Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.     

Structure and aggregation mechanism of beta(2)-microglobulin (83-99) peptides studied by molecular dynamics simulations

Many human neurodegenerative diseases are associated with amyloid fibril formation. The human 99-residue beta(2)-microglobulin (beta2m) is one of the most intensively studied amyloid-forming proteins. Recent studies show that the C-terminal fragments 72-99, 83-89, and 91-96 form by themselves amyloid fibrils in vitro and play a significant role in fibrillization of the full-length beta2m protein under acidic pH conditions. In this work, we have studied the equilibrium structures of the 17-residue fragment 83-99 in solution, and investigated its dimerization process by multiple molecular dynamics simulations. We find that an intertwined dimer, with the positions of the beta-strands consistent with the results for the monomer, is a possible structure for two beta2m(83-89) peptides. Based on our molecular-dynamics-generated dimeric structure, a protofibril model is proposed for the full-length beta2m protein.     

A role for amyloid in cell aggregation and biofilm formation

Cell adhesion molecules in Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5p(V326N) cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5p(WT) cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation.     

Heterogeneous amyloid-formed ion channels as a common cytotoxic mechanism: implications for therapeutic strategies against amyloidosis

The amyloidoses consist of human and animal chronic, progressive, and sometimes fatal diseases that are characterized by the deposition of insoluble proteinaceous amyloid fibrils in various tissues. Despite the biochemical diversity of amyloids, they share certain properties. The amphipathic and the charged nature of many amyloid-forming peptides point to their intrinsic ability to form diverse beta-sheet-based aggregates and channel types in negatively charged membranes. We hypothesize that the formation of heterogeneous channels represents a common cytotoxic mechanism that accentuates the changes in the signal transduction that underlie amyloid-induced cell malfunction. One group of amyloid-forming peptides that could mediate their action via the formation of heterogeneous channels includes the extensively examined prions and amyloid beta protein that are associated with conformational neurodegenerative diseases. The aim of this study is to examine heterogeneous channels formed in bilayers with amyloid-forming peptides as a common mechanism of malfunction of signal transduction. The observed amyloid-formed channel types include the following. (1) Natriuretic peptides: (i) 68-pS H2O2- and Ba2+-sensitive channel with fast kinetics. The fast channel had three modes (spike mode, burst mode, and open mode), which differ in their kinetics but not in their conductance properties; (ii) a 273-pS inactivating large conductance channel; and (iii) a 160-pS transiently activated channel. (2) Prions: (i) a 140-pS GSSG- and TEA-sensitive channel with fast kinetics; (ii) a 41-pS dithiothreitol (DTT)-sensitive channel with slow kinetics; (iii) a 900 to 1444-pS large channel. (3) Amyloid beta protein: (i) a 17 to 63-pS AbetaP[1-40]-formed "bursting" fast cation channel, (ii) the AbetaP[1-40]-formed "spiky" fast cation channel with a similar kinetics to the "bursting" fast channel except for the absence of the long intraburst closures, (iii) 275-pS AbetaP[1-40]-formed medium conductance channel, and (iv) 589- to 704-pS AbetaP[1-40]-formed inactivating large conductance channel. This heterogeneity is one of the most common features of these charged cytotoxic amyloid-formed channels, reflecting these channels' ability to modify multiple cellular functions. Although the diversity of these aggregated-peptide-formed channels may indicate that a stochastic mechanism governs their formation, the fact that certain channel types are often observed point to preferential channel protein conformations. In addition, the fact that other amyloids have similar structural properties (e.g. hydrophobicity, charged residues, and beta-structural linkages, suggests that, despite the intrinsic ability to form diverse conformations, certain conformations and, hence, certain channel types could be a common pathologic conformation among these amyloid-forming peptides. It is concluded that conformation-based channel diversity is an important mechanism for enhancing the toxicity of amyloid-forming peptides. The cytotoxic nature of these self-associated beta-based protein channels suggests that under normal physiological conditions cells employ well-evolved protective mechanisms against seeding and/or propagation of channel-forming peptides; for example, (a) compartmentalization of these peptides as membrane bound in internal vesicles and/or (b) degradation of these peptides by enzymes. The pharmacological diversity of the amyloid-forming channels implies that multiple therapeutic interventions may be necessary for blocking and reversing heterogeneous channel formations and preventing their associated diseases.     

A search for amyloidogenic regions in protein chain

We suggest a new method to detect amyloidogenic regions in a protein sequence. In the present work it is shown that regions enriched with amino acid residues which have a high expected packing density are responsible for the amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which positions of amyloidogenic regions have been revealed experimentally.     

Interference of low‐molecular substances with the thioflavin‐T fluorescence assay of amyloid fibrils

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type‐II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low‐molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid‐β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag‐period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Phe-Gly motifs drive fibrillization of TDP-43’s prion-like domain condensates

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

The prion-like domain of TDP-43 assembles biomolecular condensates which mature into amyloid fibrils that accumulate at the condensate/solvent interface. In vitro reconstitution of these fibrils reveals an amyloid core stabilized by residues that are not necessarily essential to create the droplet form.     

