Amyloid Fibrils and Prefibrillar Aggregates E-Book
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- Herausgeber: Wiley-VCH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Summing up almost a decade of biomedical research, this topical and eagerly awaited handbook is the first reference on the topic to incorporate recent breakthroughs in amyloid research.
The first part covers the structural biology of amyloid fibrils and pre-fibrillar assemblies, including a description of current models for amyloid formation. The second part looks at the diagnosis and biomedical study of amyloid in humans and in animal models, while the final section discusses pharmacological approaches to manipulating amyloid and also looks at its physiological roles in lower and higher organisms. For Biochemists, Molecular Biologists, Neurobiologists, Neurophysiologists and those working in the Pharmaceutical Industry.
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Seitenzahl: 950
Veröffentlichungsjahr: 2013
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Table of Contents
Related Titles
Title Page
Copyright
Preface
List of Contributors
Chapter 1: The Amyloid Phenomenon and Its Significance
1.1 Introduction
1.2 The Nature of the Amyloid State of Proteins
1.3 The Structure and Properties of Amyloid Species
1.4 The Kinetics and Mechanism of Amyloid Formation
1.5 The Link between Amyloid Formation and Disease
1.6 Strategies for Therapeutic Intervention
1.7 Looking to the Future
1.8 Summary
Acknowledgments
Chapter 2: Amyloid Structures at the Atomic Level: Insights from Crystallography
2.1 Atomic Structures of Segments of Amyloid-Forming Proteins
2.2 Stability of Amyloid Fibers
2.3 Which Proteins Enter the Amyloid State?
2.4 Molecular Basis of Amyloid Polymorphism and Prion Strains
2.5 Atomic Structures of Steric Zippers Suggest Models for Amyloid Fibers of Parent Proteins
2.6 Atomic Structures of Steric Zippers Offer Approaches for Chemical Interventions against Amyloid Formation
2.7 Summary
Acknowledgments
Chapter 3: What Does Solid-State NMR Tell Us about Amyloid Structures?
3.1 Introduction
3.2 Principles of Solid-State NMR Spectroscopy and Experiments for Structural Constraints
3.3 Amyloid Fibrils Investigated by Solid-State NMR Spectroscopy
3.4 Summary
Chapter 4: From Molecular to Supramolecular Amyloid Structures: Contributions from Fiber Diffraction and Electron Microscopy
4.1 Introduction
4.2 History
4.3 Methodology
4.4 Recent Advances in Amyloid Structure Determination
4.5 Summary
Acknowledgments
Chapter 5: Structures of Aggregating Species by Small-Angle X-Ray Scattering
5.1 Introduction
5.2 Theoretical and Experimental Aspects
5.3 Data Analysis and Modeling Methods
5.4 Studying Protein Aggregation and Fibrillation Using SAXS
5.5 General Strategies for Modeling SAXS Data from Protein Complexes
5.6 Summary and Final Remarks
Acknowledgments
Chapter 6: Structural and Compositional Information about Pre-Amyloid Oligomers
6.1 General Introduction
6.2 Biophysical Techniques to Study Amyloid Oligomers
6.3 The Structure and Composition of Amyloid Oligomers
6.4 Concluding Remarks
Acknowledgments
Chapter 7: The Oligomer Species: Mechanistics and Biochemistry
7.1 Introduction
7.2 The Structure–Toxicity Relation of Early Amyloids
7.3 The Oligomer–Membrane Complex
7.4 Biochemical Modifications Underlying Amyloid Toxicity
7.5 Summary
Chapter 8: Pathways of Amyloid Formation
8.1 Introduction
8.2 Nomenclature of the Various Conformational States
8.3 Graphical Representations of the Mechanisms Leading to Amyloid
8.4 Pathways of Amyloid Fibril Formation
8.5 Nucleation Growth versus Nucleated Conformational Conversion
8.6 Summary
Chapter 9: Sequence-Based Prediction of Protein Behavior
9.1 Introduction
9.2 The Strategy of the Zyggregator Predictions
9.3 Aggregation Under Other Conditions
9.4 Prediction of the Cellular Toxicity of Protein Aggregates
9.5 Relationship to Other Methods of Predicting Protein Aggregation Propensities
9.6 Competition between Folding and Aggregation of Proteins
9.7 Prediction of Protein Solubility from the Competition between Folding and Aggregation
9.8 Sequence-Based Prediction of Protein Interactions with Molecular Chaperones
9.9 Summary
Chapter 10: The Kinetics and Mechanisms of Amyloid Formation
10.1 Introduction
10.2 Classical Theory of Nucleated Polymerization
10.3 The Theory of Filamentous Growth with Secondary Pathways
10.4 Self-Consistent Solutions for the Complete Reaction Time Course
10.5 Summary
Chapter 11: Fluorescence Spectroscopy as a Tool to Characterize Amyloid Oligomers and Fibrils
