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
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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
Acknowledgments 108 References 109
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
Acknowledgments 135 References 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
Acknowledgments 162 References 163
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
Acknowledgments 206 References 207
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
Acknowledgments 232 References 232
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
Acknowledgments 259 References 260
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
Acknowledgments 309 References 310
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
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
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 ap- plications 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 particu- lar, 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
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
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
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
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 pro- teasome 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 pro- motes 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 propen- sity 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.
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
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
molecu- lar 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 home- ostasis. 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
6 1 The Role of Copper Ion and the Ubiquitin System in
Neurodegenerative Disorders
Cu+ Cu2+
Presynaptic cell
Endosome
Figure 1.1 A schematic model of neuronal copper home- ostasis.
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].
8 1 The Role of Copper Ion and the Ubiquitin System in
Neurodegenerative Disorders
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 transmis-
sion 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
intracel- lular 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 stabi- lized 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 disul- fide 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 neu- rotransmitter 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
degrada- tion [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].
10 1 The Role of Copper Ion and the Ubiquitin System in
Neurodegenerative Disorders
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 ubiquiti- nation 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 tar- get 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 or- ganelles 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