Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

The molecular basis of distinct aggregation pathways of islet amyloid polypeptide

Abnormal aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils is a hallmark of type 2 diabetes. In this study, we investigated the initial oligomerization and subsequent addition of monomers to growing aggregates of human IAPP at the residue-specific level using NMR, atomic force microscopy, mass spectroscopy, and computational simulations. We found that in solution IAPPs rapidly associate into transient low-order oligomers such as dimers and trimers via interactions between histidine 18 and tyrosine 37. This initial event is proceeded by slow aggregation into higher-order spherical oligomers and elongated fibrils. In these two morphologically distinct types of aggregates IAPPs adopt structures with markedly different residual flexibility. Here we show that the anti-amyloidogenic compound resveratrol inhibits oligomerization and amyloid formation via binding to histidine 18, supporting the finding that this residue is crucial for on-pathway oligomer formation.     

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

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.     

Amyloidogenicity and cytotoxicity of islet amyloid polypeptide

Insoluble amyloid formation by islet amyloid polypeptide (IAPP) in the islets of Langerhans of the pancreas is a major pathophysiological feature of noninsulin dependent diabetes mellitus (NIDDM) or type II diabetes. Because in vivo formed amyloid colocalizes with areas of cell degeneration and IAPP amyloid aggregates are cytotoxic per se, the process of IAPP amyloid formation has been strongly associated with the progressive pancreatic cell degeneration and thus much of the pathology of type II diabetes. IAPP is a pancreatic polypeptide of 37 residues that, in its soluble form, is believed to play a role as a regulator of glucose homeostasis. The molecular cause and mechanism of the conversion of soluble IAPP into insoluble amyloid aggregates in vivo and its role in disease progress still remain to be clarified. Nevertheless, in the past few years significant progress has been made in understanding the amyloidogenesis pathway of IAPP in vitro and gaining insight into the structural and conformational "requirements" of IAPP amyloidogenesis and related cytotoxic effects. Importantly, several of the studies have revealed significant similarities of the above features of IAPP to other amyloidogenic polypeptides such as the beta-amyloid polypeptide Abeta. This suggests that, at the molecular level, amyloidogenesis, and possibly related cell degeneration and disease pathogenesis by completely different polypeptide sequences, may obey to common structural and conformational "rules" and follow similar molecular pathways. This review describes studies on the structural and conformational features of IAPP amyloid formation and cytotoxicity, and the application of the obtained knowledge for the understanding of the molecular mechanism of the IAPP amyloidogenesis pathway and the related cytotoxicity.     

A single mutation in the nonamyloidogenic region of islet amyloid polypeptide greatly reduces toxicity

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide secreted with insulin by beta-cells in the islets of Langerhans. The aggregation of the peptide into either amyloid fibers or small soluble oligomers has been implicated in the death of beta-cells during type 2 diabetes through disruption of the cellular membrane. The actual form of the peptide responsible for beta-cell death has been a subject of controversy. Previous research has indicated that the N-terminal region of the peptide (residues 1-19) is primarily responsible for the membrane-disrupting effect of the hIAPP peptide and induces membrane disruption to a similar extent as the full-length peptide without forming amyloid fibers when bound to the membrane. The rat version of the peptide, which is both noncytotoxic and nonamyloidogenic, differs from the human peptide by only one amino acid residue: Arg18 in the rat version while His18 in the human version. To elucidate the effect of this difference, we have measured in this study the effects of the rat and human versions of IAPP(1-19) on islet cells and model membranes. Fluorescence microscopy shows a rapid increase in intracellular calcium levels of islet cells after the addition of hIAPP(1-19), indicating disruption of the cellular membrane, while the rat version of the IAPP(1-19) peptide is significantly less effective. Circular dichroism experiments and dye leakage assays on model liposomes show that rIAPP(1-19) is deficient in binding to and disrupting lipid membranes at low but not at high peptide to lipid ratios, indicating that the ability of rIAPP(1-19) to form small aggregates necessary for membrane binding and disruption is significantly less than hIAPP(1-19). At pH 6.0, where H18 is likely to be protonated, hIAPP(1-19) resembles rIAPP(1-19) in its ability to cause membrane disruption. Differential scanning calorimetry suggests a different mode of binding to the membrane for rIAPP(1-19) compared to hIAPP(1-19). Human IAPP(1-19) has a minimal effect on the phase transition of lipid vesicles, suggesting a membrane orientation of the peptide in which the mobility of the acyl chains of the membrane is relatively unaffected. Rat IAPP(1-19), however, has a strong effect on the phase transition of lipid vesicles at low concentrations, suggesting that the peptide does not easily insert into the membrane after binding to the surface. Our results indicate that the modulation of the peptide orientation in the membrane by His18 plays a key role in the toxicity of nonamyloidogenic forms of hIAPP.     