11.1 Introduction
11.2 Fluorescence Spectroscopy for Studies of Amyloid Reactions In vitro
11.3 Cysteine-Reactive Fluorescent Probes
11.5 Luminescent Conjugated Poly and Oligothiophenes LCPs and LCOs
11.6 Summary
Acknowledgments
Chapter 12: Animal Models of Amyloid Diseases
12.1 Introduction
12.2 Some Big Questions Regarding Amyloid Diseases and Some Answers from Animal Models
12.3 Identifying the Toxic Species in the Systemic Amyloidoses
12.4 Identifying the Toxic Species in Alzheimer's Disease
12.5 Infectious Protein Misfolding
12.6 Conclusions
Chapter 13: The Role of Aβ in Alzheimer's Disease
13.1 History of Amyloidosis
13.2 Biochemistry of Aβ
13.3 Amyloid Fibrils
13.4 The Soluble Oligomer Theory of AD
13.5 Other Factors Involved in Amyloid Plaque Formation in AD
13.6 Metal Ions in AD
13.7 Other Potential Aβ Interactions
13.8 Other Neurodegenerative Diseases
13.9 Conclusion
Chapter 14: Experimental Approaches to Inducing Amyloid Aggregates
14.1 The Need for Reproducible Fibrillation Assays
14.2 Setting Up an Assay to Monitor Fibrillation
14.3 Conditions That Promote Protein Aggregation
14.4 Processing and Batch Differences
14.5 Toward High-Throughput Assays
14.6 Summary
Chapter 15: Fibrillar Polymorphism
15.1 Detection of Fibrillar Polymorphism
15.2 The Structural Definition of Fibril Polymorphism
15.3 The Two Classes of Fibril Polymorphism
15.4 How Does Fibrillar Polymorphism Arise?
15.5 The Interconversion of Fibril Polymorphs
15.6 The Biological Implications of Fibril Polymorphism
15.7 Summary
Acknowledgments
Chapter 16: Inhibitors of Amyloid and Oligomer Formation
16.1 Introduction: Amyloidoses versus Neurodegenerative Diseases
16.2 Summary
Chapter 17: Development of Therapeutic Strategies for the Transthyretin Amyloidoses
17.1 Introduction to Transthyretin Structure and Function
17.2 Introduction to Amyloid Diseases in General
17.3 Mechanism of Transthyretin Amyloidogenesis
17.4 Kinetic Stabilization of the Transthyretin Tetramer through Small-Molecule Binding
17.5 Assessment of Diflunisal for Treatment of Transthyretin Amyloidosis
17.6 Tafamidis, the First Approved Drug for Treatment of a Transthyretin Amyloidosis
17.7 Summary
Chapter 18: Hormone Amyloids in Sickness and in Health
18.1 Introduction
18.2 Constitutive vs. Regulated Secretory Pathways
18.3 Secretory Granules Contain Aggregated Cargo
18.4 Secretory Granule Aggregation by Functional Amyloid Formation
18.5 Hormone Amyloids in Disease: Diabetes Insipidus
18.6 Conclusions
Chapter 19: Functional Amyloids in Bacteria
19.1 Introduction
19.2 Functional Amyloids are Common in Nature
19.3 Identification and Characterization of Functional Amyloids
19.4 Functional Bacterial Amyloids Play Many Roles
19.5 Biogenesis and Regulation of Functional Bacterial Amyloids
19.6 Structural Composition of Functional Amyloids
19.7 Assembly Properties of Functional Amyloid In Vitro
19.8 Diversity and Distribution of Functional Amyloid Genes
19.9 Summary
Acknowledgments
Chapter 20: Structural Properties and Applications of Self-Assembling Peptides
20.1 Introduction to Self-Assembling Peptides
20.2 The Principles of Self-Assembling Peptides
20.3 Self-Assembling Peptide Nanofibers
20.4 Diverse Applications of Self-Assembling Peptide Nanofibers Scaffolds
20.5 Summary
Acknowledgments
Chapter 21: Harnessing the Self-Assembling Properties of Proteins in Spider Silk and Lung Surfactant
21.1 Introduction
21.2 Amino Acid Sequences and Amyloid Formation
21.3 Spider Silk and How the Spiders Make It
21.4 Harnessing the Properties of Spider Silk and Its Constituent Proteins
21.5 Biosynthesis of an α-Helix from One of the Most β-Prone Sequences Known
21.6 Anti-Amyloid Properties of the BRICHOS Domain
21.7 Summary
Acknowledgments
Index
Witt, S. N. (ed.)
Protein Chaperones and Protection from Neurodegenerative Diseases
2011
Hardcover
ISBN: 978-0-470-56907-8
Jelinek, R. (ed.)
Lipids and Cellular Membranes in Amyloid Diseases
2011
Hardcover
ISBN: 978-3-527-32860-4
Ramirez-Alvarado, M., Kelly, J. W., Dobson, C. M. (eds.)
Protein Misfolding Diseases
Current and Emerging Principles and Therapies
2010
Hardcover
ISBN: 978-0-471-79928-3
Uversky, V., Longhi, S.
Assessing Structures and Conformations of Intrinsically Disordered Proteins
2010
Hardcover
ISBN: 978-0-470-34341-8
John, V.
BACE
Lead Target for Orchestrated Therapy of Alzheimer's Disease
2010
Hardcover
ISBN: 978-0-470-29342-3
The Editor
Prof. Dr. Daniel Erik Otzen
Aarhus University
iNANO (Interdisciplinary Nanoscience Centre)
Department of Molecular Biology and Genetics
Gustav Wieds Vej 14
8000 Aarhus C
Denmark
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Preface
In amyloid structures, β-strands are arranged neatly in rows to form long regular fibrils so that the β-strands are perpendicular to the fibril axis. This simple arrangement has had a profound impact on our understanding of protein structure, stability and folding. The amyloid structure challenges our established view of proteins in several ways. By demonstrating that amyloid can be formed by essentially all proteins, irrespective of their monomeric folded (“native”) structure under physiological conditions – and that these amyloid folds can also vary in subtle ways depending in both inter-strand and inter-sheet contacts – it calls into question the notion that proteins can only assume one well-defined structure, the native monomeric state. It directs our attention to the consequences of protein misfolding and the vast open spaces of conformational landscapes where proteins can form inappropriate intermolecular contacts if they are not kept under evolutionary constraint. Furthermore, there is a growing realization that the native state may not be as stable as the amyloid state and in fact is only selected because it is normally kinetically more accessible than the amyloid state. As a consequence, Nature needs to safeguard against the “inborn” temptation to lapse into the amyloid fold by providing various amyloid-inhibiting mechanisms, ranging from gatekeeper sequences to a range of different chaperones. At the same time, we are witnessing a growing number of examples of the beneficial uses of amyloid in biological contexts (at least from the perspective of the individual species), ranging from bacterial biofilm to biological warfare and melanosomes to protect us against harmful UV light.
