Perspective pubs.acs.org/JPCL
Amyloid Fibrils: Formation, Polymorphism, and Inhibition Torleif Har̈ d* Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences (SLU), Box 7015, SE-750 07 Uppsala, Sweden ABSTRACT: Amyloid fibrils with cross-β spine basic architectures are prevalent and stable forms of peptides and proteins. Recent research has provided significant contributions to our understanding of the mechanisms of fibril formation and to the surprising diversity and persistence of structural polymorphism in amyloid fibrils. There have also been successful demonstrations of how molecules can be engineered to inhibit unwanted amyloid formation by different mechanisms. Future research in these areas will include investigations of mechanisms for primary nucleation and the structure of oligomeric intermediates, the general role of secondary nucleation events (autocatalysis), elucidation of the mechanisms and implications of preservation of structural morphology in amyloid propagation, and research into the largely unexplored phenomenon of cross-seeding, by which amyloid fibrils of one species induce the formation of amyloid by another species. eptide fibrils that are a few to 20 nm wide and up to several micrometers in length are the thermodynamically most stable conformations of many proteins and peptides in water solution.1,2 Such fibrils were first described as “amyloid” when they were found in pathological deposits in human tissue. The name amyloid has since been reserved for such extracellular protein deposits if they exhibit green birefringence in the presence of the Congo red dye.3 However, amyloid-like fibrils (hereafter called amyloid fibrils) constitute a family of prevalent protein polymers with well-defined characteristics. Many, and perhaps most, proteins can form amyloid fibrils upon denaturation. Amyloid fibrils are also found with functional roles throughout Nature. They occur, for instance, as components of bacterial biofilm, as barnacle cement, for cell adhesion in diploid fungi, or as suspending fibers for lacewing eggs. Amyloid fibrils have also been recognized for their potential in nanotechnology and biotechnology. This is because of their mechanical properties and biocompatibility and because biomaterials based on amyloid fibrils undergo hierarchal selfassembly with a minimal need for external processing.
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I will, in this Perspective, provide an account of recent progress in the understanding of amyloid fibril formation and a personal view of issues that remain to be resolved. The topics include recent experimental and theoretical advances on the mechanisms of nucleation and amyloid fibril formation kinetics, including the demonstration of a secondary nucleation mechanism, structural polymorphism and its relation to effects known as amyloid strains and cross-seeding of amyloid formation, and different possibilities to design inhibitors or modulators of amyloid formation. Dozens of very good articles are written every year on these topics, and my aim has been to select and comment on representative contributions, without implying that contributions that have been left out are less significant than those mentioned. The signature of amyloid fibrils is the cross-β fiber X-ray diffraction pattern, which was first reported in an amyloid science context in 1968.4 The diffraction pattern reflects β-sheet peptide secondary structure arranged perpendicular to the long axis of the fibril. Intermolecular peptide hydrogen bonds in the β-sheet provide for the mechanical rigidity of the fibril.5 Common to amyloid fibrils is also that constituent filaments are formed with tightly packed “cross-β spine” interfaces that are devoid of water.6 Mature amyloid fibrils can then, with this underlying architecture, adopt a range of topologies and superstructures.7 Amyloid fibril polymorphism is also common, and specific morphologies can be induced by seeding and propagated. These latter qualities are suggestive of prions, and the physical chemistry of amyloid fibrils is most likely akin to the physical chemistry of prions. In fact, prions typically contain cross-β spine structures. The mechanisms and kinetics of amyloid formation have interested physical chemists for decades. The process can be
Many aspects of amyloid fibrils and their formation are intriguing to physical chemists. There are also unresolved structural and mechanistic issues surrounding their properties and formation that must be addressed using physical chemistry. Many aspects of amyloid fibrils and their formation are intriguing to physical chemists. There are also unresolved structural and mechanistic issues surrounding their properties and formation that must be addressed using physical chemistry. © 2014 American Chemical Society
Received: December 22, 2013 Accepted: January 21, 2014 Published: January 21, 2014 607
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followed by light scattering or, more frequently, by the fluorescence of dyes such as thioflavin T that preferentially bind the amyloid state. (The structural basis for such amyloid-specific binding was recently addressed.8) It was established early that the kinetics, like crystallization, is nucleation-dependent, with a characteristic lag phase, a sigmoidal growth face, and a steady state.9,10 It was also known that aggregation was accelerated and the lag phase removed when reaction mixtures were seeded with preformed fibrils.10 It was therefore assumed that the lag phase was required for aggregation nuclei to form, until more recent experiments described below indicated that this is not necessarily the case. Oligomeric intermediates thought to form on or off of the pathway to fibrils were also observed. (Such intermediates are cytotoxic, medically relevant, and subject to intense research, but they are not structurally characterized to the same extent as amyloid fibrils. See refs 11−14 for some recent reviews on oligomeric intermediates.) Still, rate-limiting mechanisms and a quantitative account of fibril formation kinetics has remained a subject of discussion. Amyloid-forming peptides are challenging to handle due to their sometimes extreme aggregation propensity. The field was therefore for many years lacking protocols that resulted in precise and reproducible kinetics data. The lack of reproducibility of kinetics experiments nurtured the notion that amyloid formation