Search

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer's disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia.

The process of amyloid formation is cytotoxic and contributes to a wide range of human diseases, but the mechanisms of amyloid-induced cytotoxicity are not well understood. It has been proposed that amyloidogenic peptides exert their toxic effects by damaging membranes. Membrane disruption is clearly not the only mechanism of toxicity, but the literature suggests that loss of membrane integrity may be a contributing factor. In this chapter we describe the measurement of in vitro membrane leakage induced by amyloidogenic proteins via the use of model vesicles. We use islet amyloid polypeptide (IAPP, amylin) as an example, but the methods are general.

The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

The hormone human islet amyloid polypeptide (hIAPP or amylin) plays a role in glucose metabolism, but forms amyloid in the pancreas in type 2 diabetes (T2D) and is associated with β-cell death and dysfunction in the disease. Inhibitors of islet amyloid have therapeutic potential; however, there are no clinically approved inhibitors, and the mode of action of existing inhibitors is not well understood. Rat IAPP (rIAPP) differs from hIAPP at six positions, does not form amyloid, and is an inhibitor of amyloid formation by hIAPP. Five of the six differences are located within the segment of residues 20-29, and three of them are Pro residues, which are well-known disruptors of β-sheet structure. rIAPP is thus a natural example of a "β-breaker inhibitor", a molecule that combines a recognition element with an entity that inhibits β-sheet formation. Pramlintide (PM) is a peptide drug approved for use as an adjunct to insulin therapy for treatment of diabetes. PM was developed by introducing the three Pro substitutions found in rIAPP into hIAPP. Thus, it more closely resembles the human peptide than does rIAPP. Here we examine and compare the ability of rIAPP, PM, and a set of designed analogues of hIAPP to inhibit amyloid formation by hIAPP, to elucidate the factors that lead to effective peptide-based inhibitors. Our results reveal, for this class of molecules, a balance between the reduced amyloidogenicity of the inhibitory sequence on one hand and its ability to recognize hIAPP on the other.

The loss of β-cell function and β-cell death are key features of diabetes. A range of mechanisms are thought to contribute to β-cell loss, including islet amyloid formation by the neuropancreatic hormone amylin (islet amyloid polypeptide, IAPP). Islet amyloid deposition also contributes to the failure of islet transplants. There are no therapeutic strategies for the treatment or prevention of islet amyloidosis. Aspirin and the nonsteroid anti-inflammatory drug (NSAID) ketoprofen, at clinically relevant doses, have been proposed to inhibit amyloid formation by amylin and thus may hold promise for treatment of islet amyloidosis. These compounds are potentially attractive given the importance of inflammation in islet amyloidosis and given the fact that there are no anti-islet amyloid agents in the clinic. We show that aspirin, even in 20-fold excess, has no effect on the kinetics of amyloid formation by amylin as judged by thioflavin-T binding, right angle light scattering, and transmission electron microscopy, nor does it alter the morphology of resulting amyloid fibrils. Aspirin showed no ability to disaggregate preformed amylin amyloid fibrils under the conditions of these studies, 25 °C and pH 7.4. Ketoprofen is similarly ineffective at inhibiting amylin amyloid formation. The compounds do, however, interfere with circular dichroism- and Congo Red-based assays of amylin amyloid formation. This study highlights the importance of using multiple methods to follow amyloid formation when screening inhibitors.

