdown of myofibrillar proteins, as measured by the formation of 3-methylhistidine. It can thus be concluded from these data that the ubiquitin system has an important role in the degradation of normal muscle proteins, probably including the long-lived myofibrillar proteins, as opposed to the view that the ubiquitin system is only involved in the breakdown of short life, nonstructural proteins.

Starvation and Malnutrition Medina et al14 showed that food deprivation (known to result in a rapid increase in muscle protein breakdown) results in an increase in ATP-dependent proteolysis that parallels an increase in the presence of ubiquitin conjugates and polyubiquitin mRNA in rat skeletal muscle after only 24 hours of starvation. Refeeding results in a reversal of the increased gene expression only 24 hours later.14 In dietary protein deficiency, the metabolic adaptations that take place are directed towards conserving protein. Thus protein malnutrition represents a metabolic situation opposed to that found during food deprivation, where important molecular mechanisms are activated and deactivated to save protein and in particular essential amino acids. Tawa et al15 found that protein breakdown in skeletal muscle decreases during dietary protein deficiency. They studied the contribution of the different proteolytic systems to this sparing of muscle protein. After submitting animals to a 72 hour protein deprivation study, they concluded that both the lysosomal and the ATP-dependent proteolytic systems were involved. Conversely, the activity of the Ca2+ -dependent proteolytic system was unaltered by protein deprivation, thus suggesting the importance of the ATP-dependent proteolytic system in regulating muscle nitrogen balance under protein malnutrition.15

Other Pathological States The role of the ubiquitin system in skeletal muscle is under extensive investigation in a number of pathological states. Remarkable progress has been made in the case of infection and acidosis where muscle wasting is often occurs. Tiao et al16 recently tested the role of the different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Using the model of cecal ligation and puncture, they reported increases of 50% and 440% in total and myofibrillar protein breakdown in septic muscle. These increases were associated with an increase in ATP-dependent proteolysis and with increases in ubiquitin conjugates and ubiquitin gene expression in the affected muscles. The other proteolytic systems (both lysosomal and Ca2+ -dependent) showed no change in activity during sepsis. Similar results have been obtained in our laboratory,17 thus demonstrating the role of the ubiquitin-dependent proteolytic system in infection. Metabolic acidosis is often associated with conditions in which there is a markedly negative nitrogen balance associated with increases in muscle proteolysis and oxidation of branched-chain amino acids.18 This seems to be the case of chronic renal failure where the

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patients are subject to extensive body wasting that can be reverted by the simple administration of sodium bicarbonate to compensate for the acidosis. Using muscles of fed acidotic rats, May et al showed an increase in muscle proteolysis without changes in protein synthesis.18 More recently, Mitch et al,19 using acidotic rats, identified the mechanism which is involved in the increased muscle proteolysis observed during metabolic acidosis. It is ATP-dependent and nonlysosomal and also seems to be related to ubiquitin conjugation since they report a two- to four-fold increase in ubiquitin mRNA in skeletal muscle of acidotic rats. Their findings strongly suggest that the accelerated proteolysis is due to a greater flux of proteins through the ubiquitin-proteasome-dependent pathway.19

Regulation of the System in Skeletal Muscle: TNF as an Activator We have seen that activation of the ubiquitin-dependent proteolytic system in skeletal muscle is associated with a considerable number of pathological states leading to body weight loss and, consequently, cachexia. A number of studies have concentrated on trying to determine the molecules that may regulate the activity of this proteolytic system. At least during fasting, the glucocorticoids seem to be involved in the activation of the proteolytic ubiquitin-dependent pathway in muscle, since adrenalectomy prevents the rise in ubiquitin conjugates after fasting.20 The mechanism is, however, unclear since the characteristic sequences found in many glucocorticoid-responsive genes are not found in the ubiquitin genes.21 In addition, recent studies carried out in our laboratory using the glucocorticoid antagonist RU486 do not support the involvement of glucocorticoids in activating the ubiquitin system during cancer cachexia.22 Recently, Wing and Banville23 presented the first evidence of hormonal control of ubiquitin conjugation. They cloned the E214k gene responsible for the synthesis of the ubiquitin conjugating enzyme. They reported increases in one of the mRNA transcripts with fasting and subsequent decrease with insulin treatment. Thus the mechanisms involving insulin and glucocorticoids in modulation of ubiquitin-dependent proteolysis may be completely independent, although insulin can suppress the stimulation of muscle proteolysis by glucocorticoids both in vivo and in vitro.24 Our group has recently reported the fact that tumor necrosis factor- α (TNF) administration to rats results in an increase in muscle proteolysis both in vivo25 and in vitro 26 and that it is associated with both an increase in the presence of ubiquitin conjugates27 and ubiquitin mRNA28 in skeletal muscle. Administration of polyclonal anti-TNF antibodies to cachectic tumor-bearing rats (with high circulating TNF levels) results in a normalization of muscle weight with no signs of muscle cachexia7 and reverts the increased muscle ubiquitin gene expression.29 These results support the involvement of the cytokine in mediating the activation of the muscle ubiquitin system during cancer cachexia. The action of this cytokine seems to be independent of that of interleukin-1 (IL-1) since treatment of the animals with IL-1 receptor antagonist (IL-1ra) is unable to reverse the increased proteolysis found after TNF treatment.30 The action of TNF could either be indirect (through another unknown mediator) or direct. Skeletal muscle has TNF receptors31 and we have observed a direct effect on ubiquitin mRNA in incubations of skeletal muscles in vitro in the presence of the cytokine.32

Anabolic Hormones and the Ubiquitin System Fasting is a physiological situation where the degradation of muscle proteins by the ubiquitin system is activated. After refeeding, muscle proteolysis is inhibited and protein synthesis is favored, basically by insulin action. According to this, it has been shown that insulin downregulates the ubiquitin-conjugating enzyme E2 which is induced by fasting.23

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So, insulin is clearly important for restoring the muscle mass in normal nutritional situations.33 However continuous fasting (starvation) can lead to insulin resistance, as it happens in other situations like cancer,34 sepsis35 or ageing,36 which could have a certain parallelism with continuous fasting in some if not all cases. In such cases, other anabolic hormones like growth hormone (GH) and insulin-like growth factor I (IGF-I) could be compromised in order to preserve and protect the muscle mass.33,37 Interestingly, whereas the insulin response depends on nutritional status, the IGF-I response is completely independent.33 Although GH may act directly on muscle through its own receptors, normally its anabolic action is mediated by the liver synthesis of IGF-I and the pancreatic synthesis of insulin in growth conditions.33 IGF-I can also be synthesized in muscle in an autocrine fashion.33 It has been clearly shown that IGF-I promotes muscle growth and protein synthesis, and inhibits protein degradation mainly in situations with a negative nitrogen balance such as diabetes or dexamethasone treatment.37 IGF-I has also been shown to be very effective in the treatment of the muscle wasting associated with myotonic dystrophy.38 This type of dystrophy causes muscle wasting and weakness and is believed to be due to a decrease in muscle protein synthesis that is secondary to insulin resistance. The same study also showed the anticatabolic effect of IGF-I, overcoming the insulin resistance with the IGF-I treatment. Such treatment also induced an increase in protein synthesis, in body weight and in lean body mass, and a decrease in the percentage of body fat. Moreover, clinical studies involving IGF-I have shown a dose dependent response improvement in manual muscle strength and neuromuscular function. In addition, some situations like cancer, sepsis or ageing are associated with muscle wasting and insulin resistance.34–36 Interestingly, in cancer or traumatic lesions, lower levels of IGF-I have been reported.37 Taking all of the above into account, insulin resistance may have a key role in preventing muscle wasting in some situations. Recently, an implication for the ubiquitin system in insulin resistance might be considered as a subject of study, from a few reports. SeppLorenzino et al39 reported that the insulin and IGF-I receptors are degraded by the ubiquitinproteolytic system. They showed both the stabilization of the receptors under restrictive conditions, after transfecting them in a temperature sensitive cell line deficient in the ubiquitin activating enzyme E1, and the stabilization with peptidyl aldehyde inhibitors for the 20S proteasome. The same authors also showed ubiquitin conjugation to the receptors. The relevance of these results is reinforced by Hong and Forsberg40 who showed a downregulation of proteasome mRNAs under IGF-I treatment in rat L8 skeletal myotube cultures. The ubiquitin system is also required for ligand-induced endocytosis and degradation of the GH receptor.41 The receptor has been shown to be ubiquitinated, and its transfection into chinese hamster ovary cells with a temperature sensitive defect in ubiquitin conjugation has been shown to lead (at nonpermissive temperatures) to the stabilization of the receptor. Although the GH receptor degradation is mediated by the endosomal-lysosomal pathway, in this case the ubiquitin conjugation seems to be quite important for the receptor internalization and degradation since it is abolished in the mutant cell line, in contrast to other membrane receptors like those of platelet-derived growth factor (PDGF) and epiderm al growth factor (EGF) wh ere th e u biqu itin con ju gation on ly en h an ces th eir internalization.42 The role of TNF in inducing muscle wasting through the activation of the ubiquitin system and also in inducing insulin resistance in different situations is well known.43,44 So, the question that arises is can a permanent activation of the ubiquitin system by TNF induce insulin resistance through the degradation of the insulin receptor? The same question could be asked for IGF-I, which probably would be more relevant to muscle wasting situations.

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Alterations in the ubiquitin system could also affect the metabolic adaptations to normal fasting, perhaps through unregulated muscle catabolism. Kornitzer et al45 have reported that Gcn4, a yeast transcriptional activator involved in the biosynthesis of amino acids and purines is rapidly turned over by the ubiquitin system. Such degradation is inhibited under conditions of amino acid starvation, probably by a single amino acid alteration in a region adjacent to the Gcn4 activation domain. In addition, the same authors have shown that the ubiquitin-dependent degradation involves two ubiquitin conjugating enzymes, UBC3 (also Cdc34) and UBC2 (also RAD6).

Ubiquitin System and Mechanotransduction in Muscle Growth Muscle strength is an important factor involved in the maintenance and increase of muscle mass. Normally, it is a consequence of exercise which is known to induce muscle hypertrophy.46 The other side of the coin is muscle wasting produced by resting (disuse) 47 or ingravity.48 So investigating the mechanotransduction mechanisms involved in muscle growth 46,49,50 and the possible involvement of the ubiquitin system might be quite interesting for muscle wasting and ubiquitin-related researchers. Mechanotransduction has been shown to play an important role in the physiology of many tissues, such as bone or endothelium.50,51 However, little is known about the biochemical pathways involved. It has been hypothesized that mechanically-sensitive ion channels and load conformation-sensitive receptors exist which could act as mechanical sensors, and it may be suggested the ubiquitin system could be involved in the regulation of mechanotransduction. Integrins have clearly been shown to act as mechanical sensors. This has been reported by Chen and Grinnell,52 who showed that integrins enhance the neurotransmitter release at motor nerve terminals by sensing mechanical stretch. Such release is suppressed by integrin antibodies or by peptides which block integrin binding. Another protein involved in mechanotransduction which senses stretch directly or indirectly is the tyrosine kinase p60src.49 Under stretch conditions the autophosphorylation of p60src is rapidly induced. p60src has been shown to link integrins with the cytoskeletal machinery, and it is believed that a displacement in these interactions could result in a conformational change in the kinase.49 In addition, both the G proteins and particularly the ras-raf interaction have been proposed to act as modulators of the kinase activity which, in turn, after signal transduction, will eventually lead to the induction of a growth response.49 Very interestingly, the integrin-p60src-cytoskeleton machinery may also be controlled by the proteolytic activity of the ubiquitin system. The results of Sepp-Lorenzino et al39 strongly suggest that p60src could be degraded by the ubiquitin-dependent pathway. They have shown that herbimycin A, an ansamycin antibiotic known to target tyrosine kinases for degradation, acts through the activation of the 20S proteasome in a ubiquitin-dependent manner. Although their results involve the IGF-I receptor and the insulin receptor, it seems very probable that the mechanism is the same for the other tyrosine kinases. Interestingly, herbimycin A has been shown not only to induce the degradation of tyrosine kinaseoncogenes such as p 60src, yes, fps, abl, erbB and ros, but also to reverse effectively the transformed phenotypes induced by them.53–33 Another interesting fact relating mechanotransduction to the ubiquitin system is that the ubiquitin system is involved in the metabolism of soluble amyloid precursor protein (APP), as previously described.56 Interestingly, APP has been shown to have high homology sequence with integrins.57 So it may be suggested that the APP isoforms could have integrinlike functions in mechanotransduction or that integrins could be partially ubiquitin-controlled as in the case of APP.

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In addition to the mechanical sensors, there are other factors involved in the stretch induced muscle hypertrophy which could be related to the ubiquitin system. These are the transcriptional activators c-jun and c-fos and the muscle growth factor IGF-I. Static stretch has been shown to cause in vivo adaptative growth of the extensor digitorum longus with increases in c-fos, c-jun and IGF-I mRNA levels, and protein and DNA synthesis.58 Also in tissue cultures, nanomolar concentrations of exogenous IGF-I stimulated growth and the autocrine IGF-I secretion in mechanically-stretched but not static cultures.59 The expression of the three major IGF-I binding proteins of 31, 36 and 43 kDa was also induced. Interestingly, the IGF-I response to stretch in vivo has been shown to be unaffected by fasting.60 In relation to the action of IGF-I we have already commented on the implication of the ubiquitin system in the degradation of its tyrosine kinase receptor. As it has been already commented, c-jun and c-fos form the early response heterodimer transcriptional factor known as AP1. There are several proteolytic systems involved in its degradation. The ubiquitin system has been shown to participate in c-jun and c-fos degradation,61–65 but the proteolytic processes related to the AP1 control are quite complex and are still being studied extensively, so the ubiquitin system is by no means the only system involved in this degradation process. Finally, it has been proposed that the transmission of signals in stretch stimulated tissues could be transmitted from sensor cells to others through gap junctions.50 Gap junctions are highly adherent plasma membrane microdomains constructed of arrays of cell-tocell channels. Such channels are constructed by connexins which oligomerize into hexamers forming an hemichannel in each cell. The link between two hemichannels of adjacent cells provides continuity between the cytoplasm, allowing for the transit of small molecules and ions. Recently, Laing and Beyer 66 clearly showed that the ubiquitin proteasome pathway is involved in the degradation of connexin 43, one of the three most widely expressed connexins. So if gap junctions were really involved in mechanotransduction, their ubiquitin-dependent degradation should be taken into account by researchers investigating stretch-induced hypertrophy and/or muscle wasting caused by resting or ingravity.

Neuromuscular Activity and the Maintenance of Muscle Mass The induction of muscle wasting by denervation is clear evidence of the importance of the neuromuscular activity in the maintenance of muscle mass. Today there is other evidence to reinforce this point of view. The involvement of ubiquitin can also be suggested. Goldspink et al reported that electrical stimulation at 10 Hz enhances the muscle growth response to stretch in vivo,58 increasing the IGF-I mRNA response from 12-fold to 40-fold, and the total rate of protein synthesis from 191% to 450%. Conversely, stretch enhances neurotransmitter release through integrins.52 So both stretch and electrical stimulation seem to collaborate in muscle growth. Handa et al67 demonstrated a therapeutic effect of electrical stimulation on muscle wasting of a patient with amyotrophic lateral sclerosis. After a month of therapy a rapid improvement of extremity motion appeared in the treated side, and more than three months of therapy increased the thickness and strength of the treated muscles. In addition, renervation is considered to be one of the main factors involved in muscle regeneration.68–70 The role of ubiquitin in this process will be discussed later in this chapter. Finally, in relation to inclusion-body myositis (IBM), a muscle pathology with wasting seems to be closely related to impairment of the regeneration of the neuromuscular junctions.

Wasting and Muscle Regeneration Muscle regeneration is a physiological mechanism activated in muscle injury, muscle myopathies and some muscle wasting situations like those related to myopathies or hindlimb

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suspension.68,71 The activity of muscle regeneration in other pathologically-associated muscle wasting situations such as cancer and sepsis is not yet understood. Muscle regeneration is a tissue repair mechanism with some similarities to the neuronal tissue repair mechanism (see chapter 3). Thus, given the involvement of the ubiquitin system in the former, it would not be strange to consider its implication in the latter. There are three major factors limiting the ability of skeletal muscle to regenerate: a viable population of myogenic precursor cells (known also as satellite cells), reinnervation and revascularization.68 When the muscle is damaged (for example by direct injury, ischemia, direct application of local anesthetics, eccentric exercise or neuromuscular disease) some areas of necrotic myofibers appear. This is followed by the release of compounds from the injured muscle that act as “wound hormones”, initiating inflammation. This process involves three stages.72 First, neutrophils rapidly invade the injury site and release cytokines to attract other inflammatory cells and oxygen free radicals in order to enhance the damage of the necrotic myofibers. This is followed by an increase in a subpopulation of ED1+ macrophages which invade damaged muscle fibers, acting as scavengers.72,73 Finally, there is an increase in a second subpopulation of ED2+ macrophages compromised in muscle regeneration.72,73 Where the process has already started, fibroblast and autocrine muscle fiber secretions also seem to be involved in regeneration.74,75 The more active and important growth factors involved in muscle regeneration are the basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGF- β1) and IGF-I.75 This has been shown by the neutralization of their activity in vivo with monoclonal antibodies,75 and also through the study of their activity and expression both in vitro and in vivo in mice models with increased muscle regeneration.76–78 These growth factors have been shown to activate the proliferation, differentiation and fusion of the satellite cells. The bFGF could be secreted by macrophages and fibroblasts whereas the IGF-I could be secreted in an autocrine way by the muscle.33,74,79,80 In fact, fibroblasts have been shown to secrete a growth factor that is involved in the activation of satellite cells and in the inhibition of their own proliferation.74 This is important because extensive fibrosis has been shown to be associated with a lack of muscle regeneration, mainly involving an extensive secretion of EGF and TGF- β1.81 Finally, th ere are oth er growth factor s th at cou ld be im p or tan t in rein n er vat ion an d revascularization,77,82 probably the most clear evidence for this being the expression of nerve growth factor (NGF) associated with muscle regeneration.82 Interestingly, the GAP43 protein, the low-affinity NGF receptor and the NGF, which are involved in the axonal growth, development and regeneration of the central and peripheral nervous systems, are also coexpressed in regenerating muscle fibers. Such expression may be related to neuromuscular junction regeneration. Concerning the muscle genes most clearly involved in muscle regeneration, it is interesting to point out that after three hours of satellite cell activation, the expression of the early genes c-jun and c-fos is detected.83 After six hours, the expression of myogenin is detected in the newly formed myotubes, but not at earlier stages.83 It has been suggested that myogenin has a role in the differentiation of myoblasts. Other genes involved in muscle regeneration are the pax-7-like gene and the Myo D gene.84,85 Muscle regeneration can be considered as another tissue repair mechanism, involving cellular proliferation, cell-extracellular matrix interactions, and new cell-cell interactions between myotubes and those associated with reinnervation and revascularization. As in other tissue repair mechanisms, different extracellular proteases and protease inhibitors are involved,86,87 for example, plasminogen activators,86 alpha-1-antichymotrypsin (ACT) and the beta amyloid precursor protein containing the Kunitz-inhibitor sequence,87 related to protease nexin 2 (see chapter 3).

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The involvement of ubiquitin in muscle regeneration is suggested by its overexpression in regenerating fibers and also by its upregulation during the myogenesis of infantile muscle fibers.88,89 If we consider some of the reviewed aspects about muscle regeneration, the involvement of the ubiquitin system in this mechanism can thus be strongly emphasized. As suggested in the chapter 3, the ubiquitin system may have a role in the inflammatory response through its action on NF- κB. This is a transcriptional activator of the immune response and may be involved in muscle regeneration, controlling the release of cytokines or the expression of cell adhesion molecules. In addition, the ubiquitin system may be involved in neuromuscular connectivity, which is essential for muscle regeneration. Its role in the degradation of the soluble APP isoform with the Kunitz-inhibitor sequence,56 which is needed for the NGF receptor activation,90 must be important. Also important is the reported existence of ubiquitin-conjugating enzymes that are involved in neuronal connectivity,91 which could also be related to neuromuscular connectivity. Another possible link between the ubiquitin system and muscle regeneration is its action on the different growth factor receptors involved in regeneration, for example on ligand-induced polyubiquitinization of the FGF receptor, EGF receptor or PDGF receptor,42,92 which may be also be involved in the same mechanism. The role of the ubiquitin system in the degradation of IGF-I receptor has to be emphasized since IGF-I has been shown to downregulate the mRNA proteasome subunits.40 Finally, it is worth pointing out that the activities of c-jun and c-fos are at least in part ubiquitin-controlled.61–65 The study of the involvement of the ubiquitin system in muscle regeneration, necrosis and degradation may be important for future genetic or pharmacological interventions. Mampuru et al93 have reported interesting sequential events in muscle degradation. They have shown in cell cultures under conditions of serum deprivation, satellite cells undergo a series of sequential events: early cell death (apoptosis), transient cell cycle traverse and delayed cell death. Interestingly they have shown that calpains are involved in the first step of apoptosis whereas the ubiquitin system is involved in the last phase.

Stress Response in Muscle Wasting As discussed in other chapters (see chapter 7), the stress response is involved in protecting different tissues against practically all types of insults. This mechanism (where the ubiquitin system is involved) may also be crucial in the prevention of muscle wasting. Recently Buck and Chojkier 94 showed that TNF (which is known to induce muscle wasting in cancer, AIDS, and sepsis),43 mediates its action in a murine model by inducing oxidative stress and activating the nitric oxide synthase (NOS). They showed that the TNF-induced muscle wasting could be prevented by antioxidant or NOS inhibitor treatment. Moreover, it was also shown that the TNF-induced oxidative stress caused muscle dedifferentiation, as measured by a decrease in the myosin creatinine phosphokinase-enhancer box (MCK-E box) binding activities. The MCK-E box is a conserved regulatory region of DNA that is critical for the expression of most skeletal muscle specific genes, such as myosin and myosin creatinine phosphokinase, and its binding and transcriptional activities have been used as indicators of muscle specific differentiation. Moreover it was shown that the decrease in the MCK-E binding activities was due to a marked decrease in junD activity as a consequence of oxidative stress (which affected the myogenin-junD transcriptional activity). At this point it could be hypothesized that the TNF-induced effects are part of a normal physiological mechanism. Only when the TNF levels were highly abnormal and continuously maintained would the stress response be necessary to control the abnormally high oxidative stress. Different efficiencies in the stress response based on genetic differences could predispose patients to different degrees of muscle wasting. Given the involvement of the stress response in muscle wasting, there are also other observations, for example, the involvement of hsp90

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and ubiquitin in myogenesis and the induction of different heat-shock proteins and ubiquitin both in degenerating and regenerating myofibers in Duchenne muscular dystrophy.88,89

The Ubiquitin System and Muscle Wasting Inducers Up to now we have considered the possible roles of the ubiquitin system in controlling the different mechanisms related to muscle protection. Its involvement in muscle degradation should also be considered. On this lines, there are signal transduction pathways activated by the muscle wasting inducers. These are TNF, glucocorticoids, acidosis, or interferon- α (IFN- α), the latter being involved in AIDS.95 In the case of glucocorticoids, the interaction of the ubiquitin conjugating enzyme Ubc9 human homolog with the glucocorticoid receptor has been identified.96 Binding of glucocorticoids to their cytosolic receptor leads to activation of the target gene and suppression of other gene transcription processes through interference with the function of several transcription factors such as AP-1 and NF- κB. Gottlicher et al96 have shown that the interaction of Ubc9 with the glucocorticoid receptor and c-jun is necessary for the negative control of the AP-1 activity by glucocorticoids. Concerning interferons, it has been shown that the ubiquitin system is involved in the degradation of a STAT protein (signal transducers and activators of transcription).97 Such proteins are latent cytoplasmatic transcription factors which are activated by Janus kinases in response to interferons. Obviously the involvement of the ubiquitin system (related to glucocorticoids or interferons) in signal transduction, does not provide conclusive evidence for its involvement in muscle wasting, but it clearly suggests the possibility.