The amyloid concept has taken a roller-coaster ride in the protein funfair. Broadly speaking, it started as an example of rather uninteresting cellular debris and then transformed itself into a dangerous pathological folding aberration before our eyes were opened to the rather bizarre beauty of the limitless arrays of β-strands and their potential in various nanotechnological applications. Along the way, we have had to expand our view of the folding funnel beyond the solipsistic behavior of the monomer to include intermolecular interactions; this leads to the concept of multiple parallel “amyloid folding” aggregation pathways in fierce kinetic competition as well as the intricacies of different nucleation mechanisms and fibrillar rearrangements at different stages of the reaction. To cap it all, Nature is starting to unveil beautiful examples of how to form appropriate amyloid under tight spatio-temporal control using evolutionarily optimized protein sequences and a cohort of ancillary transport and nucleator proteins.
These are exciting times for the amyloid field. We are in the middle of a steep “enlightenment curve” and we can expect many new developments over the next few years. The purpose of this book is to present an up-to-date view of the properties of these amyloids and their precursors from a structural, biophysical and nanotechnological perspective, highlighting current achievements and hopefully spurring new efforts where needed. We start with a general perspective on amyloids by Christopher Dobson, whose thoughts about the “generic” nature of amyloid formation have done much to stimulate research in this area. This is followed by four chapters that focus on the molecular structure of amyloid and their general architecture, as inferred from different techniques. These include X-ray crystallography (David Eisenberg and Michael Sawaya), solid state NMR (Henrike Heise), fiber diffraction and electron microscopy (Louise Serpell and Kyle Morris) and Small Angle X-ray Scattering (Jan Skov Pedersen and Cristiano Oliveira). It will be clear from these chapters how the study of amyloid goes hand in hand with new technological advances, ranging from ultra-focused micro-X-ray beams to high-field NMR magnets, advanced image analysis in cryoelectron microscopy and novel ways to model structures from SAXS spectra. We then proceed to an important pre-fibrillar species, the oligomer – or rather oligomers, since their diversity in structure and activity are clearly highlighted in the chapters by Vinod Subramanian and Niels Zijlstra (oligomer structures) and Massimo Stefani (mechanisms of formation and biochemical properties). This class of quaternary folds plays a central role in the pathology of many aggregation diseases. Their properties, currently the subject of intense research, will hold many important lessons about protein folding and assembly and highlight the multifarious nature of aggregation pathways. The mechanisms of these pathways are described in more detail at the experimental (Fabrizio Chiti and Francesco Bemporad) and analytical-theoretical (Tuomas Knowles and Sam Cohen) level, and constitute beautiful examples of how amyloid folding really has stimulated new approaches in protein biophysics, adapting classical protein folding techniques to the brave new world of protein aggregation. In silico predictions of amyloid and oligomer propensities are described by Michele Vendruscolo and Gian Gaetano Tartaglia, whose many different prediction programmes tie these simple biophysical properties with complex biological phenomena such as toxicity and chaperone interactions. Per Hammarström, Mikael Lindgren and Peter R. Nilsson describe exciting new developments in the ability to monitor the formation of different types of aggregate species, ranging from the oligomer to different types of amyloid. The fluorescent probes which they are developing may well provide the breakthrough we need to thoroughly link up in vitro understanding of the aggregation process with in vivo phenomena. This will have to be combined with a better understanding of how to induce and study amyloid in model organisms, as described succinctly by Damian Crowther and Stanislav Ott. For many people, amyloid is synonymous with Alzheimer's Disease, and this is acknowledged in the chapter by Colin Masters, Blaine Roberts and Tim Ryan, who go through the molecular history of Alzheimer's research, for many years a source of inspiration for the study of many other aggregating proteins. While there is as yet no clinically approved drug that directly interferes with the aggregation of proteins in neurodegenerative diseases, there are naturally many efforts under way using small-molecule compounds and antibodies, and these are described in the chapter by Nikolai Lorenzen, Erich Wanker and Daniel Otzen, while the development of robust fibrillation assays which may form the basis for such trials (and for the study of protein aggregation in general) is treated by Lise Giehm and Daniel Otzen. From a clinical perspective, prospects are even brighter for the treatment of amyloidosis disease. We are privileged to have a contribution from Jeff Kelly, Evan Powers and Colleen Fearns who describe the painstaking biophysical and chemical research leading to Tafamidis, an FDA-approved conformationally stabilizing drug that halts amyloid formation by transthyretin and holds potential for millions of patients. Another exciting aspect of amyloid formation is its polymorphism, i.e., the ability of one species to assume several different amyloid folds. Part of the explanation for this lies in the ability to arrange β-strands in different ways, as a described in Eisenberg and Sawaya's contribution; the chapter by Marcus Fändrich, Melanie Wulff, Jesper Søndergaard Pedersen and Daniel Otzen describes this phenomenon from a structural and mechanistic perspective. We end the book with four chapters that focus on more “useful” aspects of amyloid formation. These include the exciting discovery of the exploitation of the amyloid fold as a physiological storage state by peptide hormones (Carolin Seuring, Nadezhda Nespovitaya, Jonas Rutishauser, Martin Spiess and Roland Riek), the widespread use of amyloid in the bacterial world and the growing insight into how this process is regulated (Morten S. Dueholm, Per Halkjaer Nielsen, Matthew Chapman and Daniel Otzen), the design of self-organizing amyloid peptides and their many medical and technological uses (Shuguang Zhang and Luo Zhongli), and finally the formation and exploitation of amyloid from two very different sources, namely spider silk and lung surfactant (Jan Johansson).
