Transformation of the amyloidogenic peptide amylin(20-29) into its corresponding peptoid and retropeptoid: access to both an amyloid inhibitor and template for self-assembled supramolecular tapes

The highly amyloidogenic peptide sequence of amylin(20-29) was transformed into its corresponding peptoid and retropeptoid sequences to design a novel class of beta-sheet breaker peptides as amyloid inhibitors. This report describes the synthesis of the chiral peptoid building block of L-isoleucine, the solid phase synthesis of the peptoid and retropeptoid sequences of amylin(20-29), and the structural analysis of these amylin derivatives in solution by infrared spectroscopy, circular dichroism, and transmission electron microscopy. It was found that the peptoid sequence did not form amyloid fibrils or any other secondary structures and was able to inhibit amyloid formation of native amylin(20-29). Although the retropeptoid did not form amyloid fibrils it had only modest amyloid inhibitor properties since supramolecular tapes were formed.     

Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: Implications for the study of amyloid formation

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin(20-29)) is commonly used as a model system to study this process. Studies of amylin(20-29) and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin(20-29), which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.     

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.     

Identification of a penta- and hexapeptide of islet amyloid polypeptide (IAPP) with amyloidogenic and cytotoxic properties

Pancreatic amyloid is found in more than 95 % of type II diabetes patients. Pancreatic amyloid is formed by the aggregation of islet amyloid polypeptide (hIAPP or amylin), which is a 37-residue peptide. Because pancreatic amyloid is cytotoxic, it is believed that its formation is directly associated with the development of the disease. We recently showed that hIAPP amyloid formation follows the nucleation-dependent polymerization mechanism and proceeds via a conformational transition of soluble hIAPP into aggregated beta-sheets. Here, we report that the penta- and hexapeptide sequences, hIAPP(23-27) (FGAIL) and hIAPP(22-27) (NFGAIL) of hIAPP are sufficient for the formation of beta-sheet-containing amyloid fibrils. Although these two peptides differ by only one amino acid residue, they aggregate into completely different fibrillar assemblies. hIAPP(23-27) (FGAIL) fibrils self-assemble laterally into unusually broad ribbons, whereas hIAPP(22-27) (NFGAIL) fibrils coil around each other in a typical amyloid fibril morphology. hIAPP(20-27) (SNNFGAIL) also aggregates into beta-sheet-containing fibrils, whereas no amyloidogenicity is found for hIAPP(24-27) (GAIL), indicating that hIAPP(23-27) (FGAIL) is the shortest fibrillogenic sequence of hIAPP. Insoluble amyloid formation by the partial hIAPP sequences followed kinetics that were consistent with a nucleation-dependent polymerization mechanism. hIAPP(22-27) (NFGAIL), hIAPP(20-27) (SNNFGAIL), and also the known fibrillogenic sequence, hIAPP(20-29) (SNNFGAILSS) exhibited significantly lower kinetic and thermodynamic solubilities than the pentapeptide hIAPP(23-27) (FGAIL). Fibrils formed by all short peptide sequences and also by hIAPP(20-29) were cytotoxic towards the pancreatic cell line RIN5fm, whereas no cytotoxicity was observed for the soluble form of the peptides, a notion that is consistent with hIAPP cytotoxicity. Our results suggest that a penta- and hexapeptide sequence of an appropriate amino acid composition can be sufficient for beta-sheet and amyloid fibril formation and cytotoxicity and may assist in the rational design of inhibitors of pancreatic amyloid formation or other amyloidosis-related diseases.     

Atomic force microscopy study of human amylin (20-29) fibrils

Here we present atomic force microscopy images of the fibrils formed by human amylin(20-29). This peptide is a fragment of the polypeptide amylin, the major proteinaceous component of amyloid deposits found in cases of type-II diabetes mellitus. Our results demonstrate that the amylin(20-29) peptide fragment forms amyloid-like fibrils that display polymorphic structures. Twisting along the axis of fibrils was often observed in fibrils aged for 6 hours but disappeared in mature fibrils aged for longer time periods.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

Amyloid fibril formation from full-length and fragments of amylin

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.     