Muscle Dystrophies Muscle dystrophies are degenerative neuromuscular diseases, commonly characterized by progressive muscle fiber necrosis. Such diseases lead to muscle wasting and weakness, and eventually to death, due to cardiac or respiratory failure. The most common is the socalled Duchenne dystrophy which is caused by a lack of dystrophin. The function of dystrophin is to link the contractile apparatus to the dystrophin-associated proteinc omplex embedded in the sarcolemma. This complex in turn links the sarcolemma with the extracellular matrix through proteins like laminin. This type of regular array along the length of the myotubes is believed to have an important role in muscle work in relation to strength. It would act by preventing uncoordinated work between the contractile apparatus, the sarcolemma and the extracellular matrix, which could lead to the mechanical breakdown of the membrane or to permeability changes that would finally end in fiber necrosis. Interestingly, different muscle dystrophies have been related to protein alterations in the cytoskeletonsarcolemma-extracellular matrix connections, as is the case of the 50 kDa subunit of the dystrophin-associated protein complex, or the M subunit of laminin known as merosin.98 The clearest evidence involving the ubiquitin system in degenerative muscle disease (in relation to muscle dystrophies) comes from sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy (h-IBM).99,100 Strikingly, this evidence seems to be closely related to that already mentioned for Alzheimer’s disease (AD). s-IBM and h-IBM are both idiopathic inflammatory myopathies (s-IBM is the most common muscle disease beginning in patients 50 years of age and older) leading to severe disability. Patients suffer proximal and distal muscle weakness, thinning of the forearms (which is associated with weakness of the finger extensors, flexors or both) and prominent involvement of the quadriceps. The slow progression of the disease usually leads to severe disability and eventually to respiratory muscle weakness. The pathological features of s-IBM seen under light microscopy include mononuclear cell infiltration in the muscle at the early stages of the disease, muscle fibers with usually

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red-rimmed irregular vacuoles (on Engel-Gomori trichrome staining), which are more abundant in the middle and later stages, and atrophic angular muscle fibers that are considered to be indicative of a denervation component. At the ultrastructural level s-IBM shows cytoplasmic “twisted tubulofilaments” identical to the paired helical filaments of AD and clusters of amyloid like fibrils. h-IBM is inherited either as an autosomal recessive or an autosomal dominant trait and has clinical, morphological and immunochemical similarities with s-IBM. Perhaps the most relevant difference between them is the low presence of amyloid deposits in h-IBM samples versus the continuous presence of amyloid in the s-IBM patients. In fact, Askanas et al101 have detected the presence of beta-amyloid protein (BAP) in the muscle fibers of h-IBM, but without it being aggregated in a beta-pleated sheet structures. So they have suggested that the h-IBM fibers are an earlier stage of the pathological process that is believed to occur in the case of diffuse plaques in AD. The overexpression of ubiquitin in the vacuolated muscle fibers of s-IBM and h-IBM is associated with the same proteins which are also abnormally accumulated in AD:99,100 betaamyloid protein, the N- and C-terminal epitopes of APP, ACT, hyperphosphorylated tau protein (less present in h-IBM than s-IBM), apolipoprotein E, and fibroblast growth factor. In addition, the nicotinic acetylcholine receptor, its 43 kDa associated protein, TGF- β and prion protein are abnormally accumulated in the IBM vacuolated muscle fibers. Such parallelism between IBM and AD suggests involvement of the ubiquitin system in both pathological states. In chapter 3 we have argued a possible involvement of the ubiquitin system in neuroprotective mechanisms during stress conditions and in the regeneration of neuronal connectivity, in both cases (at least in part) through the metabolism of protease nexin 2.56 So it would not be strange to find that the ubiquitin system has a similar involvement in neuromuscular connectivity. Such involvement of the ubiquitin system could be extended to the identification of the ubiquitin-conjugating enzymes needed for such function as has been described for the neuronal connectivity in Drosophila.91 Indeed, some evidence suggests that IBM could be the result of an impaired mechanism of neuromuscular junction regeneration where the ubiquitin system may be involved.99 It has to be considered that in human neuromuscular junctions there is persistent normal accumulation of the proteins which abnormally accumulate in s-IBM and h-IBM. In contrast, nonjunctional regions of mature human innervated fibers do not have accumulation of these proteins. Moreover, when h-IBM muscle cells are cultured in vitro aneurally, they express only a partial IBM phenotype which is restored completely when they are cultured with motor neurons. Biopsy specimens from s-IBM and h-IBM patients contain small angular muscle fibers darkly stain with panesterase and NADH-tetrazolium reductase reactions, which are considered to be indicative of denervation. In spite of the above, the possibility that IBM is not the result of an impaired mechanism of muscle regeneration remains since the levels of some of the IBM proteins also increase in such situations although to a lesser extent.99 It is interesting to point out the existence of two more IBM or AD-like situations in muscle that might give additional clues in the near future concerning the involvement of ubiquitin in such diseases. One of them is the occulopharyngeal muscular dystrophy where ubiquitin deposits associated with amyloid and APP depositions have been described.99 The other is an experimental model consisting of a chloroquine-induced rat myopathy where the treatment of denervated soleus with the lysosomotropic agent leads to strikingly similar morphological and immunopathological changes to IBM.102 Apart from IBM, only a few types of muscle myopathy that apparently involve the ubiquitin system have been reported. In these, other roles for ubiquitin in muscle degeneration are suggested. For example, the supposed abnormal myofibrillar degradation in myofibrillar myopathies could be related to the ubiquitin system.103 This is suggested both by

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the fact that ubiquitin is overexpressed in the pathological structures associated with the disease,103 and also because proteasome 20S has been shown to actively degrade both actin and myosin,104 two of the major myofibrillar proteins whose metabolism is altered in myofibrillar myopathy. In addition, the accumulation of APP components and ACT suggests a blockage of muscle regeneration due to myofibrillar degradation. Therefore, investigating the signals that control the ubiquitin system in the myofibrillar degradation could have relevance for muscle integrity, function and regeneration. In the context of myofibrillar myopathies we should also consider acute steroid myopathy. This occurs in patients treated with massive doses of steroids, inducing severe respiratory and limb muscle wasting associated with myosin loss.105 Given that myosin is degraded by proteasome 20S and ubiquitin is strongly overexpressed in the atrophic fibers,104,106 acute steroid myopathy should be considered in the study of the ubiquitin system involvement in myofibrillar degradation. Finally, a new familial congenital myopathy has been reported where there is the exclusive appearance of ubiquitin, desmin and dystrophin positive plaques.107 This finding indicates the need to investigate the relationship between ubiquitin and desmin or dystrophin. It may be suggested that ubiquitin could be involved in muscle degeneration through its relationship with dystrophin functions.

References 1. Forbes GB. In: Human Body Composition: Growth, Aging, Nutrition and Activity. New York: Springer-Verlag, 1987; 171. 2. Lawson DH, Richmond A, Nixon DW et al. Metabolic approaches to cancer cachexia. Annu Rev Nutr 1982; 2:277-301. 3. DeWys HW. Management of cancer cachexia. Semin Oncol 1985; 12:452-460. 4. Tessitore L, Bonelli G, Baccino FM. Early development of protein metabolic perturbations in the liver and skeletal muscle of tumor-bearing rats. Biochem J 1987; 242:153-159. 5. Llovera M, García-Martínez C, Agell N et al. Ubiquitin gene expression is increased in skeletal muscle of tumour-bearing rats. FEBS Lett 1994; 338:311-318. 6. Llovera M, García-Martínez C, Agell N et al. Muscle wasting associated with cancer cachexia is linked to an important activation of the ATP-dependent ubiquitin-mediated proteolysis. Int J Cancer 1995; 61:138-141. 7. Costelli P, Carbó N, Tessitore L et al. Tumor necrosis factor- α mediates changes in tissue protein turnover in a rat cancer cachexia model. J Clin Invest 1993; 92:2783-2789. 8. Costelli P, García-Martínez C, Llovera M et al. Muscle protein waste in tumor-bearing rats is effectively an tagon ized by a β 2 - ad r en er gic agon ist ( clen bu ter ol) . Role of th e ATP-ubiquitin-dependent proteolytic pathway. J Clin Invest 1995; 95:2367-2372. 9. Temparis S, Asensi M, Taillandier D et al. Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats. Cancer Res 1994; 54:5568-5573. 10. Baracos VE, DeVivo C, Hoyle DHR et al. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. Am J Physiol 1995; 268:E996-E1006. 11. Grimby G, Saltin B. The aging muscle. Clin Physiol 1983; 3:209-218. 12. Carmeli E, Reznick AZ. The physiology and biochemistry of skeletal muscle atrophy as a function of age. Proc Soc Exptl Biol Med 1994; 206:103-113. 13. Riley DA, Bain JLW, Haas AL. Increased ubiquitin conjugation of proteins during skeletal muscle atrophy. J Cell Biol 1986; 103:401a. 14. Medina R, Wing SS, Haas A et al. Activation of the ubiquitin-ATP-dependent proteolytic system in skeletal muscle during fasting and denervation atrophy. Biomed Biochim Acta 1991; 50:347-356. 15. Tawa Jr NE, Kettelhut IC, Goldberg AL. Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle. Am J Physiol 1992; 263:E326-E334. 16. Tiao G, Fagan JM, Samuels N et al. Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest 1994; 94:2255-2264.

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17. García-Martínez C, Llovera M, Agell N et al. Ubiquitin gene expression in skeletal muscle is increased during sepsis: Involvement of TNF- α but not IL-1. Biochem Biophys Res Commun 1995; 217:839-844. 18. May RC, Hara Y, Kelly RA et al. Branched-chain amino acid metabolism in rat skeletal muscle: Abnormal regulation in acidosis. Am J Physiol 1987; 252:E712-E718. 19. Mitch WE, Medina R, Grieber S et al. Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes. J Clin Invest 1994; 93:2127-2133. 20. Wing SS, Goldberg AL. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting. Am J Physiol 1993; 264:E668-E676. 21. Wiborg O, Pederson MS, Wind A et al. The human ubiquitin multigene family: Some genes contain m ultiple directly repeated ubiquitin coding sequences. EMBO J 1985; 4:755-759. 22. Llovera M, García-Martínez C, Costelli P et al. Muscle hypercatabolism during cancer cachexia is not reversed by the glucocorticoid receptor antagonist RU38486. Cancer Lett 1996; 99:7-14. 23. Wing SS, Banville D. 14-kDa ubiquitin-conjugating enzyme: Structure of the rat gene and regulation upon fasting and by insulin. Am J Physiol 1994; 267:E39-E48. 24. Tomas FM, Murray AJ, Jones LM. Interactive effects of insulin and corticosterone on myofibrillar protein turnover in rats as determined by N-methylhistidine excretion. Biochem J 1984; 220:469-479. 25. Llovera M, López-Soriano FJ, Argilés JM. Effects of tumor necrosis factor- α on muscleprotein turnover in female Wistar rats. J Natl Cancer Inst 1993; 85:1334-1339. 26. García-Martínez C, López-Soriano FJ, Argilés JM. Acute treatment with tumour necrosis factor- α induces changes in protein metabolism in rat skeletal muscle. Mol Cell Biochem 1993; 125:11-18. 27. García-Martínez C, Agell N, Llovera M et al. Tumour necrosis factor- α increases the ubiquitinization of rat skeletal muscle proteins. FEBS Lett 1993; 323:211-214. 28. García-Martínez C, Llovera M, Agell N et al. Ubiquitin gene expression in skeletal muscle is increased by tumour necrosis factor- α. Biochem Biophys Res Commun 1994; 201:682-686. 29. Llovera M, Carbó N, García-Martínez C et al. Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats. Biochem Biophys Res Commun 1996; 221:653-655. 30. Costelli P, Llovera M, Carbó N et al. Interleukin-1 receptor antagonist (IL-1ra) is unable to reverse cachexia in rats bearing an ascites hepatoma (Yoshida AH-130). Cancer Lett 1995; 95:33-38. 31. Hofmann C, Lorenz K, Braithwaite SS et al. Altered gene expression for tumour necrosis factor- α and its receptors during drug and dietary modulation of insulin resistance. Endocrinology 1994; 134:264-270. 32. Llovera M, García-Martínez C, Agell N et al. TNF can directly induce the expression of ubiquitin-dependent proteolytic system in rat soleus m uscles. Biochem Biophys Res Commun 1997; 230:238-241. 33. Pell JM. Whole body and tissue protein turnover: Peptide hormone effects. In: Bond JS, Barrett AJ, eds. Proteolysis and protein turnover. London: Portland Press, 1993:133-140. 34. Tayej JA. A review of cancer cachexia and abnormal glucose metabolism in humans with cancer. J Am Coll Nutr 1992; 11:445-456. 35. Lang CH, Dobrescu C, Meszaros K. Insulin-mediated glucose uptake by individual tissues during sepsis. Metabolism 1990; 39:1096-1107. 36. Muller DC, Elahi D, Tobin JD et al. The effect of age on insulin resistance and secretion: A review. Semin Nephrol 1996; 16:289-298. 37. Tomas FM, Chandler CS, Knowles SE et al. Muscle protein turnover and insulin-like growth factor-I (IGF-I): Anti-catabolic and anabolic effects of IGFs. In: Bond JS, Barrett AJ, eds. Proteolysis and Protein Turnover. London: Portland Press, 1993:141-148.

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38. Vlachopapadopoulou E, Zachwieja JJ, Gertner JM et al. Metabolic and clinical response to recombinant human insulin-like growth factor I in myotonic dystrophy: A clinical research center study. J Clin Endocrinol Metab 1995; 80:3715-3723. 39. Sepp-Lorenzino L, Ma Z, Lebwohl DE et al. Herbimycin A induces the 20S proteasomeand ubiquitin-dependent degradation of receptor tyrosine kinases. J Biol Chem 1995; 270:16580-16587. 40. Hong D, Forsberg NE. Effects of serum and insulin-like growth factor I on protein degradation and protease gene expression in rat L8 myotubes. J Anim Sci 1994; 72:2279-2288. 41. Strous GJ, van Kerkhof P, Govers R et al. The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J 1996; 15:3806-3812. 42. Mori S, Heldin C, Claesson-Welsh L. Ligand-induced polyubiquitination of the plateletderived growth factor beta-receptor. J Biol Chem 1992; 267:6429-6434. 43. Argilés JM, López-Soriano FJ. The ubiquitin-dependent proteolytic pathway in skeletal muscle: Its role in pathological states. Trends Pharmacol Sci 1996; 17:223-226. 44. Hotamisligil GS, Spiegelman BM. Tumor necrosis factor α: A key component of the obesity-diabetes link. Diabetes 1994; 43:1271-1278. 45. Kornitzer D, Raboy B, Kulka RG et al. Regulated degradation of the transcription factor Gcn4. EMBO J 1994; 13:6021-6030. 46. Samarel AM. Mechanical load and the regulation of myofibrillar protein degradation. In: Bond JS, Barrett AJ, eds. Proteolysis and Protein Turnover. London: Portland Press, 1993:149-156. 47. Furuno K, Goodman MN, Goldberg AL. Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy. J Biol Chem 1990; 265:8550-8557. 48. Thomason DB, Booth FW. Atrophy of the soleus muscle by hindlinb unweighting. J Appl Physiol 1990; 68:1-12. 49. Banes AJ, Tsuzaki M, Yamamoto J et al. Mechanoreception at the cellular level: The detection, interpretation, and diversity of responses to mechanical signals. Biochem Cell Biol 1995; 73:349-365. 50. Duncan RL, Turner CH. Mechanotransduction and the functional response of bone to mechanical strain. Calcified Tissue Int 1995; 57:344-358. 51. Berk BC, Corson MA, Peterson TE et al. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: A hypothesis for calcium-dependent and calcium-independent events activated by flow. J Biomech 1995; 28:1439-1450. 52. Chen BM, Grinnell AD. Integrins and modulation of transmitter release from motor nerve terminals by stretch. Science 1995; 269:1578-1580. 53. Uehara Y, Hori M, Takeuchi T et al. Screening of agents which convert “transformed morphology” of Rous sarcoma virus-infected rat kidney cells to “normal morphology”: Identification of an active agent as herbimycin and its inhibition of intracellular src kinase. Jpn J Cancer Res 1985; 76:672-675. 54. Uehara Y, Hori M, Takeuchi T et al. Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus. Mol Cell Biol 1986; 6:2198-2206. 55. Uehara Y, Murakami Y, Sugimoto Y et al. Mechanism of reversion of Rous sarcoma virus transformation by herbimycin A: Reduction of total phosphotyrosine levels due to reduced kinase activity and increased turnover of p60v-src1. Cancer Res 1989; 49:780-785. 56. Gregori L, Bhasin R, Goldgaber D. Ubiquitin-mediated degradative pathway degrades the extracellular but not the intracellular form of amyloid beta-protein precursor. Biochem Biophys Res Commun 1994; 203:1731-1738. 57. Crowther RA. Structural aspects of pathology in Alzheimer’s disease. Biochim Biophys Acta 1991; 1096:1-9. 58. Goldspink DF, Cox VM, Smith SK et al. Muscle growth in response to mechanical stimuli. Am J Physiol. 1995; 268:E288-E297.

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59. Perrone CE, Fenwick-Smith D, Vandenburgh HH. Collagen and stretch modulate autocrine secretion of insulin-like growth factor-1 and insulin-like growth factor binding proteins from differentiated skeletal muscle cells. J Biol Chem 1995; 270:2099-2106. 60. Czerwinski SM, Martin JM, Bechtel PJ. Modulation of IGF mRNA abundance during stretch-in du ced skeletal m u scle hypertrophy an d regression . J Appl Physiol 1994; 76:2026-2030. 61. Hermida-Matsumoto ML, Chock PB, Curran T et al. Ubiquitinylation of transcription factors c-Jun and c-Fos using reconstituted ubiquitinylating enzymes. J Biol Chem 1996; 271:4930-4936. 62. Stancovski I, Gonen H, Orian A et al. Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: Identification and characterization of the conjugating enzymes. Mol Cell Biol 1995; 15:7106-7116. 63. Tsurumi C, Ishida N, Tamura T et al. Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases. Mol Cell Biol 1995; 15:5682-5687. 64. Jariel-Encontre I, Pariat M, Martin F et al. Ubiquitinylation is not an absolute requirem en t for degradation of c-Jun protein by the 26S proteasom e. J Biol Chem 1995; 270:11623-11627. 65. Treier M, Staszewski LM, Bohmann D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 1994; 78:787-798. 66. Laing JG, Beyer EC. The gap junction protein connexin-43 is degraded via the ubiquitin proteasome pathway. J Biol Chem 1995; 270:26399-26403. 67. Handa I, Matsushita N, Ihashi K et al. A clinical trial of therapeutic electrical stimulation for amyotrophic lateral sclerosis. Tohoku J Exp Med 1995; 175:123-134. 68. Bodine-Fowler S. Skeletal muscle regeneration after injury: An overview. J Voice 1994; 8:53-62. 69. Sesodia S, Chorski RM, Nemeth PM. Nerve-dependent recovery of metabolic pathways in regenerating soleus muscles. J Muscle Res Cell Motil 1994; 15:573-581. 70. Carlson BM, Faulkner JA. The regeneration of noninnervated muscle grafts and marcainetreated muscles in young and old rats. J Gerontol 1996; 51:B43-B49. 71. Chambers RL, McDermott JC. Molecular basis of skeletal muscle regeneration. Can J Appl Physiol 1996; 21:155-184. 72. Tidball JG. Inflammatory cell response to acute muscle injury. Med Sci Sports Exerc 1995; 27:1022-1032. 73. St. Pierre BA, Tidball JG. Differential response of macrophage subpopulations to soleus muscle reloading after rat hindlimb suspension. J Appl Physiol 1994; 77:290-297. 74. Morin S, de la Porte S, Fiszman M et al. Inhibition of proliferation in 8-week-old mdx mouse muscle fibroblasts in vitro. Differentiation 1995; 59:145-154. 75. Lefaucheur JP, Sebille A. Muscle regeneration following injury can be modified in vivo by immune neutralization of basic fibroblast growth factor, transforming growth factor-beta 1 or insulin-like growth factor I. J Neuroimmunol 1995; 57:85-91. 76. Lefaucheur JP, Sebille A. Basic fibroblast growth factor promotes in vivo muscle regeneration in murine muscular dystrophy. Neurosci Lett 1995; 202:121-124. 77. Maley MA, Davies MJ, Grounds MD. Extracellular matrix, growth factors, genetics: Their influence on cell proliferation and myotube formation in primary cultures of adult mouse skeletal muscle. Exp Cell Res 1995; 219:169-179. 78. Garrett KL, Anderson JE. Colocalization of bFGF and the myogenic regulatory gene myogenin in dystrophic mdx muscle precursors and young myotubes in vivo. Dev Biol 1995; 169:596-608. 79. Cantini M, Carraro U. Macrophage-released factor stimulates selectively myogenic cells in primary muscle culture. J Neuropathol Exp Neurol 1995; 54:121-128. 80. Kuwabara K, Ogawa S, Matsumoto M et al. Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells. Proc Natl Acad Sci USA 1995; 92:4606-4610. 81. Iannaccone S, Quattrini A, Smirne S et al. Connective tissue proliferation and growth factors in animal models of Duchenne muscular dystrophy. J Neurol Sci 1995; 128:36-44.

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82. Heuss D. Light microscopy study of low-affinity nerve growth factor receptor and phosphoprotein B-50/neuromodulin in inflammatory myopathies. Acta Neuropathol Berl 1996; 91:409-415. 83. Kami K, Noguchi K, Senba E. Localization of myogenin, c-fos, c-jun, and muscle-specific gene mRNAs in regenerating rat skeletal muscle. Cell Tissue Res 1995; 280:11-19. 84. Kay PH, Mitchell CA, Akkari A et al. Association of an unusual form of a Pax7-like gene with increased efficiency of skeletal muscle regeneration. Gene 1995; 163:171-177. 85. Megeney LA, Kablar B, Garrett K et al. MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev 1996; 10:1173-1183. 86. Akaaboune M, Verdiere-Sahuque M, Lachkar S et al. Serine proteinase inhibitors in human skeletal muscle: Expression of beta-amyloid protein precursor and alpha-1-antichymotrypsin in vivo and during myogenesis in vitro. J Cell Physiol 1995; 165:503-511. 87. Muñoz-Canoves P, Felez J. Role of plasminogen activators in muscle regeneration. Biol Clin Hematol 1994; 15:163-171. 88. Bornman L, Polla BS, Lotz BP et al. Expression of heat-shock/stress proteins in Duchenne muscular dystrophy. Muscle Nerve 1995; 18:23-31. 89. Bornman L, Polla BS, Gericke GS. Heat-shock protein 90 and ubiquitin: Developmental regulation during myogenesis. Muscle Nerve 1996; 19:574-580. 90. Streit WJ, Kincaid-Colton CA. The brain’s immune system. Scientific Amer 1995; 273:38-43. 91. Muralidhar MG, Thomas JB. The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 1993; 11:253-266. 92. Mori S, Claesson-Welsh L, Okuyama Y et al. Ligand-induced polyubiquitination of receptor tyrosine kinases. Biochem Biophys Res Commun 1995; 213:32-39. 93. Mampuru LJ, Chen SJ, Kalenik JL et al. Analysis of events associated with serum deprivation-induced apoptosis in C3H/Sol8 muscle satellite cells. Exp Cell Res 1996; 226:372-380. 94. Buck M, Chojkier M. Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. EMBO J 1996; 15:1753-1765. 95. Pallarés-Trujillo J, López-Soriano FJ, Argilés JM. TNF and AIDS: Two sides of the same coin? Med Res Rev 1995; 15:533-546. 96. Gottlicher M, Heck S, Doucas V et al. Interaction of the Ubc9 human homologue with c-Jun and with the glucocorticoid receptor. Steroids 1996; 61:257-262. 97. Kim TK, Maniatis T. Regulation of interferon-gamma-activated STAT1 by the ubiquitinproteasome pathway. Science 1996; 273:1717-1719. 98. Tinsley JM, Blake DJ, Zuellig RA et al. Increasing complexity of the dystrophin-associated protein complex. Proc Natl Acad Sci USA 1994; 91:8307-8313. 99. Askanas V, Engel WK, Mirabella M. Idiopathic inflammatory myopathies: Inclusion-body myositis, polymyositis, and dermatomyositis. Curr Opin Neurol 1994; 7:448-456. 100. Askanas V, Engel WK. New advances in the understanding of sporadic inclusion-body myositis and hereditary inclusion-body myopathies. Curr Opin Rheumatol 1995; 7:486-496. 101. Askanas V, Alvarez RB, Engel WK. Beta-amyloid precursor epitopes in muscle fibers of inclusion body myositis. Ann Neurol 1993; 34:551-560. 102. Tsuzuki K, Fukatsu R, Takamaru Y et al. Colocalization of amyloid-associated proteins with amyloid beta in rat soleus muscle in chloroquine-induced myopathy: A possible model for amyloid beta formation in Alzheimer’s disease. Brain Res 1995; 699:260-265. 103. De Bleecker JL, Engel AG, Ertl BB. Myofibrillar myopathy with abnormal foci of desmin positivity. II. Immunocytochemical analysis reveals accumulation of multiple other proteins. J Neuropathol Exp Neurol 1996; 55:563-577. 104. Taylor RG, Tassy C, Briand M et al. Proteolytic activity of proteasome on myofibrillar structures. Mol Biol Rep 1995; 21:71-73. 105. Nava S, Gayan-Ramirez G, Rollier H et al. Effects of acute steroid administration on ventilatory and peripheral muscles in rats. Am J Respir Crit Care Med 1996; 153:1888-1896. 106. Minetti C, Hirano M, Morreale G et al. Ubiquitin expression in acute steroid myopathy with loss of myosin thick filaments. Muscle Nerve 1996; 19:94-96. 107. Fidzianska A, Ryniewicz B, Barcikowska M et al. A new familial congenital myopathy in children with desmin and dystrophin reacting plaques. J Neurol Sci 1995; 131:88-95.