of, in particular, the Alzheimer-related amyloid-β (Aβ) peptide was a stochastic event. However, these issues were, in the case of Aβ, recently resolved due to substantial improvements of kinetics experiments made by Hellstrand, Linse, and collaborators, who showed that reproducible data for aggregation of Aβ indeed could be obtained. Their protocol relies on careful measures to ensure homogeneous starting materials in the aggregation reactions and to reduce unwanted surface effects from air bubbles, plastic surfaces, or indirect effects on surface tension from cosolutes.15 These and similar experimental protocols paved the way for the improved quantification of amyloid fibril formation kinetics and the examination of internal and external effectors, for example, seeding and mechanical agitation. The dominant role of secondary events, such as fibril fragmentation16 and secondary nucleation,17−20 results in highly nonlinear aggregation kinetics. Cohen, Knowles, and collaborators recently established a theoretical framework and derived closed form self-consistent solutions to the rate equations that can be used to evaluate these events.17−20 The model (Figure 1) includes homogeneous nucleation, elongation of fibrils by monomer addition, dissociation of monomers from fibrils, fragmentation of fibrils to form new ends that can be elongated, and, most important, secondary nucleation, by which mature fibrils catalyze the formation of new fibril seeds. One important feature of the theory is that it predicts a power law relationship between the lag time for the reaction and the initial monomer concentration, both of which are easily measured, with an exponent that directly reflects the reaction order with respect to the monomer concentration. This allows for an evaluation of rate-limiting mechanisms under different conditions. Cohen et al. demonstrate, for the first time, that secondary nucleation (or autocatalysis) is rate- limiting for fibril formation by the 42 residue long Aβ42 peptide under quiescent conditions but that fibril fragmentation becomes rate-limiting when shear is generated by agitating the aggregation reaction (Figure 2).20 Here, it must be recognized that neither secondary nucleation nor fragmentation in protein aggregation are new concepts. A mechanism that is formally analogous to secondary nucleation was, for instance, described in 1985 as a “double
nucleation mechanism” for the polymerization of sickle hemoglobin.21 Fibril-dependent catalysis in amyloid formation was also described earlier (though in a slightly different context; see, for instance, ref 22), and similar mechanisms are known to act in heterogeneous crystal nucleation.23 Likewise, the kinetic effects of fragmentation (breakage) in protein aggregation have also been analyzed previously.24−26 Instead, the significance of the recent work20 is that it combines protocols for precise kinetics experiments15 with a new theoretical frame of integrated rate laws that contain all microscopic events that are thought to contribute.17,18 It has, as mentioned, been thought that fibrils do not appear until late in the amyloid aggregation reaction, when the sigmoidal phase sets in, and that oligomeric states are populated during the lag phase. However, this is not true for Aβ42, and fibrils are indeed present already at the very early stages.20,27 Aggregates present early during aggregation are difficult to identify in the reaction mixture, though they may be visualized by, for instance, atomic force microscopy (as in Supplementary Figure S2 of ref 28). Arosio et al. very recently designed an amyloid chain reaction to quantify the Aβ42 “propagons” that are present during the lag phase.27 Their results confirm that both primary and secondary nucleation mechanisms act during the early stages of fibril formation. The progress in the understanding of fibril formation kinetics sets the stage for studying which primary events are affected by, for instance, a certain mutation in the aggregating peptide. Bolognesi et al. used the new methods to find evidence for how mutations in Aβ42 can switch the nature of a rate-limiting molecular event.29 They first studied the aggregation of Aβ42 peptides carrying the Arctic mutation (Aβ42E22G), associated with early onset of Alzheimer’s disease. They found that this mutation results in aggregation in which monomer-independent secondary nucleation dominates, which is contrary to wild-type Aβ42, where monomer-dependent secondary nucleation is ratelimiting. This implies that secondary nucleation in Aβ42E22G aggregation is dominated by (monomer-independent) fragmentation of fibrils rather than by fibril-catalyzed nucleation in which new nuclei form on the surface of fibrils. In an elegant experiment, Bolognesi et al. then showed that a second mutation, known as the “Arctic rescue” mutation Aβ42E22G/ I31E (ref 30) reverses the kinetics back to monomer-dependent as for wild-type Aβ42. Hence, secondary nucleation, or autocatalysis, of amyloid formation is now demonstrated by analysis of kinetics. However, it is not yet clear how secondary nucleation is related to polymorphism and conformational templating, which are described in the following discussions. It is common for amyloid-forming peptides to simultaneously assemble into fibrils with different morphologies.31−34 The beauty and complexity of amyloid structure and polymorphism were recently demonstrated in a multilaboratory study of the fibrils formed by an 11-residue peptide fragment of the protein transthyretin.35 In this tour-de-force, the authors combined data from magic angle spinning NMR spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy to derive atomic-resolution structures of three amyloid fibril polymorphs. The analyses reveal in great detail how peptides pack hierarchally into parallel β-sheets, protofilaments, filaments, and fibrils, as illustrated in Figure 3. The work highlights how fibril polymorphism can arise due to the incorporation of different numbers of filaments in the mature fibril. 608
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Figure 1. Mechanisms of amyloid fibril formation. The scheme illustrates molecular events and microscopic rate constants for (a) primary nucleation, (b) fibril elongation by monomer association, (c) dissociation of monomer, (d) fibril fragmentation, and (e) secondary nucleation. Reprinted with permission from ref 18. Copyright 2011, AIP Publishing LLC.
