ideas in chemistry and molecular sciences

Where Chemistry Meets Life
Edited by Bruno Pignataro
Sciences
Ideas in Chemistry and Molecular Sciences Advances in Synthetic Chemistry
2010
Ideas in Chemistry and Molecular Sciences Advances in Nanotechnology, Materials and Devices
2010
2010
Tomorrow’s Chemistry Today Concepts in Nanoscience, Organic Materials and Environmental Chemistry 2nd edition
2009
Chemical Biology Learning through Case Studies
2009
Redox Signaling and Regulation in Biology and Medicine
2009
RNA Purification and Analysis Sample Preparation, Extraction, Chromatography
2009
Handbook of RNA Biochemistry Student Edition
2008
Where Chemistry Meets Life
Edited by Bruno Pignataro
Prof. Bruno Pignataro University of Palermo Department of Physical Chemistry Viale delle Scienze 90128 Palermo Italy
Cover
We would like to thank Dr. Adriana Pietropaolo (ETH Zurich) for providing us with the graphic material used in the cover illustration.
All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.
2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Cover Design Adam Design, Weinheim Typesetting Laserwords Private Limited, Chennai, India Printing and Binding betz-druck GmbH, Darmstadt
Printed in the Federal Republic of Germany Printed on acid-free paper
ISBN: 978-3-527-32541-2
Part I Biochemical Studies 1
1 The Role of Copper Ion and the Ubiquitin System in Neurodegenerative Disorders 3 Fabio Arnesano
1.1 Introduction 3 1.2 Metal Ions in the Brain 4 1.3 Brain Copper Homeostasis 5 1.4 Brain Copper and Neurodegenerative Disorders 8 1.5 The Role of Ubiquitin in Protein Degradation 9 1.6 Failure of the Ubiquitin System in Neurodegenerative Disorders 13 1.7 Interaction of Ubiquitin with Metal Ions 15 1.7.1 Thermal Stability of Ubiquitin 15 1.7.2 Spectroscopic Characterization of CuII Binding 15 1.7.3 Possible Implications for the Polyubiquitination Process 17 1.7.4 CuII-Induced Self-Oligomerization of Ub 17 1.7.5 Cooperativity between CuII-Binding and Solvent Polarity 18 1.7.6 Comparison with Other Metal Ions 19 1.8 Biological Implications 21 1.8.1 The Redox State of Cellular Copper 21 1.8.2 Ubiquitin and Phospholipids 22 1.9 Conclusions and Perspectives 23
Acknowledgments 24 References 24
2 The Bioinorganic and Organometallic Chemistry of Copper(III) 31 Xavi Ribas and Alicia Casitas
2.1 Introduction 31 2.2 Bioinorganic Implications of Copper(III) 33
Ideas in Chemistry and Molecular Sciences: Where Chemistry Meets Life. Edited by Bruno Pignataro Copyright 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32541-2
VI Contents
2.2.1 Dinuclear Type-3 Copper Enzymes 33 2.2.2 Particulate Methano Monooxygenase (pMMO) 36 2.2.3 Mononuclear Monooxygenating Copper-based Enzymes 38 2.2.4 Trinuclear Copper Models for Laccase 40 2.3 Organometallic CuIII Species in Organic Transformations 41 2.3.1 C–C Bond Formation in Organocuprate(I) Catalysis 42 2.3.1.1 Conjugate Addition to α-Enones 42 2.3.1.2 Acetylene Carbocupration 43 2.3.1.3 SN2 and SN2′ Alkylations 43 2.3.2 Aryl–Heteroatom Bond Formation in Cu-mediated Cross-coupling
Processes 44 2.3.3 Aromatic and Aliphatic C–H Bond Organometallic
Functionalizations 45 2.3.3.1 Catalytic Systems 45 2.3.3.2 Stoichiometric Systems 47 2.4 Miscellany: Cuprate Superconducting Materials 51 2.5 Overview and Future Targets 51
References 52
3 Chemical Protein Modification 59 Goncalo J. L. Bernardes, Justin M. Chalker, and Benjamin G. Davis
3.1 Introducing Diversity by Posttranslational Modification 59 3.2 Chemistry: A Route to Modified Proteins 60 3.3 Challenges in Chemical Protein Modification 61 3.4 Traditional Methods for Protein Modification 61 3.4.1 Lysine Modification 62 3.4.1.1 Activated Esters 62 3.4.1.2 Isocyanates and Isothiocyanates 62 3.4.1.3 Reductive Alkylation 62 3.4.1.4 IME Reagents 63 3.4.2 Glutamic and Aspartic Acid Modification 64 3.4.3 Cysteine 64 3.4.3.1 Alkylation 65 3.4.3.2 Disulfides 66 3.4.3.3 Desulfurization at Cysteine 67 3.5 Recent Innovations in Site-Selective Protein Modification 70 3.5.1 Dehydroalanine: A Useful Chemical Handle for Protein
Conjugation 71 3.5.2 Metal-Mediated Protein Modification 71 3.5.2.1 Modification at Natural Residues 72 3.5.2.2 Iridium-Catalyzed Reductive Alkylation of Lysine 74 3.5.2.3 Modification of Unnatural Residues 74 3.5.2.4 Olefin Metathesis at S-Allyl Cysteine 77 3.5.3 Metal-Free Methods for Modifying Unnatural Amino Acids 78 3.5.3.1 Oxime Ligation at Aldehydes and Ketones 78
Contents VII
3.5.3.2 Azide and Alkyne Modification 79 3.5.3.3 Selective Modification of Tetrazole-Containing Proteins 80 3.5.3.4 Tetrazine Ligation 81 3.5.4 Dual Modification 81 3.6 Conclusion and Outlook 81
References 82
Part II Drug Delivery 93
4 Vitamin B12: A Potential Targeting Molecule for Therapeutic Drug Delivery 95 Pilar Ruiz-Sanchez
4.1 Introduction 95 4.2 Transport Mechanism 96 4.3 Metabolism of B12 97 4.3.1 Adenosylcobalamin-Dependent Reactions 97 4.3.2 Methylcobalamin-Dependent Reactions 99 4.4 Vitamin B12 Derivatives 99 4.4.1 Structure 99 4.4.2 b-, d-, e-Cobalamin Derivatives 99 4.4.3 Modifications on the Ribose Moiety 101 4.4.4 β-Axial Position 102 4.4.4.1 Cobalamin Alkylation 102 4.4.4.2 Heterodinuclear Concept 103 4.5 Outlook 108
5.1 Introduction 117 5.1.1 Cellular Delivery 117 5.1.2 Delivery Devices 117 5.1.2.1 Liposomes 117 5.1.2.2 Cell-penetrating Peptides 118 5.1.2.3 Dendrimers 118 5.1.2.4 Nanomaterials 118 5.2 Microspheres 119 5.2.1 Biodegradable Microspheres 119 5.2.1.1 Preparation 119 5.2.2 Biostable Microspheres 120 5.2.2.1 Applications of Biostable Microspheres 120 5.2.2.2 Preparation 120 5.2.3 Microspheres and Solid-phase Chemistry 121
VIII Contents
5.2.3.1 Preparation of Microspheres 121 5.2.3.2 Fmoc Chemistry on Microspheres 121 5.2.3.3 Dual Functionality of Microspheres 122 5.2.3.4 Coupling Agents 124 5.2.4 Noncleavable Link to Microspheres 126 5.2.4.1 Microsphere-based Intracellular Sensing 126 5.2.4.2 siRNA Delivery 127 5.2.5 Cleavable Linkers 130 5.2.5.1 Ester Bonds 130 5.2.5.2 Disulfide Bonds 132 5.2.6 Bioconjugation 133 5.2.6.1 Streptavidin–Biotin 134 5.3 Future Perspectives 135