CHAPTER 6

Other Pathological States Immune Response-related Diseases

T

he ability of the immune system to recognize and respond to most foreign antigens depends on the activation of T lymphocytes. However, the latter’s antigen specific T receptors (Tc receptors) are only able to recognize antigens when they have been processed to small peptide fragments and presented at the cell surface of the antigen-presenting cells (APCs), which are bound to self-molecules known as a major histocompatibility complex (MHC). Two types of MHC molecules have been described. Thus, while the MHC-II are involved in the activation of helper T lymphocytes, the MHC-I molecules are involved in the activation of cytotoxic T lymphocytes. The activation of helper T lymphocytes through MHC-II molecules is directed against extracellular antigens (the exogenous pathway), which leads to B lymphocyte activation and consequently to antibody production. Where this occurs, only certain cells (such as macrophages) act as APCs following antigen uptake and processing via the endolysosomal pathway. On the other hand, the activation of cytotoxic T lymphocytes through MHC-I molecules is directed against intracellular antigens (the endogenous pathway), which leads to the lysis of the APCs by lymphocytes. In this case, most cells are able to act as APCs, for example when they have been infected or abnormally changed like in tumor development. Interestingly, antigen processing via the endogenous pathway is mediated by the 20S proteasome.1,2 Significantly, both types of immune response to extracellular and intracellular antigens have been shown to have a role in practically all cases of immunity. This is the case, for example, of immunity to microbes (bacteria, viruses and parasites), to tissue transplants, to tumors, in autoimmunity or immunodeficiencies. Thus, an impairment in either type of immune response, or in any of the components (i.e. antigen specific T cell receptors, the MHC-II or MHC-I molecules and the processed antigens by the exogenous or endogenous pathways) taking part in T cell activation, might be expected to lead to an immune-related disease. Thus, the ubiquitin system might indeed be involved in immune-related diseases, since it would seen to be related to the expression of MHC-I molecules and the function of the antigen specific T cell receptors. In addition, antigen processing via the endogenous pathway has been shown to involve at least the 20S proteasome proteolytic activities.1,2 Thus, the ubiquitin system might be involved in the expression of MHC-I molecules. In support of this theory, it has to be pointed out that the system has been shown to activate the transcription factor NF- κB in two different ways,3 this factor being known to induce the expression of the MHC-I gene.4–6 Although a direct role for the ubiquitin system in MHC-I expression has not yet been reported, its involvement in NF-κB activation,3 through partial proteolytic processing of the p105 subunit precursor or through I- κB degradation, makes this suggestion interesting and worthy of investigation. Furthermore, the NF- κB transcription factor has also been shown to induce the expression of other immune response-related genes4–6 Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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such as the immunoglobulin light chain kappa gene, the interleukin-2 receptor α chain the interleukin-6 or the interferon- β (IFN- β) genes. This suggests other possible links between the ubiquitin system and the immune responses. The ubiquitin system has been shown to ubiquitinate multiple intracellular lysines in response to antigen-specific T receptor engagement.7 In addition, such ubiquitination has been shown to depend on receptor phosphorylation through the tyrosine kinase activity of the receptor itself.8 Taking all this into consideration, the ubiquitin system would seem to have a role in signal transduction after T cell activation. More probably, as has been reported for other receptors, its role would seem to be to induce the receptor endocytosis to the endolysosomal system for degradation.9 The role of the 20S proteasome in antigen processing via the endogenous pathway has been widely reported.1,2 However, conflicting results supporting the involvement of the ubiquitin system have been reported.10–12 In spite of this, certain regulatory aspects of the 20S proteasome in antigen processing should be taken into account since they might share certain featureswith other physiological situations in which ubiquitin is involved. Along these lines, the cytokine interferon- γ (IFN- γ) has been shown to induce the expression of new proteasome subunits called LMP2, LMP7 and MECL1, which replace their constitutive-proteolytic homologous subunits modifying the 20S proteasome proteolytic activities.1,2,13,14 In addition the IFN- γ has also been shown to induce the expression of the PA28 subunits which associate into hexameric or heptameric rings forming the proteasome regulator PA28. This regulator has been shown to bind to the 20S proteasome while also affecting its proteolytic activities.15,16 In conclusion, bearing in mind its relation with the process of T cell activation, the involvement of the ubiquitin system in immune-related diseases might be hypothesized.

The Ubiquitin System and Cancer Development Cancer development can occur as an impaired immune response to tumor cells. It has been shown that tumor cells can avoid the immune response by decreasing the expression of MHC-I molecules or the antigen processing for presentation. This would lead to a defect in T cell activation against tumor cells. Interestingly, since the ubiquitin system is involved in the expression of MHC-I molecules through NF- κB activation, impaired action of the proteolytic system may contribute to decreased MHC-I expression and consequently to cancer development. In addition, defects in the expression of the LMP2 and LMP7 proteasome subunits have been shown to be closely related to defects in antigen processing and presentation in different small cell lung carcinoma cell lines and a murine T cell lymphoma cell line.17,18 Such a relation is reinforced by the fact that transfection of IFN- γ into these cell lines has been shown to restore both the LMP2 expression and the antigen processing.18

The Ubiquitin System and Autoimmune Diseases All autoimmune disorders are associated with the production of autoantibodies and/or self-reactive T cell populations as a consequence of an impaired function in the mechanisms of self-tolerance.19–21 Such tolerance to self-antigens, which is largely due to T cell tolerance, is believed to be regulated in two different ways: first, the deletion of self-reactive T cell clones (clonal deletion) during their maturation in the thymus; and second, the inactivation of mature self-reactive T cell clones (clonal anergy) in the periphery against antigens not present in the thymus. As for the mechanisms involved in clonal anergy in the periphery, both the lack of costimulators and suppressor T cells seem to play highly important roles.19

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Fig. 6.1. Role of the ubiquitin-dependent proteolysis in antigen presentation. Ubiquitin is involved in the activation of NF-kB and therefore in the transcription of MCH-I genes and also in antigen presentation. In addition, in relation with the antigen specific T cell receptor, the ubiquitin system has been shown to ubiquitinate multiple intracellular lysines in response to the receptor engagement.

In the same way as for the involvement of the ubiquitin system in immune-related diseases, the relation of the proteolytic system with MHC-I molecules, the antigen-specific T cell receptor and antigen processing through the endogenous pathway, suggests an involvement in autoimmune diseases. This can be further supported given that the deletion of immature self-reactive T cell clones in the thymus has been shown to be closely related to the process of MHC-I-antigen presentation (Fig. 6.1). In addition, the MHC genes have also been shown to determine a genetic predisposition to the development of an autoimmune disease, and antigen specific T cell receptor V genes have been found to be highly expressed in autoimmune T cells.21

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Among those studies discussing autoimmunity,19-21 there is one particular experimental model that might be of use in seeking the possible implications for the ubiquitin system in the development of autoimmune diseases through NF- κB activation. The work of Kaltschmidt et al22 has shown that an NF- κB is activated and highly expressed in the T cells and microglia of an experimental autoimmune encephalomyelitis. Finally, although a malfunction of the ubiquitin system might be directly involved in the breakdown of self-tolerance, it should also be considered that ubiquitin has been found to be an antigenic target for an autoimmune response.23 Along these lines, autoantibodies of ubiquitin and ubiquitinated histones have been found in the serum of patients with systemic lupus erythematosus.24,25 Although in this case ubiquitin is not involved in the etiology of the disease, the deposition of their autoimmune complexes in the renal glomeruli has been shown to contribute considerably to the development of lupus nephritis, which is a severe complication of the disease. Interestingly, similar serum autoantibodies have also been found in other autoimmune diseases, such as in localized scleroderma or systemic sclerosis.26

Cataract Formation and Blindness Jahngen-Hodge et al27 have reported increases in high molecular mass ubiquitin conjugates in both aged and cataractous lenses in comparison to normal lenses of the same age. This increase in specific ubiquitin conjugates in cataractous lenses could represent altered proteins. If this is true, it would seem quite probable that the reparative action of the ubiquitin system is important in preventing the ageing process (and cataract formation) by degrading abnormal proteins as in the case of the cellular stress response. Specific proteolytic action of the ubiquitin system on the protein alpha-crystallins (either native or oxidized) has been described by Huang et al.28 Very recent work suggests that a novel ubiquitin-like peptide (ULP) could have a very important role in the control of lens cell growth.29 ULP has been detected in high concentrations in both the iris-ciliary complex and in the aqueous humor extract of rabbits,30 the peptide being a very powerful inhibitor of protein synthesis. Very interestingly, photoreceptor proteins (such as transducine and rhodopsin) are also degraded by the ubiquitin system thus suggesting a novel role for this pathway in the regulation of mammalian phototransduction events.31–33 The presence of the ubiquitinactivating enzyme (E1), different ubiquitin-carrier proteins, proteasome activity and ubiquitin isopeptidase/hydrolase activities has also been described in bovine rod outer segm en ts. This con fir m s a m ost likely involvem en t of the u biqu itin system in the phototransduction mechanisms related to vision and suggests that some types of vision defects or blindness may also be linked to the activity of the system. In connection with this, recent work has shown that perturbations in protein turnover and processing can lead to retinal disease. Indeed, overexpression of a C-terminal ubiquitin hydrolase (linked to X chromosome) has been reported 34 and may be associated with retinal disorders.

Hypertension and Ischemia The ubiquitin-dependent system may also have a role in regulating blood pressure due to its possible action on the epithelial amiloride-sensitive channel. This channel is composed of three subunits, each containing two proline-rich sequences (P1 and P2) at the C-terminus. The P2 regions in humans, which are identical to the ones in the rat, were recently shown to be deleted in patients with Liddle’s syndrome (a hereditary form of hypertension) leading to the hyperactivation of the channel. This is due to the lack of binding of a suppressor protein known as Nedd4 to the P2 regions. Staub et al35 have identified a ubiquitin ligase domain in Nedd4. Consequently, decreased ubiquitinization could lead to defects in the normal operation of the Na+ -pump, which in turn could lead to hypertension.

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Table 6.1. Genes of the ubiquitin system involved in disease Disease

E1

Angelman syndrome

Blindness

E3

Ubiquitin hydrolases

UBE2L1 (FAD3)

Alzheimer’s disease

Cancer

E2

E6AP UBE1L

Tre17 (human homolog of yeast Doa4)Unp (murine homolog of yeast Doa4), human Unp (homolog to the murine Unp) ubiquitin C-terminal hydrolase highly expressed in retina and located in chromosome X

Long lasting myocardial ischemia causes the death of myocytes despite the restoration of coronary blood flow. A short period of ischemia and reperfusion transiently injures myocytes and is followed by the prolonged but reversible contractile dysfunction called myocardial stunning. This is probably caused by injury to the heart at the molecular level, like the transient inactivation or damage of proteins of the sarcoplasmic reticulum of the contractile machinery. The presence of anti-oxidant enzymes together with the synthesis of stress proteins such as hsp70 or ubiquitin (significant increases in ubiquitin gene transcripts in stunned regions have been described) 36 may constitute the molecular defense mechanism against ischemia/reperfusion injury. In summary, adaptative myocardial response to ischemia involves the coordinated induction of heme oxygenase enzymes and ubiquitin, which may be indicative of the existence of a pathophysiologically important defense mechanism in which both degradation of denatured cellular proteins and generation of biologically active products of heme metabolism are accelerated.37 Interestingly, the ubiquitin system also degrades connexin 43, an anchor protein for cardiomyocytes.38

Liver Diseases The ubiquitin system has also been related to pathologies involving the liver. For example, alcohol consumption increases ubiquitin immunoreactivity in hepatocytes, this effect being linked either to a decreased catabolism of ubiquitin conjugates and/or to an increase in cellular stress response due to the ingestion of ethanol.39 Ethanol stabilizes the degradation of CYP2E1 (related to cytochrome P450) which is mediated by the ubiquitin system.40 This could be linked to some extent with the activation of carcinogenic compounds.

AIDS The initiation and propagation of the acquired immunodeficiency syndrome (AIDS) is totally dependent on the replication of the human immunodeficiency virus (HIV), which diminishes the population of CD4+ lymphocytes (this being the hallmark of AIDS). The

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results of intensive research have led to the suggestion that HIV replication is dependent on the activation of the nuclear factor kappa B (NF- κB), which is a transcriptional factor normally involved in lymphocyte activation by cytokines. NF- κ B is a cytosolic protein heterodimer which is composed of a 50 kDa and a 65 kDa subunit. These are normally suppressed through their binding to inhibitory proteins (I- κB). Activation of NF- κB involves dissociation from the I- κB proteins. It is then translocated to the nucleus and binds κB-specific DNA sequences, thus activating gene transcription. HIV gene transcription is controlled by its regulatory gene, named the long terminal repeat (LTR). Activated NF-κB binds to the enhancer sequences of the LTR, thus activating the promoter and beginning viral transcription. HIV infection seems to be only possible in activated cells (via cytokines), possibly because of its dependence on activated NF- κB (see ref. 15 for a review). Interestingly some cytokines, mainly tumor necrosis factor- α (TNF) seem to be the main physiological activators of the HIV-LTR through NF- κB activation. TNF activates a ph osph at idylch olin e-specific ph osph olipase C ( PC-PLC) wh ich th en gen er ates diacylglycerides. These are responsible for an acidic sphingomyelinase activation. The data suggest that the activated TNF receptor-PC-PLC complex and the diacylglycerides formed are internalized into endosomal vesicles which are then fused with others carrying the acidic sphingomyelinase. The ceramide which is formed is somehow involved in the activation of NF- κB by release of the I- κB protein. Very interestingly, Palombella et al have reported that the ubiquitin-proteasome pathway is required for processing the NF- κB (50 kDa) subunit of the heterodimeric complex and degrading the I- κB proteins.42 Thus ubiquitin could have a pivotal role in the activation of NF- κB. In addition, we have previously demonstrated that TNF is able to activate the ubiquitin-dependent pathway,43,44 so it may be suggested that TNF-dependent NF- κB activation may involve the ubiquitin system. This is the first time that a connection between ubiquitin and AIDS has been suggested. Both symptomatic and asymptomatic AIDS patients have high circulating concentrations of cytokines, particularly TNF and interferon- α (IFN- α). This latter cytokine has been associated with some of the characteristic metabolic abnormalities of AIDS patients such as hyperlipidemia.45 Very interestingly, Loeb and Haas46 have reported that IFN- α can induce the production of a ubiquitin cross-reactive protein (UCRP) which seems to be a protein serving as a transacting binding factor directing the association of ligated target proteins to intermediate cytoskeletal filaments. All together, the association between the replication of the HIV and ubiquitin/cytokines could be of relevance in the design of therapeutic strategies to stop viral spread among the human population.

The Angelman Syndrome The Angelman syndrome is a neurological disorder characterized by constant features: severe mental retardation, easily provoked laughter, ataxia, absent speech, and seizures.47 Although most cases are sporadic, severe familial cases have been reported. In most cases (60-70%) there is an interstitial deletion of the maternally inherited chromosome 15 in the region q11-q13. In only rare cases (about 2%) the Angelman syndrome is the result of paternal uniparental disomy of chromosome 15. In 30% of patients, neither maternal (by inherited deletion) nor paternal disomy nor methylation abnormalities can be found. These cases are likely to result from mutations in a gene that is expressed either exclusively or preferentially from the maternal chromosome 15. Recent studies have established that mutations in the E6-AP gene, which encodes a E3 ubiquitin-protein ligase that binds the human papillomavirus E6 oncoprotein and catalyzes the ubiquitinization of p53, are the genetic basis of the Angelman syndrome in humans.48,49 In addition, mapping data indicate that the entire transcriptional unit of the E6-AP gene

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lies within the Angelman syndrome region.50 Therefore experimental evidence demonstrates that the above-referred mutations are a possible cause of the Angelman syndrome and indicate a possible abnormality in ubiquitin-mediated protein degradation during brain development in this disease.

References 1. Monaco JJ, Nandi D. The genetics of proteasome and antigen processing. Annu Rev Genet 1995; 29:729-754. 2. Monaco JJ. Pathways for the processing and presentation of antigens to T cells. J Leukocyte Biol 1995; 57:543-547. 3. Palombella VJ, Rando OJ, Goldberg AL et al. The ubiquitin-proteasome pathway is required for processing the NF- κB1 precursor protein and the activation of NF- κB. Cell 1994; 78:773-785. 4. Baeuerle PA. The inducible transcription activator NF- κB: Regulation by distinct protein subunits. Biochim Biophys Acta 1991; 1072:63-80. 5. Liou HC, Baltimore D. Regulation of NF- κB/rel transcription factor and IkB inhibitor system. Curr Opin Cell Biol 1993; 5:477-487. 6. Grilli M, Chiu JJS, Lenardo MJ. NF- κB and Rel: Participants in a multiform transcriptional regulatory system. Int Rev Cytol 1993; 143:1-62. 7. Hou D, Cenciarelli C, Jensen JP et al. Activation-dependent ubiquitination of a T-cell antigen receptor subunit on multiple intracellular lysines. J Biol Chem 1994; 269:14244-14247. 8. Cenciarelli C, Wilhelm Jr KG, Guo A et al. T cell antigen receptor ubiquitination is a con sequ en ce of receptor-m ediated tyrosin e kin ase activation . J Biol Ch em 1996; 271:8709-8713. 9. Mori S, Heldin CH, Claesson-Welsh L. Ligand-induced polyubiquitination of the plateletderived growth factor beta-receptor. J Biol Chem 1992; 267:6429-6434. 10. Gaczynska M, Rock KL, Goldberg AL. Role of proteasomes in antigen presentation. Enzyme Protein 1993; 47:354-369. 11. Cox JH, Galardy P, Bennink JR et al. Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperature-sensitive cell lines. J Immunol 1995; 154:511-519. 12. Michalek M, Grant E, Gramm C et al. A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature, 1993; 363:552-554. 13. Aki M, Shimbara N, Takashina M et al. Interferon-gamma induces different subunit organizations and functional diversity of proteasomes. J Biochem 1994; 115:257-269. 14. Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol 1995; 7:215-223. 15. Gray CW, Slaughter CA, DeMartino GN. PA28 activator protein forms regulatory caps on proteasome stacked rings. J Mol Biol 1994; 236:7-15. 16. Groettrup M, Soza A, Eggers M et al. A role for the proteasome regulator PA28a in antigen presentation. Nature 1996; 381:166-168. 17. Restifo NP, Esquivel F, Kawakami Y et al. Identification of human cancers deficient in antigen processing. J Exp Med 1993; 177:265-272. 18. Sibile C, Gould KG, Willard-Gallo K et al. LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes. Curr Biol 1995; 5:923-930. 19. Carnaud C, Bach JF. Cellular basis of T-cell autoreactivity in autoimmune diseases. Immunol Res 1993; 12:131-148. 20. Sebastiani GD, Passiu G. The current outlook in the therapy of autoimmune diseases. Ann Ital Med Int 1992; 7:95-101. 21. Imberti L, Sottini A, Primi D. T cell repertoire and autoimmune diseases. Immunol Res 1993; 12:149-167. 22. Kaltschmidt C, Kaltschmidt B, Lanes-Vieira J et al. Transcription factor NF- κB is activated in microglia during experimental autoimmune encephalomyelitis. J Neuroimmunol 1994; 55:99-106.

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23. Muller S, Schwartz LM. Ubiquitin in homeostasis, development and disease. Bioessays 1995; 17:677-684. 24. Akashi Y, Yoshizawa N. Participation of histones and ubiquitin in lupus nephritis. Nippon Jinzo Gakkai Shi 1995; 37:462-467. 25. Stockl F, Muller S, Batsford S et al. A role for histones and ubiquitin in lupus nephritis? Clin Nephrol 1994; 41:10-17. 26. Fujimoto M, Sato S, Ihn H et al. Antiubiquitin antibody in localised and systemic scleroderma. Ann Rheum Dis 1996; 55:399-402. 27. Jahngen-Hodge J, Cyr D, Laxman E et al. Ubiquitin and ubiquitin conjugates in human lens. Exp Eye Res 1992; 55:897-902. 28. Huang LL, Shang F, Nowell Jr TR et al. Degradation of differentially oxidized alphacrystallins in bovine lens epithelial cells. Exp Eye Res 1995; 61:45-54. 29. Bagchi M, Roher AE, Bannerjee A et al. Identification of a ubiquitin-like protein in the mammalian vitreous humor. J Cell Biochem 1996; 61:26-30. 30. Bagchi M, Roher AE, Bannerjee A et al. Identification of a ubiquitin-like protein in the mammalian vitreous humor. J Cell Biochem 1996; 61-26-30 31. Obin M, Nowell T, Taylor A. The photoreceptor G-protein transducin (Gt) is a substrate for ubiquitin-dependent proteolysis. Biochem Biophys Res Commun 1994; 200:1169-1176. 32. Obin M, Nowell T, Taylor A. A comparison of ubiquitin-dependent proteolysis of rod outher segment proteins in reticulocyte lysate and a retinal pigment epithelial cell line. Curr Eye Res 1995; 14:751-760. 33. Obin MS, Jahngen-Hodge J, Nowell T et al. Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outher segments). Evidence for ubiquitinylation of Gt and rhodopsin. J Biol Chem 1996; 271:14473-14484. 34. Swanson DA, Freund CL, Ploder L et al. A ubiquitin C-terminal hydrolase gene on the proximal short arm of the X chromosome: Implications for X-linked retinal disorders. Human Mol Genet 1996; 5:533-538. 35. Staub O, Dho S, Henry P et al. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle’s syndrome. EMBO J 1996; 15:2371-2380. 36. Andres J. Molecular mechanisms for protecting the heart. Folia Med Cracov 1994; 35:22-30. 37. Sharma HS, Maulik N, Gho BC et al. Coordinated expression of heme oxygenase-1 and ubiquitin in the porcine heart subjected to ischemia and reperfusion. Mol Cell Biochem 1996; 157:111-116. 38. Laing JG, Beyer EC. The gap junction protein connexin-43 is degraded via the ubiquitin proteasome pathway. J Biol Chem 1995; 270:26399-26403. 39. Born LJ, Kharbanda KK, McVicker DL et al. Effects of ethanol administration on components of the ubiquitin proteolytic pathway in rat liver. Hepatology 1996; 23:1556-1563. 40. Roberts BJ, Song BJ, Soh Y et al. Ethanol induces CYP2E1 by protein stabilization. Role of u biqu itin con ju gation in th e r ap id d egr ad ation of CYP2E1. J Biol Ch em 1995; 270:29632-29635. 41. Pallarés-Trujillo J, López-Soriano FJ, Argilés JM.(1995) TNF and AIDS: Two sides of the same coin? Med Res Rev 1995; 15:533-546. 42. Palombella VJ, Rando OJ, Goldberg AL et al. The ubiquitin proteasome pathway is requiered for processing the NF- κB precursor protein and the activation of NF- κ B. Cell 1994; 78:773-785. 43. García-Martínez C, Agell N, Llovera M et al. Tumour necrosis factor- α increases the ubiquitinization of rat skeletal muscle proteins. FEBS Lett 1993; 323:211-214. 44. García-Martínez C, Llovera M, Agell N et al. Ubiquitin gene expression in skeletal muscle is increased by tumour necrosis factor- α. Biochem Biophys Res Commun 1994; 201:682-686. 45. Gr u n feld C, Kotler DP, Sh igen aga JK et al. Cir cu latin g in ter fer on - α levels an d hypertriglyceridem ia in the acquired im m unodeficiency syndrom e. Am J Med 1991; 90:154-162. 46. Loeb KR, Haas AL. Conjugates of ubiquitin cross-reactive protein distribute in a cytoskeletal pattern. Mol Cell Biol 1994; 14:8408-8419.

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47. Moncla A, Livet MO, Malzac P et al. The Angelm an syndrom e. Arch Pediatr 1994; 1:1118-1126. 48. Matsuura T, Sutcliffe JS, Fang P et al. De novo truncating mutations in E6-AP ubiquitinprotein ligase gene (UBE3A) in Angelman syndrome. Nature Genet 1997; 15:74-77. 49. Kishino T, Lalande M, Wagstaff J. UBE3A/E6-AP mutations cause Angelman syndrome. Nature Genet 1997; 15:70-73. 50. Sutcliffe JS, Jiang YH, Galijaard RJ et al. The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region. Genome Res 1997; 7:368-377.

