A hIAPP-derived all-d-amino-acid inhibits hIAPP fibrillation efficiently at membrane surface by targeting α-helical oligomeric intermediates

A variety of peptides and peptide derivatives have been constructed using the “β-sheet core segment” of amyloid proteins as inhibitors of amyloidogenic fibrillation. A novel all-d-amino-acid from hIAPP β-sheet core segment (hIAPP 22–27) is demonstrated to inhibit hIAPP fibril formation efficiently both at the phospholipid membrane and in bulk solution. The inhibitor terminates hIAPP aggregation to the α-helical oligomeric intermediates at the membrane surface, whereas it stops the aggregation at the stage of β-sheet oligomeric intermediates in bulk solution. This is the first evidence that the inhibition mechanism of the inhibitor at membrane surface is significantly different from that in bulk solution.     

Interfacial effect on the ability of peptide-modified gold nanoclusters to inhibit hIAPP fibrillation and cytotoxicity

Deposit of amyloid peptides in the cells is related to various amyloidosis diseases. A variety of nanomaterials have been developed to resist amyloid deposit. Most of the research on the inhibition of nanomaterials against amyloid aggregation are undertaken in solution, while the membranes that may mediate fibrillar aggregation and affect interaction of inhibitors with amyloid peptides in biotic environment are little taken into account. In this study, we synthesized three kinds of gold nanoclusters modified with cysteine (C@AuNCs), glutathione (GSH@AuNCs) and a peptide derived from the core region of hIAPP fibrillation (C-HL-8P@AuNCs), and investigated their inhibitory activities against hIAPP fibrillation in the absence and presence of lipid vesicles (POPC/POPG 4:1 LUVs) by the experiments of ThT fluorescence kinetics, AFM and CD. We also explored the inhibitions of hIAPP-induced membrane damage and cytotoxicity by peptide@AuNCs using fluorescent dye leakage and cell viability assays. Our study revealed that the inhibitory efficiency of these peptide@AuNCs against hIAPP fibrillation follows C-HL-8P@AuNCs≅GSH@AuNCs>C@AuNCs in lipid-free solution and C-HL-8P@AuNCs≫GSH@AuNCs>C@AuNCs in lipid membrane environment. Compared with the results obtained in lipid-free solution, the inhibitions of hIAPP fibrillation observed in lipid membrane environment were more associated with the inhibitions of hIAPP-induced damages of lipid vesicles and INS-1 cells (C-HL-8P@AuNCs≫GSH@AuNCs>C@AuNCs). An additional hydrophobic interaction with the homologous core region of hIAPP, which is only provided by C-HL-8P@AuNCs and largely suppressed in lipid-free solution, enhanced in the membrane environment and therefore made C-HL-8P@AuNCs much more powerful than GSH@AuNCs and C@AuNCs in the inhibitions of hIAPP fibrillation and cytotoxicity.     

Amyloidogenic Propensities and Conformational Properties of ProIAPP and IAPP in the Presence of Lipid Bilayer Membranes

Human islet amyloid polypeptide (hIAPP), which is considered the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in β-cells of the pancreas from its precursor pro-islet amyloid polypeptide (proIAPP), which may be important in early intracellular amyloid formation as well. We compare the amyloidogenic propensities and conformational properties of proIAPP and hIAPP in the presence of negatively charged lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. Circular dichroism studies verify the initial secondary structures of proIAPP and hIAPP to be predominantly unordered with small amounts of ordered secondary structure elements, and exhibit minor differences between these two peptides only. Using attenuated total reflection-Fourier transform infrared spectroscopy and thioflavin T fluorescence spectroscopy, as well as atomic force microscopy, we show that in the presence of negatively charged membranes, proIAPP exhibits a much higher amyloidogenic propensity than in bulk solvent. Compared to hIAPP, it is still much less amyloidogenic, however. Although differences in the secondary structures of the aggregated species of hIAPP and proIAPP at the lipid interface are small, they are reflected in morphological changes. Unlike hIAPP, proIAPP forms essentially oligomeric-like structures at the lipid interface. Besides the interaction with anionic membranes [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) + x1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]], interaction with zwitterionic homogeneous (DOPC) and heterogeneous (1,2-dipalmitoyl-sn-glycero-3-phosphocholine:DOPC:cholesterol 1:2:1 model raft mixture) membranes has also been studied. Both peptides do not aggregate significantly at DOPC bilayers. In the presence of the model raft membrane, hIAPP aggregates markedly as well. Conversely, proIAPP clusters into less ordered structures and to a minor extent at raft membranes only. The addition of proIAPP to hIAPP retards the hIAPP fibrillation process also in the presence of negatively charged lipid bilayers. In excess proIAPP, increased aggregation levels are finally observed, however, which could be attributed to seed-induced cofibrillation of proIAPP.     

