FTIR reveals structural differences between native beta-sheet proteins and amyloid fibrils

The presence of beta-sheets in the core of amyloid fibrils raised questions as to whether or not beta-sheet-containing proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the molecular structure of amyloid fibrils differs more generally from the beta-sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the phi/psi dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the beta-sheet twist. These data imply that amyloid fibril formation from native beta-sheet proteins can involve a substantial structural reorganization.     

Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber

Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.     

Scrapie-associated fibrils (SAF) purification method yields amyloid proteins from systemic and cerebral amyloidosis

We identified fibrils from non-transmissible systemic and cerebral amyloidosis using the purification method of scrapie-associated fibrils (SAF). The fibrils possessed the same nature of congophilia, filamentous structures and molecular weights as amyloid fibrils, and were resistant to Proteinase K digestion. This SAF method makes for a rapid extraction from amyloid-laden tissues. The method, therefore, may purify nontransmissible amyloids alone or together with SAF proteins.     

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.     

Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits

The assembly of the beta-amyloid peptide (Abeta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Abeta dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH(+) = 1825.9, the predicted mass of a fragment composed of the N-terminal Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.     

Molecular structure of amyloid fibrils: insights from solid-state NMR

Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the beta-amyloid peptide associated with Alzheimer's disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.     

Polymorphism of β2-microglobulin amyloid fibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, β(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-β core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-β structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.     

Segmental polymorphism in a functional amyloid

Although amyloid fibrils are generally considered to be causative or contributing agents in amyloid diseases, several amyloid fibrils are also believed to have biological functions. Among these are fibrils formed by Pmel17 within melanosomes, which act as a template for melanin deposition. We use solid-state NMR to show that the molecular structures of fibrils formed by the 130-residue pseudo-repeat domain Pmel17:RPT are polymorphic even within the biologically relevant pH range. Thus, biological function in amyloid fibrils does not necessarily imply a unique molecular structure. Solid-state NMR spectra of three Pmel17:RPT polymorphs show that in all cases, only a subset (∼30%) of the full amino acid sequence contributes to the immobilized fibril core. Although the repetitive nature of the sequence and incomplete spectral resolution prevent the determination of unique chemical shift assignments from two- and three-dimensional solid-state NMR spectra, we use a Monte Carlo assignment algorithm to identify protein segments that are present in or absent from the fibril core. The results show that the identity of the core-forming segments varies from one polymorph to another, a phenomenon known as segmental polymorphism.     

Peptide conformation and supramolecular organization in amylin fibrils: constraints from solid-state NMR

The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscopy and atomic force microscopy images. Solid-state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one- and two-dimensional solid-state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils and to derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four layers of parallel beta-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with a similar morphology formed by the 40-residue beta-amyloid peptide that is associated with Alzheimer's disease.     

Dimethylsulfoxide-quenched hydrogen/deuterium exchange method to study amyloid fibril structure

A general method to analyze the structure of a supramolecular complex of amyloid fibrils at amino acid residue resolution has been developed. This method combines the NMR-detected hydrogen/deuterium (H/D) exchange technique to detect hydrogen-bonded amide groups and the ability of the aprotic organic solvent dimethylsulfoxide (DMSO) to dissolve amyloid fibrils into NMR-observable, monomeric components while suppressing the undesired H/D exchange reaction. Moreover, this method can be generally applied to amyloid fibrils to elucidate the distribution of hydrogen-bonded amino acid residues in the three-dimensional molecular organization in the amyloid fibrils. In this study, we describe theoretical considerations in the H/D exchange method to obtain the structural information of proteins, and the DMSO-quenched H/D exchange method to study a supramolecular complex of amyloid fibrils. A possible application of this method to study the interaction of a protein/peptide with phospholipid membrane is also discussed.     

