Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Competition between Intramolecular and Intermolecular Interactions in an Amyloid-Forming Protein

Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human β₂-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic propensity for aggregation. We rationalise these results by analysing the effect of incorporating the conformational properties of acid-unfolded β₂-microglobulin and its variants at pH 2.5 as measured by NMR spectroscopy into the Zyggregator aggregation prediction algorithm. These results demonstrate that residual structure in the precursor state modulates the intrinsic propensity of the polypeptide chain to aggregate and that the algorithm developed here allows the key regions for aggregation to be more clearly identified and the rates of their self-association to be predicted. Given the common propensity of unfolded chains to form non-random intramolecular interactions as monomers and to self-assemble subsequently into amyloid fibrils, the approach developed should find widespread utility for the prediction of regions important in amyloid formation and their rates of self-assembly.     

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-β and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated β-strand sheets forming a cross-β configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-β amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.     

pH-dependent structural transitions of Alzheimer amyloid peptides.

To understand the molecular interactions leading to the assembly of beta/44 protein into the hallmark fibrils of Alzheimer's disease (AD), we have examined the ability of synthetic peptides that correspond to the beta/A4 extracellular sequence to form fibrils over the range of pH 3-10. Peptides included the sequences 1-28, 19-28, 17-28, 15-28, 13-28, 11-28, and 9-28 of beta/A4. The model fibrils were compared with isolated amyloid with respect to morphology, conformation, tinctorial properties, and stability under denaturing conditions. Electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and x-ray diffraction revealed that the ionization states of the amino acid sidechains appeared to be a crucial feature in fibril formation. This was reflected by the ability of several peptides to undergo fibril assembly and disassembly as a function of pH. Comparisons between different beta/A4 sequences demonstrated that the fibrillar structure representative of AD amyloid was dependent upon electrostatic interactions, likely involving His-13 and Asp-23, and hydrophobic interactions between uncharged sidechains contained within residues 17-21. The results also indicated an exclusively beta-sheet conformation for the synthetic (and possibly AD fibrils) in contrast to certain other (e.g., systemic) amyloids.     

Is it possible to predict amyloidogenic regions from sequence alone?

Identification of potentially amyloidogenic regions in polypeptide chains is very important because the amyloid fibril formation can be induced in most normal proteins. In our work we suggest a new method to detect amyloidogenic regions in protein sequence. It is based on the assumption that packing is tight inside an amyloid and therefore regions which could potentially pack well would have a tendency to form amyloids. This means that the regions with strong expected packing of residues would be responsible for the amyloid formation. We use this property to identify potentially amyloidogenic regions in proteins basing on their amino acid sequences only. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Predictions of the regions which are responsible for the formation of amyloid fibrils in proteins unrelated to disease have been also done.     

Proline periodicity modulates the self-assembly properties of elastin-like polypeptides

Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloid-like fibrils, rich in β-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in β-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized β-sheet in mediating self-assembly interactions.     

Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation

Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human β₂-microglobulin (β₂m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type β₂m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the β₂m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29-51 and 58-79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for β₂m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62-70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.     

Amyloid from a histochemical perspective. A review of the structure,properties and types of amyloid,and a proposed staining mechanism for Congo red staining

Amyloid is a diverse group of unrelated peptides or proteins that have positive functionality or are associated with various pathologies. Despite vast differences, all amyloids share several features that together uniquely define the group. 1) All amyloids possess a characteristic cross-ß pattern with X-ray diffraction typical of ß-sheet secondary protein structures. 2) All amyloids are birefringent and dichroic under polarizing microscopy after staining with Congo red, which indicates a crystalline-like (ordered) structure. 3) All amyloids cause a spectral shift in the peak wavelength of Congo red with conventional light microscopy due to perturbation of π electrons of the dye. 4) All amyloids show heightened intensity of fluorescence with Congo red, which suggests an unusual degree of packing of the dye onto the substrate. The ß portion of amyloid molecules, the only logical substrate for specific Congo red staining under histochemical conditions, consists of a stack of ß-sheets laminated by hydrophilic and hydrophobic interactions between adjacent pairs. Only the first and last ß-sheets are accessible to dyes. Each sheet is composed of numerous identical peptides running across the width of the sheet and arranged in parallel with side chains in register over the length of the fibril. Two sets of grooves are bordered by side chains. X grooves run perpendicular to the long axis of the fibril; these grooves are short (the width of the sheet) and number in the hundreds or thousands. Y grooves are parallel with the long axis. Each groove runs the entire length of the fibril, but there are very few of them. While Congo red is capable of ionic bonding with proteins via two sulfonic acid groups, physical constraints on the staining solution preclude ionic interactions. Hydrogen bonding between dye amine groups and peptide carbonyls is the most likely primary bonding mechanism, because all ß-sheets possess backbone carbonyls. Various amino acid residues may form secondary bonds to the dye via any of three van der Waals forces. It is possible that Congo red binds within the Y grooves, but that would not produce the characteristic staining features that are the diagnostic hallmarks of amyloid. Binding in the X grooves would produce a tightly packed series of dye molecules over the entire length of the fibril. This would account for the signature staining of amyloid by Congo red: dichroic birefringence, enhanced intensity of fluorescence and a shift in visible absorption wavelength.     

Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB.

Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) beta-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including alpha-helical, beta-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.     

Stem-forming regions that are essential for the amyloidogenesis of prion proteins

Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-β-structure, the initial mechanism in the conversion to a β-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-β-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains ~200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular β-sheet to a solvent through the partial transition of a native structure from an α-helix to a β-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.     

Human pancreatitis-associated protein forms fibrillar aggregates with a native-like conformation

Human pancreatitis-associated protein was identified in pathognomonic lesions of Alzheimer disease, a disease characterized by the presence of filamentous protein aggregates. Here, we showed that at physiological pH, human pancreatitis-associated protein forms non-Congo Red-binding, proteinase K-resistant fibrillar aggregates with diameters from 6 up to as large as 68 nm. Interestingly, circular dichroism and Fourier transform infrared spectra showed that, unlike typical amyloid fibrils, which have a cross-beta-sheet structure, these aggregates have a very similar secondary structure to that of the native protein, which is composed of two alpha-helices and eight beta-strands, as determined by NMR techniques. Surface structure analysis showed that the positively charged and negatively charged residues were clustered on opposite sides, and strong electrostatic interactions between molecules were therefore very likely, which was confirmed by cross-linking experiments. In addition, several hydrophobic residues were found to constitute a continuous hydrophobic surface. These results and protein aggregation prediction using the TANGO algorithm led us to synthesize peptide Thr(84) to Ser(116), which, very interestingly, was found to form amyloid-like fibrils with a cross-beta structure. Thus, our data suggested that human pancreatitis-associated protein fibrillization is initiated by protein aggregation primarily because of electrostatic interactions, and the loop from residues 84 to 116 may play an important role in the formation of fibrillar aggregates with a native-like conformation.     

Structural polymorphism in amyloids: new insights from studies with Y145Stop prion protein fibrils

The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the β-sheet core of the PrP23-144 amyloid is composed of two β-strand regions encompassing residues ～113-125 and ～130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered β-core extended N-terminally to residue ～106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal β-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.     

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.     

Usage of a dataset of NMR resolved protein structures to test aggregation versus solubility prediction algorithms

There has been an increased interest in computational methods for amyloid and (or) aggregate prediction, due to the prevalence of these aggregates in numerous diseases and their recently discovered functional importance. To evaluate these methods, several datasets have been compiled. Typically, aggregation‐prone regions of proteins, which form aggregates or amyloids in vivo, are more than 15 residues long and intrinsically disordered. However, the number of such experimentally established amyloid forming and non‐forming sequences are limited, not exceeding one hundred entries in existing databases. In this work, we parsed all available NMR‐resolved protein structures from the PDB and assembled a new, sevenfold larger, dataset of unfolded sequences, soluble at high concentrations. We proposed to use these sequences as a negative set for evaluating methods for predicting aggregation in vivo. We also present the results of benchmarking cutting edge tools for the prediction of aggregation versus solubility propensity.     

Aggregation and secondary structure of synthetic amyloid beta A4 peptides of Alzheimer's disease

The deposition of amyloid beta A4 in the brain is a major pathological hallmark of Alzheimer's disease. Amyloid beta A4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates. Using synthetic peptides corresponding to the natural beta A4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of beta A4. A comparison of amino-terminally truncated beta A4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid beta A4. Infrared spectroscopy of beta A4 peptides in the solid state shows that their secondary structure consists of a beta-turn flanked by two strands of antiparallel beta-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative beta-turn positions. Limited proteolysis of these analogs allowed a localization of the central beta-turn at residues 26 to 29 of the entire sequence. Purified beta A4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a beta-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a beta-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the beta A4 sequence. This model is sustained by the properties of beta A4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between beta A4 peptides.     

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.     

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

Amyloid-associated nucleic acid hybridisation

Nucleic acids promote amyloid formation in diseases including Alzheimer's and Creutzfeldt-Jakob disease. However, it remains unclear whether the close interactions between amyloid and nucleic acid allow nucleic acid secondary structure to play a role in modulating amyloid structure and function. Here we have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid interactions. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy we show that the polymerized charges of nucleic acids concentrate and enhance the formation of amyloid from short basic peptides, many of which would not otherwise form fibres. In turn, the amyloid component binds nucleic acids and promotes their hybridisation at concentrations below their solution K(d), as shown by time-resolved FRET studies. The self-reinforcing interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres whose properties are distinct from their component polymers. In addition to their importance in disease and potential in engineering, ANA fibres formed from prebiotically-produced peptides and nucleic acids may have played a role in early evolution, constituting the first entities subject to Darwinian evolution.     

Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences.

The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.     

What does make an amyloid toxic: Morphology,structure or interaction with membrane?

The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a “raft-like” mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.