Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies.
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ABSTRACT:
SUBMITTER: Bram Y
PROVIDER: S-EPMC3940978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (A?). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (VH) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic A?42 peptide segment (A? residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding VH domains. We find that VH expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, A? VH domains with negatively charged CDR3 mutations show significant preference for recognizing A? fibrils relative to A? monomers, whereas the same VH domains with other polar CDR3 mutations recognize both A? conformers. We observe similar behavior for a VH domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8-37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.
| S-EPMC4742750 | biostudies-literature
Project description:Islet amyloid polypeptide (IAPP, also known as amylin) is the major protein component of pancreatic amyloid fibers in type II diabetes and is normally cosecreted with insulin from the beta-cells of the pancreas. IAPP forms amyloid fibrils rapidly at concentrations well below those found in vivo, yet progression of type II diabetes occurs over many years. Insulin, a known inhibitor of IAPP fibrillogenesis, exists as a dense crystalline or near-crystalline core in the secretory vesicle, while IAPP localizes to the region between the crystal and the secretory vesicle membrane. In vitro, IAPP fibrillogenesis is both accelerated by lipid membranes and inhibited by monomeric insulin. In this work, we investigate insulin-IAPP-lipid interactions in vitro under conditions chosen to approximate native secretory vesicle physiology and the amyloid disease state. The effect of insulin on IAPP fibrillogenesis is investigated using fluorescence spectrometry. Additionally, interactions of IAPP and lipids with crystalline insulin are studied using fluorescence microscopy. We find that, while soluble states of insulin and IAPP do not interact significantly, large assemblies of either insulin (crystals) or IAPP (fibers) can lead to stable IAPP-insulin interactions. The results raise the possibility of multiple physiological interactions between these two beta-cell hormones.
| S-EPMC2548354 | biostudies-literature
Project description:Amyloidosis is a clinical disorder implicated with the formation of toxic amyloid aggregates. Despite their pathological significance, it is challenging to define the structural characteristics of amyloid oligomers owing to their metastable nature. Herein, we report structural and mechanistic investigations of human islet amyloid polypeptide (hIAPP) oligomers, found in type II diabetes mellitus, in both the absence and presence of disease-relevant metal ions [i.e., Cu(ii) and Zn(ii)]. These metal ions show suppressive effects on hIAPP fibrillation and facilitate the generation of toxic oligomers. Using circular dichroism spectroscopy, transmission electron microscopy, gel electrophoresis, small-angle X-ray scattering, and ion mobility-mass spectrometry, we investigated the assembly mechanisms of hIAPP oligomers in the presence and absence of metal ions. Oligomerization of both metal-free hIAPP and metal-associated hIAPP monomers is initiated following a similar growth model. However, in the presence of Cu(ii), hIAPP monomers self-assemble into small globular aggregates (Rg ? 45 Å) with a random coil structure. This Cu(ii)-associated hIAPP oligomer shows an off-pathway aggregation, and is suggested to be an end product which is toxic to pancreatic ?-cells. On the other hand, metal-free hIAPP and Zn(ii)-associated hIAPP monomers generate relatively less toxic aggregates that eventually grow into fibrils. We suggest that the coordination of hIAPP to Cu(ii) and the relatively high stability (Ka, ca. 108 M-1) of hIAPP-Cu(ii) complexes result in the abnormal conformation and toxicity of hIAPP oligomers. Overall, through combining multiple biophysical methods, our studies suggest that molecular interactions between hIAPP and Cu(ii) induce a different pathway for hIAPP assembly. This work will advance our knowledge of the conformational basis, assembly mechanism, and toxicity of small soluble amyloid oligomers.
| S-EPMC6020819 | biostudies-literature
Project description:The islet in type 2 diabetes (T2D) shares many features of the brain in protein misfolding diseases. There is a deficit of ? cells with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed with insulin. Small intracellular membrane-permeant oligomers, the most toxic form of IAPP, are more frequent in ? cells of patients with T2D and rodents expressing human IAPP. ? Cells in T2D, and affected cells in neurodegenerative diseases, share a comparable pattern of molecular pathology, including endoplasmic reticulum stress, mitochondrial dysfunction, attenuation of autophagy, and calpain hyperactivation. While this adverse functional cascade in response to toxic oligomers is well described, the sequence of events and how best to intervene is unknown. We hypothesized that calpain hyperactivation is a proximal event and tested this in vivo by ? cell-specific suppression of calpain hyperactivation with calpastatin overexpression in human IAPP transgenic mice. ? Cell-specific calpastatin overexpression was remarkably protective against ? cell dysfunction and loss and diabetes onset. The critical autophagy/lysosomal pathway for ? cell viability was protected with calpain suppression, consistent with findings in models of neurodegenerative diseases. We conclude that suppression of calpain hyperactivation is a potentially beneficial disease-modifying strategy for protein misfolding diseases, including T2D.