Dimethylsulfoxide-quenched hydrogen/deuterium exchange method to study amyloid fibril structure

A general method to analyze the structure of a supramolecular complex of amyloid fibrils at amino acid residue resolution has been developed. This method combines the NMR-detected hydrogen/deuterium (H/D) exchange technique to detect hydrogen-bonded amide groups and the ability of the aprotic organic solvent dimethylsulfoxide (DMSO) to dissolve amyloid fibrils into NMR-observable, monomeric components while suppressing the undesired H/D exchange reaction. Moreover, this method can be generally applied to amyloid fibrils to elucidate the distribution of hydrogen-bonded amino acid residues in the three-dimensional molecular organization in the amyloid fibrils. In this study, we describe theoretical considerations in the H/D exchange method to obtain the structural information of proteins, and the DMSO-quenched H/D exchange method to study a supramolecular complex of amyloid fibrils. A possible application of this method to study the interaction of a protein/peptide with phospholipid membrane is also discussed.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.     

A common beta-sheet architecture underlies in vitro and in vivo beta2-microglobulin amyloid fibrils

Misfolding and aggregation of normally soluble proteins into amyloid fibrils and their deposition and accumulation underlies a variety of clinically significant diseases. Fibrillar aggregates with amyloid-like properties can also be generated in vitro from pure proteins and peptides, including those not known to be associated with amyloidosis. Whereas biophysical studies of amyloid-like fibrils formed in vitro have provided important insights into the molecular mechanisms of amyloid generation and the structural properties of the fibrils formed, amyloidogenic proteins are typically exposed to mild or more extreme denaturing conditions to induce rapid fibril formation in vitro. Whether the structure of the resulting assemblies is representative of their natural in vivo counterparts, thus, remains a fundamental unresolved issue. Here we show using Fourier transform infrared spectroscopy that amyloid-like fibrils formed in vitro from natively folded or unfolded beta(2)-microglobulin (the protein associated with dialysis-related amyloidosis) adopt an identical beta-sheet architecture. The same beta-strand signature is observed whether fibril formation in vitro occurs spontaneously or from seeded reactions. Comparison of these spectra with those of amyloid fibrils extracted from patients with dialysis-related amyloidosis revealed an identical amide I' absorbance maximum, suggestive of a characteristic and conserved amyloid fold. Our results endorse the relevance of biophysical studies for the investigation of the molecular mechanisms of beta(2)-microglobulin fibrillogenesis, knowledge about which may inform understanding of the pathobiology of this protein.     

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.     

Interaction of the Molecular Chaperone DNAJB6 with Growing Amyloid-beta 42 (Aβ42) Aggregates Leads to Sub-stoichiometric Inhibition of Amyloid Formation

The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in Alzheimer disease) in a highly efficient manner. By monitoring fibril formation using Thioflavin T fluorescence and far-UV CD spectroscopy, we have found that the aggregation of Aβ42 is retarded by DNAJB6 in a concentration-dependent manner, extending to very low sub-stoichiometric molar ratios of chaperone to peptide. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of Aβ42 rather than the monomeric form of the peptide. This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. A low dissociation rate of DNAJB6 from Aβ42 aggregates leads to its incorporation into growing fibrils and hence to its gradual depletion from solution with time. When DNAJB6 is eventually depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction. These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form.     

Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self-assembled amyloid fibrils are also involved in physiological processes in several micro-organisms. Yet, the molecular basis for the recognition and self-assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self-assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid-associated disorders.     

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.     

Effects of p-benzoquinone and melatonin on amyloid fibrillogenesis of hen egg-white lysozyme

More than 20 different human proteins can fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained far from complete. In this work, utilizing hen egg-white lysozymes as a model system, two objectives were pursued: (1) to search for suitable conditions for producing amyloid fibrils and (2) to investigate inhibitory activities of two potential molecules against lysozyme fibril formation. Via numerous spectroscopic analyses and electron microscopy, our results showed that the formation of lysozyme amyloid fibrils at pH 2.0 was considerably increased by the addition of salt. Moreover, the inhibition of lysozyme amyloid formation by either p-benzoquinone or melatonin followed a concentration-dependent fashion. Furthermore, p-benzoquinone, in comparison with melatonin, served as a more effective inhibitor against amyloid fibril formation of lysozyme. We believe that a better understanding of how hen egg-white lysozymes aggregate will not only aid in deciphering the molecular mechanism of amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.     

Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms

Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12^undiff). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.     

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.