Amyloid inhibitors enhance survival of cultured human islets

Background

Amyloid fibrils created by misfolding and aggregation of proteins are a major pathological feature in a variety of degenerative diseases. Therapeutic approaches including amyloid vaccines and anti-aggregation compounds in models of amyloidosis point to an important role for amyloid in disease pathogenesis. Amyloid deposits derived from the β-cell peptide islet amyloid polypeptide (IAPP or amylin) are a characteristic of type 2 diabetes and may contribute to loss of β-cells in this disease.

Methods

We developed a cellular model of rapid amyloid deposition using cultured human islets and observed a correlation between fibril accumulation and β-cell death. A series of overlapping peptides derived from IAPP was generated.

Results

A potent inhibitor (ANFLVH) of human IAPP aggregation was identified. This inhibitory peptide prevented IAPP fibril formation in vitro and in human islet cultures leading to a striking increase in islet cell viability.

Conclusions

These findings indicate an important contribution of IAPP aggregation to β-cell death in situ and point to therapeutic applications for inhibitors of IAPP aggregation in enhancing β-cell survival.

General significance

Anti-amyloid compounds could potentially reduce the loss of β-cell mass in type 2 diabetes and maintain healthy human islet cultures for β-cell replacement therapies.     

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.     

The pathogenesis of maturity-onset diabetes mellitus: Is there a link to islet amyloid polypeptide?

The discovery of a novel polypeptide (Islet Amyloid Polypeptide: IAPP) isolated from human and cat islet amyloid and from amyloid of a human insulinoma is reviewed. Structurally, IAPP from the human and cat resembles calcitonin gene-related peptide (CGRP). The structural similarities between the neuropeptide CGRP and IAPP support the premise that IAPP is hormonal in nature. Our immunohistochemical studies also indicate that normal islet B-cells of several mammalian species (including man and cat) give strong immunoreactivity with antiserum directed to a synthetic peptide segment of IAPP. The fact that IAPP is deposited as amyloid in the pancreatic islets of type 2 (noninsulin-dependent) diabetics strongly supports an important but yet unknown link between IAPP and the development of this disease.     

Rifampicin does not prevent amyloid fibril formation by human islet amyloid polypeptide but does inhibit fibril thioflavin-T interactions: implications for mechanistic studies of beta-cell death

Amyloid formation has been implicated in more than 20 different human diseases, including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. The development of inhibitors of amyloid is a topic of considerable interest, both because of their potential therapeutic applications and because they are useful mechanistic probes. Recent studies have highlighted the potential use of rifampicin as an inhibitor of amyloid formation by a variety of polypeptides; however, there are conflicting reports on its ability to inhibit amyloid formation by islet amyloid polypeptide (IAPP). IAPP is the cause of islet amyloid in type 2 diabetes. We show that rifampicin does not prevent amyloid formation by IAPP and does not disaggregate preformed IAPP amyloid fibrils;, instead, it interferes with standard fluorescence-based assays of amyloid formation. Rifampicin is unstable in aqueous solution and is readily oxidized. However, the effects of oxidized and reduced rifampicin are similar, in that neither prevents amyloid formation by IAPP. Furthermore, use of a novel p-cyanoPhe analogue of IAPP shows that rifampicin does not significantly affect the kinetics of IAPP amyloid formation. The implications for the development of amyloid inhibitors are discussed as are the implications for studies of the toxicity of islet amyloid. The work also demonstrates the utility of p-cyanoPhe IAPP for the screening of inhibitors. The data indicate that rifampicin cannot be used to test the relative toxicity of IAPP fibrils and prefibril aggregates of IAPP.     