The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of β-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

Pancreatic islet amyloid is a characteristic feature of type 2 diabetes. The major protein component of islet amyloid is the polypeptide hormone known as islet amyloid polypeptide (IAPP, or amylin). IAPP is stored with insulin in the β-cell secretory granules and is released in response to the stimuli that lead to insulin secretion. IAPP is normally soluble and is natively unfolded in its monomeric state, but forms islet amyloid in type 2 diabetes. Islet amyloid is not the cause of type 2 diabetes, but it leads to β-cell dysfunction and cell death, and contributes to the failure of islet cell transplantation. The mechanism of IAPP amyloid formation is not understood and the mechanisms of cytotoxicity are not fully defined., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Amyloid formation plays an important role in a broad range of diseases, and the search for amyloid inhibitors is an active area of research. Amyloid formation takes places in a heterogeneous environment in vivo with the potential for interactions with membranes and with components of the extracellular matrix. Naturally occurring amyloid deposits are associated with sulfated proteoglycans and other factors. However, the vast majority of in vitro assays of amyloid formation and amyloid inhibition are conducted in homogeneous solution where the potential for interactions with membranes or sulfated proteoglycans is lacking and it is possible that different results may be obtained in heterogeneous environments. We show that variants of islet amyloid polypeptide (IAPP), which are non-amyloidogenic in homogeneous solution, can be readily induced to form amyloid in the presence of glycosaminoglycans (GAGs). GAGs are found to be more effective than anionic lipid vesicles at inducing amyloid formation on a per-charge basis. Several known inhibitors of IAPP amyloid formation are shown to be less effective in the presence of GAGs., (Copyright © 2012. Published by Elsevier Ltd.)

Amyloid formation in the pancreas by islet amyloid polypeptide (IAPP) leads to β-cell death and dysfunction, contributing to islet transplant failure and to type-2 diabetes. IAPP is stored in the β-cell insulin secretory granules and cosecreted with insulin in response to β-cell secretagogues. IAPP is believed to play a role in the control of food intake, in controlling gastric emptying and in glucose homeostasis. The polypeptide is natively unfolded in its monomeric state, but is one of the most amyloidogenic sequences known. The mechanisms of IAPP amyloid formation in vivo and in vitro are not understood; the mechanisms of IAPP induced cell death are unclear; and the nature of the toxic species is not completely defined. Recent work is shedding light on these important issues., (Copyright © 2012. Published by Elsevier Ltd.)

Antibodies hold significant potential for inhibiting toxic protein aggregation associated with conformational disorders such as Alzheimer's and Huntington's diseases. However, near-stoichiometric antibody concentrations are typically required to completely inhibit protein aggregation. We posited that the molecular interactions mediating amyloid fibril formation could be harnessed to generate antibodies with potent antiaggregation. Here we report that grafting small amyloidogenic peptides (6-10 residues) into the complementarity-determining regions of a single-domain (V(H)) antibody yields potent domain antibody inhibitors of amyloid formation. Grafted AMyloid-Motif AntiBODIES (gammabodies) presenting hydrophobic peptides from Aβ (Alzheimer's disease), α-Synuclein (Parkinson's disease), and islet amyloid polypeptide (type 2 diabetes) inhibit fibril assembly of each corresponding polypeptide at low substoichiometric concentrations (1:10 gammabody:monomer molar ratio). In contrast, sequence- and conformation-specific antibodies that were obtained via immunization are unable to prevent fibrillization at the same substoichiometric concentrations. Gammabodies prevent amyloid formation by converting monomers and/or fibrillar intermediates into small complexes that are unstructured and benign. We expect that our antibody design approach--which eliminates the need for immunization or screening to identify sequence-specific domain antibody inhibitors--can be readily extended to generate potent aggregation inhibitors of other amyloidogenic polypeptides linked to human disease.