Effect of trehalose on W7FW14F apomyoglobin and insulin fibrillization: new insight into inhibition activity

Trehalose, a disaccharide present in many nonmammalian species, protects cells against various environmental stresses. Trehalose has recently been shown to decrease aggregate formation and toxicity in cell models and to alleviate amyloid-induced diseases. The aim of our study was to use two amyloid-forming proteins, i.e., W7FW14F apomyoglobin and insulin, as model systems to elucidate the molecular mechanism by which trehalose affects the amyloid aggregation process and to investigate further its therapeutic potential. Protein aggregation was examined by far-UV circular dichroism, UV absorption, thioflavin T fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, atomic force microscopy, and Fourier transform infrared spectroscopy. Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. We found that trehalose does not inhibit protein aggregation but acts at different stages of the fibrillization process depending on the protein model used. In fact, trehalose dose-dependently inhibited fibril formation in the W7FW14F apomyoglobin model and increased the lag phase in the insulin model. In both cases, trehalose caused accumulation of toxic oligomeric species. The results suggest that trehalose may favor or inhibit the formation of "on-pathway" or "off-pathway" oligomeric intermediates depending on the nature of the aggregating protein.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

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.     

The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain

Several proteins that interact with cell surface glycolipids share a common fold with a solvent-exposed aromatic residue that stacks onto a sugar ring of the glycolipid (CH-pi stacking interaction). Stacking interactions between aromatic residues (pi-pi stacking) also play a pivotal role in the assembly process, including many cases of amyloid fibril formation. We found a structural similarity between a typical glycolipid-binding domain (the V3 loop of HIV-1 gp120) and the minimal amyloid-forming fragment of the human islet amyloid polypeptide, i.e. the octapeptide core module NFGAILSS. In a monolayer assay at the air-water interface, the NFGAILSS peptide specifically interacted with the glycolipid lactosylceramide. The interaction appears to require an aromatic residue, as NLGAILSS was poorly recognized by lactosylceramide, whereas NYGAILSS behaved like NFGAILSS. In addition, we observed that the full-length human islet amyloid polypeptide (1-37) did interact with a monolayer of lactosylceramide, and that the glycolipid film significantly affected the aggregation process of the peptide. As glycolipid-V3 interactions are efficiently inhibited by suramin, a polyaromatic compound, we investigated the effects of suramin on amyloid formation by human islet amyloid polypeptide. We found that suramin inhibited amyloid fibril formation at low concentrations, but dramatically stimulated the process at high concentrations. Taken together, our results indicate that the minimal amyloid-forming fragment of human islet amyloid polypeptide is a glycolipid-binding domain, and provide further experimental support for the role of aromatic pi-pi and CH-pi stacking interactions in the molecular control of the amyloidogenesis process.     

Prediction of amyloidogenic and disordered regions in protein chains

The determination of factors that influence protein conformational changes is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for eight of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of amyloid fibril formation is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value with which one can predict both intrinsically disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.     

What Drives Amyloid Molecules To Assemble into Oligomers and Fibrils?

We develop a theory for three states of equilibrium of amyloid peptides: the monomer, oligomer, and fibril. We assume that the oligomeric state is a disordered micellelike collection of a few peptide chains held together loosely by hydrophobic interactions into a spherical hydrophobic core. We assume that fibrillar amyloid chains are aligned and further stabilized by steric zipper interactions—hydrogen bonding, steric packing, and specific hydrophobic side-chain contacts. The model makes a broad set of predictions that are consistent with experimental results: 1), Similar to surfactant micellization, amyloid oligomerization should increase with peptide concentration in solution. 2), The onset of fibrillization limits the concentration of oligomers in the solution. 3), The extent of Aβ fibrillization increases with peptide concentration. 4), The predicted average fibril length versus monomer concentration agrees with data on α-synuclein. 5), Full fibril length distributions agree with data on α-synuclein. 6), Denaturants should melt out fibrils. And finally, 7), added salt should stabilize fibrils by reducing repulsions between amyloid peptide chains. It is of interest that small changes in solvent conditions can tip the equilibrium balance between oligomer and fibril and cause large changes in rates through effects on the transition-state barrier. This model may provide useful insights into the physical processes underlying amyloid diseases.     

Resolution of the effects induced by W?→?F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.     

Substitutions at codon 22 of Alzheimer's abeta peptide induce diverse conformational changes and apoptotic effects in human cerebral endothelial cells

Cerebral amyloid angiopathy is commonly associated with normal aging and Alzheimer's disease and it is also the principal feature of hereditary cerebral hemorrhage with amyloidosis Dutch type, a familial condition associated to a point mutation G to C at codon 693 of the amyloid beta (Abeta) precursor protein gene resulting in a Glu to Gln substitution at position 22 of the Abeta (E22Q). The patients carrying the AbetaE22Q variant usually present with lobar cerebral hemorrhages before 50 years of age. A different mutation described in several members of three Italian kindred who presented with recurrent hemorrhagic strokes late in life, between 60 and 70 years of age, also associated with extensive cerebrovascular amyloid deposition has been found at the same position 22, this time resulting in a Glu to Lys substitution (E22K). We have compared the secondary structure, aggregation, and fibrillization properties of the two Abeta40 variants and the wild type peptide. Using flow cytometry analysis after staining with propidium iodide and annexin V, we also evaluated the cytotoxic effects of the peptides on human cerebral endothelial cells in culture. Under the conditions tested, the E22Q peptide exhibited the highest content of beta-sheet conformation and the fastest aggregation/fibrillization properties. The Dutch variant also induced apoptosis of cerebral endothelial cells at a concentration of 25 micrometer, whereas the wild type Abeta and the E22K mutant had no effect. The data suggest that different amino acids at position 22 confer distinct structural properties to the peptides that appear to influence the onset and aggressiveness of the disease rather than the phenotype.     