Effects of sequential proline substitutions on amyloid formation by human amylin20-29

Amylin, also known as islet amyloid polypeptide (IAPP), is the major protein component of the fibril deposits found in the pancreas of individuals with type II diabetes. The central region of amylin, residues 20-29, has been implicated as a key determinate of amyloid formation. To establish which positions are most important for amyloid formation, the wild-type sequence of the 20-29 fragment and a set of 10 variants have been synthesized in which a proline was placed at each position. Proline is energetically unfavorable in the extended cross-beta structure found in amyloid. If a particular position is critical for amyloid formation, then substitution with a proline should inhibit amyloid formation. A proline substitution at any position inhibited aggregation and amyloid formation. Substitution of Asn22, Gly24, and residues 26-28 had the largest effect. Fourier transform infrared (FTIR) spectroscopy showed little secondary structure in these peptides, and transmission electron microscopy (TEM) showed mostly amorphous material. The peptides were much more soluble than the wild-type sequence, and no birefringence was observed with Congo Red staining. Proline substitutions at the N (residues 20 and 21) and C termini showed the least effect. These peptides showed the classic fibril morphology, a significant amount of beta-sheet structure, and exhibited green birefringence when stained with Congo Red. The results indicate that residues 22, 24, and 26-28 play a key role in formation of amyloid by amylin. Positions 23 and 25 also appear to be important, but may be less critical than positions 22, 24, and 26-28.     

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.     

Conformational preferences of the amylin nucleation site in SDS micelles: an NMR study

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic beta-cells. hIAPP constitutes approximately 90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP(20-29)) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the beta-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the beta-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP(20-29) in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP(20-29) is a distorted type I beta-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent.     

Copper(II) inhibits the formation of amylin amyloid in vitro

The amyloidogenic peptide amylin is found associated with pancreatic islet beta-cells and is implicated in the aetiology of type-2 diabetes mellitus. We have used fluorimetry and transmission electron microscopy to investigate in vitro the influence of Al(III), Fe(III), Zn(II) and Cu(II) on amylin amyloid formation under near-physiological conditions. Cu(II) at 10.0 microM inhibited amylin of 0.4 and 2.0 microM from forming amyloid fibrils while the same concentration of either Al(III) or Zn(II) promoted the formation of beta-pleated sheet structures. If amylin amyloid is cytotoxic to beta-cells then Cu(II) should protect against the degeneration of the islets in type-2 diabetes mellitus.     

Role of amino acid hydrophobicity, aromaticity, and molecular volume on IAPP(20-29) amyloid self-assembly

Aromatic amino acids strongly promote cross-β amyloid formation; whether the amyloidogenicity of aromatic residues is due to high hydrophobicity and β-sheet propensity or formation of stabilizing π-π interactions has been debated. To clarify the role of aromatic residues on amyloid formation, the islet amyloid polypeptide 20-29 fragment [IAPP(20-29)], which contains a single aromatic residue (Phe 23), was adopted as a model. The side chain of residue 23 does not self-associate in cross-β fibrils of IAPP(20-29) (Nielsen et al., Angew Chem Int Ed 2009;48:2118-2121), allowing investigation of the amyloidogenicity of aromatic amino acids in a context where direct π-π interactions do not occur. We prepared variants of IAPP(20-29) in which Tyr, Leu, Phe, pentafluorophenylalanine (F5-Phe), Trp, cyclohexylalanine (Cha), α-naphthylalanine (1-Nap), or β-naphthylalanine (2-Nap) (in order of increasing peptide hydrophobicity) were incorporated at position 23 (SNNXGAILSS-NH2), and the kinetic and thermodynamic effects of these mutations on cross-β self-assembly were assessed. The Tyr, Leu, and Trp 23 variants failed to readily self-assemble at concentrations up to 1.5 mM, while the Cha 23 mutant fibrillized with attenuated kinetics and similar thermodynamic stability relative to the wild-type Phe 23 peptide. Conversely, the F5-Phe, 1-Nap, and 2-Nap 23 variants self-assembled at enhanced rates, forming fibrils with greater thermodynamic stability than the wild-type peptide. These results indicate that the high amyloidogenicity of aromatic amino acids is a function of hydrophobicity, β-sheet propensity, and planar geometry and not the ability to form stabilizing or directing π-π bonds.     