My task as editor has been made all the easier thanks to the eminent contributions from the authors of the 21 chapters in this book, all of which are important players on the amyloid field. Thanks very much for all your efforts in making this book worth reading! It has been an immensely enjoyable and educational process to put this book together. I also thank the Wiley editorial team for approaching me with the idea for a book on amyloid and for their efficient and professional help in the editing process. Finally, my warm thanks to my own research group for providing so many stimuli and continually generating new and fascinating insights into the strange and mysterious world of amyloid.
Aarhus
Daniel Otzen
June 2012
List of Contributors
Chapter 1
The Amyloid Phenomenon and Its Significance
Christopher M. Dobson
1.1 Introduction
Interest in the topic of “amyloid” formation by peptides and proteins has increased dramatically in recent years, transforming it from a puzzling phenomenon associated with a small number of diseases into a major subject of study in disciplines ranging from chemistry and materials science to biology and medicine. The major reason for this explosion of activity is undoubtedly that many of the disorders associated with amyloid formation (see Table 1.1) [1] are no longer rare and somewhat esoteric, as they were even a generation or two ago, but are rapidly becoming the most costly, in terms of health care and social disruption, in the modern world [2]. This change is a consequence of many of these disorders being strongly associated with aging – such as Alzheimer's disease – and with lifestyle and dietary changes – such as type 2 diabetes.
Table 1.1 A selection of some of the major human diseases associated with misfolding and the formation of extracellular amyloid deposits or intracellular inclusions with amyloid-like characteristics. Taken from Ref. [1] in which a more comprehensive list is given.
These two diseases alone are having a remarkable effect on our societies, particularly in those countries where life expectancy is now 75 years or more. Thus, for example, the financial burden of Alzheimer's disease in the world each year is already estimated to be approaching $400 billion [2], and the annual cost to the US economy alone is predicted to exceed $1 trillion in 2050 (Figure 1.1) [3]. In addition, type II diabetes is predicted to bring to an end the steadily rising life expectancy in the most highly developed countries [4] and is now becoming widespread amongst the populations of the developing world. And in a quite different context, the recognition of the ability to convert a wide variety of peptides and proteins into filamentous polymers of extraordinary regularity and remarkable properties has opened up opportunities to develop novel self-assembling materials with a range of different characters and potential functions [5] (Chapters 20 and 21).
Figure 1.1 Predicted costs for care of people with Alzheimer's disease in the USA.
(Source: Alzheimer's Society [3].)
1.2 The Nature of the Amyloid State of Proteins
The amyloid state of a protein, regardless of its amino acid sequence or the structure of its native state (Table 1.1), is typically manifested in the form of thread like fibrils a few nanometers in diameter and frequently microns in length that are rich in β-sheet structure (see below). In addition to its importance in medicine and materials science, the amyloid state of polypeptides is also of fundamental significance because its existence and properties challenge many of the established concepts about the nature of the functional states of proteins, with their rich variety of distinctive three-dimensional structures, and the manner in which they have been selected through the evolution of life forms and living systems [6]. Thus, for example, experiments with a wide range of peptides and proteins in a laboratory environment has led to the realization that the ability to form amyloid structures is not a rare phenomenon associated with a small number of diseases; instead, the amyloid state emerges as an alternative well-defined structural form that can be adopted under at least some circumstances by many, in principle nearly all, polypeptide sequences [1, 6, 7] (Chapter 14).
Like the native states adopted by globular proteins, amyloid structures are highly close packed and highly ordered, but unlike native states they possess a common or “generic” main chain architecture, although the specific details and properties of the structures vary with the composition and sequence of their component amino acid side-chains, as we discuss below. Moreover, there is increasing evidence that the amyloid state might often be more stable than the functional native states of many protein molecules, even under physiological conditions, indicating that the latter may not represent the global energy minima on the free energy surfaces of the corresponding polypeptide chains in living systems but simply metastable states separated from the amyloid form by high activation barriers [8].
A consequence of these findings is that biological systems must have evolved to enable their functional peptides and proteins to remain soluble for prolonged periods of time under normal physiological conditions rather than converting into the amyloid state, except in the relatively small number of cases where this form of protein structure is utilized for functional purposes, ranging from structural templates to molecular storage devices [1, 9, 10] (Chapters 21). Some of these protective mechanisms are undoubtedly encoded in the sequence, notably through the ability of globular proteins to fold into stable and cooperative states, which sequester aggregation-prone regions of the protein in the interior of the molecule and raises the energy barriers to conversion into aggregation-prone species. Indeed, there is evidence that patterns of residues that might favor the amyloid state are commonly selected against during evolution [11, 12] (Chapter 9), and even that the large size of most protein molecules might have the advantage of favoring native folds over the amyloid state [8].
Other protective mechanisms are associated with properties of the cellular environment, such as the existence of molecular chaperones and degradation mechanisms designed to prevent the formation and accumulation of misfolded and aggregated polypeptide chains [13, 14]. Indeed, it is evident that such “housekeeping” mechanisms are vital, not just during protein folding following biosynthesis but at all the various stages in the lifecycles of proteins. This conclusion is supported by the discovery of a number of molecular chaperones that act in extracellular space [15], where many proteins in higher organisms function after being secreted from the cells in which they are synthesized, as well as the very large numbers of different types of chaperones that are now known to exist within the cellular environment where synthesis and folding take place [14].