Part III Research in Therapeutics 141
6 Fundamental Processes in Radiation Damage to DNA: How Low-Energy Electrons Damage Biomolecules 143 Ilko Bald
6.1 Radiation Damage and the Role of Low-Energy Electrons 143 6.1.1 How Chemical Bonds are Broken by Low-energy Electrons 145 6.1.2 DEA Studies of Gas-Phase DNA Building Blocks: The
Nucleobases 147 6.2 DEA Studies on Model Compounds for the DNA Backbone 148 6.2.1 Electron Attachment to d-Ribose 148 6.2.2 Cross-Ring Cleavage of d-Ribose Proceeds with Selective Charge
Retention 150 6.2.3 The Nature of the Transient Negative d-Ribose Anions 154 6.2.4 One Step Further: Tetraacetyl-d-Ribose 155 6.2.5 The Use of Laser-Induced Acoustic Desorption (LIAD) to Study DEA to
Larger Biomolecules 159 6.2.6 Sugar–Phosphate Cleavage Induced by 0 eV Electrons: DEA to
d-Ribose-5′-Phosphate 159 6.3 Outlook and Future Prospects 161
7 Structure-Based Design on the Way to New Anti-infectives 167 Anna Katharina Herta Hirsch
7.1 Introduction 167 7.2 Isoprenoids and the Nonmevalonate Pathway 169 7.2.1 4-Diphosphocytidyl-2C-methyl-d-erythritol Kinase (IspE) 170 7.2.2 Structure of IspE 170
Contents IX
7.2.3 Active Site of IspE 170 7.3 Targeting the CDP-Binding Pocket of IspE 174 7.3.1 Design 174 7.3.1.1 Possible Ribose Analogues 175 7.3.1.2 Design of the Vector 176 7.3.2 Optimization of the Ribose Analogue 176 7.3.3 Importance of the Vector 178 7.3.4 Optimization of the Filling of the Small, Hydrophobic Pocket 179 7.3.4.1 The ‘‘55% Rule’’ 179 7.3.4.2 Evaluation of Inhibitors Featuring Different Sulfone
Substituents 180 7.3.5 Summary of the First-Generation Inhibitors 182 7.4 X-ray Cocrystal Structure Analysis 182 7.4.1 Design of Water-Soluble Inhibitors 182 7.4.2 Enzyme Assays of Inhibitors Designed to be Water Soluble 183 7.4.3 Structural Analysis 184 7.4.4 Lessons Learnt from the Cocrystal Structure 185 7.5 Conclusions and Outlook 185 7.5.1 Conclusions 185 7.5.2 Outlook 186
Acknowledgments 186 List of Abbreviations 186 References 187
8 Drug–Membrane Interactions: Molecular Mechanisms Underlying Therapeutic and Toxic Effects of Drugs 191 Marlene Lucio, Jose L. F. C. Lima, and Salette Reis
8.1 Biological Membranes 191 8.1.1 Role of Membranes in Life Maintenance 191 8.1.2 Structure and Composition of Membranes 191 8.1.3 Dynamic Molecular Organization of Membranes 193 8.2 Drug–Membrane Interactions 195 8.2.1 Possible Effects of Drugs on Membranes 195 8.2.2 Clinical Relevance of the Drug–Membrane Interaction Studies 195 8.2.2.1 Contribution for Drug Development 195 8.2.2.2 Understanding Therapeutic and Toxic Effect of Drugs 197 8.2.2.3 Understanding Mechanisms of Multidrug Resistance 198 8.2.2.4 Controlling Enzymatic Inhibition 198 8.3 Analysis and Quantification of Drug–Membrane Interactions 199 8.3.1 Membrane Model Systems 199 8.3.2 Experimental Techniques 200 8.4 Drug–Membrane Interactions Applied to the Study of Nonsteroidal
Anti-inflammatory Drugs (NSAIDs) 201 8.4.1 Drug Fundamental Physical–Chemical Studies 202 8.4.2 Membrane Structural and Dynamic Studies 203
X Contents
8.4.3 Results 205 8.5 Conclusions and Future Research Directions 206
9 Targeting Disease with Small Molecule Inhibitors of Protein–Protein Interactions 215 Fedor Forafonov, Elena Miranda, Ida Karin Nordgren, and Ali Tavassoli
9.1 Introduction 215 9.2 High-Throughput Screening of Chemical Libraries 216 9.3 High-Throughput Screening of Biosynthesized Libraries 220 9.3.1 Cyclic Peptide Inhibitors of AICAR Transformylase Activity 222 9.3.2 Cyclic Peptide Inhibitors of HIV Budding 224 9.4 Future Direction 226 9.4.1 Small Molecule Inhibitors of Tumor Hypoxia Response
Network 226 9.4.2 Targeting Protein–Protein Interactions in Asthma 228 9.4.3 Targeting the Protein Interaction Networks of Influenza Virus 230
10 Cracking the Glycocode: Recent Developments in Glycomics 239 Lars Hillringhaus and Jurgen Seibel
10.1 State of the Art 239 10.1.1 Introduction 239 10.1.2 Carbohydrate-Based Drugs 239 10.1.3 Carbohydrate Synthesis 240 10.1.3.1 Chemical Synthesis 241 10.1.3.2 Enzymatic Synthesis 243 10.1.3.3 Glycoprotein Synthesis 244 10.1.4 Glycomics 245 10.1.4.1 Mass Spectrometry 245 10.1.4.2 Microarrays 246 10.1.4.3 Cell, Tissue, and Metabolic Labeling 246 10.1.4.4 Bioinformatics 247 10.2 Some New Insights in Glycomics 247 10.2.1 Microwave-Assisted Glycosylation for the Synthesis of
Glycopeptides 247 10.2.2 Highly Efficient Chemoenzymatic Synthesis of Novel Branched
Thiooligosaccharides by Substrate Direction with Glucansucrases 251 10.2.3 Identification of New Acceptor Specificities of Glycosyltransferase R
with the Aid of Substrate Microarrays 257 10.3 Future Perspectives 259
Part IV Enzyme Chemistry 265
11 O2 Reactivity at Model Copper Systems: Mimicking Tyrosinase Activity 267 Anna Company
11.1 General Introduction: O2 Activation and Model Systems 267 11.2 Copper Proteins Involved in O2 Activation 268 11.2.1 Hemocyanin 269 11.2.2 Tyrosinase 270 11.3 O2 Binding and Activation at Biomimetic Cu Complexes 272 11.3.1 Copper–Dioxygen Adducts 272 11.3.2 Ligand Architecture: Influence on Reactivity toward O2 275 11.3.3 Hydroxylation of Aromatic Rings: Mimicking Tyrosinase Activity 278 11.3.3.1 Intramolecular Aromatic Hydroxylation 278 11.3.3.2 Intermolecular ortho-Hydroxylation of Phenolic Compounds 280 11.4 Concluding Remarks 285
References 286
12 Chirality in Biochemistry: A Computational Approach for Investigating Biomolecule Conformations 293 Adriana Pietropaolo