Figure 2. Experimental kinetics for amyloid fibril formation by Aβ42. Fibril mass concentration was measured using thioflavin T fluorescence assays. Solid lines are theoretical rate laws with indicated rate constants as defined in Figure 1. (a) Fibril formation from different starting monomer concentrations at quiescent conditions. (b) Fibril formation under agitating conditions. Note the different time scales and that shear increases the rate of fibril formation. (c) Comparison of the shapes of the kinetics profiles for fibril formation under quiescent and agitating conditions at a fixed monomer concentration (note the different time scales). (d) Power-law relationships between aggregation half times and monomer concentration. The weaker monomer dependence of aggregation under agitating conditions indicates that the rate-limiting mechanism is being switched from secondary nucleation to fibril fragmentation. The figure was compiled from Figures 2 and 3 in ref 20. Copyright by the authors.
formation of fibrils in which the structural morphology of the seeding material is copied at the molecular level.37,38 A particular morphology can be retained in several generations of seeded aggregation reactions. This phenomenon might potentially be utilized in biotechnology to tune the self-assembly of designed biomaterials. More importantly, it is thought to be of considerable biological consequence as it, for instance, provides a structural explanation for the existence of different “strains” of infectious prions.39 Structural morphologies might also be directly related to the (macroscopic) pathology of a disease. In fact, Lu, Tycko, and collaborators recently analyzed the structures of Aβ40 fibrils from two patients who had suffered from Alzheimer’s disease.40
However, polymorphism can also arise at the molecular level. The so-called Iowa mutation in Aβ (AβD23N) is also associated with early onset of Alzheimer’s disease. The 40-residue Aβ40D23N aggregates rapidly, and without any lag phase, to form fibrils with multiple morphologies.36 Some of these contain the common cross-β spine architecture with parallel β-sheet packing of the protofilaments, but a majority of the fibrils actually contain antiparallel β-sheets.36 Amyloid polymorphs can be retained in subsequent aggregation, or, in other words, a particular structural morphology can be amplified by propagation. The addition of a small amount of fibrils to a solution of the same protein or peptide will seed the 609
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Figure 3. Amyloid fibril structure and polymorphism. The structures of fibrils formed by an 11-residue peptide corresponding to residues 105−115 of transthyretin [TTR(105−115)] were derived from the combined data from solid-state NMR, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy.35 (a) Hierarchy of structural motifs from which amyloid fibrils are assembled. (b) Cryoelectron microscopy images, 3D reconstructions, and 2D projections (top) and contoured density cross sections (bottom) of three fibril polymorphs of TTR(105−115). (c) Water solvation: atomic-resolution cross sections of the three fibril polymorphs with water (brown color) from molecular dynamics simulations. No waters penetrate into the cross-β spine cores of the protofilaments. The figure was compiled from Figures 3 and 5 and Supporting Information Appendix Figure S11 in ref 35. Copyright by the authors.
peptides with unrelated sequences.43 Similar effects seem to apply to the so-called cross-seeding mechanism of amyloid propagation; fibrils of one peptide can actually seed the formation of fibrils by a different peptide. It was shown, in a pioneering study, that Aβ40 fibrils are efficient seeds for fibril formation by IAPP, a polypeptide for which amyloid formation is linked to type II diabetes.44 The cross-seeding effect can, to some extent, be reconciled because the amyloid core residues 15−37 of Aβ are 65% similar and 39% identical to the amyloid-forming fragment of IAPP. However, the matter is not completely settled because fibrils of IAPP are poor seeds for fibril formation by Aβ40.44 The phenomenon of cross-seeding is attracting increased attention, with the objectives to elucidate physical mechanisms45 or to study molecular or clinical links between amyloid-forming proteins associated with disease.46 Amyloid formation is, as mentioned, associated with a number of diseases, including widespread disorders such as type II diabetes and neurological disorders such as Parkinson’s and Alzheimer’s diseases. Protein aggregation is, in general, harmful to cells, and smaller soluble protein aggregates (oligomers and protofibrils) have, in some cases, been shown to be cytotoxic via distinct molecular mechanisms. Fibrils also contribute to the formation of oligomers through secondary nucleation. It is therefore of great interest from a therapeutic point of view to develop inhibitors of both oligomer and amyloid fibril formation. We recently reviewed different strategies for intervention to modulate or completely inhibit aggregation.47 These include the use of designed small molecules or engineered proteins that act to stabilize a native protein fold to prevent it from unfolding
Hence, secondary nucleation, or autocatalysis, of amyloid formation is now demonstrated by analysis of kinetics. However, it is not yet clear how secondary nucleation is related to polymorphism and conformational templating. By adding isotopically labeled monomeric Aβ40 to sonicated brain extracts, they could propagate the morphology of amyloid fibrils from different parts of the brains and analyze these by solid-state NMR spectroscopy and electron microscopy. They made several intriguing observations, for instance, that brainderived fibrils from the two patients had different structures and that one of the brain fibril morphologies that was analyzed in more detail contained specific structural features that are not present in fibrils that form in vitro. They noted that fibrils from different parts of the brain have the same structural morphology, and they argue convincingly that this indeed suggests that it had been propagated by seeding from a single site of nucleation. The article also highlights the possible link between the structural morphology of an amyloid fibril and the clinical prognosis. The authors recognize that conformationspecific probes for amyloid imaging might be developed as a diagnostic tool, and this is indeed an area of active research.41,42 It has been known for some time that amyloid fibrils need not to be homogeneous; that is, single fibrils may contain 610