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CHAPTER 7

Ageing A

geing can be considered as a nonpathological degenerative process that causes a progressive deterioration and loss of physiological functions and, finally, death. Although there are different typical dysfunctions that finally lead to death in each animal species, it is believed that the molecular mechanism involved in the degenerative processes is similar in all cases. Two main theories have been proposed.1 One considers that ageing is a progressive accumulation of abnormalities in different macromolecules, as a consequence of the progressive dysfunction of the repair mechanisms. The other points out the possibility that ageing is the consequence of a programmed cell death mechanism (apoptosis). This mechanism is operative during tissue regression in different types of metamorphosis, in mammalian embryo development, and in the elimination of damaged cells.2–4 In fact, the main evidence supporting this theory is the existence of these mechanisms and the possibility that the apoptotic genetic information could still be present and repressed in the cellular DNA. More suggestions have been made along the same lines. For example, it has been proposed that survival could be better preserved by directing energy towards early development, growth and reproduction, instead of making the repair mechanisms more efficient. But, what do we know about the involvement of ubiquitin in the ageing process? Changes in ubiquitin have been studied by different authors in leaves of aged barley,5 in aged Drosophila melanogaster,6 in senescent fibroblasts7 and in aged human lens.8 Interestingly, in all cases a general increase in ubiquitin conjugates and specific increases and decreases in the ubiquitin conjugates have been observed. The studies reported by Jahngen-Hodge et al8 in human lens could help us to understand the meaning of the ubiquitin conjugation changes described in the aforementioned aged tissues. In this sense, they have shown that high molecular mass ubiquitin-conjugates (greater than 250 kDa) not only increase in aged lens, but also in cataractous lenses in comparison with normal ones of the same age.8 Taking this into account, it seems that the specific ubiquitin conjugate increases in aged cells could represent altered proteins, as in the case of cataractous lenses. If this is true, it would seem quite probable that the reparative action of the ubiquitin system is important in the prevention of the ageing process, degrading abnormal proteins as in the case of the cellular stress response. In relation to the apoptotic view of ageing, it has been suggested that ubiquitin has a role in this type of cell death mechanism in amphibian metamorphosis and embryo development.2,4 It could be argued that an increased ubiquitin expression in aged cells could be related to an activated apoptosis. However, a lack of increase either in the ubiquitin levels or in mRNA basal expression in senescent fibroblasts or aged hepatocytes has been reported.7,9 Thus, it seems that there is no apoptotic mechanism mediated by ubiquitin in the ageing process. Based on available data, it seems that the ubiquitin system could have an important reparative role in preventing the ageing process via a cellular stress response-like mechanism. However, there are also other reparative mechanisms where the ubiquitin system acts, which could also be important in ageing. One of them is the tissue repair mechanism that Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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we have already discussed in relation to Alzheimer’s disease (AD). Such a mechanism, where both ubiquitin and amyloid precursor protein seem to be involved in the induction of cellular growth and the restoring of cellular connections, could be important both in AD and in ageing. In fact, AD has been considered in some aspects as a model of an accelerated ageing process. The results reported by Kawarabayashi et al10 also support this hypothesis. They have shown an increase in swollen neurites throughout the central nervous system of aged rats and a widely distributed amyloid precursor protein (APP) accumulation in these abnormal neurites. Such APP accumulation could be related to enhanced activity of the repair mechanism in the aged brains. Another reparative event where ubiquitin may be important in the prevention of ageing is DNA repair. Some genes such as superoxide dismutase are clearly involved in ageing,1 and so their DNA repair could be of the utmost importance. Direct involvement of the ubiquitin system in DNA repair of aged cells may be suggested from the fact that cell cycle inhibitory proteins, such as p53, which are ubiquitin-controlled and DNA repair related, could be involved in the slower growth of senescent cells.1,11 Although many biochemical alterations have been described in aged cells, it is quite difficult to know whether they are causes or consequences of ageing. However, at present two lines of evidence may help in the understanding of the role of these changes in ageing. Diet caloric restriction extends the life-span of different animal species.1 Taking this observation into consideration, many age-related alterations have been studied in dietary restricted animals in order to see if they could be considered to be possible “causes” of ageing. Thus the heat-shock induction of the ubiquitin system seems to be age-related. This has been shown by Heydari et al9 in hepatocytes of aged rats, where the heat-shock induction of ubiquitin mRNA was impaired but was enhanced in aged food-restricted animals. These results suggest that defects in the ubiquitin system related to age are likely to be associated with its expression. In addition, it has been reported that glucocorticoids that induce ubiquitin-dependent muscle proteolysis in fasted or acidotic young rats12 do not induce such proteolysis in aged rats.13 Similarly, a reduced sensitivity to a variety of hormones and growth factors in aged tissues has been reported.14–16 It may thus be suggested that a defect in signal transduction could be related to the ubiquitin system in aged cells. A interesting way to identify ageing-related genes is the selection of long-lived mutants in Drosophila.1 Among these mutants with an extended life, a strain has been described that produces a more active isozyme of superoxide dismutase, a enzyme which protects the cell against the damage induced by oxygen radicals. The question that arises is whether this type of mutant could be useful in determining the involvement of ubiquitin-related genes in the ageing process. At present some aspects of signal transduction related to the cellular stress response and their impairment in ageing have been described.17 For example, it has been shown that the decreased expression of heat-shock proteins in several different ageing models is due to a defect in the activation of their transcriptional factor, heat-shock factor-1 (HSF1). This factor could also be involved in the expression of ubiquitin genes18,19 and is constitutively expressed and stored in a latent monomeric form in the cytosol of nonstressed cells. Under heat-shock, HSF1 activation involves the conversion of the HSF1 monomer to a sequencespecific-DNA-binding homotrimer. A delayed trimerization and DNA binding activity of HSF1 has been shown to be related to the impaired heat shock response in aged cells.17 However, more studies will no doubt be carried out in the future to determine the cellular sensors for temperature and other stress factors that promote HSF1 activation. In connection with this, a large body of evidence suggests that reactive oxygen intermediates (ROIs) could be involved in the activation of the HSF1 in the same way as they are involved in the activation of the transcriptional factors NF- κB or AP1 (formed by c-jun and c-fos). The

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Fig. 7.1. Role of HSF1 in the induction of the heat stress-response. HSF-1 is possibly involved in the expression of ubiquitin genes and is constitutively expressed and stored in a latent monomeric form in the cytosol of nonstressed cells. Under heat-shock, HSF1 activation involves the conversion of the HSF1 monomer to a sequence-specific-DNA-binding homotrimer. A delayed trimerization and DNA binding activity of HSF1 has been shown to be related to the impaired heat shock response in aged cells. A large body of evidence suggests that reactive oxygen intermediates (ROIs) could be involved in the activation of the HSF1 in the same way as they are involved in the activation of the transcriptional factors NF-kB or AP1 (formed by c-jun and c-fos). The same evidence points to ROIs as the common denominator for all stress situations, and to their imbalance as a main cause of ageing or degenerative diseases.

same evidence points to ROIs as the common denominator for all stress situations and to their imbalance as a main cause of ageing or degenerative diseases17 (Figs. 7.1 and 7.2). Studies involving signal transduction related to the stress response have also been carried out under oxidant treatments.20–24 In different cell types, such studies have shown that ROIs may act through growth factor receptors, activating the MAP-kinase signaling pathway

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Fig. 7.2. Involvement of the ubiquitin system in ageing. The ubiquitin system is involved in DNA repair through the control of p53 and also ubiquitin-related proteins participate in avoiding protein damage caused by ROIs (see the text for more details).

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(Mek-Erk2-MAPk) and finally the transcriptional factors involved in the stress response.20,21 Among them, the AP1 transcriptional factor (formed by c-jun and c-fos) promotes the transcription of the GADD153 gene, which is also involved in transcriptional events related to the stress response as a CCAAT/enhancer binding protein.21,22,24 A subgroup of MAPkinases (named stress-activated protein (SAP) kinases) has been shown to phosphorylate c-jun in response to cellular stress and to be specific for the neuronal tissue.25 Once stressinduced transcription has been activated, several different groups ofp roteins have been shown to be involved in the mammalian stress response. Increases have been observed in antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidase, these enzymes being involved in the control of levels of ROIs at physiological and nondamaging conditions.17,22,24 On the other hand, heat-shock proteins, mainly HSP70, and ubiquitin-related genes are involved in protein homeostasis, including stabilization of protein structure and function and transport of protein across organellar membranes.17– 19,22 Finally, increases associated with the stress response also affect other proteins such as the p21 cell cycle inhibitor or the heme oxygenase 1.22,26 Going back to the role of the ubiquitin system in the cellular stress response, this can be analyzed from two different points of view. On the one hand, through degradation of abnormal proteins, where the UBi4 polyubiquitin, the UBC4 and UBC5 carrier enzymes (UbcH5A, UbcH5B and UbcH5C, and UbcH6 are the human homologs) have a key role.27,28 Alternatively, through its involvement in the stress response related-signal transduction pathways. Thus, two considerations can be made. First, the ubiquitin system has recently been shown to be involved in the metabolism of secreted APP(PN2),29 which has a neuroprotective activity against different types of stress conditions related to AD development.30 This activity, as already explained (see chapter 3), is mediated through the nerve growth factor receptor and the MAP-kinase signaling pathway.31 However, although c-jun and c-fos have been shown to be degraded by different proteolytic systems, their ubiquitin-dependent degradation 32–35 could constitute an interesting and promising area of research in relation to the activation of the stress response. Several different types of DNA repair have been shown to decline with age,36–39 and caloric restriction has been shown to slow down such age-related declines.36 Moreover, many studies have reported that ROIs are one of the main causes of DNA damage and probably of the DNA impairment associated with senescence.40–42 This effect of ROIs would be a consequence of an imbalance in the stress response, affecting signal transduction events, antioxidant enzymes and ubiquitin-related enzymes, among others. Taking this into consideration, it seems quite clear that both the stress response and DNA repair are of the utmost importance for the prevention of ageing. The involvement of the ubiquitin system in DNA repair is related to two proteins: p53 and UBC2 (also RAD6) ubiquitin carrier enzyme.43,44 The tumor suppressor protein p53 senses DNA damage through specific proteins, inducing cell cycle arrest through the p21 protein, and it also participates in activating the DNA repair mechanisms.45 Its activity in senescent cells has been suggested to be involved in the slow proliferative rate, this being consistent with the already mentioned age-related DNA damage accumulation.1 Although the involvement of the ubiquitin system in the degradation of p53 has been widely studied, more studies concerning the cellular signals involved in the control of such ubiquitin-dependent degradation are needed in order to obtain a better understanding of the ageing process and possibly also cancer development (Fig. 7.3). Another way in which the ubiquitin system is involved in DNA repair is related to the action of the UBC2 ubiquitin carrier enzyme (also RAD6).44 Its activity is closely related to that of proteins that recognize DNA damage. However, little is known about the role of the ubiquitin system in DNA repair. It would thus be very interesting to find out whether the

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Fig.7.3. Hypothetical link between repair mechanisms and different pathological states. It is becoming increasingly evident that defects in DNA repair or in the stress response can result in the appearance of tumors, neurodegenerative pathologies and are also inevitably associated with the ageing process.

ubiquitin system is involved in the activation of the p53 protein. At present two human homologs of the RAD6 gene, named HHR6A and HHR6B, have been described.46 It is interesting to note that decreased activity of isopeptidases in the brain stem and medula oblongata of aged mice has recently been reported.47 Such activity was measured in brain homogenates as deubiquitination of endogenous ubiquitin-protein conjugates. In addition, an alteration of a ubiquitin C-terminal hydrolase in senescent fibroblasts has been described.48 Although the ubiquitin-carrier enzymes and the ubiquitin ligases are very important for giving specificity to the ubiquitin system, an important regulatory role has recently been suggested for the deubiquitinating enzym es known as peptidases and

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isopeptidases.49 Their relevance in disease has been widely supported by their involvement in cancer development.50 Taking all this into consideration, future research may concentrate on determining the involvement of such deubiquitinating enzymes in the ageingp rocess.

References 1. Dice JF. Cellular and molecular mechanisms of aging. Physiol Rev 1993; 73:149-159. 2. Phillips ME, Platt JE. The use of ubiquitin as a marker of thyroxine-induced apoptosis in cultured Rana catesbeiana tail tips. Gen Comp Endocrinol 1994; 95:409-415. 3. Haas AL, Baboshina O, Williams B et al. Coordinated induction of the ubiquitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle. J Biol Chem 1995; 270:9407-9412. 4. Wride MA, Sanders EJ. Potential roles for tumor necrosis factor-alpha during embryonic development. Anat Embryol 1995; 191:1-10. 5. H an dke C, Boyle C, Wettern M. Effects of agin g, abiotic an d biotic stress u pon ubiquitination in young barley plants. Angewandte Botanik 1993; 67:120-123. 6. Niedzwiecki A, Fleming JE. Heat shock induces changes in the expression and binding of ubiquitin in senescent Drosophila melanogaster. Develop Genetics 1993; 14:78-86. 7. Pan JX, Short SR, Goff SA et al. Ubiquitin pools, ubiquitin mRNA levels, and ubiquitin mediated proteolysis in aging human fibroblasts. Exp Gerontol 1993; 28:39-49. 8. Jahngen-Hodge J, Cyr D, Laxman E et al. Ubiquitin and ubiquitin conjugates in human lens. Exp Eye Res 1992; 55:897-902. 9. Heydari AR, Conrad CC, Richardson A. Expression of heat shock genes in hepatocytes is affected by age and food restriction in rats. J Nutr 1995; 125:410-418. 10. Kawarabayashi T, Shoji M, Yamaguchi H et al. Amyloid beta protein precursor accumulates in swollen neurites throughout rat brain with aging. Neurosci Lett 1993; 153, 73-76. 11. Molinari M, Milner J. p53 in complex with DNA is resistant to ubiquitin dependent proteolysis in the presence of HPV-16 E6. Oncogene 1995; 10:1849-1854. 12. Attaix D, Taillandier D, Temparis S et al. Regulation of ATP-ubiquitin dependent proteolysis in muscle wasting. Reprod Nutr Dev 1994; 34:583-597. 13. Dardevet D, Sornet C, Taillandier D et al. Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging. J Clin Invest 1995; 96:2113-2119. 14. Carlin C, Phillips P, Knowles B et al. Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts. Nature 1983; 306:617-620. 15. Harley CB, Goldstein S, Posner B. Decreased sensitivity of old and progeric human fibroblasts to a preparation of factors with insulin like activities. J Clin Invest 1981; 68:988-994. 16. Plisko A, Gilchrest B. Growth factor responsiveness of cultured human fibroblasts. J. Gerontol. 1983; 38:513-518. 17. Liu AYC, Lee YK, Manalo D et al. Attenuated heat shock transcriptional response in aging: Molecular mechanism and implication in the biology of aging. In: Feige U, Morimoto RI, Yahara I, Polla B, eds. Stress-Inducible Cellular Responses. Basel: Birkhäuser Verlag, 1996:393-408. 18. Schlesinger MJ. How the cell copes with stress and the function of heat shock proteins. Pediatr Res 1994; 36:1-6. 19. Mager WH, Moradas-Ferreira P. Stress response of yeast. Biochem J 1993; 290:1-13. 20. Guyton KZ, Liu Y, Gorospe M et al. Activation of mitogen-activated protein kinase by H 2O 2. Role in cell survival following oxidant injury. J Biol Chem 1996; 271:4138-4142. 21. Guyton KZ, Xu Q, Holbrook NJ. Induction of the mammalian stress response gene GADD153 by oxidative stress: Role of AP-1 element. Biochem J 1996; 314:547-554. 22. Guyton KZ, Spitz DR, Holbrook NJ. Expression of stress response genes GADD153, c-jun and heme oxygenase-1 in H 2O 2- and O 2-resistant fibroblasts. Free Radical Biol Med 1996; 20:735-741.

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23. Papaconstantinou J. Unifying model of the programmed (intrinsic) and stochastic (extrinsic) theories of aging. The stress response genes, signal transduction-redox pathways and aging. Ann NY Acad Sci 1994; 719:195-211. 24. Choi AM, Sylvester S, Otterbein L et al. Molecular responses to hyperoxia in vivo: Relationship to increased tolerance in aged rats. Am J Respir Cell Mol Biol 1995; 13:74-82. 25. Martin JH, Mohit AA, Miller CA. Developmental expression in the mouse nervous system of the p493F12 SAP kinase. Brain Res Mol Brain Res 1996; 35:47-57. 26. Liu Y, Martindale JL, Gorospe M et al. Regulation of p21WAF1/CIP1 expression through mitogen-activated protein kinase signaling pathway. Cancer Res 1996; 56:31-35. 27. Arnason T, Ellison MJ. Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Mol Cell Biol 1994; 14:7876-7883. 28. Jensen JP, Bates PW, Yang M et al. Identification of a family of closely related human ubiquitin conjugating enzymes. J Biol Chem 1995; 270:30408-30414. 29. Gregori L, Bhasin R, Goldgaber D. Ubiquitin-mediated degradative pathway degrades the extracellular but not the intracellular form of amyloid beta-protein precursor. Biochem Biophys Res Commun 1994; 203:1731-1738. 30. Mattson MP. Degenerative and protective signaling mechanisms in the neurofibrillary pathology of AD. Neurobiol Aging 1995; 16:447-463. 31. Van Nostrand WE, Schmaier AH, Farrow JS et al. Protease nexin-II (amyloid β-protein precursor): A platelet α-granule protein. Science 1990; 248:745-748. 32. Hermida-Matsumoto ML, Chock PB, Curran T et al. Ubiquitinylation of transcription factors c-Jun and c-Fos using reconstituted ubiquitinylating enzymes. J Biol Chem 1996; 271:4930-4936. 33. Stancovski I, Gonen H, Orian A et al. Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: Identification and characterization of the conjugating enzymes. Mol Cell Biol 1995; 15:7106-7116. 34. Tsurumi C, Ishida N, Tamura T et al. Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases. Mol Cell Biol 1995; 15:5682-5687. 35. Treier M, Staszewski LM, Bohmann D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 1994; 78:787-798. 36. Wachsman JT. The beneficial effects of dietary restriction: Reduced oxidative damage and enhanced apoptosis. Mutat Res 1996; 350:25-34. 37. Barnett YA, King CM. An investigation of antioxidant status, DNA repair capacity and mutation as a function of age in humans. Mutat Res 1995; 338:115-128. 38. Weirich-Schwaiger H, Weirich HG, Gruber B et al. Correlation between senescence and DNA repair in cells from young and old individuals and in premature aging syndromes. Mutat Res 1994; 316:37-48. 39. Kruk PA, Rampino MJ, Bohr VA. DNA damage and repair in telomeres: Relation to aging. Proc Natl Acad Sci USA 1995; 92:258-262. 40. Adams JD, Mukherjee SK, Klaidman LK et al. Apoptosis and oxidative stress in the aging brain. Ann NY Acad Sci 1996; 786:135-151. 41. Driggers WJ, Grishko VI, LeDoux SP et al. Defective repair of oxidative damage in the mitochondrial DNA of a xeroderma pigmentosum group A cell line. Cancer Res 1996; 56:1262-1266. 42. Jaruga P, Dizdaroglu M. Repair of products of oxidative DNA base damage in human cells. Nucleic Acid Res 1996; 24:1389-1394. 43. Shen Z, Pardington-Purtymun PE, Comeaux JC et al. Associations of UBE21 with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system. Genomics 1996; 37:183-186. 44. Lawrence C. The RAD6 DNA repair pathway in Saccharomyces cerevisiae: What does it do, and how does it do it. Bioessays 1994; 16:253-258. 45. Enoch T, Norbury C. Cellular responses to DNA damage: Cell-cycle checkpoints, apoptosis and the roles of p53 and ATM. Trends Biochem. Sci. 1995; 20:426-430. 46. Koken MH, Hoogerbrugge JW, Jasper-Dekker I et al. Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification. Dev Biol 1996; 10:119-132.

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47. Ohtsuka H, Takahashi R, Goto S. Age-related accumulation of high-molecular-weight ubiquitin protein conjugates in mouse brains. J Gerontol 1995; 50:B277-B281. 48. DiPaolo BR, Pignolo RJ, Cristofalo VJ. Identification of proteins differentially expressed in quiescent and proliferatively senescent fibroblast cultures. Exp Cell Res 1995; 220:178-185. 49. H ochstrasser M. Ubiquitin -depen den t protein degradation . An n u Rev Gen et 1996; 30:405-439. 50. Isaksson A, Musti AM, Bohmann D. Ubiquitin in signal transduction and cell transformation. Biochim Biophys Acta 1996; 1288:F21-F29.

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CHAPTER 8

The Role of Other Proteolytic Systems in Disease I

t is by no means the aim of this book to suggest that the ubiquitin system is the only proteolytic system which is involved in disease. Other proteolytic systems may have key roles in some diseases and even more than one system may be involved in a particular pathological state. The aim of this chapter is to describe the possible involvement of both the lysosomal and nonlysosomal proteolytic systems and to relate them to the ubiquitin-dependent proteolytic system.

Lysosomal: Cathepsins There are many human lysosomal storage diseases (both inherited and acquired) that share a partial or complete deficiency of lysosomal enzymes with the resulting accumulation of undegraded materials within the lysosomes. Although they constitute an interesting field of medical research, we will focus here on those pathological states that may be related to defects in the lysosomal proteolytic machinery. A very clear role of cathepsins is that linked with tumor invasion. The malignancy of a tumor is associated with its ability to metastasize and, therefore, invade cellular types other than the primary tumor. In order to be able to metastasize, tumor cells must be able to abandon the primary tumor and reach either the blood or the lymph. This is accomplished by the release of proteolytic enzymes that degrade the extracellular matrix, thus allowing tumor cells to expand. Among the proteolytic enzymes released, cathepsins seem to play a very important role. Sloane et al1 have proposed that malignant tumor cells are capable of establishing an acidic extracellular microenvironment in which a variety of lysososmal enzymes and glycosidases function optimally and are able to destroy the basement membrane and connective tissues, thus facilitating invasion. Secretion of cathepsin B by malignant and benign tumors has drawn a great deal of attention because the secretion of this cathepsin correlates with the metastatic potential of some experimental2 and human 3 tumors. Increased levels of cathepsin B have been found in many tumors. The alterations in cathepsin B in malignant tumors seem to be associated with changes in the synthesis, processing, subcellular localization, secretion and regulation of endogenous enzyme inhibitors.1 Indeed, cathepsin B activity is regulated by a cystatin. Cystatins have been implicated in the regulation of tumor growth and progression 4–6 and in many tumors the regulation of cathepsin activity by cystatins is decreased because of decreases in either the levels or the affinity of these compounds for the lysosomal enzymes.5,7 Altogether, the balance between abnormal synthesis of cathepsin B and cystatin levels or affinity is responsible for the expression of the malignant phenotype. Secretion of an abnormal form of cathepsin L by tumor cells has been reported by Yamaguchi and Kawai.8 Interestingly, in nontransformed cells in culture tumor promoters (such as phorbol esters) 9 and growth factors (such as platelet-derived growth factor Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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(PDGF)) 10 induce increased synthesis of procathepsin L. In addition, the levels of expression of cathepsin L in H-ras-transformed murine fibroblasts is closely associated with their metastatic potential.11 Cathepsin D has been one of the more studied proteases in relation to the metastatic ability of cancer cells because of its involvement in breast cancer. Several pieces of evidence indicate the importance of upregulated biosynthesis and increased secretion of cathepsin D in tumor proliferation and invasion.12 Clinical investigations indicate that the level of cathepsin D in primary breast cancer is correlated with recurrence and metastasis and may be one of the best prognostic markers.13,14 In other types of tumors, cathepsin D has also proven to be involved in malignancy. For example, immunohistochemical studies of gastrointestinal tumors have shown that, while in dysplastic epithelium and well-differentiated (poorly invasive) carcinomas there was no positive staining for cathepsin D, a strong and diffuse staining pattern for cathepsin D was found in the more invasive, less differentiated carcinomas.15 Garcia et al16 have provided direct evidence of the association between overexpression of cathepsin D and the increase in metastatic capacity by transfecting an adenovirus-transformed cell line that did not secrete procathepsin with a human cathepsin D cDNA vector. Transfected clones showed acceleration of cellular growth, overgrowth at high cell densities, and greater anchorage-independent growth. In addition, intravenous inoculation of transfected clones into athymic mice resulted in markedly higher liver metastasis, in comparison to the nontransfected clones.16 In summary, the balance between the levels of proteinases and the levels of their inhibitors are critical determinants in tumor growth and invasion. Interestingly, in spite of having a normal or even increased lysosomal content (together with increases in lysosomal enzymes, as stated below), neoplastic cells show lower basal protein degradation in comparison to normal cells.17 To some extent, tumor cells are more resistant to stimuli that accelerate protein degradation and may also downregulate basal proteolysis. This could be accomplished by a decrease in acidification in the lysosomes of tumor cells that would, to some extent, decrease the proteolytic capacity, as hypothesized by Anderson et al.18 The lysososmal membrane provides a physical barrier separating the degradative activity of lysosomal enzymes from the cytoplasm. Impairment of lysosomal membrane integrity and release of enzymes into the cytosol are very detrimental to cellular physiological functions and integrity. Decker et al19 suggested that an intracellular leakage of lysosomal enzymes may mediate ischemic injury in the myocardium. In accordance with this suggestion, the administration of a cysteine proteinase inhibitor decreases the degradation of cytoskeletal proteins in ischemic myocardium of dogs.20 Lysosomal proteases released from inflammatory cells degrade critical extracellular proteins and may induce injury and loss of function in various organs. For instance, increased cathepsins B and L activities play a role in the degradation of cartilage collagens in arthritis, in tenotomized muscle and in chronic periodontitis.21–23 In arthritis there is destruction of articular connective tissue linked to an inflammatory response associated with proliferating synovial cells,24 suggesting that enzymes released from these cells may have a critical role in the degradation of the extracellular matrix of cartilage and bone. Lysosomes have a key role in defending the body against microorganisms. Indeed, phagocytosis is the major route for the uptake of bacteria, fungi and protozoans in lysosomes. It starts when the IgG-opsonized microorganisms bind to specific receptors in an Fc receptor-enriched segment of plasma membrane. The cell surface gradually surrounds the particle to be ingested, and the membrane sheets fuse with each other and generate a sealed intracellular vacuole, the phagosome. This is later acidified through an ATP-dependent proton pump normally derived from the cell membrane where the phagosome originated. Later on, phagosomes fuse with lysosomes and the enzymatic machinery of these organelles takes