Recent computational studies of membrane interaction and disruption of human islet amyloid polypeptide: Monomers,oligomers and protofibrils

The amyloid deposits of human islet amyloid polypeptide (hIAPP) are found in type 2 diabetes patients. hIAPP monomer is intrinsically disordered in solution, whereas it can form amyloid fibrils both in vivo and in vitro. Extensive evidence suggests that hIAPP causes the disruption of cellular membrane, and further induces cytotoxicity and the death of islet β-cells in pancreas. The presence of membrane also accelerates the hIAPP fibril formation. hIAPP oligomers and protofibrils in the early stage of aggregation were reported to be the most cytotoxic, disrupting the membrane integrity and giving rise to the pathological process. The detailed molecular mechanisms of hIAPP-membrane interactions and membrane disruption are complex and remain mostly unknown. Here in this review, we focus on recent computational studies that investigated the interactions of full length and fragmentary hIAPP monomers, oligomers and protofibrils with anionic, zwitterionic and mixed anionic-zwitterionic lipid bilayers. We mainly discuss the binding orientation of monomers at membrane surface, the conformational ensemble and the oligomerization of hIAPP inside membranes, the effect of lipid composition on hIAPP oligomers/protofibrils-membrane interactions, and the hIAPP-induced membrane perturbation. This review provides mechanistic insights into the interactions between hIAPP and lipid bilayers with different lipid composition at an atomistic level, which is helpful to understand the hIAPP cytotoxicity mediated by membrane. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP_1-19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP_1-19 in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP_1-19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP_1-19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.     

A single mutation on the human amyloid polypeptide modulates fibril growth and affects the mechanism of amyloid-induced membrane damage

Amyloid fibril formation has been implicated in a wide range of human diseases and the interactions of amyloidogenic proteins with cell membranes are considered to be important in the aetiology of these pathologies. In type 2 diabetes mellitus (T2DM), the human islet amyloid polypeptide (hIAPP) forms amyloid fibrils which impair the functionality and viability of pancreatic β cells. The mechanisms of hIAPP cytotoxicity are linked to the ability of the peptide to self-aggregate and to interact with membranes. Previous studies have shown that the N-terminal part of hIAPP from residues 1 to 19 is the membrane binding domain. The non-amyloidogenic and nontoxic mouse IAPP differs from hIAPP by six residues out of 37, among which a single one, residue 18, lies in the membrane binding region. To gain more insight into hIAPP-membrane interactions we herein performed comprehensive biophysical studies on four analogues (H18R-IAPP, H18K-IAPP, H18E-IAPP and H18A-IAPP). Our data reveal that all peptides are able to insert efficiently in the membrane, indicating that residue 18 is not essential for hIAPP membrane binding and insertion. However, only wild-type hIAPP and H18K-IAPP are able to form fibrils at the membrane. Importantly, all peptides induce membrane damage; wild-type hIAPP and H18K-IAPP presumably cause membrane disruption mainly by fibril growth at the membrane, while for H18R-IAPP, H18E-IAPP and H18A-IAPP, membrane leakage is most likely due to high molecular weight oligomeric species. These results highlight the importance of the residue at position 18 in IAPP for modulating fibril formation at the membrane and the mechanisms of membrane leakage.     

Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity

The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.     