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and exposing aggregation-prone regions, bind a peptide to sequester aggregation-prone regions, bind and stabilize protein aggregates that are off-pathway compared to amyloid or toxic aggregate formation, or engage amyloid fibril ends to prevent elongation of fibrils. (For completeness, it should be mentioned that there also are biological machineries, such as molecular chaperones and protein homeostasis systems, that retain a balance between protein production, folding and breakdown, and the immune system, which can clear a tissue from protein aggregates.) Several of these strategies to prevent amyloid fibril formation have been successfully demonstrated. An advanced application, in a therapeutic sense, of an amyloid inhibitor is the drug tafamidis, which in many countries has been approved for treatment of a rare but deadly neurological disorder called familial amyloid polyneuropathy. Patients suffering from this disease carry mutations in the gene for the thyroid hormone transporter protein transthyretin (TTR), which destabilizes the native tetrameric form of TTR. Dissociation into monomers then results in pathological amyloid formation. The drug prevents this by binding to the native TTR tetramer (Figure 4a). Interestingly, the mechanism of action is not primarily thermodynamic, that is, to stabilize native tetrameric TTR relative to aggregated TTR, but rather kinetic to slow down tetramer dissociation, as shown by Hammarström et al.48 TTR stabilizing compounds of this type have therefore been named “kinetic stabilizers”.49 It is also possible to inhibit, or at least dramatically delay, fibril formation by blocking the ends of growing fibrils. Peptidebased “β-sheet breakers” that inhibit fibril growth have been studied for many years (for instance, ref 50), and this research has been reviewed elsewhere.51 It is, however, essential to point to the use of non-natural amino acids for structure-based design of such inhibitors (Figure 4b).52 Still, amyloid-fibril-catalyzed secondary nucleation would, if it turns out to be a general mechanism, potentially undermine the applicability of inhibitors of fibril elongation. This is because they might not block the sites for secondary nucleation (as also discussed below), and also, a small pool of amyloid fibrils would, in this case, even when elongation is inhibited, still act to catalyze the formation of new and potentially toxic oligomeric seeds. Hence, any pharmaceutical strategy aiming at fibril growth termination should involve a consideration of the actual rate-limiting steps of the aggregation reaction.
The only way to inhibit protein or peptide aggregation that is independent of the aggregation mechanism is to bind the monomeric state to sequester aggregation-prone regions.
Figure 4. Strategies to inhibit amyloid fibril formation. (a) Kinetic stabilization of the native tetramer state of TTR carrying the diseaseassociated V122I mutation by the compound AG-10, which binds in two ligand binding cavities at the interfaces between monomeric subunits (from ref 65. Copyright by the authors). (b) Inhibition of fibril formation by proteolytic peptide fragments containing residues 248−286 of prostatic acid phosphatase (248PAP286). The designed non-natural peptide (in cyan) binds to the end of the elongating fibril (purple spheres). Reprinted by permission from Macmillan Publishers Ltd.: Nature (ref 52, Figure 4b). Copyright 2011. (c) Sequestering of aggregation-prone Aβ peptide regions (orange) within the hydrophobic cavity of the engineered ZAβ3 Affibody binding protein (blue and cyan backbone; van de Waals surface in white). From ref 54. Copyright by the authors.
The only way to inhibit protein or peptide aggregation that is independent of the aggregation mechanism is to bind the monomeric state to sequester aggregation-prone regions. Such inhibition has, in the case of Aβ, been achieved by an Affibody binding protein (Figure 4c). The Affibody, called ZAβ3, was selected by phage display,53 and the structure of the Affibody in complex with Aβ40 shows that it binds Aβ within a large tunnellike cavity that protects the peptide from fibril formation.54 This Affibody was also found to be an efficient aggregation
inhibitor of Aβ in vivo in a fly model of Alzheimer’s disease.55 The concept of engineered binding proteins may therefore be worth pursuing as a therapeutic strategy, given that the issue of pharmacological half-life, that is, the degradation of protein therapeutics in the kidney and liver, can be satisfactorily addressed. On the basis of this progress in our understanding of mechanisms, structure, and inhibition of amyloid fibril formation, 611
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and seeding, and perhaps, eventually, prion transmissibility across species. A similar area that is equally intriguing concerns the mechanisms for cross-seeding where fibril seeds of one peptide initiate amyloid formation by another protein or peptide. Cross-seeding has been demonstrated in animal models.61 It is medically relevant because it would imply that amyloids and prions are physically analogous and that amyloids, like prions, could be infective and even transmissible across species. Little is however known about physical mechanisms even though some key aspects have been identified.62 The molecular basis for cross-seeding of amyloid formation is therefore in need of further attention from physical chemists and structural biologists. For instance, the molecular requirements for peptide “compatibility” and the mechanisms for nucleation must be established. The design of inhibitors and lead compounds for drug discovery can also be expected to be further developed based on recent insight into secondary nucleation mechanisms and cross-seeding. Hence, if secondary nucleation occurs along the sides of fibrils, these might constitute “drugable” surfaces. Or, in other words, molecules that cover the fibril might act to inhibit secondary nucleation. It would not be surprising if molecules that act to delay fibril formation at substoichiometric concentrations (for instance, ref 63) actually are found to inhibit secondary nucleation. Cross-seeding mechanisms may also represent an opportunity for the design of inhibitors and drug discovery. As mentioned, Aβ40 can seed amyloid formation of IAPP, but fibrils of IAPP are poor seeds for formation of Aβ40 fibrils,44 and an IAPP analogue has indeed been shown to be a promising lead to block cytotoxic self-assembly and fibril formation of Aβ.64 In summary, the physical chemistry of amyloid fibril formation has matured into a field that is fascinatingly rich in detailed structural knowledge, elegant analyses of complex kinetics, intriguing biological consequences, and the successful demonstrations of designed inhibitors, but many issues remain in which physical chemical thinking and methodology can be expected to make further contributions.