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser20-to-Gly missense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early-onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism, and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 (transformed rat insulinoma-1) β-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in transplanted islets. The flavanol (-)-epigallocatechin-3-gallate [EGCG; (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] is an effective inhibitor of amyloid formation by IAPP; however, the interactions required for the inhibition of IAPP amyloid formation and for the remodeling of amyloid fibers are not known. A range of features have been proposed to be critical for EGCG protein interactions, including interactions with aromatic residues, interactions with amino groups, or sulfhydryls. Using a set of IAPP analogues, we show that none of these are required. Studies in which EGCG is added to the lag phase of amyloid formation shows that it interacts with intermediates as well as with monomers and amyloid. The features of EGCG required for effective inhibition were examined. The stereoisomer of EGCG, (-)-gallocatechin gallate (GCG), is an effective inhibitor, although less so than EGCG. Removing the gallate ester moiety leads to EGC which is a less effective inhibitor. Removing only the 3-hydroxyl group of the trihydroxyphenyl ring leads to a compound that has more pronounced effects on the lag phase than EGC but is less effective at reducing the amount of amyloid. Elimination of both the 3-hydroxy group and the gallate ester results in loss of activity. EGCG remodels IAPP amyloid fibers but does not fully resolubilize them to unstructured monomers, and the remodeling is not the reverse of amyloid assembly. The ability of the compounds to remodel IAPP amyloid closely follows their relative ability to inhibit amyloid formation.

Amyloid formation has been implicated in the pathology of over 20 human diseases, but the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labelling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin (the peptide responsible for islet amyloid formation in type 2 diabetes) with a known inhibitor (rat amylin). Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal β-sheet instead. At 24 h after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet, most probably on the outside of the human fibrils. This is striking, because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

The polypeptide hormone Islet Amyloid Polypeptide (IAPP, amylin) is responsible for islet amyloid formation in type-2 diabetes and in islet cell transplants, where it may contribute to graft failure. Human IAPP is extremely amyloidogenic and fewer inhibitors of IAPP amyloid formation have been reported than for the Alzheimer's Aβ peptide or for α-synuclein. The ability of a set of hydroxyflavones to inhibit IAPP amyloid formation was tested. Fluorescence detected thioflavin-T-binding assays are the most popular methods for measuring the kinetics of amyloid formation and for screening potential inhibitors; however, we show that they can lead to false positives with hydroxyflavones. Several of the compounds inhibit thioflavin-T fluorescence, but not amyloid formation; a result which highlights the hazards of relying solely on thioflavin-T assays to screen potential inhibitors. Transmission electron microscopy (TEM) and right-angle light scattering show that Morin hydrate (2',3,4',5,7-Pentahydroxyflavone) inhibits amyloid formation by human IAPP and disaggregates preformed IAPP amyloid fibers. In contrast, Myricetin, Kaempferol, and Quercetin, which differ only in hydroxyl groups on the B-ring, are not effective inhibitors. Morin hydrate represents a new type of IAPP amyloid inhibitor and the results with the other compounds highlight the importance of the substitution pattern on the B-ring., (Copyright © 2012 The Protein Society.)

The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

Amyloid formation and aberrant protein aggregation are hallmarks of more than 30 different human diseases. The proteins that form amyloid can be divided into two structural classes: those that form compact, well-ordered, globular structures in their unaggregated state and those that are intrinsically disordered in their unaggregated states. The latter include the Aβ peptide of Alzheimer's disease, islet amyloid polypeptide (IAPP, amylin) implicated in type 2 diabetes and α-synuclein, which is linked to Parkinson's disease. Work in the last 10 years has highlighted the potential role of pre-amyloid intermediates in cytotoxicity and has focused attention on their properties. A number of intrinsically disordered proteins appear to form helical intermediates during amyloid formation. We discuss the spectroscopic methods employed to detect and characterize helical intermediates in homogenous solution and in membrane-catalyzed amyloid formation, with the emphasis on the application of circular dichroism (CD). IAPP is used as an example, but the methods are generally applicable.

The process of amyloid formation is cytotoxic and contributes to a wide range of human diseases, but the mechanisms of amyloid-induced cytotoxicity are not well understood. It has been proposed that amyloidogenic peptides exert their toxic effects by damaging membranes. Membrane disruption is clearly not the only mechanism of toxicity, but the literature suggests that loss of membrane integrity may be a contributing factor. In this chapter we describe the measurement of in vitro membrane leakage induced by amyloidogenic proteins via the use of model vesicles. We use islet amyloid polypeptide (IAPP, amylin) as an example, but the methods are general.