In order to begin to understand the detailed manner in which biological systems are able to ensure that individual proteins adopt the state appropriate to the needs of the organism under specific circumstances (e.g., to enable them to carry out a specific function or to be targeted for degradation) it is necessary to be able to define the nature and properties of the multiple states that are, in principle, accessible to a given sequence [7, 15]. In particular, to understand how living systems are generally able to avoid the conversion of proteins into the amyloid state in contexts where it can cause disease, it is necessary to define the nature of the amyloid structure, the mechanism by which it forms, and the manner in which it can induce pathogenic behavior [1, 7, 16]. In recent years huge strides have been made in each of these areas. Much of this progress has come from the introduction of new techniques and approaches, many of which have been adapted from methods developed for studying molecular systems in other areas of science, for example, microfluidics and nanotechnology [5, 17], as we discuss below.
1.3 The Structure and Properties of Amyloid Species
Unlike the intricate and widely differing structures formed by proteins when folded into their native states, the amyloid forms of proteins look remarkably similar [1, 18]. Amyloid fibrils examined by electron microscopy (EM) or atomic force microscopy (AFM) are typically thread-like structures that are a few nanometers in diameter but can be microns in length. They are typically composed of a number of protofilaments that twist around each other to form the mature fibril. The core of each protofilament adopts a cross-β structure, in which β-strands are oriented perpendicularly to the axis of the protofilament to form effectively continuous hydrogen-bonded β-sheets running along the length of the fibril (Figure 1.2) [18, 19]. Developments in X-ray diffraction studies of peptide microcrystals [20] (Chapter 2), solid-state NMR spectroscopy [21, 22] (Chapter 3) and cryo-EM [19, 22, 23] (Chapter 4), complemented by other data, for example from real-time small-angle X-ray scattering measurements that provide information about the shapes and populations of different aggregate species present during the process of fibril formation (Chapter 5), have resulted in a steady increase in our detailed knowledge of the molecular structures of amyloid fibrils and reveal that they represent variations on a common theme, as a result of the manner in which the differing side-chains are incorporated into the cross-β fibril architecture that is determined primarily by the properties of the common polypeptide main chain [1, 24]. This generic architecture gives very great stability to the fibrils that are, weight for weight, as strong as steel and highly resistant to degradation by proteolytic enzymes or chemical denaturants [25].
Figure 1.2 Model of one of the polymorphs of the amyloid fibrils formed from insulin as defined from cryo-EM analysis. This particular fibril contains four protofilaments that twist around each other to form the mature fibril. Each of the protofilaments has a pair of nearly flat β-sheets, with the component strands oriented perpendicularly to the main fibril axis. See Chapter 15 for more information about fibrillar polymorphism.
(Reproduced with permission from Ref. [19].)
A wide variety of studies has shown that the process of conversion from a soluble, usually monomeric, state of a protein to the polymeric fibrillar state involves a series of elementary steps and a variety of precursor species, commonly including approximately spherical assemblies that are visible in EM and AFM images, and also a variety of shorter protofibrils whose widths are significantly less than those of the mature fibrils (Figure 1.3) [26]. Studies, in particular by mass spectrometry [27, 28] and single molecule optical methods [29, 30], reveal directly that the initial stages of the aggregation process involve the formation of a broad array of oligomeric species. In at least one case that has been studied, that of α-synuclein associated with Parkinson's disease, such oligomers have been observed to undergo a slow transition between relatively disorganized species to more stable structures that are likely to possess at least a rudimentary cross-β structure and, hence, to be readily able to grow into fibrillar species [30].
Figure 1.3 AFM images of aggregates formed during the conversion of α-synuclein from its soluble monomeric form into amyloid fibrils. The series of images show the appearance initially of approximately spherical oligomers prior to more numerous aggregates that include thin protofibrils and then mature fibrils.
(Reproduced with permission from Ref. [26].)
1.4 The Kinetics and Mechanism of Amyloid Formation
A major advance in our understanding of the nature of amyloid fibrils has come from careful experimental studies of the kinetics of their formation (Chapter 8) along with the application of mathematical methods that allow the individual rates of the various microscopic processes that underlie the overall aggregation reaction to be defined (Chapter 10). These methods have revealed that the typical sigmoidal curve describing the conversion of a soluble protein into the amyloid structure depends not simply on a single primary nucleation step that is then followed by growth and elongation, but normally also involves at least one secondary process that is dependent on the degree of aggregation that has occurred at any given stage in the reaction (Figure 1.4) [31] (Chapter 10). One well-defined example of such a secondary process is fragmentation, where each breakage event doubles the number of growing fibril ends and, therefore, results in a rapid proliferation of fibrillar species [31, 32].
Figure 1.4 (a) Simplified model of the processes involved in the mechanism of formation of amyloid fibrils showing (top to bottom) primary nucleation, growth, and one example of a secondary process (here fibril breakage). (b) A typical sigmoidal curve describing the kinetics of amyloid formation that involves a lag phase, in which primary nucleation occurs, followed by rapid proliferation that is dominated by secondary processes and growth, and then a plateau resulting from the depletion of the soluble form of the protein. Such curves can now be fitted using an analytical expression to extract the microscopic rate constants for the individual steps in the reaction (Chapter 10).
(Reproduced with permission from Ref. [31].)
In addition to this ability to extract the microscopic processes that contribute to the overall aggregation process, developments in microfluidics techniques have enabled additional events to be characterized, including the diffusion of aggregates, a process that represents a crucial step in the spatial propagation of amyloid species [17]. In addition, by varying the volume of the solution in the droplets it has proved possible to observe the primary nucleation step that is essential to initiate the aggregation process and is likely to result in the array of oligomers observed particularly clearly in the single molecule experiments. Such studies also reveal that “molecular confinement” within a small volume, such as will occur in a cell or a cellular compartment, can result in a dramatic reduction in the probability of the initiation of aggregation, hence increasing significantly the kinetic stability of the soluble states of peptides and proteins in biological systems [17].
