The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

A molecular dynamics study of the interaction of D-peptide amyloid inhibitors with their target sequence reveals a potential inhibitory pharmacophore conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.     

Apolipoproteins and amyloid fibril formation in atherosclerosis

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures. The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases. A number of plasma apolipoproteins, including apolipoprotein (apo) A-I, apoA-II, apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins. We review present knowledge of amyloid formation by apolipoproteins in disease, with particular focus on atherosclerosis. Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions. Additionally, we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis, and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.     

Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.     

Structural characterisation of islet amyloid polypeptide fibrils

Islet amyloid is found in many patients suffering from type 2 diabetes. Amyloid fibrils found deposited in the pancreatic islets are composed of a 37-residue peptide, known as islet amyloid polypeptide (IAPP) (also known as amylin) and are similar to those found in other amyloid diseases. Synthetic IAPP peptide readily forms amyloid fibrils in vitro and this has allowed fibril formation kinetics and the overall morphology of IAPP amyloid to be studied. Here, we use X-ray fibre diffraction, electron microscopy and cryo-electron microscopy to examine the molecular structure of IAPP amyloid fibrils. X-ray diffraction from aligned synthetic amyloid fibrils gave a highly oriented diffraction pattern with layer-lines spaced 4.7 A apart. Electron diffraction also revealed the characteristic 4.7 A meridional signal and the position of the reflection could be compared directly to the image of the diffracting unit. Cryo-electron microscopy revealed the strong signal at 4.7 A that has been previously visualised from a single Abeta fibre. Together, these data build up a picture of how the IAPP fibril is held together by hydrogen bonded beta-sheet structure and contribute to the understanding of the generic structure of amyloid fibrils.     

Consensus features in amyloid fibrils: sheet-sheet recognition via a (polar or nonpolar) zipper structure

Amyloid fibrils characterized as highly intractable thread-like species are associated with many neurodegenerative diseases. Although neither the mechanism of amyloid formation nor the origin of amyloid toxicity is currently completely understood, the detailed three-dimensional atomic structures of the yeast protein Sup35 and Abeta amyloid protein determined by recent experiments provide the first and important step towards the comprehension of the pathogenesis and aggregation mechanisms of amyloid diseases. By analyzing these two amyloid peptides which have available crystal structures and other amyloid sequences with proposed structures using computational simulations, we delineate three common features in amyloid organizations and amyloid structures. These could contribute to an improved understanding of the molecular mechanism of amyloid formation, the nature of the aggregation driving forces that stabilize these structures and the development of potential therapeutic agents against amyloid diseases.     

Structure-activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross-β-sheet structure are the pathological hallmarks of several diseases including Alzheimer’s disease, but are also associated with functional states such as the fungal HET-s prion. This review aims to summarize the recent high-resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross-β-sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity.     

Interpreting the aggregation kinetics of amyloid peptides

Amyloid fibrils are insoluble mainly beta-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. beta-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the beta-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable beta-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable beta-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation.     

Distinguishing the cross-beta spine arrangements in amyloid fibrils using FRET analysis

The recently published microcrystal structures of amyloid fibrils from small peptides greatly enhanced our understanding of the atomic-level structure of the amyloid fibril. However, only a few amyloid fibrils can form microcrystals. The dansyl-tryptophan fluorescence resonance energy transfer (FRET) pair was shown to be able to detect the inter-peptide arrangement of the Transthyretin (105-115) amyloid fibril. In this study, we combined the known microcrystal structures with the corresponding FRET efficiencies to build a model for amyloid fibril structure classification. We found that fibrils with an antiparallel structural arrangement gave the largest FRET signal, those with a parallel arrangement gave the lowest FRET signal, and those with a mixed arrangement gave a moderate FRET signal. This confirms that the amyloid fibril structure patterns can be classified based on the FRET efficiency.     

Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.     

Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils

Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer's disease, Creutzfeldt-Jakob and type II diabetes. Alzheimer's amyloid fibrils consist of amyloid-β (Aβ) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Aβ(1-40) amyloid fibrils, all formed under the same solution conditions, were determined by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The observed fibril polymorphism implies that amyloid formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.     

Molecular dynamics simulations of Alzheimer's beta-amyloid protofilaments

Filamentous amyloid aggregates are central to the pathology of Alzheimer's disease. We use all-atom molecular dynamics (MD) simulations with explicit solvent and multiple force fields to probe the structural stability and the conformational dynamics of several models of Alzheimer's beta-amyloid fibril structures, for both wild-type and mutated amino acid sequences. The structural models are based on recent solid state NMR data. In these models, the peptides form in-register parallel beta-sheets along the fibril axis, with dimers of two U-shaped peptides located in layers normal to the fibril axis. Four different topologies are explored for stacking the beta-strand regions against each other to form a hydrophobic core. Our MD results suggest that all four NMR-based models are structurally stable, and we find good agreement with dihedral angles estimated from solid-state NMR experiments. Asp23 and Lys28 form buried salt-bridges, resulting in an alternating arrangement of the negatively and positively charged residues along the fibril axis that is reminiscent of a one-dimensional ionic crystal. Interior water molecules are solvating the buried salt-bridges. Based on data from NMR measurements and MD simulations of short amyloid fibrils, we constructed structural models of long fibrils. Calculated X-ray fiber diffraction patterns show the characteristics of packed beta-sheets seen in experiments, and suggest new experiments that could discriminate between various fibril topologies.     

The Common Architecture of Cross-β Amyloid

Amyloid fibril deposition is central to the pathology of more than 30 unrelated diseases including Alzheimer's disease and Type 2 diabetes. It is generally accepted that amyloid fibrils share common structural features despite each disease being characterised by the deposition of an unrelated protein or peptide. The structure of amyloid fibrils has been studied using X-ray fibre diffraction and crystallography, solid-state NMR and electron paramagnetic resonance, and many different, sometimes opposing, models have been suggested. Many of these models are based on the original interpretation of the cross-β diffraction pattern for cross-β silk in which β-strands run perpendicular to the fibre axis, although alternative models include β-helices and natively structured proteins. Here, we have analysed opposing model structures and examined the necessary structural elements within the amyloid core structure, as well as producing idealised models to test the limits of the core conformation. Our work supports the view that amyloid fibrils share a number of common structural features, resulting in characteristic diffraction patterns. This pattern may be satisfied by structures in which the strands align close to perpendicular to the fibre axis and are regularly arranged to form β-sheet ribbons. Furthermore, the fibril structure contains several β-sheets that associate via side-chain packing to form the final protofilament structure.     

Expanding the Repertoire of Amyloid Polymorphs by Co-polymerization of Related Protein Precursors

Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human β₂-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.     

Domain swapping and amyloid fibril conformation

For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins β₂-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.