12.1 Introduction 293 12.1.1 Molecular Chirality in Living Systems 293 12.1.2 Protein Secondary Structures 295 12.1.3 Protein Secondary Structure Assignment 296 12.1.4 Intrinsic Chirality and Protein Secondary Structures 297 12.2 Computational Techniques for Studying Protein Dynamics 297 12.3 Employing Chirality to Analyze Protein Motions 298 12.3.1 The Chirality Index 298 12.3.2 Using Chirality to Understand Protein Structure 300 12.3.3 Chirality Index as a Tool for Monitoring Protein Dynamics 302 12.3.4 Chirality and Circular Dichroism 305 12.4 Perspectives 308
13 Collisional Mechanism–Based E-DNA Sensors: A General Platform for Label-Free Electrochemical Detection of Hybridization and DNA Binding Proteins 313 Francesco Ricci
13.1 Introduction 313 13.2 E-DNA Signaling Mechanism 315 13.3 E-DNA Sensor for DNA Binding Proteins Detection 319
XII Contents
References 324
Index 327
XIII
Preface
The idea of publishing books based on contributions given by emerging young chemists arose during the preparation of the first EuCheMs (European Association for Chemical and Molecular Sciences) Conference in Budapest. In this conference I cochaired the competition for the first European Young Chemist Award aimed at showcasing and recognizing the excellent research being carried out by young scientists working in the field of chemical sciences. I then proposed to collect in a book the best contributions from researchers competing for the Award.
This was further encouraged by EuCheMs, SCI (Italian Chemical Society), RSC (Royal Society of Chemistry), GDCh (Gesellschaft Deutscher Chemiker), and Wiley-VCH and brought out in the book ‘‘Tomorrow’s Chemistry Today’’ edited by myself and published by Wiley-VCH.
The motivation gained by the organization from the above initiatives was, to me, the trampoline for co-organizing the second edition of the award during the second EuCheMs Conference in Torino. Under the patronage of EuCheMs, SCI, RSC, GDCh, the Consiglio Nazionale dei Chimici (CNC), and the European Young Chemists Network (EYCN), the European Young Chemist Award 2008 was again funded by the Italian Chemical Society.
In Torino, once again, I personally learned a lot and received important inputs from the participants about how this event can serve as a source of new ideas and innovations for the research work of many scientists. This is also related to the fact that the areas of interest for the applicants cover many of the frontier issues of chemistry and molecular sciences (see also Chem. Eur. J. 2008, 14, 11252–11256). But, more importantly, I was left with the increasing feeling that our future needs of new concepts and new technologies should be largely in the hands of the new scientific generation of chemists.
In Torino, we received about 90 applications from scientists (22 to 35 years old) from 30 different countries all around the world (Chem. Eur. J. 2008, 14, 11252–11256).
Most of the applicants were from Spain, Italy, and Germany (about 15 from each of these countries). United Kingdom, Japan, Australia, United States, Brazil, Morocco, Vietnam, as well as Macedonia, Rumania, Slovenia, Russia, Ukraine, and most of the other European countries were also represented. In terms of applicants, 63% were male and about 35% were PhD students; the number of
XIV Preface
postdoctoral researchers was only a small percentage, and only a couple of them came from industry. Among the oldest participants, mainly born between 1974 and 1975, several were associate professors or researchers at Universities or Research Institutes and others are lecturers, assistant professors, or research assistants.
The scientific standing of the applicants was undoubtedly very high and many of them made important contributions to the various symposia of the 2nd EuCheMs Congress. A few figures help to substantiate this point. The, let me say, ‘‘h index’’ of the competitors was 20, in the sense that more than 20 applicants coauthored more than 20 publications. Some patents were also presented. Five participants had more than 35 publications, and, h indexes, average number of citations per publication, and number of citations, were as high as 16, 35.6, and 549, respectively. Several of the papers achieved further recognition as they were quoted in the reference lists of the young chemists who where featured on the covers of top journals. The publication lists of most applicants proudly noted the appearance of their work in the leading general chemistry journals such as Science, Nature, Angewandte Chemie, Journal of the American Chemical Society, or the best niche journals of organic, inorganic, organometallic, physical, analytical, environmental, and medicinal chemistry.
All of this supported the idea of publishing a second book with the contributions of these talented chemists.
However, in order to have more homogeneous publications and in connection to the great number of interesting papers presented during the competition, we decided to publish three volumes.
This volume represents indeed one of the three edited by inviting a selection of young researchers who participated in the European Young Chemist Award 2008. The other two volumes concern the two different areas of synthetic chemistry and nanotechnology/materials-science and are, respectively, entitled ‘‘Ideas in Chemistry and Molecular Science: Advances in Synthetic Chemistry’’ and ‘‘Ideas in Chemistry and Molecular Sciences: Advances in Nanotechnology, Materials, and Devices’’.
It is important to mention that the contents of the books are a result of the work carried out in several topmost laboratories around the world both by researchers who already lead their own group and by researchers who worked under a supervisor. I would like to take this occasion to acknowledge all the supervisors of the invited young researchers for their implicit or explicit support to this initiative that I hope could also serve to highlight the important results of their research groups.
The prospect of excellence of the authors was evident from the very effusive recommendation letters sent by top scientists supporting the applicants for the Award.