These structural and mechanistic studies have largely been carried out in the “test tube,” and it is of great interest and importance to be able to relate such studies to the events occurring within living systems, and hence to begin to explore the molecular basis of aberrant phenomena that can lead to disease [33, 34]. Exploration of the mechanism of aggregate propagation through analysis of the reaction kinetics can, in principle, be carried out in living systems in a manner analogous to that described above for “test tube” experiments, provided that parameters such as the concentrations of the various protein species can be monitored. Indeed, application of such a procedure in studies of transgenic mice provides direct evidence that a dominant contribution to mammalian prion propagation results from fragmentation processes [31].
In addition, studies of amyloid formation within relatively simple model organisms, such as fruit flies and nematode worms, also demonstrate that it is possible to relate the findings of detailed experiments that can be carried out in vitro to the events taking place in vivo [35] (Chapter 12). There are also opportunities to apply biophysical techniques, for example, those exploiting fluorescence labeling, directly within living systems, and hence to compare the events occurring in the different environments [36] (Chapter 11). Of particular interest in this context is to understand the ways in which the different types of aggregates interact with the various components of the cell and their consequences for the cellular viability and the onset of disease, as we discuss below.
1.5 The Link between Amyloid Formation and Disease
Although the general link between pathogenicity and the appearance of amyloid deposits in the family of misfolding diseases has been clear for many years, the specific mechanism by which pathogenesis is induced has been the subject of intense debate [1, 37, 38]. For the systemic amyloidoses, the burden of large quantities, literally kilograms in some cases, of fibrillar deposits in vital organs, such as the liver or spleen, is likely to be highly damaging and the major cause of their failure to function normally [1] (Chapter 17). In the case of the neurodegenerative diseases in particular, however, the amyloid burden can be quite low, and there may be little correlation between the quantity of the deposits and the severity of the symptoms [35, 39].
This evidence and the results from a wide variety of experiments over the past decade or so have resulted in the finger of blame, in neurodegenerative disorders at least, being pointed at smaller pre-fibrillar species rather than mature amyloid fibrils [33, 37, 38] (Chapters 6 and 7). Indeed, there is now a mass of evidence that indicates that the oligomeric aggregates discussed above, and which are almost universally observed to be present as intermediates during the interconversion of the soluble and fibrillar forms of peptides and proteins, are “generically” damaging to cells; indeed, such cellular toxicity has been observed to be induced by such oligomeric species both for molecules that are associated with disease and those that are not linked to any known pathology (Figure 1.5) [40].
Figure 1.5 Plot of the effects on cell viability (measured using the MTT test) against the length of time that the protein PI3-SH3 had been incubated (during which it converts into amyloid fibrils) prior to the addition of aliquots of the solution to neuronal cells. The different colors represent different volumes of the added aliquots (i.e., the addition of different concentrations of aggregates), with black being the smallest and dark blue the largest volumes. The zero time point represents the monomeric protein prior to aggregation, and the final time points the addition of solutions containing mature fibrils. The aliquots added after 40 h of aggregation contain the highest concentrations of pre-fibrillar (oligomeric) species, as indicated by EM analysis of the solutions. The data show that the oligomeric species are highly toxic, even when formed from a protein with no connection to any known amyloid disease.
(Reproduced with permission from Ref. [40].)
The origin of such toxicity is likely to arise fundamentally from the fact that these oligomers are inherently misfolded, and therefore have the potential to interact inappropriately with many of the functional components of the highly complex and crowded environments with which the latter have co-evolved; the high surface-to volume ratios of these species relative to larger aggregates will also serve to enhance the ability of a given quantity of misfolded material to generate cellular damage [41]. The observation discussed above of an array of oligomers of different sizes strongly suggests that there is no unique “toxic species” that is formed during the aggregation process; indeed, it is likely that almost any misfolded species will have the potential to generate at least some level of toxicity [42].
It is increasingly clear, however, that different forms of oligomers can differ significantly in the degree of toxicity that they exhibit [30, 43]. Indeed, it seems likely that the oligomers formed initially during the aggregation process are relatively disordered species whose formation is triggered by the drive to sequester hydrophobic and other aggregation-prone regions of unfolded or misfolded polypeptides away from solvent water molecules. In some cases, at least, it then appears that there is a conformational change associated with the formation of precursors to the amyloid cross-β structure that have both enhanced stability and a more highly hydrophobic surface [30, 41, 44]; such a surface is, however, likely to increase the probability of aberrant interactions with other components of the cell, notably membranes. As these oligomeric species grow in size, and ultimately form mature fibrils and plaques, their surface-to-volume ratios will decrease and many of their more hydrophobic regions may be concealed within the larger assembly, giving rise to a relative reduction in their potential to generate pathological effects.
As discussed earlier in this chapter, the generation of misfolded species with the potential to aggregate is an inherent danger at virtually all times during the lifespan of proteins, and so it is no surprise that these hazardous oligomeric species appear to be a primary target for molecular chaperones, which are likely to target hydrophobic regions on their surfaces, either to induce correct refolding or to sequester the misfolded forms of proteins and to target them for degradation [13, 14, 29]. It will then only be under exceptional circumstances when potentially toxic species are free to generate damage, for example, when the quantities of misfolded and aggregation prone species reach a level where they overwhelm the defensive “housekeeping” systems, leading to cellular malfunction and, ultimately, to apoptosis or other forms of cell death. Of particular interest is the possibility that such a situation can lead to a widespread loss of cellular regulation and a more general breakdown of proteostasis [13, 45]; the initial aggregation of one protein may, therefore, lead to a range of downstream processes that contribute substantially to the onset of disease.