| S-EPMC5085608 | biostudies-literature
Project description:The self-assembly of human islet amyloid polypeptide (hIAPP) into ?-sheet rich amyloid aggregates is associated with pancreatic ?-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of ?-helical intermediates before the rapid conversion into ?-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more ?-helices than ?-sheets, consistent with ensemble-based experimental measurements. However, in ~4-6% independent simulations, ?-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These ?-rich oligomers could adopt ?-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these ?-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. ?-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.
| S-EPMC6310638 | biostudies-literature
Project description:A leading hypothesis for the decimation of insulin-producing ?-cells in type 2 diabetes attributes the cause to islet amyloid polypeptide (IAPP) for its deleterious effects on the cell membranes. This idea has produced extensive investigations on human IAPP (hIAPP) and its interactions with lipid bilayers. However, it is still difficult to correlate the peptide-lipid interactions with its effects on islet cells in culture. The hIAPP fibrils have been shown to interact with lipids and damage lipid bilayers, but appear to have no effect on islet cells in culture. Thus, a modified amyloid hypothesis assumes that the toxicity is caused by hIAPP oligomers, which are not preamyloid fibrils or protofibrils. However, so far such oligomers have not been isolated or identified. The hIAPP monomers also bind to lipid bilayers, but the mode of interaction is not clear. Here, we performed two types of experiments that, to our knowledge, have not been done before. We used x-ray diffraction, in conjunction with circular dichroism measurement, to reveal the location of the peptide bound to a lipid bilayer. We also investigated the effects of hIAPP on giant unilamellar vesicles at various peptide concentrations. We obtained the following qualitative results. Monomeric hIAPP binds within the headgroup region and expands the membrane area of a lipid bilayer. At low concentrations, such binding causes no leakage or damage to the lipid bilayer. At high concentrations, the bound peptides transform to ?-aggregates. The aggregates exit the headgroup region and bind to the surface of lipid bilayers. The damage by the surface bound ?-aggregates depends on the aggregation size. The initial aggregation extracts lipid molecules, which probably causes ion permeation, but no molecular leakage. However, the initial ?-aggregates serve as the seed for larger fibrils, in the manner of the Jarrett-Lansbury seeded-polymerization model, that eventually disintegrate lipid bilayers by electrostatic and hydrophobic interactions.
| S-EPMC3296043 | biostudies-literature
Project description:Aggregation of the islet amyloid polypeptide (IAPP) to form fibrils and oligomers is important in the progression of type 2 diabetes. This article describes X-ray crystallographic and solution-state NMR studies of peptides derived from residues 11-17 of IAPP that assemble to form tetramers. Incorporation of residues 11-17 of IAPP (RLANFLV) into a macrocyclic ?-sheet peptide results in a monomeric peptide that does not self-assemble to form oligomers. Mutation of Arg11 to the uncharged isostere citrulline gives peptide homologues that assemble to form tetramers in both the crystal state and in aqueous solution. The tetramers consist of hydrogen-bonded dimers that sandwich together through hydrophobic interactions. The tetramers share several features with structures reported for IAPP fibrils and demonstrate the importance of hydrogen bonding and hydrophobic interactions in the oligomerization of IAPP-derived peptides.
| S-EPMC7532939 | biostudies-literature
Project description:Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two ?-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing ?-sheets that twist around each other. The two ?-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (?15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.
| S-EPMC3285303 | biostudies-literature
Project description:The abnormal self-assembly of a number of proteins or peptides is a hallmark of >20 amyloidogenic diseases. Recent studies suggest that the pathology of amyloidogenesis can be attributed primarily to cytotoxic, soluble, intermediate oligomeric species rather than to mature amyloid fibrils. Despite the lack of available structural information regarding these transient species, many therapeutic efforts have focused on inhibiting the formation of these aggregates. One of the most successful approaches has been to use small molecules, many of which have been found to inhibit toxic species with high efficacy. A significant issue that remains to be resolved is the mechanism underlying the inhibitory effects of these molecules. In this article, we present extensive replica-exchange molecular dynamics simulations to study the early aggregation of the human islet amyloid polypeptide segment 22-27 in the presence and absence of the small-molecule inhibitor resveratrol. The simulations indicate that aggregation of these peptides was hindered by resveratrol via a mechanism of blocking the lateral growth of a single-layered ?-sheet oligomer (rather than preventing growth by elongation along the fibril axis). Intersheet side-chain stacking, especially stacking of the aromatic rings, was blocked by the presence of resveratrol molecules, and the overall aggregation level was reduced.
| S-EPMC3059578 | biostudies-literature
Project description: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.
| S-EPMC3753189 | biostudies-literature