Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane

Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet β cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this β cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins

Exendin-4 increases islet amyloid deposition but offsets the resultant beta cell toxicity in human islet amyloid polypeptide transgenic mouse islets

Aims/hypothesis In type 2 diabetes, aggregation of islet amyloid polypeptide (IAPP) into amyloid is associated with beta cell loss. As IAPP is co-secreted with insulin, we hypothesised that IAPP secretion is necessary for amyloid formation and that treatments that increase insulin (and IAPP) secretion would thereby increase amyloid formation and toxicity. We also hypothesised that the unique properties of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 to maintain or increase beta cell mass would offset the amyloid-induced toxicity.Methods Islets from amyloid-forming human IAPP transgenic and control non-transgenic mice were cultured for 48 h in 16.7 mmol/l glucose alone (control) or with exendin-4, potassium chloride (KCl), diazoxide or somatostatin. Human IAPP and insulin release, amyloid deposition, beta cell area/islet area, apoptosis and AKT phosphorylation levels were determined.Results In control human IAPP transgenic islets, amyloid formation was associated with increased beta cell apoptosis and beta cell loss. Increasing human IAPP release with exendin-4 or KCl increased amyloid deposition. However, while KCl further increased beta cell apoptosis and beta cell loss, exendin-4 did not. Conversely, decreasing human IAPP release with diazoxide or somatostatin limited amyloid formation and its toxic effects. Treatment with exendin-4 was associated with an increase in AKT phosphorylation compared with control and KCl-treated islets.Conclusions/interpretation IAPP release is necessary for islet amyloid formation and its toxic effects. Thus, use of insulin secretagogues to treat type 2 diabetes may result in increased islet amyloidogenesis and beta cell death. However, the AKT-associated anti-apoptotic effects of GLP-1 receptor agonists such as exendin-4 may limit the toxic effects of increased islet amyloid.<br /

Sequence-dependent oligomerization of the neu transmembrane domain suggests inhibition of "conformational switching" by an oncogenic mutant

Membrane-spanning epidermal growth factor receptor ErbB2 is of key importance in cell division, in which a dimeric complex of the protein is responsible for tyrosine kinase activation following ligand binding. The rat homologue of this receptor (Neu) is prone to a valine to glutamic acid mutation in the transmembrane domain (TM), resulting in permanent activation and oncogenesis. In this study, the TM domains of Neu and the corresponding oncogenic mutant Neu*, which contains a V to E mutation at position 664 in the TM domain, have been analyzed to improve our understanding of the structural effects of the oncogenic V664E mutation. Building on previous work, we have focused here on understanding the sequence dependence of TM helix helix interactions and any differences in behavior upon introduction of the V664E mutation. Using a variety of biochemical and biophysical methods, we find that the rat Neu TM domain forms strong oligomers and, similar to previous observations for the human ErbB2 TM domain, the oncogenic mutation results in a reduced level of self-association. Our data also strongly indicate that the proto-oncogenic Neu TM domain can adopt multiple (at least two) oligomeric conformations in the membrane, possibly corresponding to the active and inactive forms of the receptor, and can "switch" between the two. Further, the oncogenic Neu* mutant appears to inhibit this "conformational switching" of TM dimers, as we observe that dimerization of the Neu* TM domain in the Escherichia coli inner membrane strongly favors a single conformation stabilized by an I XXXV motif (I-659-XXX-V-663) originally identified by site-specific infrared spectroscopic studies

Alzheimer's Aß42 oligomers but not fibrils simultaneously bind to and cause damage to ganglioside containing lipid membranes.

International audienceAmyloid-Beta peptide (Ab) assembles to form amyloid fibres that accumulate in senile plaques associated with Alzheimer's disease (AD). The major constituent, a 42-residue Ab peptide, has the propensity to assemble and form soluble and potentially cytotoxic oligomers as well as ordered, stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity maybe associated with the ability of oligomers to associate with and cause permeation of lipid membranes. Here, we have investigated the ability of oligomeric and fibrillar Ab42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface Plasmon field enhanced Fluorescence spectroscopy reveals that the binding of the freshly dissolved, oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity and their addition results in around 2-fold less fluorophore leakage compared to oligomeric Ab. Binding of the oligomers may be in part mediated by the GM1 ganglioside receptors showing a 1.8-fold increase in oligomeric Ab binding and a 2-fold increase in permeation compared to when GM1 is not present. Atomic Force Microscopy reveals the formation of defects and holes in response to oligomeric Ab, but not preformed fibrillar Ab. Our results indicate significant membrane disruption arises from association of low molecular weight Ab and this may be mediated by mechanical damage to the membranes by Ab aggregation. This membrane disruption may play a key role in the mechanism Ab-related cell toxicity in Alzheimer's disease

Activation of innate immunity by lysozyme fibrils is critically dependent on cross-β sheet structure

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