A flavor of these letters is given by the extracts from some of the sentences below: ‘‘The candidate creativity in polymer design for therapeutic impact is extremely
impressive. I believe that he is an excellent example of an outstanding young European chemist’’; ‘‘Is an energetic, enthusiastic, articulate, bright researcher’’; ‘‘Talented and enthusiastic scientist who has a lot of determination and persistence. I was impressed by the strong preparation, enthusiasm and motivation. First rate
Preface XV
researcher’’; ‘‘. . .outstanding, very broad and far reaching research achievements’’; ‘‘The personality and the scientific research achievements will certainly pave to the candidate the way as a future leader of an independent and creative research group. Due to his original and uncommon approach together with the high quality of results, he will become well known in the field of medicinal chemistry. All this exciting work relied on his ability as a chemist and on his bright intellect, which allows him to move across disciplines seamlessly’’; ‘‘The candidate is a very articulate, knowledgeable, driven, clear minded, and extremely personable young scientist’’; ‘‘Application in this work has been outstanding, strong, powerful and very dedicated. The candidate will be in a very good position to make tremendous impact in the area of therapeutics and to become a leader in the area of organic chemistry in the next generation’’; ‘‘As one of the most talented graduate students to have emerged for our group he has the vision, skill and drive to make a real difference to the national research community’’; ‘‘. . .outstanding young scientist and a charming person’’; ‘‘. . .has an astonishing level of productivity’’; ‘‘. . .work of very high caliber’’; ‘‘Such deep and insightful investigations are refreshing and invigorating in my view especially in times that tend to favor simple, phenomenological results’’; ‘‘The candidate has been the intellectual driver and forged the necessary collaborations to address this complex problem. He deft integration of the various experimental and theoretic disciplines needed for the work is impressive’’; ‘‘The candidate performed outstanding research projects’’; ‘‘He should be a compelling and deserving candidate of the European Young Chemist award’’; ‘‘The candidate is for sure one of the most outstanding and promising young researchers that I know in the field of molecular science. Despite being young, he has a lot of experience and high motivation. He is excellent in the lab and extremely smart person’’; ‘‘The candidate has performed work of top quality and originality and of great breadth’’; ‘‘. . .a special individual who ranks among the very best’’; ‘‘Exceptional in many respect. The candidate commands superior experimental and intellectual skills and has shown great chemical structure intuition in the project’’. ‘‘. . .outstanding scientific debater. The personality is impeccable’’. ‘‘Extremely gifted young researcher who has already developed an outstanding track record of achievement while working in one of the world’s premier laboratories in the USA’’; ‘‘Has the skills, expertise and leadership potential to discover new compounds that will revolutionize the therapy of some difficult diseases. The work is well-aligned to the University’s strategy of supporting cross disciplinary research of the highest quality’’; ‘‘. . .unusually broad dimension of experimental skills’’; ‘‘This is a rare combination of expertise’’; ‘‘enables to undertake a diverse scope of key problems. In my opinion these are the type of individuals who will drive the field forward’’; ‘‘. . .has an engaging, outgoing, attractive personality to which others instinctively respond’’; ‘‘Brilliant young chemist with a great potential’’.
The chapters written by the various contributors cover several areas of life science and range from more or less typical biochemical studies, to research relevant in the therapeutic field, to the enzyme or drug activity, to drug delivery, to computational structure–activity relation or sensors.
XVI Preface
In the area of biochemical studies, the book collects a contribution in the field of inorganic chemistry of the brain with the chapter on the role of metal ions (Cu(II) in particular) and the ubiquitin–proteasome system on neurodegenerative disorder (Arnesano). In another chapter, the strategies for chemical modification of proteins as well as selective installation of biochemical probes and tethering of therapeutic cargo to proteins are highlighted (Bernardes et al.). Another chapter (Ribas et al.) is centered on Cu(III) ion and its bioinorganic and organometallic chemistry. In particular, this contribution turns out to be relevant for understanding copper-dependent metalloenzymes.
In the drug delivery domain, a chapter is dedicated to vitamin B12 as potential targeting molecule for therapeutic drug delivery (Ruiz Sanchez). This potentiality is vividly illustrated by the author with the following definition: vitamin B12 is an attractive ‘‘Trojan horse’’ for therapeutic drug delivery.
A second contribution starts from the consideration that the cellular membrane poses a formidable barrier to the intracellular flux of materials circulating extra- cellularly due to its selective permeability based predominantly on ionic charge, hydrophobicity, and size. As such, many drugs, nucleic acids, proteins, and other investigative constructs are not able to translocate the cellular membrane alone and often require a delivery vehicle for function and/or activity. After consid- ering various vehicles this contribution focuses on a highly competitive cellular delivery technology based on polymeric microspheres-mediated cellular delivery (Sanchez-Martin). In particular, the author presents different strategies that can be applied for the attachment of biomolecules to the microspheres and their applications as a delivery system. Monodispersed populations of robust cross-linked microspheres of defined sizes (from 200 nm to 2 µm) have been synthesized and standard solid phase multistep protocols have been applied to them.
Several contributions are primarily dedicated to research that has relevance to the therapeutic field.
The chapter (Lucio et al.) on molecular mechanisms, underlying therapeutic and toxic effects of drugs, highlights the importance of membrane composition and the dynamic molecular organization of membranes in drug–membrane interaction. Also, the work aims to exemplify an application of drug–membrane interaction studies in the evaluation of the nonsteroidal anti-inflammatory drug effect on membranes. These studies may prove valuable in the design of novel drug formulations with increasing efficacy and reduced side effects.
A second contribution in the area deals with targeting diseases with small molecule inhibitors of protein–protein interactions (Tavassoli et al.). The chapter starts from the consideration that there are several thousand protein–protein interactions that control the majority of biological processes. There is therefore great potential for small molecule therapeutics that affect disease through modulation of protein–protein interactions. In this chapter, there is then a discussion on recent approaches taken toward controlling protein interactions with small molecules, from logical design using structural data to high-throughput screening of chemical and biological libraries. The authors go on to outline their own efforts in this
Preface XVII
field, and end with a detailed discussion of some of the important protein–protein interactions currently being targeted.
Another chapter (Hirsh) is dedicated to the design and synthesis of inhibitors of the kinase IspE as potential antimalarians opening a number of research avenues summarized in the chapter itself, while another contribution (Hillringhaus) deals with recent developments in glicomics – a technology that can be anticipated that it will strongly influence tomorrow’s therapy in the areas of various diseases.
A last contribution (Bald) in this area deals with the important studies on the electron-induced modification of biomolecules, which may have potential implications for the design of new chemotherapeutic and radiosensitizing drugs and for the development of more efficient protocols in cancer therapy.
Understanding how nature works in the area of enzyme chemistry is the goal of a chapter (Company) dedicated to the tyrosinase reactivity. In this chapter, in particular, it is shown how the model chemistry approach has allowed to reproduce and to better understand the mechanisms by which O2 is activated in the dinuclear copper protein just called tyrosinase.
The studies summarized in another chapter (Ricci) refer to electrochemical biosensors, termed E-DNA sensors, that are based on the target binding–induced folding of electrode-bound DNA probes and gives contribution on E-DNA signaling mechanisms. In addition, studies on E-DNA sensors for DNA binding protein detection are also reported. The E-DNA sensing platform has then demonstrated to be not only a promising and appealing approach for the sequence-specific detection of DNA and RNA but also to be flexible enough to be adaptable for the detection of DNA–protein interaction.