Protein Glycation by Glyoxal Promotes Amyloid Formation by Islet Amyloid Polypeptide

Protein glycation, also known as nonenzymatic glycosylation, is a spontaneous post-translational modification that would change the structure and stability of proteins or hormone peptides. Recent studies have indicated that glycation plays a role in type 2 diabetes (T2D) and neurodegenerative diseases. Over the last two decades, many types of advanced glycation end products (AGEs), formed through the reactions of an amino group of proteins with reducing sugars, have been identified and detected in vivo. However, the effect of glycation on protein aggregation has not been fully investigated. In this study, we aim to elucidate the impact of protein glycation on islet amyloid polypeptide (IAPP, also known as amylin) aggregation, which was strongly associated with T2D. We chemically synthesized glycated IAPP (AGE-IAPP) to mimic the consequence of this hormone peptide in a hyperglycemia (high blood sugar) environment. Our data revealed that AGE-IAPP formed amyloid faster than normal IAPP, and higher-molecular-weight AGE-IAPP oligomers were also observed in the early stage of aggregation. Circular dichroism spectra also indicated that AGE-IAPP exhibited faster conformational changes from random coil to its β-sheet fibrillar states. Moreover, AGE-IAPP can induce normal IAPP to expedite its aggregation process, and its fibrils can also act as templates to promote IAPP aggregation. AGE-IAPP, like normal IAPP, is capable of interacting with synthetic membranes and also exhibits cytotoxicity. Our studies demonstrated that glycation modification of IAPP promotes the amyloidogenic properties of IAPP, and it may play a role in accumulating additional amyloid during T2D progression.     

Lipid membranes modulate the structure of islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.     

Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species

Amyloid deposits in the islets of Langerhans occur in association with type 2 diabetes mellitus (DM) in humans and cats and consist of a 37-amino-acid polypeptide known as islet amyloid polypeptide (IAPP). In order to find an explanation for the situation that islet amyloid (IA) does not develop in common rodent species, we have deduced the amino acid sequence of the IAPP molecule in mouse, rat and hamster. We find that a specific region of the molecule diverges to a high degree. Synthetic peptides corresponding to this region of human and hamster IAPP were compared for their ability to form amyloid fibrils in vitro. Whereas the human peptide readily formed fibrils with amyloid character, the hamster peptide completely lacked this property. We suggest this to be a likely explanation for the differences in IA formation between humans and rodents and discuss our findings in relation to the type 2 DM syndrome.     

Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment

Human islet amyloid polypeptide is a hormone coexpressed with insulin by pancreatic beta-cells. For reasons not clearly understood, hIAPP aggregates in type II diabetics to form oligomers that interfere with beta-cell function, eventually leading to the loss of insulin production. The cellular membrane catalyzes the formation of amyloid deposits and is a target of amyloid toxicity through disruption of the membrane's structural integrity. Therefore, there is considerable current interest in solving the 3D structure of this peptide in a membrane environment. NMR experiments could not be directly utilized in lipid bilayers due to the rapid aggregation of the peptide. To overcome this difficulty, we have solved the structure of the naturally occurring peptide in detergent micelles at a neutral pH. The structure has an overall kinked helix motif, with residues 7-17 and 21-28 in a helical conformation, and with a 3(10) helix from Gly 33-Asn 35. In addition, the angle between the N- and C-terminal helices is constrained to 85°. The greater helical content of human IAPP in the amidated versus free acid form is likely to play a role in its aggregation and membrane disruptive activity.     

IAPP in type II diabetes: Basic research on structure,molecular interactions,and disease mechanisms suggests potential intervention strategies

Islet amyloid polypeptide (a.k.a. IAPP, amylin) is a 37 amino acid hormone that has long been associated with the progression of type II diabetes mellitus (TIIDM) disease. The endocrine peptide hormone aggregatively misfolds to form amyloid deposits in and around the pancreatic islet β-cells that synthesize both insulin and IAPP, leading to a decrease in β-cell mass in patients with the disease. Extracellular IAPP amyloids induce β-cell death through the formation of reactive oxygen species, mitochondrial dysfunction, chromatin condensation, and apoptotic mechanisms, although the precise roles of IAPP in TIIDM are yet to be established. Here we review aspects of the normal physiological function of IAPP in glucose regulation together with insulin, and its misfolding which contributes to TIIDM, and may also play roles in other pathologies such as Alzheimer's and heart disease. We summarize information on expression of the IAPP gene, the regulation of the hormone by post-translational modifications, the structural properties of the peptide in various states, the kinetics of misfolding to amyloid fibrils, and the interactions of the peptide with insulin, membranes, glycosaminoglycans, and nanoparticles. Finally, we describe how basic research is starting to have a positive impact on the development of approaches to circumvent IAPP amyloidogenesis. These include therapeutic strategies aimed at stabilizing non-amyloidogenic states, inhibition of amyloid growth or disruption of amyloid fibrils, antibodies directed towards amyloid structures, and inhibition of interactions with cofactors that facilitate aggregation or stabilize amyloids.     