one can identify several open issues and envision directions for future research. I will, in the following, point to some of these. For instance, the mechanisms for the very early formation of aggregation nuclei from natively folded proteins are probably not yet understood. It is frequently assumed that nucleation occurs from a pool of unfolded peptides that expose aggregation-prone regions. Such ensembles can accumulate in vivo due to imbalances in protein production and degradation, destabilizing mutations that shift the protein folding equilibrium, or defective molecular chaperone action. However, other mechanisms are probably also at work. For instance, in a pioneering paper, Eichner, Radford, and their collaborators showed that a rare conformation of a (folded) protein actually can act to catalytically convert a nonamyloid-forming ensemble of correctly folded proteins into an amyloidogenic state.56 It should be important to study to which extent such mechanisms operate in other cases, both in vitro and, if possible, in vivo. A related field of research concerns the actual structure of aggregation nuclei and oligomeric and protofibrillar states that are precursors to amyloid fibrils or off-pathway intermediates. This field is of considerable interest from a medical point of view. Some structural information has been obtained for oligomers and protofibrils57−59 but the instability and heterogeneity of protein aggregate intermediates make structural biology studies of these a formidable challenge. Another important issue concerns the mechanisms of aggregation. In particular, how general is the Aβ42 aggregation mechanism and secondary nucleation among other amyloidforming proteins and peptides? This is important not only for our basic understanding of protein aggregation but also because it would imply that mechanisms of inhibition of aggregation by designed compounds or proteins need to be quantitatively evaluated in kinetics experiments. As mentioned, β-sheet blockers of fibril elongation may not be effective if and when secondary nucleation sets in. It would also be interesting to learn if and to what extent secondary nucleation actually contributes to prion assembly, for which the alternative fragmentation mechanism is believed to be vital.60 The mechanism of secondary nucleation must, if it is general, also be understood in relation to the preservation of structural morphology in seeded reactions. Propagation of morphology appears straightforward when seeded aggregation occurs by elongation and breakage of fibrils, when monomers attach to fibril ends adopt conformations that are compatible with the structure of the fibril that is being elongated. However, the preservation of morphology represents an enigma if and when aggregation is dominated by secondary nucleation, which perhaps is the case. Do new seeds (with identical morphology) in this case form by interactions at the surface of a catalyzing fibril and by what mechanisms is then the morphology preserved? Or does the secondary nucleation still rely on the presence of fibril ends that can serve as templates for a particular morphology? The kinetics of fibril formation of amyloid-β does not seem to rule out this possibility; it only implies that there is a monomer-concentration-dependent secondary mechanism, but how can one envision templated nucleation that is not simple elongation? Physical mechanisms, conditions, and biological consequences of formation and self-propagation of amyloid fibril polymorphism is therefore an area that is attracting increasing attention.33 Such research will hopefully establish connections between structural polymorphism, amyloid formation kinetics
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AUTHOR INFORMATION
Corresponding Author
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[email protected]. Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Swedish Research Council (VR Grant No. 621-2011.5812). The author would like to thank Dr. Jun-Xia Lu and Dr. Robert Tycko at NIH for providing the electron microscope image shown in the table of contents graphic.