Taking into account that chirality selection is a key issue in many important biochemical phenomena such as protein folding and enzyme recognition, another chapter discusses the role of chirality in the chemistry of the life sciences. In particular, a novel methodology for the study of local chirality is presented, which provides one with a deeper understanding of the connection between secondary structure and protein flexibility. This computational study for investigating biomolecule con- formation reported in the chapter (Pietropaolo) suggests that chirality is a central organizing principle in life that confers order at every length scale up to the full cell.
Probably, everyone who reads this book will have their own opinion on what is relevant for the future of life chemistry, and in this respect I would like to notify that, due to its peculiar genesis, the book reflects the opinions of a select group of young chemists and therefore does not pretend to cover the whole area.
The main aim is just to offer a variety of individual views that will provoke thought possibly giving an attractive insight into the minds and research areas for the next generation of chemical and molecular sciences, especially those related with life sciences and technologies.
Starting from this point, I hope that the many ideas that can be grasped sailing through the various contributions by the young authors of the book should be very useful for helping to make several step forward in the field of chemistry and molecular sciences to solve problems related to our health or more generally to our
XVIII Preface
life as well as to discover new tools in medicinal chemistry needed for the present and the future society.
I cannot end this preface without acknowledging all the authors and the persons who helped me in the book project together with all the societies (see the book cover) that motivated and sponsored the book. I’m personally grateful to Professors Giovanni Natile, Francesco De Angelis and Luigi Campanella for their motivation and support in this activity.
Palermo, October 2009 Bruno Pignataro
XIX
List of Contributors
Lois M. Alexander University of Edinburgh Chemical Biology Section School of Chemistry Joseph Black Building West Mains Road Edinburgh EH9 3JJ Scotland
Fabio Arnesano University of Bari ‘‘A. Moro’’ Department of Farmaco-Chimico Via E. Orabona 4 70125 Bari Italy
Ilko Bald Freie Universitat Berlin Institut fur Chemie und Biochemie–Physikalische und Theoretische Chemie Takustraße 3 D-14195 Berlin Germany
and
Interdisciplinary Nanoscience Center (iNANO) Aarhus University Ny Munkegade 8000 Aarhus C Denmark
Goncalo J. L. Bernardes University of Oxford Department of Chemistry Chemistry Research Laboratory 12 Mansfield Road Oxford OX1 3TA United Kingdom
Mark Bradley University of Edinburgh Chemical Biology Section School of Chemistry Joseph Black Building West Mains Road Edinburgh EH9 3JJ Scotland
Alicia Casitas Universitat de Girona Department de Qumica Grup de Qumica Inorganica i Supramolecular (QBIS) Campus Montilivi 17071 Girona – Catalonia Spain
Justin M. Chalker University of Oxford Department of Chemistry Chemistry Research Laboratory 12 Mansfield Road Oxford OX1 3TA United Kingdom
XX List of Contributors
Anna Company Technische Universitat Berlin Institut fur Chemie. Sekretariat C2 Straße des 17.Juni 135 D-10623 Berlin (Germany)
Benjamin G. Davis University of Oxford Department of Chemistry Chemistry Research Laboratory 12 Mansfield Road Oxford OX1 3TA United Kingdom
Fedor Forafonov University of Southampton School of Chemistry University Road, Highfield Southampton SO17 1BJ United Kingdom
Lars Hillringhaus University of Oxford Department of Chemistry Chemistry Research Laboratory 12 Mansfield Road Oxford OX1 3TA United Kingdom
Ida Karin Nordgren University of Southampton School of Chemistry University Road, Highfield Southampton SO17 1BJ United Kingdom
Anna Katharina Herta Hirsch Universite de Strasbourg Institut de Science et d’Ingenierie Supramoleculaires 8 Allee Gaspard Monge Strasbourg 67000 France
Jose L. F. C. Lima Universidade do Porto Faculdade de Farmacia Requimte, Servico de Qumica-Fsica Rua Anbal Cunha, 164 Porto 4099-030 Portugal
Marlene Lucio Universidade do Porto Faculdade de Farmacia Requimte, Servico de Qumica-Fsica Rua Anbal Cunha, 164 Porto 4099-030 Portugal
Juan Manuel Cardenas-Maestre University of Edinburgh Chemical Biology Section School of Chemistry Joseph Black Building West Mains Road Edinburgh EH9 3JJ Scotland
Elena Miranda University of Southampton School of Chemistry University Road, Highfield Southampton SO17 1BJ United Kingdom
List of Contributors XXI
and
ETH Zurich Department of Chemistry and Applied Biosciences Institute of Computational Science USI Campus Via Giuseppe Buffi 13 CH-6900 Lugano Switzerland
Salette Reis Universidade do Porto Faculdade de Farmacia, Requimte, Servico de Qumica-Fsica Rua Anbal Cunha, 164 Porto 4099-030 Portugal
Xavi Ribas Universitat de Girona Department de Qumica Grup de Qumica Inorganica i Supramolecular (QBIS) Campus Montilivi 17071 Girona – Catalonia Spain
Francesco Ricci University of Rome Tor Vergata Dipartimento di Scienze e Tecnologie Chimiche Via della Ricerca Scientifica 00133 Rome Italy
Pilar Ruiz-Sanchez Universitat Zurich Anorganisch-chemisches Institut Winterthurerstr. 190 8057 Zurich Switzerland
Rosario M. Sanchez-Martin University of Edinburgh Chemical Biology Section School of Chemistry Joseph Black Building West Mains Road Edinburgh EH9 3JJ Scotland
Jurgen Seibel University of Wurzburg Institute of Organic Chemistry Am Hubland D-97074 Wurzburg Germany
Ali Tavassoli University of Southampton School of Chemistry University Road, Highfield Southampton SO17 1BJ United Kingdom
1
Part I Biochemical Studies
3
1 The Role of Copper Ion and the Ubiquitin System in Neurodegenerative Disorders Fabio Arnesano
1.1 Introduction
Ubiquitin (Ub) plays a crucial role in intracellular protein degradation via the proteasome and the autophagy–lysosome pathways [1]. Failure to eliminate misfolded proteins can lead to the formation of toxic aggregates and cell death [2]. Insoluble protein aggregates enriched with Ub are a hallmark of most neurodegenerative disorders including Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis and prion diseases [3, 4]. All of these disorders have been linked to metal accumulation and disturbance of redox and metal homeostasis in the brain [5–7], and metal ions have been implicated in the aggregation of disease-related, amyloidogenic proteins [8–12]. The potential role of metal ions in the aggregation of Ub has recently been examined [13, 14]. CuII is different from ZnII, NiII, AlIII, or CdII
in that it binds to the N-terminal end of Ub, destabilizes the protein, and promotes its oligomerization into spherical particles. By mimicking the condition of low dielectric constant experienced near a membrane surface, the assembly of spherical oligomers of Ub yields a series of intermediate species leading to an extended nonfibrillar filament network. Aggregate disassembly is triggered by CuII
chelation or reduction [14]. Intermediate annular and porelike structures, stabilized by the interaction of CuII-induced Ub oligomers with lipid bilayers, resemble toxic protofibrillar species produced by amyloidogenic proteins, which cause membrane permeabilization and disruption of metal homeostasis [15–19]. Susceptibility to aggregation of Ub represents a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates. CuII binding and proximity to biological membranes appear to dramatically increase the aggregation propensity of Ub and other disease-related proteins, thus emphasizing the importance of preserving cellular compartmentalization and metal homeostasis for the correct functioning of protein degradation systems. Recent findings reinforce the vision of metal ions as key factors and promising therapeutic targets in protein confor- mational disorders [20, 21]. New strategies are being developed that will help to investigate their functional and pathogenic interactions in vivo.
