Molecular Insights into the Mechanism and Regulation of Insulin-Degrading Enzyme

描述(申请人提供)：胰岛素降解酶(IDE)是一种锌金属蛋白酶，参与胰岛素和淀粉样蛋白-p的清除，这两种蛋白质分别与糖尿病和阿尔茨海默病的发展有关。此外，IDE可以结合某些阿片肽，这些阿片肽主要作用于疼痛神经生理学。蛋白水解酶抑制剂可以用于治疗疼痛，因为它们可以延长阿片肽在体内的寿命，并降低成瘾的风险。此外，在阿片肽结合后，IDE被选择性地激活以降解淀粉样蛋白-p，但被抑制以降解胰岛素。然而，没有 IDE的晶体结构与阿片肽家族中的任何成员都存在，因此，我建议以每类阿片肽、脑啡肽、强啡肽和内啡肽为代表的复合体来解决IDE的晶体结构问题。我还计划用IDE来表征每个阿片肽的动力学参数和裂解位点。此外，我建议检查IDE是否可以影响阿片肽的代谢，以及它是否参与了模型培养细胞中阿片介导的信号传递。阿片肽与IDE相互作用的特征可能揭示IDE在阿片肽结合时所经历的构象变化，从而促进对AP清除的选择性激活，并可能为开发专用于特定多肽底物的新型IDE抑制剂提供基础。越来越多的证据有力地表明，氧化和亚硝化是糖尿病、阿尔茨海默病和心血管疾病的发病因素，部分原因是酶的翻译后修饰。在这一应用中，我还建议评估IDE对氧化和亚硝化修饰的脆弱性及其对酶活性的影响。我将研究在培养的神经母细胞瘤细胞N2a中是否可以发生IDE的氧化/亚硝化抑制，N2a过度表达(3-淀粉样前体蛋白)。定点突变将被用来鉴定被修饰的残基。我还建议进行X射线结晶学研究，以确定氧化和亚硝化使IDE失活的结构含义。这一目标的成功将确定IDE氧化和亚硝化的分子基础，并可能成为设计治疗策略的工具，以保留IDE的活性，以便有效清除AP和胰岛素等多肽。公共卫生相关性：胰岛素降解酶被认为是治疗糖尿病、阿尔茨海默病和心血管疾病的一个令人兴奋的可能靶点。这项拟议的研究可能会提供一种通过操纵这种关键酶的活性来治疗这些疾病的方法。

DESCRIPTION (provided by applicant): Insulin-degrading enzyme (IDE) is a zinc-metalloprotease that is involved in the clearance of insulin and amyloid-p, two key proteins for the development of diabetes and Alzheimer's disease, respectively. Additionally, IDE can bind certain opioid peptides, which function mainly in pain neurophysiology. Protease inhibitors could be used to treat pain because they may increase the lifetime of opioid peptides in vivo and there is a decreased risk for the development of addiction. Furthermore, upon opioid peptide-binding, IDE is selectively activated towards amyloid-p degradation but inhibited towards insulin hydrolysis. However, no crystal structure exists of IDE in complex with any member of the opioid peptide family; thus, I propose to solve the crystal structures of IDE in complex with a representative of each class of opioid peptide, enkephalins, dynorphins, and endorphins. I also plan to characterize the kinetic parameters and the cleavage sites of each opioid peptide by IDE. Moreover, I propose to examine whether IDE can affect the metabolism of opioid peptides and whether it is involved in opioid-mediated signaling in model cultured cells. The characterization of the interaction of opioid peptides with IDE may reveal clues about the conformational changes IDE undergoes upon opioid peptide binding that promote the selective activation toward Ap clearance and may provide a basis for the development of a new class of inhibitors of IDE designed to exclusively catabolize certain peptide substrates. Accumulating evidence strongly suggests that oxidation and nitrosylation are factors involved in the development of diabetes, Alzheimer's disease and cardiovascular disease partially due to post-translational modification of enzymes. In this application, I also propose to assess the vulnerability of IDE to oxidative and nitrosative modification and its effects on enzymatic activity. I will examine whether oxidative/nitrosative inhibition of IDE can occur in cultured neuroblastoma cells, N2a, which overexpress (3-amyloid precursor protein. Site-directed mutagenesis will be used to identify the residues that are modified. I also propose to perform X-ray crystallography to identify the structural implications of IDE inactivation by oxidation and nitrosylation. Success in this aim will define the molecular basis for oxidation and nitrosylation of IDE and may serve as a tool for the design of a therapeutic strategy to reserve IDE activity for the effective clearance of peptides like Ap and insulin. PUBLIC HEALTH RELEVANCE: Insulin-degrading enzyme is considered to be an exciting possible target for treatment of diabetes, Alzheimer's disease and cardiovascular disease. The proposed research may provide an approach to treating these ailments by manipulating the activity of this critical enzyme.