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REFERENCES
(1) Dobson, C. Protein Misfolding, Evolution and Disease. Trends Biochem. Sci. 1999, 24, 329−332. (2) Baldwin, A.; Knowles, T.; Tartaglia, G.; Fitzpatrick, A.; Devlin, G.; Shammas, S.; Waudby, C.; Mossuto, M.; Meehan, S.; Gras, S.; et al. Metastability of Native Proteins and the Phenomenon of Amyloid Formation. J. Am. Chem. Soc. 2011, 133, 14160−14163. (3) Sipe, J.; Benson, M.; Buxbaum, J.; Ikeda, S.-I.; Merlini, G.; Saraiva, M.; Westermark, P. Amyloid Fibril Protein Nomenclature: 2010 Recommendations from the Nomenclature Committee of the International Society of Amyloidosis. Amyloid 2010, 17, 101−104. 612
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Perspective
(4) Eanes, E.; Glenner, G. X-ray Diffraction Studies on Amyloid Filaments. J. Histochem. Cytochem. 1968, 16, 673−677. (5) Knowles, T.; Fitzpatrick, A.; Meehan, S.; Mott, H.; Vendruscolo, M.; Dobson, C.; Welland, M. Role of Intermolecular Forces in Defining Material Properties of Protein Nanofibrils. Science 2007, 318, 1900−1903. (6) Nelson, R.; Sawaya, M.; Balbirnie, M.; Madsen, A.; Riekel, C.; Grothe, R.; Eisenberg, D. Structure of the Cross-β Spine of AmyloidLike Fibrils. Nature 2005, 435, 773−778. (7) Eisenberg, D.; Jucker, M. The Amyloid State of Proteins in Human Diseases. Cell 2012, 148, 1188−1203. (8) Schütz, A.; Soragni, A.; Hornemann, S.; Aguzzi, A.; Ernst, M.; Böckmann, A.; Meier, B. The Amyloid−Congo Red Interface at Atomic Resolution. Angew. Chem., Int. Ed. 2011, 50, 5956−5960. (9) Colon, W.; Kelly, J. Partial Denaturation of Transthyretin is Sufficient for Amyloid Fibril Formation In Vitro. Biochemistry 1992, 31, 8654−8660. (10) Jarrett, J.; Lansbury, P. Amyloid Fibril Formation Requires a Chemically Discriminating Nucleation Event: Studies of an Amyloidogenic Sequence from the Bacterial Protein OsmB. Biochemistry 1992, 31, 12345−12352. (11) Benilova, I.; Karran, E.; de Strooper, B. The Toxic Aβ Oligomer and Alzheimer’s Disease: An Emperor in Need of Clothes. Nat. Neurosci. 2012, 15, 349−357. (12) Härd, T. Protein Engineering to Stabilize Soluble Amyloid βProtein Aggregates for Structural and Functional Studies. FEBS J. 2011, 278, 3884−3892. (13) Roychaudhuri, R.; Yang, M.; Hoshi, M.; Teplow, D. Amyloid βProtein Assembly and Alzheimer Disease. J. Biol. Chem. 2009, 284, 4749−4753. (14) Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. ATR-FTIR: A “Rejuvenated” Tool to Investigate Amyloid Proteins. Biochim. Biophys. Acta 2013, 1828, 2328−2338. (15) Hellstrand, E.; Boland, B.; Walsh, D. M.; Linse, S. Amyloid βProtein Aggregation Produces Highly Reproducible Kinetic Data and Occurs by a Two-Phase Process. ACS Chem. Neurosci. 2010, 1, 13−18. (16) Knowles, T. P.; Waudby, C. A.; Devlin, G. L.; Cohen, S. I.; Aguzzi, A.; Vendruscolo, M.; Terentjev, E. M.; Welland, M. E.; Dobson, C. M. An Analytical Solution to the Kinetics of Breakable Filament Assembly. Science 2009, 326, 1533−1537. (17) Cohen, S.; Vendruscolo, M.; Dobson, C.; Knowles, T. Nucleated Polymerization with Secondary Pathways. II. Determination of Self-Consistent Solutions to Growth Processes Described by NonLinear Master Equations. J. Chem. Phys. 2011, 135, 065106. (18) Cohen, S.; Vendruscolo, M.; Welland, M.; Dobson, C.; Terentjev, E.; Knowles, T. Nucleated Polymerization with Secondary Pathways. I. Time Evolution of the Principal Moments. J. Chem. Phys. 2011, 135, 065105. (19) Lorenzen, N.; Cohen, S.; Nielsen, S.; Herling, T.; Christiansen, G.; Dobson, C.; Knowles, T.; Otzen, D. Role of Elongation and Secondary Pathways in S6 Amyloid Fibril Growth. Biophys. J. 2012, 102, 2167−2175. (20) Cohen, S.; Linse, S.; Luheshi, L.; Hellstrand, E.; White, D.; Rajah, L.; Otzen, D.; Vendruscolo, M.; Dobson, C.; Knowles, T. Proliferation of Amyloid-β42 Aggregates Occurs through a Secondary Nucleation Mechanism. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 9758− 9763. (21) Ferrone, F.; Hofrichter, J.; Eaton, W. Kinetics of Sickle Hemoglobin Polymerization. II. A Double Nucleation Mechanism. J. Mol. Biol. 1985, 183, 611−631. (22) Ruschak, A.; Miranker, A. Fiber-Dependent Amyloid Formation as Catalysis of an Existing Reaction Pathway. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12341−12346. (23) Cacciuto, A.; Auer, S.; Frenkel, D. Onset of Heterogeneous Crystal Nucleation in Colloidal Suspensions. Nature 2004, 428, 404− 406. (24) Oosawa, F.; Asakura, S. Thermodynamics of the Polymerization of Protein; Academic Press: New York, 1975.