4 1 The Role of Copper Ion and the Ubiquitin System in Neurodegenerative Disorders
1.2 Metal Ions in the Brain
The brain is a specialized organ that controls cognitive and motor functions. To carry out its functions the brain requires the highest concentrations of metal ions in the body and the highest per-weight consumption of body oxygen [22].
Metal ions in the brain fulfill catalytic and structural roles, which include the stabilization of biomolecules (e.g., MgII in nucleic acids, ZnII in Zn-finger transcription factors) or dynamic processes (e.g., NaI and KI in ion channels, CaII
in neuronal cell signaling) [23]. The dynamic partitioning of these metal ions is controlled by ion-specific
channels that selectively allow passage of ions in and out of cells. In the brain, the uneven distribution of NaI and KI ions across a cell membrane creates a potential that enables transmission of nervous pulses. CaII is also a key modulator of molecular information transfer within and between cells during neurotransmission; most eukaryotic cells either export or store CaII within membrane-enclosed vesicles to maintain cytosolic- free CaII levels at 100–200 nM, roughly 10 000-fold less than in the extracellular space [23].
More recently, considerable attention has been directed to the role of transition metal ions in the brain [22, 24]. Zn, Fe, Cu, and related d-block metals are emerging as significant players in both neurophysiology and neuropathology, particularly with regard to aging and neurodegenerative diseases [25]. Relatively high concentrations of these d-block metals are present within the different cellular compartments, the values ranging from 100 to 1000 µM [22]. The metal concentrations in brain tissue are up to 10 000-fold higher than those in common neurotransmitters and neuropeptides. Not only do these metals serve as components of various proteins and enzymes essential for normal brain function, but, in the labile form, are also involved in specialized brain activities; therefore, if misregulation of their homeostasis occurs, toxicity, mediated also by oxidative stress in the case of Cu and Fe [26], could ensue. Oxidative stress has been identified in many neurodegenerative diseases, and is commonly associated with increased levels of at least one of these transition metal ions in specific brain regions [27].
Transporters for Cu, Zn, Fe, and Mn play an important part in the intracellular distribution of these metals [28], such that defects in their regulation, which could possibly occur with aging, may create an environment that could result in protein misfolding and aggregation, thereby accelerating degenerative conditions [2, 29, 30]. Notably, brain homeostasis of metals is intertwined with changes in one metal leading to changes in the levels of other metals [24]. This is well established for Cu and Fe, where decreased Cu bioavailability may result in altered Fe levels, and for Fe and Mn, where Fe deficiency leads to a significant increase in brain Mn.
The following discussion will be focused on basic aspects of brain Cu homeostasis. The widespread distribution and mobility of Cu required for normal brain function, along with the numerous correlations between Cu misregulation and a variety of neurodegenerative diseases, have prompted interest in studying its roles in neurophysiology and neuropathology [26, 31].
1.3 Brain Copper Homeostasis 5
1.3 Brain Copper Homeostasis
Copper is the third-most abundant transition metal in the brain, after Fe and Zn, with average neuronal Cu concentrations of ∼0.1 mM. This redox-active nutrient is distributed unevenly within brain tissue, as Cu levels in the gray matter are two- to threefold higher than those in the white matter. Cu is particularly abundant in the locus coeruleus (1.3 mM), the neural region responsible for physiological responses to stress and panic, as well as the substantia nigra (0.4 mM), the center for dopamine production in the brain [32]. The major oxidation states for Cu ions in biological systems are cuprous CuI and cupric CuII; the former is more common in the reducing intracellular environment, and the latter is dominant in the more oxidizing extracellular environment [33]. Levels of extracellular CuII
vary from 10−25 µM in blood serum, 0.5−2.5 µM in cerebrospinal fluid (CSF), and 30 µM in the synaptic cleft. Intracellular Cu levels within neurons can reach concentrations higher than 2–3 orders of magnitude [32]. Like Zn and Fe, brain Cu is partitioned into tightly bound and labile pools. Owing to its redox activity, Cu is an essential cofactor in numerous enzymes that handle the chemistry of oxygen or its metabolites, including cytochrome c oxidase (CcO), Cu, Zn superoxide dismutase (Cu,Zn-SOD1), ceruloplasmin (CP), dopamine β
monooxygenase (DβM), peptidylglycine α-hydroxylating monooxygenase (PHM), and tyrosinase [31].
Because of its propensity to trigger aberrant redox chemistry and oxidative stress when unregulated, the brain maintains strict control over its Cu levels and distributions. An overview of homeostatic Cu pathways in the brain is given in Figure 1.1.
Many of the fundamental concepts for neuronal Cu homeostasis are derived from studies in yeast, but the brain provides a more complex system with its own unique and largely unexplored inorganic physiology. There is little ‘‘free’’ Cu in the yeast cytoplasm, which is due to the tight regulation of metallochaperones [35, 36]; however, many open questions remain concerning the homeostasis of organelle Cu stores, particularly in higher organisms with specialized tissues. Some data suggest that yeast and mammals possess pools of labile Cu in the mitochondrial matrix [37].
Uptake of Cu by the blood–brain barrier (BBB) is considered to occur through the P-type ATPase ATP7A, which can pump Cu into the brain [38]. Mutations in the related gene lead to Menkes disease, an inherited neurodegenerative disorder that is globally characterized by brain Cu deficiency. This phenotype is mirrored by Wilson disease, which involves mutations in the ATP7B gene responsible for excretion of excess Cu from the liver into the bile. Loss of ATP7B function leads to abnormal increase of Cu in the liver [39].
The extracellular trafficking of brain Cu differs from that in the rest of the body. CSF, the extracellular medium of the brain and central nervous system, possesses a distinct Cu homeostasis from blood plasma, which carries Cu to organs in the rest of the body. Cp, a multicopper oxidase that is essential for Fe metabolism, is
Cu+ Cu2+
Presynaptic cell
Endosome
Figure 1.1 A schematic model of neuronal copper homeostasis. Reprinted with permission from [34]. Copyright 2008 American Chemical Society.
the major carrier of CuII in the plasma, but houses less than 1% of Cu in CSF [32]. The primary protein or small-molecule ligands for Cu in CSF remain unidentified. Uptake of Cu into brain cells requires reduction of CuII to CuI. Steap proteins may fulfill this role like the yeast ferric and cupric reductases Fre1 and Fre2 [40]. Following reduction, CuI ions can be transported into cells through a variety of trafficking pathways [41, 42].