That such a situation is very likely to be at least broadly correct relates to the fact that proteins can only be selected during evolution only to be good enough for their purpose. It appears from a range of experimental and theoretical studies that many, at least, of our proteins are close to their “solubility” limit [46] and, therefore, even small changes – induced, for example, by mutation, post-translational modifications, changes of concentration, or a reduced effectiveness of the protective machinery with age – can be enough to permit self-association to be initiated and then lead to the proliferation of aggregates to the levels that generate pathogenicity (Chapter 9). In this context the growing level of understanding of the aggregation process sheds further light on how such a situation is likely to occur. We can speculate, for example, that chaperones may be very effective at neutralizing the species formed by primary nucleation, particularly before any conversion to amyloid-like forms occurs. But once this stage is passed, secondary process, such as fragmentation and aggregate-dependent catalysis of oligomer formation, coupled with aggregate growth, can give rise to both a rapid rise in the quantities of aggregates and a continuing generation of potentially toxic oligomers [47].
1.6 Strategies for Therapeutic Intervention
Amyloid-related disorders differ from other more well known forms of medical conditions, such as bacterial and viral diseases, or even cancer and heart disease, as they are triggered by the failure of control and regulatory processes to prevent individual protein molecules reverting from their functional states to a persistent misfolded state whose interactions can disrupt the normal processes of life. Pharmaceutical intervention will therefore require different strategies from those applied to “conventional” diseases where the selective targeting of specific biological processes, along with other factors such as improved diet and increased standards of hygiene, have proved to be extremely effective in reducing the incidence of disease and also in limiting its effects on the individuals concerned. Ironically, it is advances in the prevention and treatment of these other conditions that has resulted in our increased lifespans that increase dramatically the probability of the onset of amyloid diseases, particularly neurodegenerative conditions such as Alzheimer's disease.
The advances that are being made in understanding the mechanism of aggregation to form amyloid structures, however, offer tremendous opportunities to intervene therapeutically in a rational manner [48, 49] (Chapters 14, and 16). Indeed, there appear to be unique opportunities for such an approach because such therapies need not involve the requirement to affect differentially analogous processes occurring within different types of cells (e.g., bacterial vs human, virally infected vs normal, damaged vs undamaged), but can address the underlying differences between proteins in their functional state and those that are misfolded and which possess fundamentally different properties. Moreover, it is evident that the protective systems that have emerged in biological systems are extraordinarily effective under the conditions for which they have evolved, notably to enable us to live long enough to pass on our genes and look after our offspring, but then become less effective as we age and become a potential burden for the rest of society. Thus, for example, the occurrence of Alzheimer's disease under the age of 65 is less than 1 in 1000, but at ages above 85 is approximately 1 in 3 [2, 3]. These diseases can be considered to be “post-evolutionary,” as they are become epidemic in modern societies where we have effectively moved out of the realm of continuing natural selection [50].
In order to address the treatment or prevention of these diseases it is possible to imagine intervention at different stages of the misfolding and aggregation process, as indicated in Figure 1.6 [48]. There are several general ways in which one can, in principle, perturb individual steps in the aggregation process, and it is interesting to examine the manner in which biology has generated such effective means of avoiding (at least for most of the “three score years and ten” that has traditionally been the maximum lifespan to which any individual might optimistically aspire) the events that lead to a catastrophic breakdown of the ability of organs such as the brain to function.
Figure 1.6 Schematic diagram indicating various stages of the lifecycle of a protein in which the propensity to form toxic oligomeric precursors to amyloid fibrils can potentially be inhibited for therapeutic purposes. Therapeutic strategies include (A) stabilizing the native state; (B) inhibiting enzymes that process proteins into peptides with a propensity to aggregate; (C) altering protein synthesis; (D) stimulating clearance of misfolded proteins, for example, by boosting their proteasomal degradation; (E) inhibiting fibril assembly; and (F) preventing accumulation of fibril precursors.
(Reproduced with permission from Ref. [48].)
It is clear that the primary strategy for therapy should ideally be prevention not cure, as once the initial stages of aggregation have taken place the further proliferation of aggregates is likely to be effectively uncontrollable [47]; one excellent example of such a preventative strategy is that designed for the treatment of the amyloid-related conditions based on the aggregation of transthyretin [51] (Chapter 17). This highly β-sheet-rich protein, whose primary natural mechanism of action is to transport the thyroid hormone thyroxin, is a homotetramer in its native state. But this state can be destabilized by a large number of mutations, and the dissociated monomers are highly aggregation prone, giving rise to familial diseases that include both systemic and neurological conditions (Table 1.1). By binding a substrate analog, however, the native state of the protein can be stabilized, thereby reducing the probability of aggregation [51, 52]; indeed this strategy has been developed into a small molecule drug that has now been approved for clinical use.
Another facet of this general strategy is to use the ability of antibodies [53], or artificially generated analogs such as affibodies [54], to bind selectively to the native states of aggregation-prone proteins, as binding generally results in enhanced stability and, hence, to a reduction in aggregation propensity (Chapter 17). In some cases it might be possible to use related approaches to reduce the level of highly aggregation-prone species (such as Aβ1-42) by stimulating clearance [55]. But antibodies and their analogs have other possibilities, one of which could be to mimic the action of natural chaperones by targeting the aberrant misfolded species that give rise to cellular damage. A very exciting step in this general direction has come from the discovery that antibodies can be raised that bind selectively to the oligomeric species that are, as we have discussed above, likely to be the dominant cause of disease, particularly in the case of neurodegenerative conditions [56]. If such “artificial chaperones” can be developed, and can be targeted to the appropriate location (e.g., by enhancing their ability to cross the blood–brain barrier), then they could represent a highly effective therapy.