(25) Bishop, M.; Ferrone, F. Kinetics of Nucleation-Controlled Polymerization. A Perturbation Treatment for Use with a Secondary Pathway. Biophys. J. 1984, 46, 631−644. (26) Wegner, A.; Savko, P. Fragmentation of Actin Filaments. Biochemistry 1982, 21, 1909−1913. (27) Arosio, P.; Cukalevski, R.; Frohm, B.; Knowles, T.; Linse, S. An Amyloid Chain Reaction Assay Quantifies the Concentration of Aβ42 Propagons During the Lag-Phase. J. Am. Chem. Soc. 2014, 136, 219− 225. (28) Dubnovitsky, A.; Sandberg, A.; Rahman, M.; Benilova, I.; Lendel, C.; Härd, T. Amyloid-β Protofibrils: Size, Morphology and Synaptotoxicity of an Engineered Mimic. PloS ONE 2013, 8, e66101. (29) Bolognesi, B.; Cohen, S.; Terol, P.; Esbjörner, E.; Giorgetti, S.; Mossuto, M.; Natalello, A.; Brorsson, A.; Knowles, T.; Dobson, C. Single Point Mutations Induce a Switch in the Molecular Mechanism of the Aggregation of the Alzheimer’s Disease Associated Aβ42 peptide. ACS Chem Biol 2013, DOI: 10.1021/cb400616y. (30) Luheshi, L.; Tartaglia, G.; Brorsson, A.-C.; Pawar, A.; Watson, I.; Chiti, F.; Vendruscolo, M.; Lomas, D.; Dobson, C.; Crowther, D. Systematic In Vivo Analysis of the Intrinsic Determinants of Amyloid β Pathogenicity. PLoS Biol. 2007, 5, e290. (31) Fändrich, M.; Schmidt, M.; Grigorieff, N. Recent Progress in Understanding Alzheimer’s β-Amyloid Structures. Trends Biochem. Sci. 2011, 36, 338−345. (32) Jiménez, J.; Guijarro, J.; Orlova, E.; Zurdo, J.; Dobson, C.; Sunde, M.; Saibil, H. Cryo-Electron Microscopy Structure of an SH3 Amyloid Fibril and Model of the Molecular Packing. EMBO J. 1999, 18, 815−821. (33) Toyama, B.; Weissman, J. Amyloid Structure: Conformational Diversity and Consequences. Annu. Rev. Biochem. 2011, 80, 557−585. (34) Volpatti, L.; Vendruscolo, M.; Dobson, C.; Knowles, T. A Clear View of Polymorphism, Twist, and Chirality in Amyloid Fibril Formation. ACS Nano 2013, 7, 10443−10448. (35) Fitzpatrick, A.; Debelouchina, G.; Bayro, M.; Clare, D.; Caporini, M.; Bajaj, V.; Jaroniec, C.; Wang, L.; Ladizhansky, V.; Müller, S.; et al. Atomic Structure and Hierarchical Assembly of a Cross-β Amyloid Fibril. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 5468− 5473. (36) Tycko, R.; Sciarretta, K.; Orgel, J.; Meredith, S. Evidence for Novel β-Sheet Structures in Iowa Mutant β-Amyloid Fibrils. Biochemistry 2009, 48, 6072−6084. (37) Petkova, A.; Leapman, R.; Guo, Z.; Yau, W.-M.; Mattson, M.; Tycko, R. Self-Propagating, Molecular-Level Polymorphism in Alzheimer’s β-Amyloid Fibrils. Science 2005, 307, 262−265. (38) Qiang, W.; Kelley, K.; Tycko, R. Polymorph-Specific Kinetics and Thermodynamics of β-Amyloid Fibril Growth. J. Am. Chem. Soc. 2013, 135, 6860−6871. (39) Collinge, J.; Clarke, A. A General Model of Prion Strains and Their Pathogenicity. Science 2007, 318, 930−936. (40) Lu, J.-X.; Qiang, W.; Yau, W.-M.; Schwieters, C.; Meredith, S.; Tycko, R. Molecular Structure of β-Amyloid Fibrils in Alzheimer’s Disease Brain Tissue. Cell 2013, 154, 1257−1268. (41) Nilsson, K.; Aslund, A.; Berg, I.; Nyström, S.; Konradsson, P.; Herland, A.; Inganäs, O.; Stabo-Eeg, F.; Lindgren, M.; Westermark, G.; et al. Imaging Distinct Conformational States of Amyloid-β fibrils in Alzheimer’s Disease Using Novel Luminescent Probes. ACS Chem. Biol. 2007, 2, 553−560. (42) Nyström, S.; Psonka-Antonczyk, K.; Ellingsen, P.; Johansson, L.; Reitan, N.; Handrick, S.; Prokop, S.; Heppner, F.; BM WegenastBraun, M. J.; Lindgren, M.; et al. Evidence for Age-Dependent In Vivo Conformational Rearrangement within Aβ Amyloid Deposits. ACS Chem. Biol. 2013, 8, 1128−1133. (43) MacPhee, C.; Dobson, C. Formation of Mixed Fibrils Demonstrate the Generic Nature and Potential Utility of Amyloid Nanostructures. J. Am. Chem. Soc. 2000, 122, 12707−12713. (44) O’Nuallain, B.; Williams, A.; Westermark, P.; Wetzel, R. Seeding Specificity in Amyloid Growth Induced by Heterologous Fibrils. J. Biol. Chem. 2004, 279, 17490−17499. 613
dx.doi.org/10.1021/jz4027612 | J. Phys. Chem. Lett. 2014, 5, 607−614
The Journal of Physical Chemistry Letters
Perspective