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care of the invading microorganism. In some cases, there may be failure of the antimicrobial defense system due to either the escape of microorganisms from endocytic compartments, a failure in the fusion mechanism between phagosomes and lysosomes, deficient lysosomal acidification or a reduction in the enzymatic machinery (lysosomal proteases and other enzymes) that actually degrades the microorganisms. In macrophages, for example, bacterial lipopolysaccharide (LPS) reduces the activity of lysosomal enzymes by nearly half by decreasing enzyme biosynthesis. As mentioned previously (see chapter 5), skeletal muscle protein degradation is stimulated during infection. Although we now have a lot of evidence concerning the role of the ubiquitin-dependent proteolytic system in this catabolic process, other authors have described a role for the lysosomal machinery in this process. For example, Fagan et al25 hypothesized that the enhanced protein degradation occurs through the macroautophagic pathway and that calcium probably exerts an effect in the autophagic sequestration and fusion. In other situations where there is a loss of skeletal muscle protein such as trauma or denervation, activation of cathepsins may also occur. Odessey26 reported that burn injury caused elevations in the activity of cathepsins B, D and H, suggesting that lysosomal proteolysis may modulate muscle proteolysis during trauma. Bird and Roisen 27 described an elevation in the activity of lysosomal proteinases during muscle atrophy induced by denervation, suggesting that an increase in autophagic lysosomal proteolysis may be partially responsible for the atrophy. Similarly, Tischler et al28 showed that denervation atrophy is primarily caused by an enhanced lysosomal pathway, possibly due to greater lysosomal permeability. Turner et al29 proposed that both lysosomal cathepsins and Ca2+ -dependent proteases play a role in the enhanced protein degradation. Stimulation of autophagy and/or increased levels of cathepsins have been found in various myopathies such as Duchenne muscular dystrophy30 and sex-linked hereditary myopathy.31 In cardiomyopathy caused by hereditary muscular dystrophy in rodents, an elevated thiol protease activity has also been described.32 Similarly, Warner and Ryan 33 reported that in cardiomyopathy caused by hereditary muscular dystrophy, an imbalance in the ratio of activity of thiol protease to that of the inhibitor occurs. It has to be pointed out, however, that, at least in vitro, the lysosomal pathway appears to play a secondary role in the degradation of normal skeletal muscle proteins.34 In addition, the involvement of other proteolytic systems seems fairly clear (see chapter 5). Defects in lysosomal function are also related to the pathogenesis of atherosclerosis. In this process, alterations in cholesterol metabolism seem to play a key role. Cholesterol can either be synthetized de novo in most cell types or taken up from the circulation in the form of plasma lipoproteins. The uptake of circulating low density lipoproteins (LDL) by tissues is modulated by receptor-mediated endocytosis and lysosomal degradation of LDL.35 The degradation of this plasma lipoprotein fraction involves the breakdown of the outer protein coat, cholesteryl esters being hydrolyzed by lysosomal lipases to free cholesterol.35 It is precisely a rise in the concentration of free cholesterol (either derived from LDL or endogenously synthesized) that results in the inhibition of cholesterol biosynthesis at the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) downregulation of the LDL receptors on the cellular surface and activation of a microsomal acyl-CoA cholesterol acyl-transferase (ACAT), that facilitates the reesterification of cholesterol. Defects in the endocytosis of LDL or in the enzymatic machinery described above can lead to an abnormal accumulation of cholesterol. It is interesting to note that there is an accumulation of cholesterol in the lysosomes of atherosclerotic lesions.36 Indeed, in atherosclerotic lesions, the arterial intima is infiltrated with either the so-called “foam cells”, macrophages that have taken up a considerable amount of lipid through LDL uptake, or smooth muscle cells from the arterial media. Initially, the lipid is cytosolically localized but as the disease progresses, intracellular lipid deposits become massive and the site of accumulation shifts

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to the lysosomes.36 The atherosclerotic process is favored by the oxidation of LDL, since the generation of foam cells is induced by this modified type of lipoprotein. It has been reported that an impairment in the ability of lysosomal cathepsins to degrade the oxidized LDL may be responsible for the massive generation of foam cells during the atherosclerotic process. Concerning the involvement of lysosomal cathepsins in neurodegenerative diseases, several facts are interesting to note. First, immunochemical studies of lysosomal proteases in brains of Alzheimer’s disease (AD) patients have shown that cathepsins can be identified in senile plaques and in degenerating neurons.37,38 Indeed, cathepsin D expression is very elevated in astrocytes within senile plaques.39 Second, amyloid precursor protein (APP) is present in lysosomes of normal neurons and becomes more abundant in degenerating neuronal components in senile plaques.40 Therefore, a role of lysosomes in the proteolytic processing of APP cannot be discarded. Third, there are several pieces of evidence indicating that lysosomes may have a role in the development of prion-related encephalopathies. Thus, gold particles corresponding to a lysosomal marker enzyme (beta-glucuronidase) are seen in lysosome-related dense bodies adjacent to large spongiform lesions in areas of spongiosis.41 In addition, gold particles specific for hsc 70 (involved in the direct transport of some proteins into lysosomes of stressed cells) can be seen in dense bodies beside small areas of rarefaction and also associated with debris in large spongiform areas.41 Therefore, release of proteins from lysosomes may lead to the generation of spongiform lesions in the prionrelated encephalopathies. The reason for the lysosomal disruption in scrapie-infected brain may be the accumulation of the abnormal isoform of the prion protein.42

Nonlysosomal: Calpains The involvement of the calpain system in pathological states is of particular clinical interest since further research may lead to eventual therapeutic implications. In addition, the involvement of calpain in disease allows approaches with which to analyze the physiological function of the calpain system. A fairly clear relationship has been established between ischemic injury and activation of the calpain system. In the rabbit, in pyramidal and granular cells of the dorsal hippocampus (a region susceptible to ischemic injury) there is positive calpain system immunostaining, and proteolysis can easily be induced by hypoxia. In those regions that are less susceptible to ischemia, there is increased immunostaining for calpastatin.43,44 It may thus be proposed that calpain inhibitors decrease neuronal injury caused by global ischemia in the central nervous system. Similarly, the result of blocking calpain-dependent proteolysis (with leupeptin, for instance) is decreased neuronal death in the hippocampus.45 In addition, hypoxia activates cytoskeletal protein degradation by calpain activation during transient brain ischaemia.45 In neurosurgery, basilar artery vasospasm is accompanied by calpain activation 46 in addition to the transient forebrain ischaemia-induced neuronal degeneration. The induction of vasospasm results in continuous autolytic activation of µ-calpain as well as reduction in calpastatin activity. The involvement of both protein kinase C and cytoskeletal proteins in this process seems fairly clear. The calpain system has also been reported to be activated in brain trauma and in the development of amyloid plaques in AD.47 The calpain system may also be involved in cardiac ischemia and could therefore be related to myocardial injury. It appears that, as a result of hypoxia, the increased intracellular calcium mobilization activates calpain-mediated proteolysis.48 In hypoxic cardiomyocytes calpain activity is increased several-fold,48,49 this contributing, no doubt, to the cell damage following the ischemic period. The increase in calpain-mediated proteolysis following cardiac hypoxia can be prevented either by the use of inhibitors or by calcium antagonists. Other types of cardiomyocyte pathologies also seem to involve activation of the calpain

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system. Studies carried out in rodent cardiomyopathy or hypertension-induced cardiac hypertrophy have also revealed increased levels of calpain-mediated proteolysis.50,51 In addition, rats subjected to hypothermia or starvation are affected by an increase in calpain activity in the myocardium.52 It can thus be suggested that many pathological conditions involving heart damage are associated with an abnormal performance of the calpain system. In patients with essential hypertension, erythrocyte calpastatin levels are decreased, probably causing unregulated degradation of cell membrane proteins and cytoskeleton.53–55 Following the same lines, antihypertensive therapy restores calpastatin to normal values.56 Concerning skeletal muscle, activation of the calpain system has been reported during ageing,57 diabetic amyotrophy,58 sepsis,59 denervation 60 and muscular dystrophies.29 In fact, in dystrophic hamster muscles there are very low levels of calpastatin, probably as a result of enhanced breakdown.38 Interestingly, Badalamente et al61 have reported that the application of leupeptin (an inhibitor of the calpain system) results in an improvement in the neuromuscular recovery process. Antibodies against calpastatin have been identified in the serum of rheumatoid arthritis patients.62 Interestingly, Yamamoto et al described a Ca2+ -dependent proteinase in human arthritic synovial joints.63 Sasaki et al64 also suggested the involvement of the calpain system in many inflammatory processes. These observations could be related to the role that calpain plays in the processing and secretion of interleukin-1 (IL-1), an important inflammatory mediator.65 It has been suggested that IL-1 plays a role in the development of rheumatoid arthritis.66 Although it is still not very clear how the calpain-catalyzed cleavage of the IL-1 precursor protein takes place, it is likely to be one of the rate-limiting steps controlling the plasma cytokine levels. In addition, the IL-1 precursor does not possess a signal peptide sequence, therefore it may represent a secretory pathway distinct from those involving signal peptidases. Interestingly, calpain inhibitors have been reported to have a positive effect in the prevention of the invasion of human erythrocytes by the parasite Plasmodium falciparum and, therefore, the calpain system may also be involved in the development of malaria.67 The calpain system also seems to be involved in the degradation of abnormal cytosolic and membrane proteins in red blood cells damaged by phenylhydrazine.68 Platelets from individuals with the Montreal platelet syndrome (a defect in platelet size) show lower calpain activity.69 An abnormal activity of the calpain system has also been observed in the WiskottAldrich 70,71 and in the Chediak-Higashi72 syndromes. Limited proteolysis of crystallins by abnormally activated calpain is likely to be associated with lens protein insolubilization (and thus cataract formation) during ageing.73,74

Interaction Between the Ubiquitin System and Others Although it was originally thought that the ubiquitin system was regulated completely independently from the calpain system and from the lysosomal proteolytic enzymes, a considerable amount of evidence points to important interactions between the different proteolytic systems, both in healthy and diseased states. Ciechanover et al75 have shown that the ubiquitin and the lysosomal systems are linked to each other: stress-induced degradation of cellular proteins within lysososmes is dependent upon the activity of the ubiquitin-activating enzyme E1. Using mouse ts85 mutant cells (which possess a thermolabile mutated E1 and which, after inactivation, are unable to degrade short-lived proteins), they have shown that when cells are shifted to the restrictive temperature, there is no change in the rate of degradation of long-lived proteins. In contrast, shifting the wild-type FM3A cells to the high temperature is accompanied by a two- to three-fold increase in the rate of proteolysis in this group of proteins. The heat-induced accelerated degradation can be com pletely inhibited by the lysosom otropic agents

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Table 8.1. Pathological involvement of the non-ubiquitin-dependent proteolytic systems DISEASE

Tumor invasion Hypertension Myocardial injury Brain ischemia Alzheimer’s disease Neurodegenerative disorders Aging Cataract formation Muscular dystrophies Diabetes Sepsis Atherosclerosis Arthritis Malaria Wiskott-Aldrich syndrome Montreal platelet syndrome Chediak-Higashi syndrome

References Ca2+-dependent

Lysosomal

— 53 48 45 47 — 57 74 29 58 59 — 62 67 71 69 72

1 — 33 — 38 41 37 — 30 — 25 36 24 — — — —

ammonium chloride and chloroquine. A functional ubiquitin system may be required for the formation and maturation of autophagic vacuoles or for their conversion into residual bodies. Alternatively, conjugated proteins may be digested preferentially by the lysosomal proteases; as conjugation is inhibited in the mutant cells at the restrictive temperature, lysosomes may be loaded with proteins that cannot be proteolyzed efficiently.75 Therefore, ubiquitinization of some proteins is a prerequisite or signal for their uptake into the autophagic vacuoles or other lysosomal compartments. It has been postulated that some nonlysosomal pathways may function by partially processing native proteins and by targeting them for final degradation into the lysosomes. Supporting this, ubiquitinized proteins accumulate in the lysosomes of both fibroblasts and hepatoma cells.76–78 Conversely, evidence of incomplete degradation of protein by the lysosomes has also been presented. Botbol and Scornik found that specific di- and tripeptidic proteolytic intermediates distributed in particulate and cytosolic subcellular fractions originated mainly from autophagic vacuoles, suggesting that oligopeptides derived from the intralysosomal degradation of proteins translocate from this compartment into the cytosol where the complete degradation of the oligopeptides is achieved.79 As presented in Table 8.1, there is a clear overlap in the involvement of the different proteolytic systems in different pathological states. A paradigm of this is found in neurodegenerative diseases, in particular AD. Ubiquitin-protein deposits have been immunohistochemically shown in association with neurofibrillary tangles in AD (see chapter 3). The calpain system has also been reported to be activated in the development of amyloid plaques in AD.47 Immunochemical studies of lysosomal proteases in brains of AD

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patients have shown that cathepsins can be identified in senile plaques and in degenerating neurons.37,38 In addition, lysosomes may have a role in the development of prion-related encephalopathies. Thus, gold particles corresponding to a lysosomal marker enzyme (betaglucuronidase) are seen in lysosome-related dense bodies adjacent to large spongiform lesions in areas of spongiosis,41 suggesting that release of proteins from lysosomes may lead to the generation of spongiform lesions in the prion-related encephalopathies. It is therefore obvious that, in spite of the differences in subcellular localization, sensitivity to inhibitors, substrate specificity, optimum pH and participation in the degradation of short- or long-lived proteins, the different proteolytic systems share common physiological functions and, despite the intrinsic difficulties confronting protease researchers, a better understanding of many pathophysiological states could be accomplished by an integrated study.

References 1. Sloane BF, Moin K, Krepela E et al. Cathepsin B and its endogenous inhibitors. The role in tumor malignancy. Cancer Metast Rev 1990; 9:333-352. 2. Sloane BF, Honn KV, Sadler JG et al. Cathepsin B activity in B-16 melanoma cells: A possible marker for metastatic potential. Cancer Res 1982; 42:980-986. 3. Murnane MJ, Sheahan K, Ozdemirli M et al. Stage-specific increases in cathepsin B mRNA content in human colorectal carcinoma. Cancer Res 1991; 51:1137-1142. 4. Lah TT, Buck MR, Honn KV et al. Degradation of laminin by human tumor cathepsin B. Clin Exp Metast 1989; 7:461-468. 5. Lah TT. Clifford JL, Helmer KM et al. Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma. Biochim Biophys Acta 1989; 993:63-73. 6. Rozhin J, Gomez AP, Ziegler GH et al. Cathepsin B to cysteine proteinase inhibitor balance in metastatic cell subpopulations isolated from murine tumors. Cancer Res 1990; 50:6278-6284. 7. Jarvinen M, Rinne A, Hopsu-Havu VK. Human cystatins in normal and diseased tissues: A review. Acta Histochem 1987; 82:5-18. 8. Yamaguchi N, Kawai K. Acid protease secreted from human pancreatic carcinoma cell line HPC-YT into serum-free, chemically-defined medium. Cancer Res 1986; 46:5353-5359. 9. Gal S, Willingham MC, Gottesman MM. Processing and lysosomal localization of a glycoprotein whose secretion is transformation-stimulated. J Cell Biol 1985; 100:535-544. 10. Hiwasa T, Sawada T, Tanaka K et al. Colocalization of ras gene products and cathepsin L in cytoplasmic vesicles in v-Ha-ras-transformed NIH3T3 mouse fibroblasts. Biomed Biochim Acta 1991; 50:576-585. 11. Denhardt DT, Greenberg AH, Egan SE et al. Cysteinase proteinase cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts. Oncogene 1987; 2:55-59. 12. Rochefort H, Capony F, Garcia M. Cathepsin D in breast cancer: From molecular and cellular biology to clinical applications. Cancer Cells 1990; 2:383-388. 13. Brouillet JP, Theillet C, Maudelonde T et al. Cathepsin D assay in primary breast cancer and lymph nodes. Relationship with c-myc, c-erbB2 and int-2 oncogene amplification and node invasiveness. Eur J Cancer 1990; 26:437-441. 14. Granata G, Coradini D, Cappelletti V et al. Prognostic relevance of cathepsin D versus estrogen receptors in node negative breast cancers. Eur J Cancer 1991; 27:970-972. 15. Saku T, Sakai H, Tsuda N et al. Cathepsin D and cathepsin E in normal, metaplastic, dysplastic, and carcinomatous gastric tissue. An immunohistochemical study. Gut 1990; 31:1250-1255. 16. Garcia M, Derocq D, Pujol P et al. Overexpression of transfected cathepsin D in transformed cells increasese their malignant phenotype and metastatic potency. Oncogene 1990; 5:1809-1814.

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17. Lee HK, Jones RT, Myers RA et al. Regulation of protein degradation in normal and transform ed hu m an bron chial epithelial cells in cu ltu re. Arch Biochem Biophys 1992; 296:271-278. 18. Andersson GN, Torndal UB, Eriksson LC. Decreased vacuolar acidification capacity in drugresistent rat liver preneoplastic nodules. Cancer Res 1989; 49:3765-3769. 19. Decker RS, Poole AR, Crie JS et al. Lysosomal alterations in hypoxic and reoxygenated hearts. 2. Immunohistochemical and biochemical changes in cathepsin D. Am J Pathol 1980; 98:445-446. 20. Tsuchida K, Aihara M, Isogai K et al. Degradation of myocardial structural proteins in myocardial infarcted dogs is reduced by EP459, a cysteine proteinase inhibitor. Biol Chem Hoppe Seyler 1986; 367:39-45. 21. Harris CI, Baillie AGS. The localized elevation of cathepsins B and L in rat gastrocnemius muscle following tenotomy. Biochem Soc Trans 1990; 18:1254-1255. 22. Eley BM, Cox SW. Cathepsin B-like and L-like activities at local gingival sites of chronic periodontitis patients. J Clin Periodontol 1991; 18:499-504. 23. Maciewicz RA, Wotton SF. Degradation of cartilage matrix components by the cysteine proteinases cathepsins B and cathepsins L. Biomed Biochim Acta 1991; 50:561-564. 24. Tanaka A, O’Sullivan FX, Koopman WJ et al. Etiopathogenesis of rheumatoid arthritis-like disease in MRL/1 mice. 2. Ultrastructural basis of joint destruction. J Rheumatol 1988; 15:10-16. 25. Fagan JM, Baracos V, Goldberg AL. In: Glaumann H, Ballard FJ, eds: Lysosomes: Their Role in Protein Breakdown. London: Academic Press, 1987:659-678. 26. Odessey R. Regulation of lysosomal proteolysis in burn injury. Metabolism 1987; 36:670-676. 27. Bird JWC, Roisen FJ. In: Engel A, Banker B, eds: Myology, vol. 1. New York: McGraw Hill, 1986:745-768. 28. Tischler ME, Rosenberg S, Satarug S et al. Different mechanisms of increased proteolysis in atrophy induced by denervation of unweighting of rat soleus muscle. Metabolism 1990; 39:756-763. 29. Turner PR, Westwood T, Regen CM et al. Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature 1988; 335:735-738. 30. Kominami EH, Kunio II, Katunuma N. Activation of the intramyofibral autophagic-lysosomal system in muscular dystrophy. Am J Pathol 1987; 127:461-466. 31. Kalimo H, Savontaus ML, Lang H et al. X-linked myopathy with excessive autophagy. A new hereditary muscle disease. Ann Neurol 1988; 23:258-265. 32. Gopalan P, Dufresne MJ, Warner AH. Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice. Can J Physiol Pharmacol 1987; 65:124-129. 33. Warner AH, Ryan MA. Thiol protease-thiol protease inhibitor imbalance in cardiac tissue of aging cardiomyopathic hamsters. J Mol Cell Cardiol 1990; 22:577-586. 34. Zeman RJ, Kameyama T, Matsumoto K et al. Regulation of protein degradation in muscle by calcium. Evidence for enhanced nonlysosomal proteolysis associated with elevated cytosolic calcium. J Biol Chem 1985; 260:13619-13624. 35. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986; 232:34-47. 36. Jerome WG, Minor LK, Glick JM et al. Lysosomal lipid accumulation in vascular smooth muscle cells. Exp Mol Pathol 1991; 54:144-158. 37. Cataldo AM, Nixon RA. Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain. Proc Natl Acad Sci USA 1990; 87:3861-3865. 38. Nakamura Y, Takeda M, Suzuki H et al. Abnormal distribution of cathepsins in the brain of patients with Alzheimer’s disease. Neurosci Lett 1991; 130:195-198. 39. Diedrich JF, Minnigan H , Carp RI et al. Neuropathological changes in scrapie and Alzheimer’s disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes. J Virol 1991; 65:4759-4768. 40. Cole G, Masliah E, Huynh TV et al. An antiserum against amyloid-beta protein precursor detects a unique peptide in Alzheimer brain. Neurosci Lett 1989; 100:340-346.

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41. Laszlo L, Lowe J, Self T et al. Lysosomes are key organelles in the pathogenesis of prion encephalopathies. J Pathol 1992; 166:333-341. 42. Mayer RJ, Laszlo L, Middleton A et al. Ubiquitin, lysosomes and neurodegenerative diseases. Biochem Soc Trans 1992; 20:645-648. 43. Arai A, Vanderklish P, Kessler M et al. A brief period of hypoxia causes proteolysis of cytoskeletal proteins in hippocampal slices. Brain Res 1991; 555:276-280. 44. Fukuda T, Adachi E, Kawashima S et al. Immunohistochemical distribution of calciumactivated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus. J Comp Neurol 1990; 302:100-109. 45. Lee KS, Frank S, Vanderklish P et al. Inhibition of proteolysis protects hippocampal neurons from ischemia. Proc Natl Acad Sci USA 1991; 88:7233-7237. 46. Yamaura I, Tani E, Saido TC et al. Calpain-calpastatin system of canine basilary artery in vasospasm. J Neurosurg 1993; 79:537-543. 47. Saito K, Elce JS, Hamos JE et al. Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer’s disease: A potential molecular basis for neuronal degeneration. Proc Natl Acad Sci USA 1993; 90:2628-2632. 48. Iizuka K, Kawaguchi H, Yasuda H. Calpain is activated by β-adrenergic receptor stimulation under hypoxic myocardial cell injury. Jpn Circ J 1991; 55:1086-1093. 49. Toyo-Oka T, Morita M, Shin WS et al. Contribution of calcium-activated neutral protease to the degradation process in ischemic heart. Jpn Circ J 1991; 55:1124-1126. 50. Spalla M, Tsang W, Kuo TH et al. Purification and characterization of two distinct Ca2+ -activated proteinases from hearts of hypertensive rats. Biochim Biophys Acta 1985; 830:258-266. 51. Spalla M, Kuo TH, Wiener J. Calcium-activated proteinase in hamster cardiomyopathy. Muscle Nerve 1987; 10:54-59. 52. Tolnai S, von Althen I. Calcium-dependent proteolysis in the myocardium of rats subjected to stress. Life Sci 1987; 41:1117-1122. 53. Pontremoli S, Melloni E, Sparatore B et al. Erythrocyte deficiency in calpain inhibitor activity in essential hypertension. Hypertension 1988; 12:474-478. 54. Pontremoli S, Sparatore B, Salamino F et al. The role of calpain in the selective increased phosphorylation of the anion transport protein in red cell of hypertensive subjects. Biochem Biophys Res Commun 1988; 151:590-597. 55. Pontremoli S, Salamino F, Sparatore B et al. Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension. Biochem Biophys Res Commun 1988; 157:867-874. 56. Salamino F, Sparatore B, De Tullio R et al. The calpastatin defect in hypertension is possible due to a specific degradation by calpain. Biochim Biophys Acta 1991; 1096:265-269. 57. Johnson P, Hammer JL. Cardiac and skeletal muscle enzyme levels in hypertensive and aging rats. Comp Biochem Physiol B 1993; 104:63-67. 58. Kobayashi S, Fujihara M, Hoshino N et al. Diabetic state-induced activation of calciumactivated neutral proteinase in mouse skeletal muscle. Endocrinol Japon 1989; 36:833-844. 59. Bhattacharyya J, Thompson K, Sayeed MN. Calcium-dependent and calcium-independent protease activities in skeletal muscle during sepsis. Circ Shock 1991; 35:117-122. 60. Hussain H, Dudley GA, Johnson P. Effects of denervation on calpain and calpastatin in hamster skeletal muscles. Exp Neurol 1987; 97:635-643. 61. Badalamente MA, Hurst LC, Stracher A. Neuromuscular recovery using calcium protease inhibitor after median nerve repair in primates. Proc Natl Acad Sci USA 1989; 86:5983-5987. 62. Despres N, Talbot G, Plouffe B et al. Detection and expression of a cDNA clone that encodes a polypeptide containing inhibitory domains of human calpastatin and its recognition by rheumatoid arthritis sera. J Clin Invest 1995; 95:1891-1896. 63. Yamamoto S, Shimizu K, Shimizu K et al. Calcium-dependent cysteine proteinase (calpain) in human arthritic synovial joints. Arthritis Rheum 1992; 35:1309-1317. 64. Sasaki M, Kunimatsu M, Tada T et al. Calpain and kininogen mediated inflammation. Biomed Biochim Acta 1991; 50:499-508.

