Dataset Information

The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

ABSTRACT: Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

SUBMITTER: Guan H

PROVIDER: S-EPMC4507941 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

Guan Hanjun H Chow K Martin KM Song Eunsuk E Verma Nirmal N Despa Florin F Hersh Louis B LB

PloS one 20150720 7

Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypep ...[more]

PMID: 26191799

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Project description:Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-Î²-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-ÎºB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis. The INS-1 cells were grown as 5 separate clones for 10 passages before plating in a 12-well plate (Nunc) at 100.000 cells per well and grown in complete RPMI 1640 medium to 70% confluency for approximately 48 h. The cells were then challenged by adding 77 Î¼M monomeric IAPP alone or together with C4BP (0.6 Î¼M). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 Î¼M) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).

Project description:Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in β-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid β peptide (Aβ1-42) as a successful strategy to inhibit its aggregation involved in Alzheimer's disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic β-hairpin like structures built on a piperidine-pyrrolidine β-turn inducer are described. By linking to this β-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.

Dataset Information

The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

Publications

The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

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