(45) Surmacz-Chwedoruk, W.; Nieznańska, H.; Wójcik, S.; Dzwolak, W. Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains. Biochemistry 2012, 51, 9460−9469. (46) Botelho, H.; Leal, S.; Cardoso, I.; Yanamandra, K.; MorozovaRoche, L.; Fritz, G.; Gomes, C. S100A6 Amyloid Fibril Formation is Calcium-Modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation. J. Biol. Chem. 2012, 287, 42233−42242. (47) Härd, T.; Lendel, C. Inhibition of Amyloid Formation. J. Mol. Biol. 2012, 421, 441−465. (48) Hammarström, P.; Wiseman, R.; Powers, E.; Kelly, J. Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics. Science 2003, 299, 713−716. (49) Connelly, S.; Choi, S.; Johnson, S.; Kelly, J.; Wilson, I. StructureBased Design of Kinetic Stabilizers that Ameliorate the Transthyretin Amyloidoses. Curr. Opin. Struct. Biol. 2010, 20, 54−62. (50) Tjernberg, L.; Näslund, J.; Lindqvist, F.; Johansson, J.; Karlström, A.; Thyberg, J.; Terenius, L.; Nordstedt, C. Arrest of βAmyloid Fibril Formation by a Pentapeptide Ligand. J. Biol. Chem. 1996, 271, 8545−8548. (51) Sciarretta, K.; Gordon, D.; Meredith, S. Peptide-Based Inhibitors of Amyloid Assembly. Methods Enzymol. 2006, 413, 273−312. (52) Sievers, S.; Karanicolas, J.; Chang, H.; Zhao, A.; Jiang, L.; Zirafi, O.; Stevens, J.; Münch, J.; Baker, D.; Eisenberg, D. Structure-Based Design of Non-Natural Amino-Acid Inhibitors of Amyloid Fibril Formation. Nature 2011, 475, 96−100. (53) Grönwall, C.; Jonsson, A.; Lindstrom, S.; Gunneriusson, E.; Ståhl, S.; Herne, N. Selection and Characterization of Affibody Ligands Binding to Alzheimer Amyloid β Peptides. J. Biotechnol. 2007, 128, 162−183. (54) Hoyer, W.; Grönwall, C.; Jonsson, A.; Ståhl, S.; Härd, T. Stabilization of a β-Hairpin in Monomeric Alzheimer’s Amyloid-β Peptide Inhibits Amyloid Formation. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5099−5104. (55) Luheshi, L.; Hoyer, W.; de Barros, T. P.; Härd, I. v. D.; Brorsson, A.-C.; Macao, B.; Persson, C.; Crowther, D.; Lomas, D.; Ståhl, S.; et al. Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo. PLoS Biol. 2010, 8, e1000334. (56) Eichner, T.; Kalverda, A.; Thompson, G.; Homans, S.; Radford, S. Conformational Conversion During Amyloid Formation at Atomic Resolution. Mol. Cell 2011, 41, 161−172. (57) Cerf, E.; Sarroukh, R.; Tamamizu-Kato, S.; Breydo, L.; Derclaye, S.; Dufrêne, Y.; Narayanaswami, V.; Goormaghtigh, E.; Ruysschaert, J.M.; Raussens, V. Antiparallel β-Sheet: A Signature Structure of the Oligomeric Amyloid β-Peptide. Biochem. J. 2009, 421, 415−423. (58) Sandberg, A.; Luheshi, L.; Söllvander, S.; de Barros, T. P.; Macao, B.; Knowles, T. J.; Biverstål, H.; Lendel, C.; Ekholm-Petterson, F.; Dubnovitsky, A.; et al. Stabilization of Neurotoxic Alzheimer Amyloid-β Oligomers by Protein Engineering. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 15595−15600. (59) Laganowsky, A.; Liu, C.; Sawaya, M.; Whitelegge, J.; Park, J.; Zhao, M.; Pensalfini, A.; Soriaga, A.; Landau, M.; Teng, P.; et al. Atomic View of a Toxic Amyloid Small Oligomer. Science 2012, 335, 1228−1231. (60) Tanaka, M.; Collins, S.; Toyama, B.; Weissman, J. The Physical Basis of how Prion Conformations Determine Strain Phenotypes. Nature 2006, 442, 585−589. (61) Lundmark, K.; Westermark, G.; Olsén, A.; Westermark, P. Protein Fibrils in Nature Can Enhance Amyloid Protein A Amyloidosis in Mice: Cross-Seeding as a Disease Mechanism. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6098−6102. (62) Ma, B.; Nussinov, R. Selective Molecular Recognition in Amyloid Growth and Transmission and Cross-Species Barriers. J. Mol. Biol. 2012, 421, 172−184. (63) Lowe, T.; Strzelec, A.; Kiessling, L.; Murphy, R. Structure− Function Relationships for Inhibitors of β-Amyloid Toxicity Containing the Recognition Sequence KLVFF. Biochemistry 2001, 40, 7882−7889. (64) Andreetto, E.; Yan, L.-M.; Caporale, A.; Kapurniotu, A. Dissecting the Role of Single Regions of an IAPP Mimic and IAPP
in Inhibition of Aβ40 Amyloid Formation and Cytotoxicity. ChemBioChem 2011, 12, 1313−1322. (65) Penchala, S.; Connelly, S.; Wang, Y.; Park, M.; Zhao, L.; Baranczak, A.; Rappley, I.; Vogel, H.; Liedtke, M.; Witteles, R.; et al. AG10 Inhibits Amyloidogenesis and Cellular Toxicity of the Familial Amyloid Cardiomyopathy-Associated V122I Transthyretin. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 9992−9997.
614
dx.doi.org/10.1021/jz4027612 | J. Phys. Chem. Lett. 2014, 5, 607−614