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65. Kobayashi Y, Yamamoto K, Saido TC et al. Identification of calcium-activated neutral protease as a processing enzyme of human interleukin 1α. Proc Natl Acad Sci USA 1990; 87:5548-5552. 66. Arend WP, Dayer JM. Inhibition of the production and effects of interleukin-1 and tumor necrosis factor- α in rheumatoid arthritis. Arth Rheum. 1995; 38:151-160. 67. Olaya P, Wasserman M. Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium falciparum. Biochim Biophys Acta 1991; 1096:217-222. 68. Mortensen AM, Novak RF. Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells. Toxicol Appl Pharmacol 1991; 110:435-449. 69. Okita JR, Frojmovic MM, Kristopeit S et al. Montreal platelet syndrome. A defect in calcium-activated neutral proteinase (calpain). Blood 1989; 74:715-721. 70. Remold-O’Donnell E, Van Brocklyn J, Kenney DM. Effect of platelet calpain on normal T-lymphocyte CD43:hypothesis of events in the Wiskott-Aldrich syndrome. Blood 1992; 79:1754-1762. 71. Kenney DM, Reid R, Parent DW et al. Evidence implicating calpain (Ca2+ -dependent neutral protease) in the destructive therbocytopenia of Wiskott-Aldrich syndrome. Br J Hematol 1994; 87:773-781. 72. Ito M, Sato A, Tanabe F et al. The thiol proteinase inhibitors improve the abnormal rapid down-regulation of protein kinase C and the impaired natural killer cell activity in (ChediakHigashi syndrome) beige mouse. Biochem Biophys Res Commun 1989; 160:433-440. 73. David LL, Shearer TR, Shih M. Sequence analysis of lens β-crystallins suggests involvement of calpain in cataract formation. J Biol Chem 1993; 268:1937-1940. 74. Iwasaki N, David LL, Shearer TR. Crystallin degradation and insolubilization in regions of young rat lens with calcium ionophore cataract. Invest Ophthalmol Vis Sci 1995; 36:502-509. 75. Ciechanover A, Dunn Jr WA, Schwartz AL. The ubiquitin-mediated proteolytic system: Studies on the degradation of N- α-acetylated proteins and on the linkage to the lysosomal proteolytic pathway. In: Bond JS, Barret AJ, eds: Proteolysis and Protein Turnover. London: Portland Press, 1993:89-96. 76. Schwartz AL, Ciechanover A, Brandt RA et al. Immunoelectron microscopic localization of ubiquitin in hepatoma cells. EMBO J 1988; 7:2961-2966. 77. Mayer RJ, Arnold J, László L et al. Ubiquitin in health and disease. Biochim Biophys Acta 1991; 1089:141-157. 78. Mayer RT, Lowe J, Landon M et al. Ubiquitin and the lysosome system: Molecular immunopathology reveals the connection. Biomed Biochim Acta 1991; 50:333-341. 79. Botbol V, Scornik OA. Measurement of instant rates of protein degradation in the livers of in tact m ice by the accum ulation of bestatin -in duced peptides. J Biol Chem 1991; 266:2151-2157.

CHAPTER 9

Cell Injury and Cell Death: Apoptosis Apoptosis and Disease lthough cell death can occur as a consequence of either ageing or accidental injury, it can also be the consequence of a genetically programmed active cell suicide, known as apoptosis. Necrosis and apoptosis are the two modes of cell death in living organisms. While necrosis is a pathological form of cell death and occurs during cell injury and repair, apoptosis is a normal and physiological form of cell death that occurs in all living organisms in their embryonic or adult development, thus eliminating unwanted, functionally abnormal or harmful cells. Although apoptosis in normal embryonic or adult development appears to be a programmed event, there is compelling evidence that shows the existence of external factors that trigger or control apoptosis. Some agents (such as cytokines and growth factors) are able to induce apoptosis and therefore alter the “programmed” suicide. In some cases, nonphysiological events, such as radiation, toxins and drugs have also been shown to induce apoptosis. At the mechanistic level, there are marked differences between the two types of cell death. In necrosis there is swelling of the cytoplasm and organelles as a result of a loss of selective permeability of the cell membrane (due to a decrease in ion-pumping activities or direct physical or chemical membrane damage). In the necrotic cell there is dissolution of the organelles, leaking of cellular contents into the extracellular space and disappearance of the cytoplasmic membrane. It affects groups of contiguous cells and elicits an inflammatory reaction in the adjacent viable tissues in response to the released cell debris. In apoptosis, coordinate changes take place in the nucleus, the cytoplasm and the cell surface. Organelles remain largely unaffected while there is a loss of cell volume and condensation of the nucleus associated with changes in chromatin which leads to dense granular caps under the intact nuclear membrane. Finally, the nucleus breaks up into several membrane-fragments, and the cell splits into multiple membrane-bound apoptotic bodies (some of which contain nuclear fragments) which are phagocytosed almost immediately by either neighbor cells or macrophages without generating an inflammatory reaction. The mechanism of apoptosis has long been neglected in clinical research. Nevertheless, programmed cell death offers an understanding of a number of pathological syndromes and clinical observations, which otherwise cannot be explained by well-known biological processes. Such pathological states relate to inflammation, cancer, neurodegenerative diseases, AIDS, autoimmune diseases, hematological diseases and ischaemic injury, among others. The significance and implications of apoptosis in these circumstances have only recently been recognized. In some cases the above mentioned pathologies are associated with an inhibition of the apoptotic process; in others there is an increase in the rate of apoptosis (Table 9.1).

A

Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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Table 9.1. Diseases associated with apoptosis Changes in: DISEASE

Apoptosis

ubiquitin conjugation

ubiquitin gene expression

Cancer Viral infections Autoimmune diseases Neurodegenerative disorders AIDS Ischemic injury Polycystic kidney disease Toxin-induced liver disease

Decreased Decreased Decreased Increased Increased Increased Increased Increased

YES (87,88) YES (89,90) YES (91) YES (92-95) YES (89) ? ? YES (96-100)

YES (101,102) YES (103,104) ? ? ? ? ? ?

Cancer Cancer researchers have increasingly become aware that the growth of a tumor is regulated not only by the rate at which tumor cells divide but more importantly by the rate at which they die. Indeed, genetic alterations that disregulate the physiological cell death process appear to contribute to the clonal expansion of malignant cells. From this point of view, triggering of apoptosis in cancer cells may turn out to be more important to cancer therapy and to limiting of tumor invasion than inhibition of proliferation with the use of conventional cancer treatments such as radiotherapy. Indeed, a number of oncogenes and tumor suppressor genes have been found to be involved in the control of apoptosis. Oncogenes that promote cell proliferation and those that inhibit cell death (such as p53, which in its mutated form is one of the most common oncogenic lesions detected in human tumors) 1 could cooperate to induce a neoplastic lesion. One of the best examples is the socalled apoptosis suppressor gene bcl-2 which was originally described at the site of translocations typical of follicular lymphoma.2 Due to these translocations the bcl-2 gene is situated adjacent to the Ig heavy chain locus on chromosome 14. Driven by the Ig heavy chain promotors, the bcl-2 gene is transcribed at a much higher level, blocking apoptosis and thus contributing to the development of the tumors. In cultured cells, upregulation of bcl-2 oncogene expression specifically inhibits apoptosis induced by a wide range of insults and stimuli such as growth factor deprivation, loss of contact with extracellular matrix, cytotoxic T cells, cytokines, chemotherapeutic drugs and radiation.3,4 The mechanism by which bcl-2 blocks apoptosis is unknown but it may be related to the fact that the gene product resides at the inner side of mitochondria, which remain intact for a large part of the apoptotic process. An elevated level and aberrant pattern of bcl-2 expression has been found in a wide variety of human tumors, including lymphomas, leukemias, adenocarcinomas, neuroblastomas, renal and lung cancers and melanomas.5 In most of these tumors, disregulation of bcl-2 gene expression is not the consequence of the previously mentioned translocation since structural alterations are not present in most leukemias or solid tumors. This suggests that trans-regulatory rather than cis-regulatory mechanisms account for bcl-2 overexpression. In fact, one of the potential trans-regulators of the bcl-2 gene is the protein of the p53 gene which functions primarily to suppress neoplastic growth by inducing apoptosis. In addition to repressing bcl-2 gene expression, p53 trans-activates the expression of bax, a homolog of bcl-2.6 In contrast to bcl-2, the protein encoded by bax functions as a promoter of cell death.

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In fact, both bax and bcl-2 are members of a larger family of proteins, including bcl-x, mcl-1, bak, bad and bag-1, which can interact through heterodimerization and finally control the balance between cell growth and cell death. But the behavior of bcl-2 in human cancer is much more complicated than can be explained by taking into account these transregulatory elements. For instance, bcl-2 protein appears to be virtually undetectable by immunohistochemistry in the majority of androgen-dependent human prostate cancers. In contrast, all androgen-independent cancers show bcl-2 positive suggesting that failure to undergo apoptosis is involved in the androgen-independency.7 In breast cancer, however, bcl-2 expression has been found to correlate with the expression of the estrogen and progesterone receptor, this finding suggesting a role for hormonal factors in the expression of bcl2. Therefore, the role of bcl-2 in human cancer is much more complex than can be explained by direct regulation of apoptosis. bcl-2 overexpression has been associated with resistance to cell death in response to chemotherapeutic drugs.8 This implies that chemotherapeutic drugs do not only kill cells by inflicting irreversible metabolic damage but through creating aberrations in cellular physiology that result in the activation of a cell death program in the target cell. Consistent with this hypothesis, overexpression of bcl-2 can result in a multidrug resistant phenotype in vitro. Other chemotherapeutic drugs are effective by initiating DNA damage. In fact, DNA damage may lead to upregulated p53 expression. The tumor suppressor protein recognizes the damage and blocks the cell cycle to allow repair or initiates apoptosis. Cells deficient in p53 may therefore become resistant to chemotherapy because they cannot undergo apoptosis.7 This may contribute to the development and/or progression of neoplasia.

Inflammation The inflammatory response to injury or infection has considerable potential to damage tissue and is therefore tightly regulated. Leukocytes, monocytes and macrophages are selectively eliminated from inflammatory tissues by apoptosis. One of the major cellular events in the onset of an inflammatory reaction is the migration of large numbers of neutrophils to the inflammatory site. Neutrophil granule contents amplify the immune response by enzymatic cleavage of matrix proteins.9 Removal of neutrophils from the inflammatory site involves apoptosis.10 Therefore removal of cells without release of granule contents is of paramount importance for the cessation of inflammation. In fact, for inflammatory tissues to return to normal all the events involved in the development of the inflammation must be reversed. These include removal of the initiating stimulus, cessation of neutrophil accumulation, cessation of further release of proinflammatory mediators, return of microvascular permeability to normal and cessation of monocyte emigration from blood vessels. Defects in these mechanisms or in the clearance of apoptotic cells may underly some chronic inflammatory diseases due to inappropriate persistence of inflammatory cells, and continued release of toxic cellular contents, and thus perpetuation of tissue injury and inflammation. In conclusion, activation of apoptosis in neutrophils could lead to limitation of tissue injury associated with inflammation and could be very useful for the treatment of chronic inflammatory diseases such as rheumatoid arthritis. Therefore the regulation of an apoptotic drug after an inflammatory process offers a new approach for promoting rapid healing and reduction of unwanted consequences of inflammatory processes.

Viral Infections and AIDS Cells that have been infected by a virus can undergo apoptosis as a defense mechanism to prevent viral propagation. Cytotoxic T cells are able to recognize viral peptides in combination with MHC class II molecules on the surface of infected cells.11 Cytotoxic T cells carry the Fas ligand on their plasma membrane and this triggers the fas receptor on the membrane

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of the infected cell, leading to apoptosis through the induction of the expression of intracellular proteases such as granzyme B.12 T cells can also induce apoptosis by using perforin to introduce proteases into the target cell.13 However, viruses have developed strategies which serve to prolong the life of their target cells and, therefore, promote virus replication. To reach this goal, viral genes sometimes encode inhibitory proteins, most of which target one of the two main checkpoints of the pathway leading to apoptosis. The Epstein-Barr virus BHRF1 protein is a bcl-2 homolog whilst the LMP-1 protein not only upregulates bcl-2 expression but also induces the A20 zinc finger protein that confers resistance to tumor necrosis factor- α (TNF) cytotoxicity.14,15 The cowpox virus gene crmA encodes a protease inhibitor that prevents apoptotic cell death by specifically inhibiting the interleukin-1- β converting enzyme (ICE), a key protease in the cytotoxic pathway associated with both fas and TNF and that ultimately leads to cell death.16,17 The adenovirus EIB gene encodes a functional homolog of bcl-2 and a protein that inactivates the p53 oncosuppressor.18,19 In fact, several viruses (SV40, Epstein-Barr virus, some human papilloma viruses and the hepatitis B virus) inhibit p53 function, reinforcing the crucial role of the protein in the control of abnormal cell proliferation. Sometimes, however, viruses can induce apoptosis of T cells thus depleting their population and also contributing to viral spread. Perhaps the most dramatic example of virus associated cell depletion is AIDS. HIV promotes a depletion of CD4+ T cells. Surprisingly, most T cells that die during HIV infection do not appear to be infected with the virus. Some studies indicate that binding of the soluble viral product gp120 to the CD4 receptor may cross-link the receptor. If this occurs prior to T cell receptor binding the cells may undergo apoptosis which leads to CD4+ T cell depletion and loss of the cell-mediated immune response. CD4+ cells are normally activated by simultaneous engagement of the T cell receptor and CD4 by antigen-MHC class II complexes on the surface of the antigen-presenting cell. Both CD4+ T cells from normal individuals as well as from individuals with HIV undergo apoptosis in vitro if cell-surface CD4 is cross-linked before engagement of T cell receptors.20 Recently, HIV-1 Tat protein has been shown to induce cell death by apoptosis in a T cell line and in mononuclear peripheral blood cells from uninfected donors.21 In addition, Tat protein has also been shown to induce a premature activation of cyclin-dependent kinases (CDKs) in T cells, an event that has been associated with apoptosis induction in several other cell systems. Tat-activated T cells would be depleted when either the T cell receptor is activated by an antigen or when gp120 binds to the CD4 receptor. Fas could also be involved in the death of CD4+ T cells during the course of an HIV infection.22 Human T cell lines, transformed with HIV, are more sensitive to Fas-mediated apoptosis than the parental cells. Fas is highly expressed on T cells in mice with retrovirus-induced immunodeficiency syndrome and on T lymphocytes of HIV infected children. The current hypothesis is that HIV, Tat and gp120 accelerate Fas-mediated, activation-induced T cell apoptosis, therefore contributing to CD4+ T cell depletion during the course of the syndrome.21 One important question concerning this model is why would the virus develop a mechanism to selectively deplete its host cell. The answer to this question, which accounts for the unlimited nature of viral replication, lies in the fact that the viral protein Nef specifically downregulates the CD4 receptor of infected T cells, thus preventing both viral reinfection and CD4-mediated apoptosis.20

Autoimmunity Apoptosis has a very important role in the development, function and regulation of the immune system. Autoimmune diseases may be related to defective regulation of apoptosis in the immune system. Under normal conditions, autoreactive lymphocytes formed due to random gene recombination and somatic hypermutation die by apoptosis. However, a de-

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fect in their deletion could predispose a patient to autoimmune disease. Alterations in the susceptibility of lymphocytes to death by apoptosis in vitro have been reported in several autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune diabetes mellitus and inflammatory bowel disease.23 As an example, a critical molecule in the regulation of cell death in lymphocytes is the cell surface receptor Fas. The Fas and Fas ligand have important homologies with members of the TNF receptor and TNF, respectively.24,25 The Fas ligand binds to its receptor, thus inducing apoptosis in the Fasbearing target cell. Triggering of this apoptotic pathway requires the cross-linking of Fas with either the purified Fas ligand, cells expressing the Fas-ligand or antibodies to Fas.26 It has recently been demonstrated that activated Fas interacts with several cellular proteins such as FAP-1,27 FADD,28 and RIP29 and activates a sphingomyelinase-dependent pathway.30 Fas-mediated apoptosis is basically triggered by ICE or ICE-like proteases.31 Stimulation of Fas on activated lymphocytes can induce apoptosis. In mice, two forms of hereditary autoimmune disease have been attributed to alterations in Fas-mediated apoptosis. MRL-Ipr mice, which develop fatal systemic lupus erythematosus by six months of age, have a mutation in the Fas receptor.32 In contrast GLD mice, which develop a similar illness, have a mutation in the Fas ligand.33 Bone marrow transplantation performed between the Ipr mutant and wild-type MRL has suggested that the Fas system may be involved in graft-vshost disease in allogenic bone marrow transplantation.26 In humans, a secreted form of Fas has been identified.34 Interestingly, patients with lupus erythematosus have elevated levels of soluble Fas which may competitively inhibit Fas ligand-Fas interactions. In addition, two recent studies have reported mutations affecting the Fas gene in children who develop a rare autoimmune, lymphoproliferative disorder, characterized by massive, nonmalignant lymphadenopathy, autoimmune phenomena and expanded populations of immature lymphocytes.35,36 Finally, transgenic mice overexpressing bcl-2 in their B lymphocytes have been reported to develop an immune complex nephritis.37 Failure to remove autoimmune cells that arise during development or that develop as a result of somatic mutation during an immune response can result in autoimmune disease. For example, in the thymus, 97% of thymocytes are deleted in their first few days of life.38 This occurs as a result of a complex weeding-out process in which immature thymocytes die by apoptosis unless they are able to recognize and bind antigen presented in association with self-MHC molecules (known as positive selection for MHC restriction) and yet die if the receptor occupancy is too great (negative selection). The principle is that high receptor occupancy is likely to reflect reactivity to self-antigen since most antigen presented in the thymus is self-derived. However, negative selection is certainly more complex than that and further mechanisms involving other surface signals are likely.

Neurodegenerative Diseases Apoptosis may prove to be relevant in certain neurodegenerative disorders, as the major consequences of stroke and trauma are from neuronal cell death. Indeed, a variety of neurological disorders are characterized by the gradual loss of specific sets of neurons. Such disorders (involving alterations in movement and central nervous system function) include Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and various forms of cerebellar degeneration. Oxidative stress, excitatory toxicity, calcium toxicity and survival factor deficiency have been implicated as being responsible for neuronal cell death. It is likely that all these factors induce apoptosis in neurons. Interestingly, transgenic mice overexpressing bcl-2 in neural cells are less prone to the action of these apoptotic agents.39 Neurotrophic growth factors and the extracellular matrix also prevent cells from apoptotic engagement.

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Retinitis pigmentosa occurs as a result of retinal degeneration associated with specific mutations in one of the three photoreceptor genes, rhodopsin, the beta subunit of cyclic GMP diesterase and the peripherin gene.40 Interestingly, intraocular injection of different neurotrophic and growth factors enhances photoreceptor survival in a hereditary rat model of light-induced retinal degeneration.41 These data suggest a model in which the threshold of cell death is dynamically regulated and is determined by the combined effects of external and internal survival factors. Another good example is amyotrophic lateral sclerosis. This pathological state results from mutations in the gene encoding copper-zinc superoxide dismutase, leading to a decrease in the ability of cells to detoxify free radicals. This results in an induction of apoptosis that can be specifically inhibited by treatment with survival growth factors or antioxidants.42 In Alzheimer’s disease there is a characteristic formation of senile plaques where beta-amyloid is accumulated (see chapter 3). It has been demonstrated that this peptide is able to induce neurons to undergo apoptosis.43 Mutations in the neuronal apoptosis inhibitory protein ( NAIP) gene, a gene homologous to IAP from baculovirus, have been identified in spinal muscular atrophy (a neurodegenerative disorder of childhood characterized by progressive spinal cord motor neuron depletion) and may decrease the apoptotic threshold of spinal cord neurons.44

Hematological Disorders The same mechanisms could apply for the increased apoptosis of hematopoietic cells. Mature blood cells are constantly being produced from hematopoietic stem cells located in the bone marrow. Proliferation and differentiation of hematopoietic precursor cells is regulated through various growth factors such as stem cell factor, granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin and thrombopoietin. These factors are not only required for proliferation and maturation of these cells but are also required for adequate cell survival, since in their absence the terminally differentiated cells undergo apoptosis. It has been suggested that hematopoietic differentiation is primarily determined by the precursor cell rather than as a result of the inductive effects of hematopoietic growth factors. Consistent with this view, overexpression of bcl-2 prevents apoptosis of hematopoietic cells induced by growth factor withdrawal.45 Bone marrow-derived stromal cells also prevent apoptotic death of normal and malignant hematopoietic cells. Together, these data suggest that hematopoietic growth factors control blood cell production at least in part by inhibiting apoptosis during the expansion and differentiation of intrinsically committed progenitors. Thus chronic hematological disorders characterized by insufficient production of blood cells, such as aplastic anemia, chronic neutropenia, beta-thalassemia and myelodysplastic syndromes, may be caused by activation of cell death genes through stromal factors from the bone marrow or deficiencies in hematopoietic growth factors essential for cell survival.46

Other Diseases Myocardial infarctions and stroke are two common disorders associated with death that are triggered by ischemia. In ischaemic injury there is a rapid death of cells within the central area of ischemia by necrosis. Outside this area, cells die over a more protracted time period by apoptosis. Agents known to be inhibitors of apoptosis in vitro have been shown to limit infarct size in these disorders.47 Apoptosis has also been described in atheroma, its pathological role still remaining to be ascertained.48 Degenerative disorders of the musculoskeletal system, such as osteoporosis and arthritis, could be the result of increased apoptosis of osteocytes and chondrocytes, respectively. Apoptosis is also a pathological feature in polycystic kidney disease49 and toxic-induced liver diseases50 and maybe central to the pathogenesis of these diseases.