Inhibition of human IAPP fibril formation does not prevent beta-cell death: evidence for distinct actions of oligomers and fibrils of human IAPP

Type 2 diabetes mellitus (T2DM) is characterized by an approximately 60% deficit in beta-cell mass, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (hIAPP) forms oligomers, leading to either amyloid fibrils or toxic oligomers in an aqueous solution in vitro. Either application of hIAPP on or overexpression of hIAPP in cells induces apoptosis. It remains controversial whether the fibrils or smaller toxic oligomers induce beta-cell apoptosis. Rifampicin prevents hIAPP amyloid fibril formation and has been proposed as a potential target for prevention of T2DM. We examined the actions of rifampicin on hIAPP amyloid fibril and toxic oligomer formation as well as its ability to protect beta-cells from either application of hIAPP or endogenous overexpression of hIAPP (transgenic rats and adenovirus-transduced beta-cells). We report that rifampicin (Acocella G. Clin Pharmacokinet 3: 108-127, 1978) prevents hIAPP fibril formation, but not formation of toxic hIAPP oligomers (Bates G. Lancet 361: 1642-1644, 2003), and does not protect beta-cells from apoptosis induced by either overexpression or application of hIAPP. These data emphasize that toxic hIAPP oligomers, rather than hIAPP fibrils, initiate beta-cell apoptosis and that screening tools to identify inhibitors of amyloid fibril formation are likely to be less useful than those that identify inhibitors of toxic oligomer formation. Finally, rifampicin and related molecules do not appear to be useful as candidates for prevention of T2DM.     

Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide (hIAPP)

The dramatic expansion of nanotechnology applications, particularly the advent of nanomaterials and nanoparticles (NPs) into the consumer economy, have led to heightened awareness of their potential health risks. This study examines the impact of several NPs upon membrane-induced aggregation and bilayer interactions of the human Islet amyloid polypeptide (hIAPP). We report that several NPs – polymeric NPs, TiO₂ NPs, and Au NPs displaying coating layers exhibiting different electrostatic charges - did not significantly interfere with the fibrillation process and fibril morphology of hIAPP, both in buffer or in biomimetic DMPC:DMPG vesicle solutions. Spectroscopic and microscopic analyses suggest, in fact, that the NPs promoted membrane-induced fibrillation. Importantly, we find that all the NPs examined, regardless of composition or surface properties, gave rise to more pronounced, synergistic bilayer interactions when co-incubated with hIAPP. NP-enhanced bilayer interactions of hIAPP might point to possible toxicity and pathogenicity risks of amyloidogenic peptides in the presence of NPs.     

Antimicrobial activity of human islet amyloid polypeptides: an insight into amyloid peptides' connection with antimicrobial peptides

Abstract Human islet amyloid polypeptide (hIAPP) shows an antimicrobial activity towards two types of clinically relevant bacteria. The potency of hIAPP varies with its aggregation states. Circular dichroism was employed to determine the interaction between hIAPP and bacteria lipid membrane mimic. The antimicrobial activity of each aggregate species is associated with their ability to induce membrane disruption. Our findings provide new evidence revealing the antimicrobial activity of amyloid peptide, which suggest a possible connection between amyloid peptides and antimicrobial peptides.     

Cholesterol modulates the interaction of the islet amyloid polypeptide with membranes

The deposition of insoluble amyloid fibrils resulting from the aggregation of the human islet amyloid polypeptide (hIAPP) within the islet of Langerhans is a pathological feature of type 2 diabetes mellitus (T2DM). Increasing evidence indicates that biological membranes play a key role in amyloid aggregation, modulating among others the kinetics of amyloid formation, and being the target of toxic species generated during amyloid formation. In T2DM patients, elevated levels of cholesterol, an important determinant of the physical state of biological membranes, are observed in β-cells and are thought to directly impair β-cell function and insulin secretion. However, it is not known whether cholesterol enhances membrane-interaction or membrane-insertion of hIAPP. In this study, we investigated the effect of cholesterol incorporated in zwitterionic and anionic membranes. Our circular dichroism and liquid state NMR data reveal that 10–30% of cholesterol slightly affects the aggregational and conformational behaviour of hIAPP. Additional fluorescence results indicate that 10 and 20% of cholesterol slightly slow down the kinetics of oligomer and fibril formation while anionic lipids accelerate this kinetics. This behavior might be caused by differences in membrane insertion and therefore in membrane binding of hIAPP. The membrane binding affinity was evaluated using ¹H NMR experiments and our results show that the affinity of hIAPP for membranes containing cholesterol is significantly smaller than that for membranes containing anionic lipids. Furthermore, we found that hIAPP-induced membrane damage is synchronized to fibril formation in the absence and in the presence of cholesterol.     