One of the challenges in exploring possible ways of preventing or treating the major neurodegenerative disorders such as Alzheimer's (Chapter 13) and Parkinson's diseases is that the soluble precursors (Aβ and α-synuclein) are not stable globular proteins but, at least under many conditions, they are “natively unfolded” or “intrinsically disordered,” members of a class of peptides and proteins that is now known to be more common than was previously thought [57]. Strategies based on stabilizing a globular fold are, therefore, not applicable to such situations; but exciting opportunities arise from our increasing knowledge of the conformational properties of such species [58] (Chapter 6) and the increasing evidence of the importance of the kinetic stability of the functional states of proteins. Thus, for example, it may be possible to maintain the level of toxic oligomeric species below those that can be managed by the cellular “housekeeping” mechanisms for longer periods of time and, hence, postpone the onset of disease [48, 59].
1.7 Looking to the Future
There are very considerable grounds for optimism in the “amyloid field” both in the opportunities that its further study offers in developing a deep understanding of the nature of functional biological states, and in the quest to prevent or treat the rapidly growing numbers of our populations that are suffering, or otherwise will suffer, from the debilitating diseases with which its formation is related [1–4]. In addition, there will undoubtedly be progress in developing applications in a wide range of fields for these most remarkable self-assembling materials [5]. The increasing evidence for the “generic” nature of the amyloid state indicates that it will be possible both to generate a vast range of sequences that are designed to adopt this state and to have a wide range of properties and functionalities. In addition, the “generic” factors associated with the mechanism of its formation and the nature and properties of the precursors to the fibrillar state promise to enable common approaches to be adopted to tackle the increasing number of diseases that are now recognized to be associated with its formation [1, 7, 48].
Research into the amyloid phenomenon is at an exceptionally exciting stage. We are beginning to understand at a molecular level the origins of the structures and properties of both the fibrillar and the prefibrillar states of proteins, as well as their functional native states, and the mechanism by which they are formed under different conditions. The enhanced knowledge in both these areas of research provides unprecedented opportunities to perturb in a rational manner the effects of the aberrant behavior of proteins so as to enhance human health and longevity. In order for these advances to be exploited to their full it is important continually to develop rational methods of building on present progress. As we have discussed above, a range of exciting developments is underway, including the introduction of new means of probing the structures of the wide range of amyloid-related states formed from different polypeptide sequences, the exploitation of new techniques for monitoring the microscopic events involved in amyloid formation, such as microfluidics and single molecule methodologies, an enhanced ability to simulate the conformational transitions using advanced computational methods, the development of high resolution imaging techniques that make it possible to monitor the development and fate of aggregates of different types in vivo as well as in vitro, the extension of a molecular understanding of structure and mechanism from in vitro to in vivo situations, and the exploitation of new approaches to perturb these processes using both small and large molecules.
In terms of the medical impact of amyloid formation, with such enhanced knowledge and an extension of our understanding of the types of molecules that will interact with, and perturb, the aggregation process, will come novel diagnostics that are desperately needed for detecting the onset and progression of amyloid-associated diseases, for identifying new targets for drug discovery, and for defining the efficacy of therapeutic strategies. To achieve effective and affordable therapies on the timescale that is necessary to avoid a situation where a large fraction of the human race is suffering from highly debilitating and incurable conditions that will place a huge burden on the financial resources of the populations of the world it will be necessary to increase dramatically and rapidly the funds available for research into the amyloid field. At present, for example, the spending on research into amyloid-related diseases is less than one tenth of that directed to cancer, even though the health care cost of just one type of the former, Alzheimer's disease, is much greater than all forms of the latter [2, 3].
With increasing resources should come new ways of carrying out research, that bring together different scientific disciplines, basic scientists and clinicians, theoreticians and experimentalists, and academic and industrial organizations. We need a “war on dementia” to match the “war on cancer” that came into being with the US National Cancer Act of 1971 [60]. As with cancer, the increasing generation of even rudimentary forms of therapy will make it possible to talk about the disease openly, because diagnosis will cease to be considered a sentence of inevitable decline and untimely death. The chapters that follow, however, reveal the astonishing progress made already in understanding the fundamental process of amyloid formation and its consequences, provide ample evidence for optimism both for controlling the “amyloid phenomenon” for medical purposes and potentially exploiting it for the development of new materials and devices for the world of the future.
1.8 Summary
Interest in the phenomenon of amyloid formation by polypeptide molecules has developed with extraordinary rapidity in recent years, such that it is now a major topic of research across a wide range of disciplines. The reasons for this surge of interest arise primarily from (i) the links between amyloid formation and a range of rapidly proliferating medical disorders, including Alzheimer's disease and type II diabetes, (ii) the insights that studies of the amyloid state provide about the nature of the functional forms of peptides and proteins within living systems, and (iii) the opportunities that could exist for generating new therapeutic strategies and for devising new types of materials with novel and potentially useful properties. In this chapter I have given a personal overview of the “amyloid phenomenon,” and have discussed some priorities for further investigation and also opportunities for the future. I hope that this chapter serves as an introduction to the remaining chapters in this volume that describe specific aspects of the “amyloid phenomenon” in much greater detail.
Acknowledgments
I would like to acknowledge the many graduate students, post-doctoral fellows, colleagues, and collaborators whose discoveries and ideas are reflected in this chapter, many of whose names appear in the citations to published work. I am also very grateful to the many organizations who have funded our research over many years including the Wellcome Trust, the Leverhulme Trust, the Alzheimer's Research Trust, Parkinson's UK, the European Commission, UK Research Councils (EPSRC, BBSRC and MRC), and Elan Pharmaceuticals.
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