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The Role of Ubiquitin in Apoptosis Little is known about the role of protease(s) involved in the apoptotic death, although preliminary work has suggested the involvement of calpains (calcium-dependent proteases).51 The nature of their involvement is very controversial since, in some reports, inhibitors of calpain blocked apoptosis, showing a positive function of calpain in the induction of apoptosis,51–53 while other work showed that calpain inhibitors accelerated apoptosis,54 thus suggesting a negative role. Additional work is necessary to determine the role of calpain in apoptosis, since the so-called calpain inhibitors used in the study mentioned were not specific to calpain but were also effective on the proteasome. A clearer involvement in apoptosis has been shown for apopsin (the ICE homolog) which induces apoptosis via the degradation of poly(ADP-ribose) polymerase.55,56 Kobayashi et al57 examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation and found that the proteasome is a major ICE-like protease in these cells and may be involved in the neural differentiation and the apoptotic pathway. Shinohara et al58 studied the involvement of proteases in apoptosis by examining the effects of different protease inhibitors on X-ray-induced apoptosis in MOLT-4 cells. ZLLL, a potent proteasome inhibitor, caused a marked induction of apoptosis while ZLL (a specific inhibitor of calpain) did not. Interestingly, the levels of p53 increased with time in the cells incubated with ZLLL, suggesting that the accumulation of the tumor-suppressor protein due to proteasome inhibition was responsible for the induction of apoptosis. The increase in p53 results primarily from the inhibition of the ubiquitin-dependent proteolytic pathway, which degrades p53, and not from an increase in p53 production, since it has been demonstrated that the increase in p53 protein is not accompanied by an increase in mRNA.59 Similarly, Drexler has shown that inhibition of proteasome-mediated proteolysis by specific proteasomal inhibitors leads to the rapid induction of apoptosis in human HL60 leukemic cells.60 In another study it has been shown that lactacystin, a specific proteasome inhibitor (see chapter 2) induces DNA fragmentation and apoptosis in a T-cell hybridoma in a dose-dependent manner, as judged by morphological changes as well as by nuclear condensation and DNA fragmentation.61 Ubiquitin-dependent proteolysis plays an important role in the degradation of proteins involved in the control of the cellular cycle (such as p53, c-myc, c-fos, c-jun) and, therefore, in apoptosis. Conjugation of these proteins to ubiquitin could be very important in the control of apoptosis. Machiels et al62 have shown an increase in proteasomes in lung tumor cells after the induction of apoptosis with a specific CDK inhibitor, thus reinforcing a possible role of the proteasome in the control of the cell cycle. Similarly, specific inhibitors of the proteasome were able to block cell death induced by ionizing radiation, glucocorticoids or phorbol esters in a thymocyte cell line.63 The diverging effects on apoptosis observed following inhibition of the ubiquitin-dependent proteolytic pathway (either induction or inhibition) leads to the suggestion that the proteasome may either degrade regulatory protein(s) that normally inhibits the apoptotic pathway or may proteolytically activate protein(s) that promotes cell death. Another example of the ubiquitin-apoptosis connection is found in muscle tissue after exercise. Following eccentric exercise, myofibers of both normal and dystrophic mice show an increase in both apoptotic rate (as measured by a technique of DNA fragmentation) and ubiquitin, thus reinforcing the role of ubiquitin in apoptosis in muscle fibers.64 Conversely, after spontaneous exercise, no changes are found in ubiquitin expression in normal mice but dystrophic mice show an increase that is also accompanied by a decrease in bcl-2 protein and an increased apoptotic rate.65 This suggests that the ubiquitin system plays an important role in the progression of dystrophinopathies (see chapter 5). Ubiquitin conjugation of nuclear proteins appears to be important in some cases of apoptosis and may regulate some of the chromatin structural changes.66 Marushige and

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Marushige67 studied chromatin condensation during apoptosis induced by transforming growth factor- β1 in T24 glioma and 476-16 trigeminal neurinoma cells and compared this with that occurring during mitosis. While mitosis-specific hyperphosphorylation of histones H1 and phosphorylation of histone H3 was not observed in apoptotic cells, apoptotic chromatin lacked ubiquitinated histone H2A, as did metaphase chromosomes, thus suggesting that the ubiquitin-conjugating machinery for histone H2A may be specifically perturbed during the chromatin condensation occurring in apoptosis. In addition, using the human epidermoid carcinoma-derived cell line MAI (established by introduction of the adenovirus EIA 12S cDNA linked to the mouse mammary tumor virus long terminal repeat), Nakajima et al68 showed that the degradation of topoisomerase II-alpha during adenovirus EIA-induced apoptosis is mediated by the activation of the ubiquitin proteolytic system. Another interesting example of the possible link between ubiquitin and apoptosis is found when analyzing TNF-induced apoptosis. TNF can bind to its cellular p55 receptor and trigger apoptosis through TRADD, a protein that binds its cytoplasmic “death domain” and that when overexpressed triggers ICE-mediated apoptosis.69 The cytosolic conserved domain of the p55 TNF receptor, known as a “death domain”, is also present in the cell death proteins FADD, RIP and TRADD.70 Death domains appear to be a subset of ankyrin repeat motifs, hence, rather than being specific mediators of cell death, they probably mediate homotypic protein-protein interactions. Deletion studies have shown that these motifs are required for the binding of these cell death proteins to their receptors. Interestingly, Boldin et al71 described a novel protein that binds specifically to the intracellular death domain of the p55 TNF receptor which has significant similarities with SEN3, the yeast equivalent of the p112 subunit of the 26S proteasome.

Manipulation of Apoptosis as a Therapeutic Strategy Because apoptosis is such a prevalent event in the control of specific cell populations, it is easy to predict that the ability to regulate apoptosis could have many therapeutic applications. Potential strategies fall into three categories: direct triggering of apoptosis by cytotoxic agents, enhancing susceptibility to apoptosis to increase the efficiency of other therapies, and boosting the efficiency of normal cells to apoptosis with the utilization of survival factors. Those diseases char acterized by apoptotic rem oval of n orm al cells (su ch as neurodegenerative disorders or AIDS) (Table 9.1) may benefit from treatments that increase the resistance of the cell against apoptosis. Preventing cell death may also be useful in mitigating against ischemia associated with stroke and myocardial infarctions. Drugs that inhibit apoptosis (such as cysteine protease inhibitors) may be effective even when administered to ischemic tissues some time after the stroke. These treatments may be beneficial even in the absence of specific alterations in the genes involved in cell death regulation. Current evidence suggests that the susceptibility of cells to undergo apoptosis is regulated continuously. This is the case for bcl-2 overexpression which is able to increase the resistance of cells to almost all apoptotic stimuli. Therefore, treatments that can increase the apoptotic thresholds of specific cells may be beneficial in the treatment of disorders associated with cell loss. Examples of this are the use of growth factors to stimulate recovery of hematopoietic cells after cancer chemotherapy, the use of neurotrophic factors to support survival of neurons in neurodegenerative disorders72 and treatment of HIV infected patients with antioxidants (such as N-acetylcysteine) 73 to prevent apoptosis of CD4+ T cells. This last treatment is based on the fact that early HIV infection is associated with reduced glutathione levels in blood cells. The pro-oxidant state associated with HIV infection may result in perturbations of activation-induced signal transduction in T lymphocytes, which

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may be related to the enhanced tendency of these cells to undergo apoptosis.74 Another way of decreasing the apoptotic rate associated with pathological states is to inhibit the action of cell surface effectors such as the Fas ligand. Therefore, as well as neutralizing antibodies to Fas or Fas ligand, the soluble form of Fas could prove clinically useful. In fact, Fas-based therapies have been considered for the treatment of AIDS in combination with other drugs, since anti-Fas monoclonal antibodies are cytotoxic to HIV-infected cells, without increasing HIV infection.75 Removal of abnormal (cancer or immunocompetent) cells might be stimulated through activation of the cellular death program. Indeed, effective tumor therapy may involve the induction of apoptosis in cancer cells rather than inhibition of tumor cell proliferation. The study of apoptosis has provided some understanding of how cancer cells evade treatment and may be useful for the design of novel chemotherapeutic drugs that either target apoptotic suppressors, such as cellular death suppressors, or activate apoptotic effectors. It is clear that most tumors contain zones of ongoing apoptosis, presenting an obvious target for induced regression. Since many tissues are dependent on growth factors, and certain cytokines and hormones can induce apoptosis, it is sometimes possible to therapeutically manipulate these to induce apoptosis. Hormone-responsive tumors can undergo apoptosis in vivo if the right stimulus is applied. Examples include glucocorticoid-induced death of chronic B lymphocytic leukemia cells,76 MXT mammary carcinoma death induced by analogs of LHRH and somatostatin,77 and regression and MCF-7 mammary adenocarcinoma and PC82 prostate adenocarcinoma after removal of estrogen and androgen, respectively.78,79 In this last case, however, the treatment often leads to selection of androgen-independent clonal populations within the tumors, in some cases due to increased expression of androgen receptors. This raises the prospects of using drugs that target the androgen receptors and associated signal transduction pathways to kill androgen-resistant cells. Similar strategies might be used to induce apoptosis in other cancer cell types that have lost their requirement for constant survival signals from hormones or growth factors, by targeting the associated receptors or their downstream signal transduction elements. Another possibility for targeting to neoplastic cells for apoptosis is the use of monoclonal antibodies to induce cell death. B lymphoma cells undergo apoptosis when treated with antimembrane immunoglobulins.80 Sometimes hormones can induce apoptosis of tumor cells. This is the case for the beta chain of human chorionic gonadotrophin ( β-HCG), a pregnancy hormone that has been shown to induce both in vitro and in vivo cell death, supposedly by be apoptosis, in a neoplastic Kaposi’s sarcoma cell line.81 In fact, it was proposed that the low rate of Kaposi’s sarcoma in women could be due to the interplay of hormones in the regulation of vascular proliferation. Specific regulation of apoptosis by gene therapy may also be possible. In vitro experiments using the expression of sense and antisense RNA or treatment with antisense oligonucleotides for various apoptotic genes has been shown to influence cell death. In the case of human B cell lymphomas bearing bcl-2 translocations, these can be specifically inhibited in vitro by antisense oligonucleotides targeted against the bcl-2 gene.82 An adenovirus carrying p53 has been used to kill osteosarcoma cells in vitro.83 In inflammation, activation of apoptosis in neutrophils could lead to limitation of tissue injury and could be very useful for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis. Autoimmune diseases are characterized by the proliferative expansion of lymphocytes that are reactive to self-antigens. Several groups have been exploring methods to induce selective apoptosis in the autoreactive cells that cause disease. It has been shown that repetitive treatment with an antigen can result in the selective death of antigen-reactive lymphocytes in vivo. Although the exact mechanism by which such treatments induce apoptosis are unclear, the treatments may prime cells for Fas-mediated death.84 Specific deletion of lymphocytes by repetitive treatment with a disease-associated autoantigen

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has been shown to be effective in the treatment of experimental autoimmune encephalitis in mice.85 Similar treatment strategies may prove effective in human autoimmune disease if the specific antigens involved in the autoimmune reaction can be identified. A better understanding of the tolerance induced by high doses of antigen could also help immunologists to improve their protocols for inducing specific tolerance to transplantation antigens. Another apoptosis-related strategy for the immunotherapy of autoimmune diseases in vivo would be to deliver an antigen-specific signal to the T cells in the absence of the second costimulatory signal; a strategy that was recently shown to induce specific apoptosis of autoreactive T cells.86 Concerning viral infections, induction of apoptosis in viral-infected cells may prove useful for the treatment of viral infections or viral-induced tumors. For example, a drug that could inhibit the action of the Epstein-Barr virus may be useful in treating nasopharyngeal carcinoma or Burkitt’s lymphoma. A drug that could block the association of the E6 protein of human papilloma virus with p 53 may be useful in treating cervical carcinoma by restoring the ability of the cells to undergo apoptosis. As we have seen for many pathological states, specific manipulation of the regulators of apoptosis, in order to inhibit or stimulate cell death, would seem a logical way forward. This is, however, not a straightforward solution since these regulators may have pleiotropic functions, and selective interference with the apoptotic-regulating properties will not be easy. In addition, apoptosis is a very general mechanism, and specific manipulation of a targeted cell population may prove to be very difficult. Furthermore, some of these regulators (most notably bcl-2) belong to a family of proteins, together constituting a complex regulating network, which may prove difficult to influence with a predictable result. To determine whether therapeutic manipulation of apoptosis is likely to be worthwhile, it is also important to consider whether aberrant apoptotic mechanisms are causal in a disease or whether apoptosis is just an indirect consequence of some other fault. In cases where the eventual loss of cells is apoptotic, merely inhibiting cell death may not restore cellular function. In the case of Alzheimer’s disease, for example, it is difficult to know whether inhibition of cell death by interference with apoptotic mechanisms would be an effective long-term treatment since it is unlikely to prevent the further development of beta-amyloid deposits.

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The Regulation of the Ubiquitin System he regulation of the ubiquitin system does not differ greatly from that of other metabolic pathways. First messengers, including hormones, cytokines or growth factors, have been shown to up- or downregulate certain ubiquitin system components in various metabolic situations. As in other cases, ubiquitin system activity has been shown to be modulated at the transcription level through degradation of ubiquitin system components or by direct stimulation or inhibition of ubiquitin system and/or proteasome-related activities. In addition, the ubiquitination of substrates has been shown to depend on regulatory changes which release specific signal sequences needed for the ubiquitin transfer. Although the amount of published data on the regulation of the ubiquitin system is still small, more detailed research of these mechanisms of control in different physiological and/or pathological situations may result in suggestions concerning useful therapeutic strategies for the treatment of various diseases. Our interest in these strategies is reinforced by the fact that even today no anti-ubiquitin system drugs have been tested in clinical trials, with the exception of a number of antitumoral drugs (see chapter 2). This is due in the main to the lack of specificity and toxicity of the known anti-ubiquitin system chemicals.

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Mechanisms Controlling the Ubiquitin System Control at the Transcription Level The genetic expression of the ubiquitin system components has been reported to be influenced by first messengers, particularly in different pathophysiological situations such as fasting, muscle wasting and cellular growth. During fasting an increase of muscle proteolysis occurs due to an increased activity of the ubiquitin system. Related to this, increased expression of the E2 14kDa enzyme has been described.1 Interestingly, insulin has been shown to downregulate its expression in vitro, while decreasing in parallel the amount of ubiquitinated proteins and the rate of muscle proteolysis.1 In addition, insulin-like growth factor-I (IGF-I), which is a muscle growth factor that acts independently of the nutritional status, has also been shown to induce the same change, not only in the E2 14kDa mRNA but also in proteasome mRNAs.2,3 Muscle wasting situations might be considered as being a result of an impairment in the regulatory signals involved in promoting or inhibiting muscle proteolysis. Tumor necrosis factor- α (TNF) is clearly involved in promoting muscle proteolysis in muscle wasting situations through an increased expression of ubiquitin system components and proteasome subunits (see chapter 5 for more information). The same effect has also been reported for corticosteroids in the muscle wasting associated with metabolic acidosis4,5 but not with aging6 or cancer.7,8 Ubiquitin and Disease, by Josep M. Argilés, Francisco J. López-Soriano, Javier Pallarés-Trujillo. ©1998 R.G. Landes Company.

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Cellular growth is a further physiological situation where cytokines are involved in inducing the expression of ubiquitin system components. Interleukin-2 (IL-2) has been shown to induce in T cells the expression of the DUB2 gene, which is an early-response gene that is rapidly downregulated, and which belongs to the deubiquitinating family of enzymes.9 Similarly, the DUB1 gene, which is an erythroid cell specific gene, has been shown to be induced by interleukin-3 (IL-3).10 Interestingly, additional DUB genes have been identified in the same chromosomal region, which suggests the presence of a larger family of cytokineinducible DUB genes.9 In the near future, in addition to the identification of further first messengers involved in the control of the ubiquitin system in various physiological situations, greater details about the signals involved in inducing transcription will probably be reported. The results reported by Choi et al11 suggest that proteasome itself may be involved in the signal transduction leading to alterations in transcription. The yeast MIP224/human TBP7 protein (which is an ATPase proteasome regulatory subunit) has been shown to interact specifically with both the yeast MB67 protein, which is an orphan member of the nuclear hormone receptor superfamily, and with the human TRIP1 protein associated with transcriptional activation. Finally, testosterone has also been reported to decrease the immunoreactive ubiquitin in the nucleus of rat pituitary luteinizing hormone (LH) cells.12

Direct Modulation of Ubiquitin-related or Proteasome Activities The activity of first messengers in the control of the ubiquitin system not only seems to act at the transcription level, but it also seems to directly modulate the ubiquitin transfer or proteasome related activities. At least three different mechanisms have been suggested as being operative in this direct control. These mechanisms are based on modulating the activity of kinases, the action of specific binding proteins and the influence of calcium levels. A) Kinases Kong and Chock13 have reported changes in the activities of various kinases on enzymes involved in the process of ubiquitin transfer. Along these lines, they have shown that the protein kinase C (PKC) phosphorylates in vitro an E1 enzyme, purified from rabbit reticulocytes, increasing 2-fold its ubiquitin activating activity; whereas a tyrosine kinase phosphorylates a purified E2 32 kDa enzyme from the same source, increasing 2.4-fold its ubiquitin conjugating activity to the histone H2A. In another study, reported by the same authors, a protein kinase of 300 kDa active in the cytosolic fraction of HeLa cells has been shown to phosphorylate a 20 kDa E2 enzyme on serine residues.14 Thus, it seems quite probable that more ubiquitin-conjugating enzymes will be found in the near future as being activated by kinases. Kinases also seem to be involved in the regulation of the proteasome activity. Thus, a casein kinase II copurifies with the 20S proteasome of human erythrocytes and has been shown to phosphorylate serine residues within a single 30 kDa proteasome subunit.15 Interestingly, a 32 kDa subunit of the 20S proteasome (which seems to be important for the latency of this core protease) has been suggested as being phosphorylated several times.16 Furthermore, tyrosine phosphorylation and cyclic AMP/cyclic GMP-dependent phosphorylation sites have also been predicted by computer analysis on the 20S proteasome subunits.17 Taking all this into consideration, it seems feasible that any type of proteasomal change might be regulated by phosphorylation. B) Binding proteins The activity of the ubiquitin system could also be directly modulated through the binding of specific proteins to ubiquitin system components. This might be the case

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as two types of specific proteins have been reported to bind to the proteasome resulting in a probable modulating activity. The first type refers to receptors of first messengers which have been shown to bind to 26S proteasome regulatory subunits. The protein associated to type 1 TNF receptor (known as TRAP2/55.11) has been shown to be identical to the human p97 regulatory subunit of the 26S proteasome, as well as being important for cellular growth.18 Similarly, the yeast MB67 protein (which is an orphan member of the nuclear hormone receptor superfamily) has been shown to interact with a regulatory subunit of the 26S proteasome known as MIP224/TPB7 and with the TRIP1 protein involved in transcriptional activation.11 Both cases illustrate the direct regulation of the proteasome activity by first messengers. Perhaps they may activate the ubiquitin transfer through kinases, on the one hand, and the proteasome activity, on the other. In addition, these results have suggested that the proteasome represents a first step in the signal transduction of first messengers. Such transduction through the proteasome might be closely related to transcriptional events, including the transcription of ubiquitin system genes, since some proteasome regulatory subunits have been reported as being related to transcriptional proteins.19 A second type of binding proteins are those which have been reported to bind to the 20S proteasome with activating or inhibiting activity. The activating proteins are the 19S cap complex, also known as PA700 (which forms the 26S proteasome), and the PA28 and 11S regulators (which bind to the 20S proteasome at both ends modulating its peptidase activities in the process of antigen presentation).20,21 In addition, several proteins that are not part of the 26S or the 20S proteasome have been reported to inhibit the 20S proteasome activities. These include a 31 kDa protein that forms multimers,22 a 60 kDa and a 200 kDa proteins,23 all of which might play a major regulatory role in the proteasome metabolism. C) Calcium levels Several authors have reported that the 19S cap complex and the 20S proteasome are found free in cellular extracts and seem to be in equilibrium with the 26S proteasome.24–26 Interestingly, Kawahara and Yokosawa27 reported that the 26S proteasome activity is activated periodically during the ascidian mitotic division cycle. Along these lines, they have also shown that an increase in calcium levels promotes the assembly of the 26S proteasome as well as its proteolytic activity during the ascidian meiotic metaphase-anaphase transition and egg activation.27,28 Thus, these authors have suggested that 26S proteasome activity can be regulated through interconversion between the 26S and the 20S proteasomes induced by intracellular calcium mobilization.

Degradation of Ubiquitin System Components As with certain cellular enzymatic activities, the ubiquitin system may also be regulated via an increased degradation. Although there is little data to support this, the autoubiquitination of ubiquitin conjugating enzymes does seem to support this hypothesis.29 In addition, the studies of Wing and Bedard 30 have shown that even the regulation might occur via the degradation of specific mRNA transcripts of ubiquitin system genes, since the IGF-I has been shown to enhance the rate of degradation of the 1.2 kb mRNA transcript of the E2 14 kDa increased in muscle as a consequence of fasting.

Activation of the Ubiquitin Transfer Through Signal Sequences The activation of the ubiquitin transfer can occur at the transcriptional level of E2 and/or E3 enzymes or by direct activation of these enzymes. However, the activation of signal sequences on protein subtrates is also a requisite for the ubiquitin transfer. Such signal

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sequences are thought to be directly or indirectly involved in substrate recognition by the E2 and/or E3 enzymes, as the ubiquitin acceptors are specific lysines (sometimes distant from signal sequences).21,31 The activation of these sequences has been shown to be induced by substrate modification, of which phosphorylation is the most widely reported.21,32 Thus, the activation of signal sequences on protein substrates is believed to be an important regulatory signal ensuring that preceding and necessary reactions have been completed. Among the various signal sequences, it was the nature of the N-terminal residue that was first described as being involved in determining the rate of protein degradation (“N-end rule” pathway).33,34 In this way, stabilizing and unstabilizing N-terminal residues were described. Interestingly, unstabilizing N-terminal residues have recently been shown to be recognition sites for enzymes in the ubiquitin system.35 Taking into consideration the reported data on the “N-end rule” pathway, it would seem to constitute a minor proteolytic pathway.31 In addition, in spite of the fact that most cellular proteins are N-alpha acetylated, they are known to be degraded without prior removal of the modifying group.31 As for the functional involvement of the “N-end rule” pathway, a role in peptide import has recently been suggested in yeast cells.36 Interestingly, more roles might be proposed from the experimental model reported by Chakraborty and Ingoglia,37 which shows an increased degradation of N-terminal arginilated proteins in nerve regeneration. The N-terminal arginilation is known as a tRNA-dependent reaction on stabilizing N-terminal acidic residues which renders stable proteins to good substrates for the ubiquitin system. Other signal sequences involved in inducing protein degradation are the PEST elements32 and the “destruction boxes”.38,39 The PEST elements are protein sequences enriched in proline (P), glutamic acid (E), serine (S), and threonine (T) known to be present in certain regulatory proteins such as G1 cyclins (Cln3 and Cln2), transcription related proteins (c-jun, c-fos, Gcn4, IKB, p53, yeast MAT-alpha 2 repressor), metabolic enzymes (fructose-1,6-bisphosphatase) or acute-phase proteins. Although it was initially controversial as to which proteolytic system was linked to the PEST signalling, it now seems quite clear that the ubiquitin system is the most highly active in response to these signal sequences.32 The “destruction boxes” represent a further type of signal sequence for protein degradation. They were initially described as being essential for ubiquitination of mitotic cyclins and comprise a moderately conserved nine-residue sequence in the N-terminal region of these proteins.38,39 Recently, a “destruction box”-like sequence has also been shown to be critical for ubiquitination of uracil permease in yeast.40 As for the activation of PEST elements or the “destruction boxes”, the most widely reported reaction has been phosphorylation. This has been reported at least in Cln3 and Cln2 cyclins and I- κB in the case of PEST elements and in mitotic cyclins in the case of “destruction boxes”. As for the involvement of signal sequences in the mechanism of protein degradation, they may act as recognition sites exposed by phosphorylation or rather that they act indirectly as phosphorylation targets for protein kinases, subsequently becoming exposed binding sites and the ubiquitin accepting lysines.41 Interestingly, the E3 enzyme Ubr1 has been shown to interact with N-terminal residues.35 Thus, other signal sequences might also be recognized by E3 or E2 enzymes. The cyclosome/APC, an E3-like factor, has been proposed as being involved in the ubiquitination of proteins with “destruction boxes”,42,43 and the Npi1/Rsp5 protein, which is an E3 hect domain protein, has been reported as being involved in the ubiquitination of a protein with a “destruction box”-like sequence.40 Other signal sequences have also been reported for the yeast MAT-alpha 2 repressor, c-jun and c-mos. In the first case, two degradation signals, one requiring residues 53-67 in the N-terminal region (Deg1) and the other requiring residues 136-140 in the C-terminal domain (Deg2), have been reported.44,45 In the case of c-jun, its degradation has been shown to involve residues 31-57 (delta region) and an additional sequence between amino acids

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224 and 331 in the DNA-binding/dimerization domain.46 The degradation of c-mos has been reported to involve a Pro-2 and the dephosphorylation of its neighboring Ser-3, where Lys-34 is the ubiquitin acceptor.47

Proposed Rate-limiting Steps in the Regulation of the Ubiquitin System The processes of ubiquitin transfer and protein degradation require the activation of different enzymes. Among these, the enzyme acting as a rate-limiting step in protein degradation is the most critical and highly regulated step. Knowledge of the nature of this enzyme and the characterization of the different subtypes involved in each proteolytic pathway is of utmost importance from a pharmacological point of view. Although the evidence is sketchy, the 26S proteasome regulatory subunits seem to be good candidates as the rate limiting step. Temparis et al have shown a relation between the increase of the C8 and C9 proteasome subunits and the development of muscle wasting in rats bearing a Yoshida sarcoma.48 This relation is reinforced as the initial increase of ubiquitin and E2 14 kDa mRNA transcripts was not accompanied by muscle wasting. Interestingly, the suggested role for certain deubiquitinating enzymes in the control of the proteasome activity may also be closely related to this question.49 The expression of the Doa4 deubiquitinating enzyme, which is part of the 26S proteasome, has also been shown to be rate-limiting for the turnover of certain proteins.50 It is more than likely that other deubiquitinating enzymes will be found to act as Doa4 in other physiological situations. Along these lines, the highly regulated family of cytokine-inducible DUB genes of deubiquitinating enzymes should not be overlooked.9 Finally, other possibilities can still be excluded. Along these lines, Wing and Banville1 have reported a relation between the E2 14 kDa changes and the rates of proteolysis in muscle cells as a consequence of fasting. When considering possible pathways involved in the control of the ubiquitin system, those related to the metabolism of the cyclic AMP (cAMP) and calmodulin should not be forgotten. The ubiquitin system has been shown to degrade the regulatory subunit of the cAMP-dependent kinase51 and to specifically ubiquitinate calmodulin.52 In addition, ubiquitin and calmodulin genes have been shown to be controlled by the same promoter.53 All these observations would seem to suggest the involvement of the ubiquitin system in cAMP and calmodulin related pathways. However, the reciprocal situations needs also to be considered.