Interplay between human islet amyloid polypeptide aggregates and micro-heterogeneous membranes

Human islet amyloid polypeptides (hIAPP) aggregate into amyloid deposits in the pancreatic islets of Langerhans, contributing to the loss of β-cells of patients with type 2 diabetes. Despite extensive studies of membrane disruption associated with hIAPP aggregates, the molecular details regarding the complex interplay between hIAPP aggregates and raft-containing membranes are still very limited. Using all-atom molecular dynamics simulations, we investigate the impact of hIAPP aggregate insertion on lipid segregation. We have found that the domain separation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is enhanced upon hIAPP membrane permeabilization in the absence of cholesterol, while within our simulation timescale, we cannot provide definitive evidence regarding the impact of hIAPP insertion on domain segregation in the ternary mixture (DOPC/DPPC/cholesterol). When the lipid domains are perturbed, their restoration occurs rapidly and spontaneously in the presence of hIAPP aggregates. hIAPP insertion affects membrane thickness in its immediate surroundings. On average, hIAPP causes the fluidity of lipids to increase and even cholesterol shows enhanced diffusivity. The acyl chain packing of the lipids near hIAPP is disrupted as compared to that further away from it. Cholesterol not only modulates membrane mobility and ordering but also hIAPP aggregates' structure and relative orientation to the membrane. Our investigations on the interaction between hIAPP aggregates and raft-containing membranes could lead to a better understanding of the mechanisms of amyloid cytotoxicity.     

Nucleobindin 1 caps human islet amyloid polypeptide protofibrils to prevent amyloid fibril formation

Many human diseases are associated with amyloid fibril deposition, including type 2 diabetes mellitus where human islet amyloid polypeptide (hIAPP) forms fibrils in the pancreas. We report here that engineered, soluble forms of the human Ca(2+)-binding protein nucleobindin 1 (NUCB1) prevent hIAPP fibril formation and disaggregate preexisting hIAPP fibrils. Scanning transmission electron microscopy (STEM) and atomic force microscopy indicate that NUCB1 binds to and stabilizes heterogeneous prefibrillar hIAPP species. The NUCB1-stabilized prefibrillar species were isolated by size-exclusion chromatography and analyzed by STEM, dynamic light scattering, and multi-angle light scattering. The stabilized prefibrillar species show a size range of 2-6 million Da and have other similarities to hIAPP protofibrils, but they do not progress to become mature fibrils. The effects of NUCB1 are absent in the presence of Ca(2+). We postulate that the engineered forms of NUCB1 prevent hIAPP fibril formation by a mechanism where protofibril-like species are "capped" to prevent further fibril assembly and maturation. This mode of action appears to be different from other protein-based inhibitors, suggesting that NUCB1 may offer a new approach to inhibiting amyloid formation and disaggregating amyloid fibrils.     

Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes

A key factor in the development of Type II diabetes is the loss of insulin producing pancreatic β-cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill β-cells by disrupting the cellular membrane. In this study, we report membrane fragmentation by hIAPP using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes. In a narrow concentration range of hIAPP, an isotropic ³¹P chemical shift signal indicative of the peptide-induced membrane fragmentation was detected. Solid-state NMR results suggest that membrane fragmentation is related to peptide aggregation as the presence of Congo Red, an inhibitor of amyloid formation, prevented membrane fragmentation and the non-amyloidogenic rat-IAPP did not cause membrane fragmentation. The disappearance of membrane fragmentation at higher concentrations of hIAPP suggests an alternate kinetic pathway to fibril formation in which membrane fragmentation is inhibited.     

Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes

A key factor in the development of Type II diabetes is the loss of insulin producing pancreatic beta-cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill beta-cells by disrupting the cellular membrane. In this study, we report membrane fragmentation by hIAPP using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes. In a narrow concentration range of hIAPP, an isotropic (31)P chemical shift signal indicative of the peptide-induced membrane fragmentation was detected. Solid-state NMR results suggest that membrane fragmentation is related to peptide aggregation as the presence of Congo Red, an inhibitor of amyloid formation, prevented membrane fragmentation and the non-amyloidogenic rat-IAPP did not cause membrane fragmentation. The disappearance of membrane fragmentation at higher concentrations of hIAPP suggests an alternate kinetic pathway to fibril formation in which membrane fragmentation is inhibited.     

Recovery and purification of highly aggregation-prone disulfide-containing peptides: application to islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a 37-residue pancreatic hormone. It is responsible for the formation of islet amyloid in vivo and is very insoluble and aggregation-prone in vitro, particularly at basic pH. The peptide contains a disulfide bridge between residues two and seven and an amidated C terminus. There is no reported expression system for the production of amidated IAPP. The peptide is difficult to synthesize and formation of the disulfide by traditional methods is problematic. We have found that the use of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or dimethyl sulfoxide (DMSO) significantly improves disulfide formation and purification of highly aggregation-prone IAPP sequences. The use of these organic solvents increases the solubility of the hydrophobic peptides, avoids the use of aqueous basic solutions, and eliminates the need for continuous stirring during oxidation to form the Cys-2 to Cys-7 disulfide bridge. Elimination of the stirring step and basic solution helps to reduce aggregation and allows for more consistent high-performance liquid chromatography (HPLC) retention times. Formation of the intramolecular disulfide using DMSO was found to be the most effective method for IAPP oxidation, reducing the reaction time from 24 to 5 h. Aggregated IAPP can be resolubilized by HFIP or DMSO and recovered by HPLC with very good yield.     

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.     

Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement

Protein aggregation and amyloid fibrillation can lead to several serious diseases and protein drugs ineffectiveness; thus, the detection and inhibition of these processes have been of great interest. In the present study, the inhibition of insulin amyloid fibrillation by laser irradiation was investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD), and thioflavin T (ThT) fluorescence. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1 M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1 M NaCl for 45 h incubation. Moreover, our CD results showed that the laser-irradiated insulin solution maintained mainly an α-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in β-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.     

Molecular basis of the anchoring and stabilization of human islet amyloid polypeptide in lipid hydroperoxidized bilayers

The molecular structure of membrane lipids is formed by mono- or polyunsaturations on their aliphatic tails that make them susceptible to oxidation, facilitating the incorporation of hydroperoxide (R-OOH) functional groups. Such groups promote changes in both composition and complexity of the membrane significantly modifying its physicochemical properties. Human Langerhans islets amyloid polypeptide (hIAPP) is the main component of amyloid deposits found in the pancreas of patients with type-2 diabetes (T2D). hIAPP in the presence of membranes with oxidized lipid species accelerates the formation of amyloid fibrils or the formation of intermediate oligomeric structures. However, the molecular bases at the initial stage of the anchoring and stabilization of the hIAPP in a hydroperoxidized membrane are not yet well understood. To shed some light on this matter, in this contribution, three bilayer models were modeled: neutral (POPC), anionic (POPS), and oxidized (POPC_OOH), and full atom Molecular Dynamics (MD) simulations were performed. Our results show that the POPC_OOH bilayer increases the helicity in hIAPP when compared to POPC or POPS bilayer. The modification in the secondary structure covers the residues of the so-called amyloidogenic core of the hIAPP. Overall, the hydroperoxidation of the neutral lipids modifies both the anchoring and the stabilization of the peptide hIAPP by reducing the random conformations of the peptide and increasing of hydrogen bond population with the hydroperoxidized lipids.     

Blockade of islet amyloid polypeptide fibrillation and cytotoxicity by the secretory chaperones 7B2 and proSAAS

The deposition of fibrillated human islet β-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure–function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.