The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.
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ABSTRACT:
SUBMITTER: Guan H
PROVIDER: S-EPMC4507941 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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-β-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-κB 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 μM monomeric IAPP alone or together with C4BP (0.6 μM). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 μM) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).
2016-08-25 | E-GEOD-79224 | biostudies-arrayexpress
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-β-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-κB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis.
2016-08-25 | GSE79224 | GEO
Project description:Epidemiological studies support a connection between the two common disorders, type-2 diabetes and Alzheimer's disease. Both conditions have local amyloid formation in their pathogenesis, and cross-seeding between islet amyloid polypeptide (IAPP) and amyloid β (Aβ) could constitute the link. The bimolecular fluorescence complementation (BiFC) assay was used to investigate the occurrence of heterologous interactions between IAPP and Aβ and to compare the potential toxic effects of IAPP/Aβ, IAPP/IAPP, and Aβ/Aβ expression in living cells. Microscopy was used to confirm the fluorescence and determine the lysosomal, mitochondrial areas and mitochondrial membrane potential, and a FACS analysis was used to determine ROS production and the role for autophagy. Drosophila melanogaster expressing IAPP and Aβ was used to study their co-deposition and effects on longevity. We showed that the co-expression of IAPP and Aβ resulted in fluorophore reconstitution to the same extent as determined for homologous IAPP/IAPP or Aβ/Aβ expression. The BiFC(+)/BiFC(-) ratio of lysosomal area calculations increased in transfected cells independent of the vector combinations, while only Aβ/Aβ expression increased mitochondrial membrane potential. Expression combinations containing Aβ were necessary for the formation of a congophilic amyloid. In Drosophila melanogaster expressing IAPP/Aβ, co-deposition of the amyloid-forming peptides caused reduced longevity. The BiFC results confirmed a heterologous interaction between IAPP and Aβ, while co-deposits in the brain of Drosophila suggest mixed amyloid aggregates.
| S-EPMC8541034 | biostudies-literature
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.
| S-EPMC8481668 | biostudies-literature
Project description:Assembly of amyloid-beta peptide (A?) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with A? 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 A?. Here we investigate the interaction of IAPP-GI with A?40 and A?42 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 A?--that is both regions of the A? sequence that are converted into ?-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with A? resulted in a concentration-dependent co-aggregation of A? and IAPP-GI that was enhanced for the more aggregation prone A?42 peptide. On the basis of the reduced toxicity of the A? peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects A? into nontoxic "off-pathway" aggregates.
| S-EPMC3102090 | biostudies-literature
Project description:A direct observation of amyloid aggregation from isolated peptides to cross-? fibrils is crucial for understanding the nucleation-dependence process, but the corresponding macroscopic timescales impose a major computational challenge. Using rapid all-atom discrete molecular dynamics simulations, we capture the oligomerization and fibrillization dynamics of the amyloid core sequences of amyloid-? (A?) in Alzheimer's disease and islet amyloid polypeptide (IAPP) in type-2 diabetes, namely A?16-22 and IAPP22-28. Both peptides and their mixture spontaneously assemble into cross-? aggregates in silico, but follow distinct pathways. A?16-22 is highly aggregation-prone with a funneled free energy basin toward multi-layer ?-sheet aggregates. IAPP22-28, on the other hand, features the accumulation of unstructured oligomers before the nucleation of ?-sheets and growth into double-layer ?-sheet aggregates. In the presence of A?16-22, the aggregation of IAPP22-28 is promoted by forming co-aggregated multi-layer ?-sheets. Our study offers a detailed molecular insight to the long-postulated oligomerization-nucleation process in the amyloid aggregations.
| S-EPMC5657190 | biostudies-literature
Project description:Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. Although the cellular toxicity of IAPP has been established, the structure of the fibrillar form found in these deposits is unknown. Here we have crystallized two segments from IAPP, which themselves form amyloid-like fibrils. The atomic structures of these two segments, NNFGAIL and SSTNVG, were determined, and form the basis of a model for the most commonly observed, full-length IAPP polymorph.
