Investigating IAPP Aggregation and Toxicity using Small Molecule Interference

描述（由申请人提供）：胰岛淀粉样蛋白多肽（IAPP）是胰腺β细胞与胰岛素共同分泌的37残留激素。IAPP聚集成淀粉样纤维与2型糖尿病中产生胰岛素的β细胞死亡有关。IAPP淀粉样蛋白形成和毒性的机制尚不清楚。脂质双分子层是淀粉样蛋白形成的有效促进剂。IAPP已被证明会引起膜渗漏，这被认为是细胞毒性的来源。我建议使用小分子来研究这些过程，这些小分子是我之前通过一种新的筛选试验发现的。这些化合物对淀粉样蛋白形成和膜渗漏过程的干扰将有助于更好地理解控制这些现象的机制。首先，将小分子进行表征，以确定抑制淀粉样蛋白形成的原因。脂质催化和新生（无脂质存在）条件将用于区分特异性抑制脂质催化的小分子和一般淀粉样蛋白抑制剂。CD将用于监测脂质结合和识别由于小分子相互作用而导致的二级结构的变化。15N IAPP的核磁共振将用于确定小分子与IAPP之间的特定相互作用。HSQC光谱将用于识别受这些相互作用扰动的单个残基。通过将核磁共振和CD数据与动力学进行比较，我将分配对淀粉样蛋白形成和脂质结合重要的区域和残基。其次，我将使用膜渗漏作为细胞毒性的模拟物来识别能够减少IAPP毒性作用的化合物。先前对引起淀粉样蛋白形成和脂质结合的分子相互作用的了解，将有助于识别淀粉样蛋白形成中的有毒物质，并可能为2型糖尿病提供新的治疗靶点。

DESCRIPTION (provided by applicant): Islet Amyloid Polypeptide (IAPP) is a 37 residue hormone that is co-secreted with insulin from the Beta cells of the pancreas. IAPP aggregation into amyloid fiber has been implicated in the death of insulin producing Beta cells in Type 2 Diabetes. The mechanisms of IAPP amyloid formation and toxicity are not well understood. Lipid bilayers are potent accelerants of amyloid formation. IAPP has been shown to cause membrane leakage, which is believed to be the source of cell toxicity. I propose to study these processes using small molecules that I have previously identified through a novel screening assay. These compounds' interference with the processes of amyloid formation and membrane leakage will lead to a better understanding of the mechanisms that govern these phenomena. First, small molecules will be characterized to determine the causes of the inhibition of amyloid formation. Lipid catalyzed and de novo (no lipid present) conditions will be used to distinguish between small molecules that specifically inhibit lipid catalysis and general amyloid inhibitors. CD will be used to monitor lipid binding and to identify changes in secondary structure due to small molecule interactions. NMR of 15N IAPP will be used to identify specific interactions between small molecules and IAPP. HSQC spectra will be used to identify individual residues perturbed by these interactions. By comparing NMR and CD data with kinetics, I will assign regions and residues that are important for amyloid formation and lipid binding. Second, I will use membrane leakage as a mimic for cell toxicity to identify compounds that are able to reduce the toxic effects of IAPP. Previous knowledge of the molecular interactions that give rise to amyloid formation and lipid binding will allow identification of the toxic species in amyloid formation, and potentially lead to new therapeutic targets for Type 2 Diabetes. IAPP is a small protein made in the pancreas, in the same cells as insulin. Aggregation of IAPP into amyloid fibers has been implicated in the death of these cells that make insulin in type 2 diabetes. Using small molecules I will investigate how IAPP aggregates and kills cells, as well as open new avenues for the treatment of type 2 diabetes.