References 1 Wing SS, Banville D. 14-kDa ubiquitin-conjugating enzyme: Structure of the rat gene and regulation upon fasting and by insulin. Am J Physiol 1994; 267:E39-E48. 2. Wing SS, Banville D. Regulation of 14 kDa ubiquitin conjugating enzyme (E2) in rat muscle upon fasting and by insulin and IGF-1. In: Proceedings of the 9th ICOP Conference on Proteolysis and Protein Turnover. Williamsburg, USA (1991), abstract 161. 3. Wing SS, Bedard N. Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme. Biochem J 1996; 319:455-461. 4. Mitch WE, Medina R, Grieber S et al. Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes. J Clin Invest 1994; 93:2127-2133. 5. Isozaki U, Mitch WE, England BK et al. Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification. Proc Natl Acad Sci USA 1996; 593:1967-1971.

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6. Dardevet D, Sornet C, Taillandier D et al. Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging. J Clin Invest 1995; 96:2113-2119. 7. Llovera M, García-Martínez C, Costelli P et al. Muscle hypercatabolism during cancer cachexia is not reversed by the glucocorticoid receptor antagonist RU38486. Cancer Lett 1996; 99:7-14. 8. Rallière C, Tauveron I, Taillandier D et al. Glucocorticoids do not regulate the expression of proteolytic genes in skeletal muscle from Cushing’s syndrome patients. J Clin Endocrinol Metab 1997; 82:3161-3164. 9. Zhu Y, Lambert K, Corless C et al. DUB-2 is a member of a novel family of cytokineinducible deubiquinating enzymes. J Biol Chem 1997; 272:51-57. 10. Zhu Y, Carroll M, Papa FR et al. DUB-1, a novel deubiquitinating enzyme with growthsuppressing activity. Proc Natl Acad Sci USA 1996; 93:3275-3279. 11. Choi HS, Seol W, Moore DD. A component of the 26S proteasome binds on orphan member of the nuclear hormone receptor superfamily. J Steroid Biochem Mol Biol 1996; 56:23-30. 12. Ohtani-Kanero R, Hattori A, Hara M et al. Increase in ubiquitin-immunoreactive nuclei in pituitary luteinizing hormone cells after castration. Endocrinology 1992; 130:2345-2348. 13. Kong SK, Chock PB. Protein ubiquitination is regulated by phosphorylation: An in vitro study. J Biol Chem 1992; 267:14189-14192. 14. Kong SK, Chock PB. Purification and characterization of a ubiquitin carrier protein kinase from HeLa cells. Proc Natl Acad Sci USA 1994; 91:11601-11605. 15. Lundem ann R, Lerea KM, Etlinger JD. Copurification of casein kinase II with 20S proteasomes and phosphorylation of a 30-kDa proteasome subunit. J Biol Chem 1993; 268:17413-17417. 16. Etlinger JD, Li SX, Guo GG et al. Phosphorylation and ubiquitination of the 26S proteasome complex. Enzyme Protein 1993; 47:325-329. 17. Rivett AJ. Proteasomes: Multicatalytic proteinase complexes. Biochem J 1993; 291:1-10. 18. Tsurumi C, Shimizu Y, Saeki M et al. cDNA cloning and functional analysis of the p97 subunit of the 26S proteasome, a polypeptide identical to the type-a tumor necrosis factor receptor-associated protein 2/55.11.Eur J Biochem 1996; 239:912-921. 19. Hilt W, Wolf DH. Proteasomes: Destruction as a programme. Trends Biochem Sci 1996; 21:96-102. 20. Peters, JM. Proteasomes: Protein degradation machines of the cell. Trends Biochem Sci 1994; 19:377-382. 21. H ochstrasser M. Ubiquitin -depen den t protein degradation . An n u Rev Gen et 1996; 30:405-439. 22. Chu-Ping M, Slaughter CA, DeMartino GN. Purification and characterization of a protein inhibitor of the 20S proteasome (macropain). Biochim Biophys Acta 1992; 1119:303-311. 23. Li X, Gu M, Etlinger JD. Isolation and characterization of a novel endogenous inhibitor of the proteasome. Biochemistry 1991; 30:9709-9715. 24. Udvardy A. Purification and characterization of a multiprotein component of the Drosophila 26S (1500 kDa) proteolytic complex. J Biol Chem 1993; 268:9055-9062. 25. Chu-Ping M, Vu SH, Proske RJ et al. Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20S proteasome. J Biol Chem 1994; 269:3539-3547. 26. Peters JM, Franke WW, Kleinschmidt JA. Distinct 19S and 20S subcomplexes of the 26S proteasome and their distribution in the nucleus and the cytoplasm. J Biol Chem 1994; 269:7709-7718. 27. Kawahara H, Yokosawa H. Intracellular calcium mobilization regulates the activity of 26S proteasome during the metaphase-anaphase transition in the ascidian meiotic cell cycle. Dev Biol 1994; 166:623-633. 28. Aizawa H, Kawahara H, Tanaka K et al. Activation of the proteasome during Xenopus egg activation implies a link between proteasome activation and intracellular calcium release. Biochem Biophys Res Commun 1996; 218:224-228.

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29. Banerjee A, Gregori L, Xu Y et al. The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein. J Biol Chem 1993; 268:5668-5675. 30. Wing SS, Bedard N. Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme. Biochem J 1996; 319:45-61. 31. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell 1994; 79:13-21. 32. Rechsteiner M, Rogers SW. PEST sequences and regulation by proteolysis. Trends Biochem Sci 1996; 21:267-271. 33. Hershko A, Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem 1992; 61:761-807. 34. Varshavsky A. The N-end rule. Cell 1992; 69:725-735. 35. Bartel B, Wünning I, Varshavsky A. The recognition component of the N-end rule pathway. EMBO J 1990; 9:3179-3189. 36. Alagramam K, Naider F, Becker JM. A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae. Mol Microbiol 1995; 15:225-234. 37. Chakraborty G, Ingoglia NA N-terminal arginylation and ubiquitin-mediated proteolysis in nerve regeneration. Brain Res Bull 1993; 30:439-445. 38. Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature 1992; 349:132-138. 39. Amon A, Irniger S, Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell 1994; 77:1037-1050. 40. Hein C, Springael JY, Volland C et al. NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur1 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol 1995; 18:77-87. 41. Yaglom J, Linskens HK, Sadis S et al. p34Cdc28-mediated control of Cln3 cyclin degradation. Mol Cell Biol 1995; 15:731-741. 42. King RW, Peters JM, Tugendreich S et al. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 1995; 81:279-288. 43. Sudakin V, Ganoth D, Daham A et al. The cyclosome, a large complex containing cyclinselective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol Biol Cell 1995; 6:185-197. 44. Hochstrasser M, Varshavsky A. In vivo degradation of a transcriptional regulator: The yeast α2 repressor. Cell 1990; 61:697-708. 45. Chen P, Johnson P, Sommer T et al. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor. Cell 1993; 74:357-369. 46. Treier M, Staszewski LM, Bohmann D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the δ domain. Cell 1994; 78:787-798. 47. Nishizawa M, Furuno N, Okazaki K et al. Degradation of Mos by the N-terminal proline (Pro2)-dependent ubiquitin pathway on fertilization of Xenopus eggs: Possible significance of natural selection for Pro2 in Mos. EMBO J 1993; 12:4021-4027. 48. Temparis S, Asensi M, Taillandier D et al. Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats. Cancer Res 1994, 54:5568-5573. 49. Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol 1995; 7:215-223. 50. Papa F, Hochstrasser M. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene Nature 1993; 366:313-319. 51. Hedge AN, Goldberg AL, Schwartz JH. Regulatory subunits of cAMP-dependent protein kinases are degraded after conjugation to ubiquitin: A molecular mechanism underlying long-term synaptic plasticity. Proc Natl Acad Sci USA 1993; 90:7436-7440. 52. Laub M, Jennissen HP. Ubiquitination of endogenous calmodulin in rabbit tissue extracts. FEBS Lett 1991; 294:229-233. 53. Wong S, Morales TH, Neigel JE et al. Genomic and transcriptional linkage of the genes for calmodulin, EF-hand5 protein and ubiquitin extension protein 52 in Trypanosoma brucei. Mol Cell Biol 1993; 13:207-216.

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The Design of Therapeutic Strategies s we have seen, ubiquitin-mediated proteolysis is involved in the turnover of many key regulatory proteins. Any pharmacological intervention which is able to alter the halflives of these cellular proteins may have wide-ranging therapeutic potential. Manipulating or inhibiting members of the enzymatic cascade responsible for conjugating ubiquitin to target substrates could result in a promising new strategy for therapeutic intervention. Inhibition of enzymes that remove, rather than add, ubiquitin from protein substrates could also be a promising strategy. Ubiquitin isopeptidases may have potential roles as key regulators for stability of individual substrates.1 Therefore for a given target protein that is subject to ubiquitin-mediated regulation, depending on its cellular function, one could choose to increase (e.g. tumor suppressor) or decrease (e.g. oncogene) the cellular levels of the protein by designing small molecules that inhibit either the ubiquitinating or the deubiquitinating enzyme for that particular substrate. In fact, selective inhibition of a single peptidase, as opposed to general inhibition of the proteasome, can sometimes be sufficient to induce a specific cellular process. Thus, selective inhibition might be useful in managing diseases where only one activity is involved, without completely inhibiting the proteasome. An important therapeutic area where inhibition of the ubiquitin-dependent pathway may prove to be very useful is that of cellular proliferation and therefore malignant diseases. As discussed previously, the control of the cell cycle is associated with ubiquitin-dependent processes. Taking p53, for example, this protein appears to be able to control proliferation by either imposing a block on cell cycle progression at G1 or by inducing apoptosis in transformed cells.2 p53 is degraded by a ubiquitin-dependent process and therefore the search for a specific inhibitor of ubiquitin conjugation to p53 could halt its proteolytic breakdown, increasing the intracellular levels of the regulatory protein. This could lead to an induction of apoptosis in transformed cells, especially if the Rb pathway is inactivated. Another interesting group of molecules involved in the control of the cell cycle are cyclins. They are required for the activation of the cyclin-dependent kinases (CDKs). In particular, cyclin B is an activator of Cdc2 (a protein which is involved in mitosis). Degradation of cyclin B is necessary to stop mitosis and, consequently, entry into interphase of the next cell cycle.3 Proteolytic degradation of cyclin B is dependent on ubiquitinization.4,5 Interestingly, among the ubiquitin-conjugating enzymes involved, one of them (E3) is highly specific. This is a multiprotein complex (involving Cdc16, Cdc23 and Cdc27) which is only active during mitosis. Therefore the discovery of a specific inhibitor of this enzyme (and thus possible prevention of cyclin B degradation) may prove to be very useful since it would have very powerful cytostatic activity. Apart from cyclins, CDKs can also associate with other regulatory proteins such as p21, p27 and p57. As we have previously emphasized (see chapter 4), p27 is a powerful inhibitor of both CDK4 and CDK2 and is involved in the regulation of the G1 phase of the cell cycle. Since p27 is degraded by ubiquitinization, a specific inhibitor of this process would be expected to raise the intracellular levels of the protein and should lead

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to a block in proliferation. Tan et al6 have shown that the levels of p27 can be a prognostic factor in colorectal carcinomas. Interestingly, tumors expressing low p27 levels contained more p27 specific degradation activity, suggesting that the p27 protein is eliminated because of increased degradation.1 Therefore, blocking p27 ubiquitinization may lead to a reversal in disease progression, especially in those tumors that have low to undetectable p27 protein levels as a result of an “activated” ubiquitinization pathway. Among other areas of cancer biology where involvement of the ubiquitin pathway may be relevant are growth factor receptors and their transduction pathways. Several cell surface receptors have been shown to be ubiquitinated, suggesting that proteasome-mediated proteolysis could be involved in their normal turnover. Involvement of proteasomes in the degradation of cell surface receptors might have increasing relevance in cancer chemotherapy, as new compounds modulating growth factor transduction pathways are discovered. These compounds could have a tremendous therapeutic potential. Herbimycin A, a compound with antitumor activity based on its capacity to inhibit tyrosine kinases associated with growth factor receptors (PDGF, EGF) signalling pathways, also acts by activating receptor degradation via the proteasome/ubiquitin pathway.7 Proteasome inhibitors have been shown to counteract the effects of herbimycin A in vitro 7 and it is conceivable that modulation of proteasome function might influence its antitumor activity. A similar case is that of bryostatin 1, a compound which is able to downregulate protein kinase C through the activation of its degradation by the ubiquitin pathway.8 Another important area where blocking ubiquitin-dependent proteolysis may prove to be extremely useful is that of inflammatory and immune diseases. B and T cell proliferation is closely associated with ubiquitin-dependent proteolysis. It should be remembered here that NF- κB plays a central role in the signal transduction pathways leading to the activation of the immune responses. The protein is synthesized as a 105 kDa precursor which is posttranslationally processed to a 50 kDa mature form (p50) by means of a ubiquitin-dependent reaction (see chapter 3). Interestingly, p50 can bind to several related regulators (p65, ReIB or c-Rel) to generate distinct heterodimeric transcriptional activators. Another molecule, I- κB, can associate with each of these heterodimers, thus inactivating them. The inactivation of I- κB is mediated by phosphorylation by means of a specific kinase that requires the presence of E1, Ubc4, Ubc5 and ubiquitin.9 A variety of inflammatory agents (viral proteins, cytokines, antigens) are able to activate a signal transduction pathway that leads to the inactivation of I- κB. Therefore, either inactivation of the processing of the NF- κB or activation of the I- κB kinase could prove to be a very promising therapeutic tool in several pathological conditions such as inflammation, autoimmune diseases and viral infections. Schow and Joly10 reported that the proteasome inhibitor ALL is able to inhibit the activation of NF- κB in macrophage cultures stimulated with LPS. As a result, tumor necrosis factor- α (TNF) synthesis, a reaction dependent upon NF- κB activation, is also blocked by the inhibitor. In addition, administration of the inhibitor to mice pretreated with LPS (which normally results in induction of TNF and interleukin-6 (IL-6) within 90 minutes, followed by lethal shock at 24 hours) considerably lowered the levels of circulating cytokines, thus reducing the incidence of septic shock. The proteasome may also be involved in drug resistance. Overexpression of the fission yeast protein Pad1 confers multidrug resistance to unrelated compounds such as stausporin, caffeine and leptomycin B through the activation of the yeast transcription factor Pap1, a homolog of human AP-1.11 Dubiel et al12 have cloned the human homolog of Pad1, POH1, a molecule that can confer multidrug resistance in mammalian cells when it is overexpressed. The POH1 molecule displays a significant similarity to the S12/p40 subunit of the 26S proteasome.12 In addition, it has a certain sequence similarity with JAB1, a protein that interacts with c-jun to activate AP-1 transcription factors.13 Various independent data, namely

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the dependence of the Pad1 multidrug resistance phenotype in fission yeast on the activation of an AP-1-like factor, the sequence similarity between POH1 and JAB1, and the importance of proteasome degradation for c-jun regulation, support a model whereby overexpression of POH1 could upregulate AP-1 factors resulting ultimately in drug resistance. Therefore, the proteasome could be involved in conferring drug resistance on human tumors and the development of efficient and very specific inhibitors could prove a useful tool for coadjuvant chemotherapy. The ability of our immune system to specifically recognize and respond to most foreign proteins is dependent on the activation of T cells by peptide fragments of antigen bound to a cell surface molecule. These molecules are encoded by a cluster of linked genes called the major histocompatibility complex (MHC). MHC class I molecules bind and display on the cell surface a sample of peptides from intracellularly synthesized proteins. Although the significance of ubiquitinization on peptide generation remains to be established, there is little question that proteasomes are of substantial importance in this process (see chapter 6). Viruses have evolved diverse strategies to evade immunological detection of virus-infected cells. Recently published data suggest that the human cytomegalovirus can thwart the immune system by reversing the translocation into the endoplasmic reticulum of the MHC class I heavy chain, a transmembrane protein that is required for antigen presentation to T cells.14 Apparently, synthesis of a single viral protein, US11, is sufficient to cause the dislocation. US11 is also a transmembrane glycoprotein that localizes to the endoplasmic reticulum. Unlike in control cells, the MHC class I molecules are extremely short-lived in US11-transfected cells. Interestingly, the use of protease inhibitors known to inhibit the proteasome strongly inhibited turnover of a deglycosylated form of the class I heavy chain. It has been suggested that, by an unknown mechanism, the viral protein causes the ejection of class I molecules (but not other molecules) from the endoplasmic reticulum back into the cytosol where they are deglycosylated by an N-glycanase and degraded by the proteasome.14 Therefore, inhibition of the proteasome might result in an efficient therapeutic strategy against this type of viral infection. Another particular therapeutic area where manipulating ubiquitin-dependent proteolysis could be promising concerns that of cardiovascular diseases, atherosclerosis in particular. This type of disease is one of the most important causes of mortality in the Western world. Atheroma formation appears to be intimately associated with an abnormal low density lipoprotein (LDL) metabolism. Interestingly, lactacystin, a specific proteasome inhibitor of microbial origin, increases the LDL receptor level in HepG2 cells.15 The proteasome could therefore play an important down-regulatory role in LDL receptor expression. In addition, Yeung et al16 have proposed that the ubiquitin-proteasome pathway is involved in the degradation of apoB in mammalian cells. They based their suggestion on the fact that the proteasome inhibitor ALLN inhibited the degradation of newly synthesized apoB in liver cells and also on the fact that intracellular human apoB isolated by immunoprecipitation reacted specifically with anti-ubiquitin antibody by immunoblotting.16 It should be remembered that apoB constitutes the apoprotein moiety of the LDL and therefore, an abnormal degradation of the apoprotein could lead to an abnormal cellular cholesterol metabolism and, consequently, may be involved in atherosclerosis. Due to the broad involvement of proteasomes in normal cellular physiology, any attempt to target the proteasome nonspecifically might be associated with dangerous in vivo toxicity. However, the complexity and specificity of proteasome regulation indicate that specific inhibitors of individual proteasome-mediated processes might ultimately become available. Moreover, the rapidly expanding knowledge about the role of proteasomes in normal and tumor cells could provide in the future a rational basis for the use of proteasometargeting drugs.

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References 1. Rolfe M, Chiu MI, Pagano M. The ubiquitin-mediated proteolytic pathway as a therapeutic area. J Mol Med 1997; 75:5-17. 2. White E. p53, guardan of Rb. Nature 1994; 371:21-22. 3. Murray AW, Solomon MJ, Kirschner MW. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature 1989; 339:280-286. 4. Glotzer M, Murray A, Kirschner M. Cyclin is degraded by the ubiquitin pathway. Nature 1991; 349:132-138. 5. Lorca T, Galas S, Fesquet D et al. Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: Requirement for a Ca2+ -calmodulin dependent effect. EMBO J 1991; 10:2087-2093. 6. Tan P, Cady B, Wanner M et al. The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinomas. Cancer Res 1997; 57:1259-1263. 7. Sepp Lorenzino L, Ma Z, Lebwohl DE et al. Herbimycin A induces the 20S proteasomeand ubiquitin-dependent degradation of receptor tyrosine kinases. J Biol Chem 1995; 270:16580-16587. 8. Lee HW, Smith L, Pettit GR et al. Ubiquitination of protein kinase C-alpha and degradation by proteasome. J Biol Chem 1996; 271:20973-20976. 9. Ch en Z, Paren t L, Man iatis T. Site-specific ph osph orylation of I κ Bα by a n ovel ubiquitination-dependent protein kinase activity. Cell 1996; 84:853-862. 10. Schow SR, Joly A. N-acetyl-leucinyl-leucinyl-norleucinal inhibits lipopolysaccharide-induced NF- κB activation and prevents TNF and IL-6 synthesis in vivo. Cell Immunol 1997; 175:199-202. 11. Shimanuki M, Saka Y, Yanagida M et al. A novel essential fission yeast gene pad1+ positively regulates pap1(+)-dependent transcription and is implicated in the maintenance of chromosome structure. J Cell Sci 1995; 108:569-579. 12. Dubiel W, Ferrell K, Dumdey R et al. Molecular cloning and expression of subunit 12: A non-MCP and non-ATPase subunit of the 26S protease. FEBS Lett 1995; 363:97-100. 13. Claret FX, Hibi M, Dhut S et al. A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature 1996; 383:453-457. 14. Wiertz EJHJ, Jones TR, Sun L et al. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 1996; 84:769-780. 15. Miura H, Tomoda H, Miura K et al. Lactacystin increases LDL receptor levels in HepG2 cells. Biochem Biophys Res Commun 1996; 227:684-687. 16. Yeung SJ, Chen SH, Chan L. Ubiquitin-proteasome pathway mediates intracellular degradation of apolipoprotein B. Biochemistry 1996; 35:13843-13848.

Index A

D

ACT 61, 63, 65-69, 79, 107, 110, 111 AIDS 108, 109, 121, 122, 147, 149, 150, 154, 155 Alpha-1-antichymotrypsin 61, 107 Alzheimer 34, 38, 61, 67, 75, 109, 128, 140, 151, 152, 156 Amyloid precursor protein 34, 61, 97, 105, 107, 128 Amyotrophic lateral sclerosis 79, 81, 106, 151, 152 Antigen processing 4, 117-119 Apoptosis 1, 8, 108, 127, 147-156, 169 APP 34, 61-64, 66-68, 70, 73, 74, 76-79, 82, 97, 105, 108, 110, 111, 128, 131, 140 Arsenoxides 49 Autoimmune diseases 118-120, 147, 150, 155, 156, 170

DNA repair 9, 22, 34-36, 38-40, 42, 77, 78, 94-96, 128, 131 Doa4 32, 33, 41, 42, 46, 96, 165 Duchenne dystrophy 109

Hect domain 16, 17, 22, 25, 39, 164 Hyperphosphorylated tau protein 61, 110

B

I

BAP 61-70, 74, 75, 77, 78, 82, 110 Bcl-2 148-156 Beta amyloid protein 75, 78

IL-1 63, 68, 75, 103, 141 Inflammation 107, 147, 149, 155, 170 Insulin 103-105, 161 Interleukin-1 6, 63, 103, 141, 150

E E2-F1 22, 38, 40, 95

F FAD3 38, 78

H

C c-fos 36, 38, 94, 95, 97, 106-108, 114, 128, 131, 153, 164 c-jun 36, 95, 97, 106-109, 128, 131, 153, 164, 170, 171 Cachexia 101, 103 Calpain 5-8, 49, 101, 108, 140-142, 153 Cathepsin B 4, 137 Cathepsin D 66, 67, 138, 140 Cathepsin G 63-68, 79 Cathepsin L 137, 138 Corticosteroids 161 Cyclin-dependent kinases 91, 150 Cyclins 35, 36, 37, 38, 40, 91, 92, 93, 94, 95, 164, 169 Cytokine 6, 43, 62, 67, 68, 77, 103, 107, 108, 118, 122, 141, 147, 148, 155, 161, 162, 165, 170

L Lactacystin 48, 153, 171

M Major histocompatibility complex (MHC) 4, 5, 79, 117-119, 149-151, 171 MAP kinase 71, 73, 74, 77 Metalloprotease 64, 65, 67 Multicatalytic protease complex 15, 25

N N-terminal threonine 25, 28, 44, 48 Nerve growth factor (NGF) 62, 63, 68, 71, 74, 76, 107, 108, 131 Neurofibrillary tangles 61, 69-71, 75, 81, 82, 142 Neuronal regeneration 73, 76, 77, 79 NF- κB 36, 38, 40, 79, 94, 108, 109, 117, 118, 120, 122, 128, 170

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174

P

T

p53 22, 36, 38-40, 49, 94, 95, 122, 128, 131, 132, 148-150, 153, 155, 156, 164, 169 Paired helical filaments 61, 69, 75, 78, 81, 110 Parkinson 79, 81, 151 Peptide aldehydes 48 PEST 77, 164 PN2 62, 63, 65-67, 74, 77, 131 Polyubiquitin linear chains 29 Protease nexin 2 62, 97, 107, 110

Thiolester link 16, 22, 39 Tre2 33, 42 Tumor necrosis factor(TNF) 62, 63, 68, 77, 103, 104, 108, 109, 122, 150, 151, 154, 161, 163, 170

U UBC domain 16, 17, 22, 37, 38 Ubp family 41

R Reactive oxygen intermediates (ROIs) 70, 128, 129, 131

S Sepsis 102, 104, 107, 108, 141 Skeletal muscle 6, 37, 101-103, 107, 108, 139, 141 Spongiform encephalopathies 79, 82 Sporadic inclusion 109 Stress response 34, 36, 37, 39, 42, 43, 46, 77, 78, 108, 120, 121, 127-129, 131

Y Yuh1 32