| S-EPMC2525530 | biostudies-literature
Project description:Here, we investigated transcriptional and trafficking mechanisms of human islet amyloid polypeptide (hIAPP) in normal and stressed β-cells. In high glucose-challenged human islets and rat insulinoma cells overexpressing hIAPP, cell fractionation studies revealed increased accumulation of hIAPP. Unexpectedly, a significant fraction (up to 22%) of hIAPP was found in the nuclear soluble and chromatin-enriched fractions of cultured human islet and rat insulinoma cells. The nucleolar accumulation of monomeric forms of hIAPP did not have any adverse effect on the proliferation of β-cells nor did it affect nucleolar organization or function. However, intact nucleolar organization and function were essential for hIAPP expression under normal and ER-stress conditions as RNA polymerase II inhibitor, α-amanitin, reduced hIAPP protein expression evoked by high glucose and thapsigargin. Promoter activity studies revealed the essential role of transcription factor FoxA2 in hIAPP promoter activation in ER-stressed β-cells. Transcriptome and secretory studies demonstrate that the biosynthetic and secretory capacity of islet β-cells was preserved during ER stress. Thus, the main reason for increased intracellular hIAPP accumulation is its enhanced biosynthesis under these adverse conditions.
| S-EPMC8011634 | biostudies-literature
Project description:Aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils in islets of Langerhans is associated with type 2 diabetes, and formation of toxic IAPP species is believed to contribute to the loss of insulin-producing beta cells. The BRICHOS domain of integral membrane protein 2B (Bri2), a transmembrane protein expressed in several peripheral tissues and in the brain, has recently been shown to prevent fibril formation and toxicity of Aβ42, an amyloid-forming peptide in Alzheimer disease. In this study, we demonstrate expression of Bri2 in human islets and in the human beta-cell line EndoC-βH1. Bri2 colocalizes with IAPP intracellularly and is present in amyloid deposits in patients with type 2 diabetes. The BRICHOS domain of Bri2 effectively inhibits fibril formation in vitro and instead redirects IAPP into formation of amorphous aggregates. Reduction of endogenous Bri2 in EndoC-βH1 cells with siRNA increases sensitivity to metabolic stress leading to cell death while a concomitant overexpression of Bri2 BRICHOS is protective. Also, coexpression of IAPP and Bri2 BRICHOS in lateral ventral neurons of Drosophila melanogaster results in an increased cell survival. IAPP is considered to be the most amyloidogenic peptide known, and described findings identify Bri2, or in particular its BRICHOS domain, as an important potential endogenous inhibitor of IAPP aggregation and toxicity, with the potential to be a possible target for the treatment of type 2 diabetes.
| S-EPMC5866560 | biostudies-literature
Project description:Amyloid aggregation of amyloid-beta (Aβ) and islet amyloid polypeptide (IAPP) is associated with Alzheimer's disease (AD) and type-2 diabetes (T2D), respectively. With T2D being the risk factor for AD and the ability of IAPP to cross the blood-brain barrier, the coaggregation of Aβ and IAPP has been explored to understand the cross-talk between the two diseases. Recent studies demonstrated that soluble IAPP could significantly accelerate the aggregation of Aβ while preformed amyloids of IAPP were poor "seeds" for Aβ aggregation. Here, we apply all-atom discrete molecular dynamics simulations to investigate possible molecular mechanisms for the accelerated coaggregation of IAPP and Aβ42 comparing to Aβ42 aggregation alone, which was confirmed by the complementary thioflavin-T fluorescence assay. Our simulation results suggest that peptides in the mixture tend to form heterodimers as the first step toward their coaggregation. Strong interpeptide interactions with IAPP in the heterodimer shift the helical conformation of Aβ42 in its amyloidogenic central hydrophobic core, residues 16-22 (Aβ16-22), to the extended conformation ready to form β-sheets. Our study suggests that the unfolding of Aβ16-22 helix contributes to the aggregation free-energy barrier and corresponds to the rate-limiting conformational change for Aβ42 aggregation. Therefore, we propose that soluble IAPP promotes the aggregation of Aβ42 by binding-induced conformational change of Aβ42 in its amyloidogenic core and thus reduced aggregation free-energy barrier.
| S-EPMC5955824 | biostudies-literature