Evidence for a Partially Structured State of the Amylin Monomer

Islet amyloid polypeptide (amylin) is the main component in amyloid deposits formed in type II diabetes. We used triplet quenching to probe the dynamics of contact formation between the N-terminal disulfide loop and a C-terminal tryptophan in monomeric amylins from human and rat. Quenching rates measured in the absence of denaturant are four times larger than those in 6 M guanidinium chloride, indicating a decrease in the average end-to-end distance (collapse) at low denaturant concentrations. We were surprised to find an even greater (sevenfold) increase in quenching rates on removal of denaturant for a hydrophilic control peptide containing the disulfide loop compared to the same peptide without the loop (twofold change). These results suggest that collapse is driven by backbone-backbone and backbone-side chain interactions involving the disulfide loop portion of the chain rather than by the formation of side-chain hydrophobic contacts. Molecular dynamics simulations of the control peptide show that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop. The quenching experiments also indicate that the monomer of the human, amyloidogenic form of amylin is more compact than the rat form, which does not form amyloid. We discuss these newly observed differences between human and rat amylin in solution and their possible relation to aggregation and to the physiological function of amylin binding to the calcitonin receptor.     

Supramolecular assembly of collagen triblock peptides

The relationship between primary sequence and collagen triple-helix formation is relatively well characterized, while higher levels of structural assembly from these sequences is poorly understood. To address this gap, a new collagen-like triblock peptide design was used to study the relationship between amino acid sequence and supramolecular assembly. Four collagen-like peptides with the sequence (Glu)(5)(Gly-Xaa-Hyp-Gly-Pro-Hyp)(6)(Glu)(5) and corresponding to Xaa = alanine, proline, serine, or valine, and an analogous peptide without the glutamic acid end blocks, were solubilized in water at high concentrations (20-150 mg/mL) and analyzed in optical polarizing microscopy and transmission electron microscopy. Some of the peptides self-assembled into supramolecular structures, the nature of which was determined by the core collagen-like sequence. The globular end blocks appeared necessary for these short triple-helix-forming peptides to spontaneously organize into supramolecular structures in solution and also provided enhanced thermal stability based on CD analysis. The results indicate a strong dependence of the peptide triblock assembly behavior on the identity of the guest residue Xaa; nematic order when Xaa was valine, no organization when Xaa was serine, and banded spherulites displaying a cholesteric-like twist when Xaa was proline or alanine. According to these results, the identity of the amino acid in position Xaa of the triplet Gly-Xaa-Yaa dramatically determined the type of supramolecular assembly formed by short triple helices based on collagen-triblock like sequences. Moreover, the structural organization observed for these collagen-triblock peptides was analogous to some assemblies observed for native collagen in vivo and in vitro. The amino acid sequence in the native collagen proteins may therefore be a direct determinant of the different supramolecular architectures found in connective tissues.     

The dye SYPRO orange binds to amylin amyloid fibrils but not pre‐fibrillar intermediates

Amyloid deposition underlies a broad range of diseases including multiple neurodegenerative diseases, systemic amyloidosis and type‐2 diabetes. Amyloid sensitive dyes, particularly thioflavin‐T, are widely used to detect ex‐vivo amyloid deposits, to monitor amyloid formation in vitro and to follow the kinetics of amyloid self‐assembly. We show that the dye SYPRO‐orange binds to amyloid fibrils formed by human amylin, the polypeptide responsible for islet amyloid formation in type‐2 diabetes. No fluorescence enhancement is observed in the presence of pre‐fibrillar species or in the presence of non‐amyloidogenic rat amylin. The kinetics of human amylin amyloid formation can be monitored by SYPRO‐orange fluorescence and match the time course determined with thioflavin‐T assays. Thus, SYPRO‐orange offers an alternative to thioflavin‐T assays of amylin amyloid formation. The implications for the interpretation of SYPRO‐orange‐based assays of protein stability and protein‐ligand interactions are discussed.     

Amylin proprotein processing generates progressively more amyloidogenic peptides that initially sample the helical state

Human amylin, or islet amyloid polypeptide, is a peptide cosecreted with insulin by the beta cells of the pancreatic islets of Langerhans. The 37-residue, C-terminally amidated human amylin peptide derives from a proprotein that undergoes disulfide bond formation in the endoplasmic reticulum and is then subjected to four enzymatic processing events in the immature secretory granule. Human amylin forms both intracellular and extracellular amyloid deposits in the pancreas of most type II diabetic subjects, likely reflecting compromised secretory cell function. In addition, amylin processing intermediates, postulated to initiate intracellular amyloidogenesis, have been reported as components of intracellular amyloid in beta cells. We investigated the amyloidogenicity of amylin and its processing intermediates in vitro. Chaotrope-denatured amylin and amylin processing intermediates were subjected to size exclusion chromatography, affording high concentrations of monomeric peptides. NMR studies reveal that human amylin samples helical conformations. Under conditions mimicking the immature secretory granule (37 degrees C, pH 6), amylin forms amyloid aggregates more rapidly than its processing intermediates, and more rapidly than its reduced counterparts. Our studies also show that the amyloidogenicity of amylin and its processing intermediates is negatively correlated with net charge and charge at the C-terminus. Although our conditions may not precisely reflect those of amyloidogenesis in vivo, the lower amyloidogenicity of the processing intermediates relative to amylin suggests their presence in intracellular amyloid deposits in the increasingly stressed beta cells of diabetic subjects may be a consequence of general defects in protein homeostasis control known to occur in diabetes rather than serving as amyloid initiators.     

Delayed fibril formation of amylin(20-29) by incorporation of alkene dipeptidosulfonamide isosteres obtained by solid phase olefin cross metathesis

The synthesis of a new peptidomimetic structure, the alkene dipeptidosulfonamide isostere, is described. The synthesis is based on a cross metathesis reaction between two allylic building blocks, both in solution and on the solid phase. This method was also applicable to the solid phase synthesis of alkene dipeptide isosteres. Derivatives of amylin(20-29) containing the alkene dipeptidosulfonamide isostere as well as the alkene dipeptide isostere were successfully synthesized using the solid phase cross metathesis method. Investigation of relations between structure and fibril formation of these amylin(20-29) derivatives showed retardation of fibril formation and altered secondary structures, compared to native amylin(20-29).     

Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic β-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured β-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured β-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.     

Cholesterol Regulates Assembly of Human Islet Amyloid Polypeptide on Model Membranes

Amylin, a 37-aa pancreatic hormone, is the major constituent of islet amyloid, a hallmark of type II diabetes mellitus. Recent studies have revealed a pivotal role of anionic phospholipids in membrane-catalyzed amylin fibrillogenesis and aggregation. However, cholesterol, an integral component of eukaryotic cell membranes, also could have a role. In this study, we have examined the effect of cholesterol on amylin polymerization both on planar membranes and in solution. Using time-lapse atomic force microscopy, we have studied the dynamics and macromolecular organization of amylin on anionic and neutral planar membranes that lack or include cholesterol. On cholesterol-depleted planar membranes, amylin formed highly symmetrical tetrameric and pentameric pore-like supramolecular structures composed of 25- to 35-nm intermediate-sized globular structures or oligomers. Conversely, on membranes incorporating cholesterol, amylin formed highly compact ∼ 200- to 500-nm protein clusters that constituted seeds or nuclei for continuing amylin binding and aggregation. However, cholesterol inhibited amylin nucleation with a 7-fold decrease in the number of amylin particles. Consequently, cholesterol-containing membranes accumulated significantly less amyloid with some membrane areas completely free of amyloid particles. The inhibitory effect of cholesterol on amylin aggregation in solution was also demonstrated as a 16-fold decrease in the aggregation rate. Consistent with this, circular dichroism spectroscopy revealed a stable, soluble random-coil conformation for amylin in the presence of cholesterol that could explain the inhibitory effect of cholesterol on amylin polymerization in solution and on membranes. The modulatory effect of cholesterol was largely independent of membrane charge or phospholipids, suggesting a novel cholesterol-regulated amylin polymerization process.     

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.