Investigating the Link between IAPP Amyloidogenicity and Diabetes Propensity within the Animal Kingdom

项目摘要/摘要 由37个氨基酸组成的人胰岛淀粉样多肽(hIAPP， 胰淀素)，作为不溶性淀粉样蛋白或作为小寡聚体，似乎在 2型糖尿病患者胰腺β-胰岛细胞死亡。已知有几种生物表达 IAPP的非淀粉样变异体(如大鼠和小鼠)尚未发现 糖尿病是自然而然的。相反，几种生物表达高度淀粉样变异体 IAPP(如人类、猫和灵长类)，并已知会患糖尿病。尽管 虽然有大量的遗传信息，但还没有进行全面的研究 进行IAPP聚合潜力与发病倾向的直接关联 动物王国里的糖尿病。在这项工作中，我们将比较聚合势 以及已知或已知生物体中自然产生的IAPP变异体的细胞毒性 不会，会患上糖尿病。我们建议(1)构建和筛选IAPP变异体文库 基因融合到增强型绿色荧光蛋白。在这个屏幕上，IAPP的基因是 基因融合到增强型绿色荧光蛋白(EGFP)基因。当 IAPP-EGFP融合蛋白在大肠杆菌中的表达IAPP的天然聚集倾向 阻止绿色荧光蛋白折叠和发光。然而，抵抗聚合的变体将 让绿色荧光蛋白折叠并发出明亮的荧光。(2)研究人工合成的聚集势。 IAPP变种。使用硫代黄素T结合、原子力显微镜、圆二色谱和抗体 在检测分析中，我们将量化IAPP变体的聚集能力。(3)量化 IAPP变异体对哺乳动物细胞的毒性。我们将孵化IAPP的变种 并用四甲基偶氮唑盐比色法测定细胞活力。(4)识别变种 能够抑制人IAPP聚集。我们将与每个人孵化HIAPP 合成IAPP变异体以确定哪些变异体能够抑制 有毒的HIAPP物种。我们相信这些实验有可能(I)进一步发展我们的 了解IAPP聚集的序列决定因素(II)确定 IAPP聚集与糖尿病发病倾向及(Iii)识别新肽 能够抑制有毒hIAPP形成的序列。

Project Summary/Abstract The aggregation of the 37-amino acid polypeptide human Islet Amyloid Polypeptide (hIAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-islet cells in type 2 diabetes. It is known that several organisms express non-amyloidogenic variants of IAPP (such as rat and mouse) and are not known to develop diabetes naturally. Conversely, several organisms express highly amyloidogenic variants of IAPP (such as human, cat and primates) and are known to develop diabetes. Despite the significant amount of genetic information available, no comprehensive study has been conducted to directly correlate IAPP aggregation potential to the propensity to develop diabetes within the animal kingdom. In this work, we will compare the aggregation potential and cellular toxicity of naturally occurring IAPP variants from organisms known to, or known not to, develop diabetes. We propose to (1) construct and screen a library of IAPP variants genetically fused to enhanced green fluorescent protein. In this screen, the gene for IAPP is genetically fused to the gene for enhanced green fluorescent protein (EGFP). When the IAPP-EGFP fusion protein is expressed in E. coli the natural propensity of IAPP to aggregate precludes EGFP from folding and fluorescing. However, variants that resist aggregation, will allow the EGFP to fold and fluoresce brightly. (2) Investigate the aggregation potential of synthetic IAPP variants. Using Thioflavin T binding, atomic force microscopy, circular dichroism and antibody detection assays, we will quantitate the ability of the IAPP variants to aggregate. (3) Quantitate the toxicity of IAPP variants on mammalian cells. We will incubate the IAPP variants with mammalian cells and determine cell viability using MTT assays. (4) Identify variants capable of inhibiting human IAPP aggregation. We will incubate hIAPP with each synthetic IAPP variant to determine which variants are capable of inhibiting the formation of toxic hIAPP species. We believe these experiments have the potential to (i) further our understanding of the sequence determinants of IAPP aggregation (ii) identify the link between IAPP aggregation and the propensity to develop diabetes and (iii) identify new peptide sequences capable of inhibiting the formation of toxic hIAPP.