A major class of proteins involved in cellular Cu uptake is the copper transport (Ctr) protein family. Ctr1 is a representative member that is ubiquitously expressed. It resides predominantly in the plasma membrane and is essential for the survival of mammalian embryos and for Cu import into neurons and astrocytes [43]. Elevated Cu stimulates rapid endocytosis and degradation of Ctr [44]. Ctr1 contains three transmembrane helices, an N-terminal extracellular domain, and a C-terminal cytosolic domain. Electron crystallography revealed that Ctr1 is trimeric and possesses the type of radial symmetry associated with the structure of certain ion channels [45]. A region of low protein density at the center of the trimer is consistent with the existence of a Cu permeable pore. Mutagenesis studies have established that a methionine(Met)-rich motif in the N-terminal domain and a Met-rich motif at the extracellular end of the second transmembrane helix of Ctr1 play a pivotal
1.3 Brain Copper Homeostasis 7
role in the mechanism of Cu uptake [46, 47]. The mechanisms of Cu translocation across cellular membranes, however, remain largely unknown.
In addition to Ctr1, prion protein (PrP) and amyloid precursor protein (APP) are two other abundant Cu-binding proteins, found specifically at brain cell sur- faces, implicated in Cu uptake/efflux [48, 49]. In particular, PrP is localized in synaptic membranes of presynaptic neurons. Mammalian PrP contains at least four octapeptide repeats in the N-terminal region that can bind CuII. Millimolar concentrations of CuII induce endocytosis of PrP, suggesting that PrP may act as a buffer for Cu in the synaptic cleft, maintaining presynaptic Cu concentrations while preventing CuII-related toxicity in the extracellular space [49, 50].
Upon its entry into brain cells, CuI can be funneled to its ultimate intracellular destinations through the use of Cu chaperone proteins or buffering by metalloth- ioneins (MTs), such as MT1 (ubiquitously expressed) and MT3 (expressed in the brain) [51]. The metallochaperones function not only as intracellular Cu delivery proteins but also as protective agents against toxicity resulting from unbound and unregulated Cu ions [35, 36].
Three human Cu chaperones have been characterized so far: Atox1, CCS, and Cox17. Atox1 loads CuI into the Menkes and Wilson P-type ATPases, ATP7A and ATP7B, which mediate Cu delivery to the secretory pathway from the trans-Golgi network (TGN) to the plasma membrane [41]. Both Atox1 and ATP7A/B contain CXXC sequence motifs that are essential for CuI binding and exchange of CuI
between the two partner proteins [52]. The combination of available structural and biochemical data suggests a docking model that involves CuI transfer through two- and three-coordinate intermediates [53–56].
ATP7A and ATP7B play multiple roles in neurons from the delivery of Cu to cuproenzymes involved in neurotransmitter synthesis and metabolism, such as DβM, to the removal of excess Cu via secretion or vesicular sequestration [39]. To carry out this function, ATP7A undergoes Cu-stimulated translocation from the Golgi to the plasma membrane [57]. Metabolic studies also revealed that translocation of ATP7A after N-methyl d-aspartate (NMDA) receptor activation is associated with rapid release of Cu from hippocampal neurons [58, 59], a finding that suggests a role for Cu in the modulation of synaptic activity [60].
The copper chaperone for superoxide dismutase (CCS) inserts Cu into SOD [61]. Cu,Zn-SOD1 is a ubiquitous component of the cellular antioxidant system, which catalyzes disproportionation of the superoxide anion to oxygen and hydrogen peroxide [62]. Active Cu,Zn-SOD1 is a dimer; each subunit binds one Cu and one Zn ion, and contains an intramolecular disulfide bond [63]. On the contrary, in the immature, apo form of SOD1, cysteines are in the reduced state and the protein is a monomer [64, 65]. CCS docks with and transfers the Cu ion to the latter form of SOD [66], and also catalyzes disulfide bond formation [67, 68]. CCS is made of three domains: the N-terminal domain I has a fold similar to Atox1 and contains a conserved CXXC motif, domain II has a fold similar to SOD1 and participates in target recognition, domain III is constituted by ∼30 residues and contains a CXC Cu-binding motif [69]. While domain III is essential for CCS activity in vivo, the requirement for domain I is only apparent under Cu-limiting conditions [69].
A third Cu chaperone, Cox17, is involved in Cu delivery to CcO in mitochondria [70, 71]. CcO is a 13-subunit complex embedded in the mitochondrial inner membrane and a key component of the respiratory chain that reduces oxygen to water [72]. Two Cu ions form a dicopper cluster, designated CuA, in a CcO subunit, while a third Cu ion, designated CuB, forms a dinuclear site with heme a in another CcO subunit [72]. Cox17 acts as CuI donor for Sco1 and Cox11 [73]. Sco1, in turn, transfers Cu to CuA [74], while Cox11 is involved in CuB assembly [75]. Cox17 contains two CX9C motifs implicated in two intramolecular disulfide bonds [76] and a conserved CC motif essential for CuI binding [77]. Fully reduced Cox17 binds up to four CuI ions in a polycopper cluster and undergoes oligomerization [76, 78].
1.4 Brain Copper and Neurodegenerative Disorders
Disruption of Cu homeostasis is implicated in a number of neurodegenerative diseases, including Alzheimer’s disease (AD), prion diseases, Parkinson’s disease (PD), familial amyotrophic lateral sclerosis (ALS), Menkes disease, and Wilson disease [5]. In all these disorders, the deleterious effects of Cu stem from its dual abilities to bind ligands and trigger uncontrolled redox chemistry. A dominant risk factor associated with most neurodegenerative diseases is increasing age. A positive correlation with chronic occupational exposure to Cu and other metal ions in industrialized countries has also been recognized [79, 80].
Several studies have reported a rise in the levels of brain Cu from youth to adulthood [32]. However, biologically available Cu levels drop markedly with advanced age and in AD brain [20, 81]. The connection between Cu and AD pathology is due mainly to its reactions with APP and its β-amyloid cleavage product (Aβ), that result in imbalance of extracellular and intracellular brain Cu pools [20]. Aberrant binding of CuII to APP triggers its reduction to CuI with concomitant disulfide bond formation; this intermediate can then participate in reactive oxygen species (ROS) production [82]. Extracellular Aβ deposits from AD brains (amyloid plaques) are rich in Cu, in addition to Zn and Fe [83]. The MT3, released in the synaptic cleft by neighboring astrocytes, has the potential to ameliorate this adverse interaction, but is downregulated in AD [84]. Moreover, the β-secretase β-site of amyloid precursor protein cleaving enzyme (BACE1), involved in APP cleavage, possesses a CuI-binding site in its C-terminal cytosolic domain through which it interacts with domain I of CCS, indicating that intracellular Cu levels can have an impact on Aβ generation [85]. Altered brain Cu distribution in AD, with abnormal accumulation of Cu in amyloid plaques and Cu deficiency in neighboring cells, is accompanied by a loss of Cu-dependent enzymes (e.g., CcO, Cu,Zn-SOD1, CP). Therefore, administration of Cu chelators such as clioquinol, that can reverse Aβ
aggregation and redistribute brain Cu pools acting as ionophores, can have dual beneficial effects [20]. It is also found that Cu-bound clioquinol and other Cu complexes can exhibit proteasome-inhibitory abilities [86, 87].
1.5 The Role of Ubiquitin in Protein Degradation 9
Prion diseases are also linked to brain Cu misregulation, where opposing CuII and MnII levels may influence the conversion of PrP into the toxic, protease-resistant form, PrPSc [88, 89]. PrP may act as a Cu-chelating agent, when extracellular Cu reaches high concentration peaks (15–300 µM) such as during synaptic transmission and depolarization [8]. Another hypothesis is that the binding of Cu to PrP could act directly to detoxify ROS, performing SOD-like activity [90]. In one pro- posal for prion toxicity, excess free Cu further exacerbates the disease by promoting oxidative stress [91].
Onset of PD is accompanied by death of dopaminergic neurons and intracellular accumulation of Lewy bodies [92], which are protein aggregates containing α-synuclein (α-syn), an abundant protein in the brain whose function is still unclear [93]. Monomeric α-syn, which has no persistent structure in aqueous solution, is known to bind anionic lipids [94] with a resulting increase in α-helix structure [95, 96]. Factors including oxidative stress and presence of various metal ions promote its fibrillation [97, 98]. CuII also promotes the self-oligomerization of α-syn [10] and its oxidation and aggregation in the presence of H2O2 [99]. Although α-syn is widely expressed in the brain, inclusions of α-syn are commonly localized in the substantia nigra, locus coeruleus, and cerebral cortex, which are the regions where Cu is abundant [32]. In PD brain, increased levels of Cu are found in the CSF [100].
Familial ALS is an inherited neurodegenerative disorder stemming from mutations in Cu,Zn-SOD [101, 102]. Three main hypotheses exist regarding the molecular mechanisms of this disease: (i) the loss-of-function mechanism, which results in toxic accumulation of superoxide by lack of SOD1 protection, (ii) the gain-of-function mechanism, in which SOD1 exhibits enhanced peroxidase activity by aberrant redox chemistry, and (iii) the aggregation mechanism, where SOD1 aggregates are induced by decreased availability of Cu and Zn ions and are stabilized by intermolecular disulfide bonds [103, 104]. The role of Cu homeostasis in this disease remains uncertain, however molecular machineries controlling redox homeostasis in mitochondria [68] appear to be essential for intramolecular disulfide bond formation and correct metal incorporation into SOD1, two processes involving the CCS metallochaperone [105].
1.5 The Role of Ubiquitin in Protein Degradation
The unique morphology of neurons (with specialized zones for presynaptic neurotransmitter release and postsynaptic receptor activation) and the plasticity of synapses (which is tightly coupled to changes in the synaptic proteome) impose special challenges on the cellular machinery for both protein synthesis and degradation [106]. Protein degradation has important roles in both neuronal development and long-term synaptic plasticity. Moreover, many neurodegenerative diseases are associated with abnormal protein aggregates, implicating degradative dysfunction [4, 107].
Major proteases in eukaryotic cells are confined to specialized protein complexes (proteasomes) and organelles (lysosomes) to prevent nonspecific proteolysis. The ubiquitin-proteasome system (UPS) is responsible for degrading most intracellular, soluble proteins, but it can also degrade transmembrane proteins if they are extracted from the membrane into the cytosol (Figure 1.2). The lysosome degrades most membrane and endocytosed proteins, but it can also digest cytosolic proteins through autophagy (Figure 1.3) [1]. In both cases ubiquitin (Ub) plays a crucial role.
Ub is a small protein of 76 aminoacids folded in a compact globular structure in which a mixed parallel/antiparallel β-sheet packs against an α-helix generating a hydrophobic core [108]. Not found in bacteria, this protein is ubiquitous in eukaryotes and has highly conserved sequences, the human and the yeast proteins differing by only three residues. The remarkable degree of sequence conservation underscores its important physiological role [109].
Ubiquitination is a posttranslational modification that forms an isopeptide bond between a lysine residue on the protein and the C-terminus of Ub. The ubiquitination requires four different classes of enzymes: E1–E4 [110, 111]. First, Ub is covalently conjugated to E1 (Ub-activating enzyme) in an ATP-dependent reaction, and then it is transferred to E2 (Ub-conjugating enzyme). E3 (Ub-protein ligase) then transfers Ub from E2 to the substrate protein and is largely responsible for target recognition through physical interactions with the substrate. After the first Ub has been attached (monoubiquitination), E3 can also elongate the Ub chain by cre- ating Ub–Ub isopeptide bonds. Finally, E4 enzymes (chain elongation factors) are a subclass of E3-like enzymes that only catalyze chain extension (Figure 1.2) [110, 111].
Ub has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), all of which are available and indeed used in vivo for chain extension. The significance of complex ubiquitination patterns is only partially understood: K48 chains lead to degradation of the substrate by the 26S proteasome, whereas monoubiquitination and K63 chains are known to activate cell signals in several pathways including tolerance of DNA damage, inflammatory response, ribosomal protein synthesis, endocytosis, and protein trafficking [111].
The 26S proteasome is a large multisubunit complex of ∼2 MDa, localized in the cytosol and nucleus, and composed of a 20S proteolytic core and one or two 19S regulatory caps. After substrate–proteasome association, deubiquitinating enzymes (DUBs) and ATP-dependent unfoldase activities help the substrate to enter the proteolytic lumen of the 20S core by regenerating monomeric Ub [110, 111]. Notably, the cleavage of the isopeptide bond between the substrate and the most proximal Ub of the polyUb chain requires the metalloprotease activity of a 19S proteasome subunit, which contains a JAMM (JAB1 (Jun activation domain-binding protein-1)/MPN (Mpr1 Pad1 N-terminal domain)/Mov34 metalloenzyme) domain with a coordinated ZnII ion [112, 113].
Ub is also involved in the lysosomal degradation pathway. Lysosomes are organelles that contain acid hydrolases that break down biomolecules. The hydrolases in the lumen of lysosomes (pH 4–5) and late endosomes (pH 5–6) are highly active in acidic environments but loose their activities in the cytosol (pH ∼ 7.2) [114]. Many types of signals can regulate endocytosis and sorting of lysosomes, including
1.5 The Role of Ubiquitin in Protein Degradation 11
E2 E2
Target protein

ideas in chemistry and molecular sciences

Documents