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid‐β peptide 1‐42 but not amylin and insulin fibrils can grow under quiescent conditions

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides – Alzheimer's amyloid‐β (Aβ) 1‐40 and 1‐42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aβ₄₀ substantially decreased, whereas the fibrillization of Aβ₄₂ peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Deacylated pulmonary surfactant protein SP-C transforms from alpha-helical to amyloid fibril structure via a pH-dependent mechanism: an infrared structural investigation

Bovine pulmonary surfactant protein C (SP-C) is a hydrophobic, alpha-helical membrane-associated lipoprotein in which cysteines C4 and C5 are acylated with palmitoyl chains. Recently, it has been found that the alpha-helix form of SP-C is metastable, and under certain circumstances may transform from an alpha-helix to a beta-strand conformation that resembles amyloid fibrils. This transformation is accelerated when the protein is in its deacylated form (dSP-C). We have used infrared spectroscopy to study the structure of dSP-C in solution and at membrane interfaces. Our results show that dSP-C transforms from an alpha-helical to a beta-type amyloid fibril structure via a pH-dependent mechanism. In solution at low pH, dSP-C is alpha-helical in nature, but converts to an amyloid fibril structure composed of short beta-strands or beta-hairpins at neutral pH. The alpha-helix structure of dSP-C is fully recoverable from the amyloid beta-structure when the pH is once again lowered. Attenuated total reflectance infrared spectroscopy of lipid-protein monomolecular films showed that the fibril beta-form of dSP-C is not surface-associated at the air-water interface. In addition, the lipid-associated alpha-helix form of dSP-C is only retained at the surface at low surface pressures and dissociates from the membrane at higher surface pressures. In situ polarization modulation infrared spectroscopy of protein and lipid-protein monolayers at the air-water interface confirmed that the residual dSP-C helix conformation observed in the attenuated total reflectance infrared spectra of transferred films is randomly or isotropically oriented before exclusion from the membrane interface. This work identifies pH as one of the mechanistic causes of amyloid fibril formation for dSP-C, and a possible contributor to the pathogenesis of pulmonary alveolar proteinosis.     

The role of G protein activation in the toxicity of amyloidogenic Abeta-(1-40), Abeta-(25-35), and bovine calcitonin

More than 16 different proteins have been identified as amyloid in clinical diseases; among these, beta-amyloid (Abeta) of Alzheimer's disease is the best characterized. In the present study, we performed experiments with Abeta and calcitonin, another amyloid-forming peptide, to examine the role of G protein activation in amyloid toxicity. We demonstrated that the peptides, when prepared under conditions that promoted beta-sheet and amyloid fibril (or protofibril) formation, increased high affinity GTPase activity, but the nonamyloidogenic peptides had no discernible effects on GTP hydrolysis. These increases in GTPase activity were correlated to toxicity. In addition, G protein inhibitors significantly reduced the toxic effects of the amyloidogenic Abeta and calcitonin peptides. Our results further indicated that the amyloidogenic peptides significantly increased GTPase activity of purified Galpha(o) and Galpha(i) subunits and that the effect was not receptor-mediated. Collectively, these results imply that the amyloidogenic structure, regardless of the actual peptide or protein sequence, may be sufficient to cause toxicity and that toxicity is mediated, at least partially, through G protein activation. Our abilities to manipulate G protein activity may lead to novel treatments for Alzheimer's disease and the other amyloidoses.     

Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.     

Prevention and promotion effects of apolipoprotein E4 on amylin aggregation

The misfolding of islet amyloid polypeptide (IAPP, amylin) results in the formation of islet amyloid, which is one of the most common pathological features of type 2 diabetes (T2D). Amylin, a 37-amino-acid peptide co-secreted with insulin and apolipoprotein E (ApoE) from the β-cells of pancreatic islets, is thought to be responsible for the reduced mass of insulin-producing β-cells. However, neither the relationship between amylin and ApoE nor the biological consequence of amylin misfolding is known. Here we have characterized the interaction between ApoE4 and amylin in vitro. We found that ApoE4 can strongly bind to amylin, and insulin can hardly inhibit amylin-ApoE binding. We further found that amylin fibrillization can be prevented by low concentration of ApoE4 and promoted by high concentration of ApoE4. Taken together, we propose that under physiological conditions ApoE4 efficiently binds and sequesters amylin, preventing its aggregation, and in T2D the enhanced ApoE4-amylin binding leads to the critical accumulation of amylin, facilitating islet amyloid formation.