Analysis of the minimal amyloid-forming fragment of the islet amyloid polypeptide. An experimental support for the key role of the phenylalanine residue in amyloid formation.

The development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin). Recently, a short functional hexapeptide fragment of IAPP (NH(2)-NFGAIL-COOH) was found to form fibrils that are very similar to those formed by the full-length polypeptide. To better understand the specific role of the residues that compose the fragment, we performed a systematic alanine scan of the IAPP "basic amyloidogenic units." Turbidity assay experiments demonstrated that the wild-type peptide and the Asn(1) --> Ala and Gly(3) --> Ala peptides had the highest rate of aggregate formation, whereas the Phe(2) --> Ala peptide did not form any detectable aggregates. Dynamic light-scattering experiments demonstrated that all peptides except the Phe(2) --> Ala form large multimeric structures. Electron microscopy and Congo red staining confirmed that the structures formed by the various peptides are indeed amyloid fibrils. Taken together, the results of our study provide clear experimental evidence for the key role of phenylalanine residue in amyloid formation by IAPP. In contrast, glycine, a residue that was suggested to facilitate amyloid formation in other systems, has only a minor role, if any, in this case. Our results are discussed in the context of the remarkable occurrence of aromatic residues in short functional fragments and potent inhibitors of amyloid-related polypeptides. We hypothesize that pi-pi interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation.     

胰岛淀粉样多肽的研究进展

由蛋白错误折叠后聚集所产生的淀粉样蛋白沉积是导致老年痴呆症、疯牛病、2型糖尿病等多种疾病的重要因素。由胰岛淀粉样多肽(islet amyloid polypeptide,IAPP)所形成的淀粉样蛋白沉积,具有破坏胰岛β细胞膜结构、诱导β细胞凋亡和损伤β细胞功能的作用,被认为是2型糖尿病的重要致病原因之一。对IAPP的聚集性、聚集体的结构,以及其对β细胞的毒性作用研究,不但有助于明确2型糖尿病的发病机制,而且最新研究也表明抑制IAPP的聚集可有效减少β细胞的凋亡,提高胰岛移植的成功率。因此,IAPP已成为2型糖尿病治疗中一个具有良好前景的靶点。该文对IAPP研究的最新进展进行了简要介绍。     

Structural characterisation of islet amyloid polypeptide fibrils

Islet amyloid is found in many patients suffering from type 2 diabetes. Amyloid fibrils found deposited in the pancreatic islets are composed of a 37-residue peptide, known as islet amyloid polypeptide (IAPP) (also known as amylin) and are similar to those found in other amyloid diseases. Synthetic IAPP peptide readily forms amyloid fibrils in vitro and this has allowed fibril formation kinetics and the overall morphology of IAPP amyloid to be studied. Here, we use X-ray fibre diffraction, electron microscopy and cryo-electron microscopy to examine the molecular structure of IAPP amyloid fibrils. X-ray diffraction from aligned synthetic amyloid fibrils gave a highly oriented diffraction pattern with layer-lines spaced 4.7 A apart. Electron diffraction also revealed the characteristic 4.7 A meridional signal and the position of the reflection could be compared directly to the image of the diffracting unit. Cryo-electron microscopy revealed the strong signal at 4.7 A that has been previously visualised from a single Abeta fibre. Together, these data build up a picture of how the IAPP fibril is held together by hydrogen bonded beta-sheet structure and contribute to the understanding of the generic structure of amyloid fibrils.     

Drosophila melanogaster as a model system for studies of islet amyloid polypeptide aggregation

Background

Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death.

Methodology/Principal Findings

We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elav^C155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures.

Conclusions/Significance

These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology.