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

胰岛素降解酶机制和调节的分子洞察

• 批准号: 7613626
• 负责人: Luis Abel Ralat
• 金额: $ 4.72万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7613626
• 关键词: Address; Adopted; Affect; Affinity; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein Precursor; Binding; Carboxypeptidase; Cardiac; Cardiovascular Diseases; Catabolism; Cells; Cleaved cell; Complex; Cultured Cells; Data; Development; Diabetes Mellitus; Disease; Dynorphins; Endorphins; Enkephalins; Enzymes; Exposure to; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Hormones; Human; Hydrogen Peroxide; Hydrolysis; Immunity; In Vitro; Insulin; Insulinase; Kinetics; Laboratories; Leucine Enkephalin; Mediating; Metabolism; Metalloproteases; Modeling; Modification; Molecular; Molecular Conformation; Neprilysin; Neuroblastoma; Neurotransmitters; Non-Insulin-Dependent Diabetes Mellitus; Opioid; Opioid Peptide; Organogenesis; Pain; Peptide Hydrolases; Peptides; Physiological; Post-Translational Protein Processing; Predisposition; Protease Inhibitor; Proteins; Regulation; Relative (related person); Reporting; Research; Risk; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Therapeutic; Time; X-Ray Crystallography; Zinc; addiction; alanine aminopeptidase; autocrine; base; design; enzyme activity; in vivo; inhibitor/antagonist; insight; member; neurophysiology; novel; overexpression; oxidation; pain behavior; paracrine; peptide I; peptide hormone; public health relevance; success; tool

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.

描述（由申请人提供）：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β蛋白的清除，这两种蛋白分别是糖尿病和阿尔茨海默病发展的关键蛋白。此外，IDE 可以结合某些阿片肽，这些肽主要在疼痛神经生理学中发挥作用。蛋白酶抑制剂可用于治疗疼痛，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，在与阿片肽结合时，IDE 会选择性地激活淀粉样蛋白-β 的降解，但会抑制胰岛素的水解。然而，没有 IDE 的晶体结构与阿片肽家族的任何成员形成复合物；因此，我建议解决 IDE 与每类阿片肽、脑啡肽、强啡肽和内啡肽的代表复合物的晶体结构。我还计划通过 IDE 表征每种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，以及它是否参与模型培养细胞中阿片介导的信号传导。阿片肽与 IDE 相互作用的表征可能揭示有关 IDE 在阿片肽结合时发生构象变化的线索，从而促进选择性激活 Ap 清除，并可能为开发专门用于分解某些肽底物的新型 IDE 抑制剂提供基础。越来越多的证据强烈表明，氧化和亚硝基化是糖尿病、阿尔茨海默氏病和心血管疾病发生的相关因素，部分原因是酶的翻译后修饰。在此应用中，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 IDE 的氧化/亚硝化抑制是否会发生在培养的神经母细胞瘤细胞 N2a 中，该细胞过度表达（3-淀粉样前体蛋白）。定点诱变将用于识别被修饰的残基。我还建议进行 X 射线晶体学，以确定氧化和亚硝基化导致 IDE 失活的结构影响。这一目标的成功将 定义了 IDE 氧化和亚硝基化的分子基础，并可作为设计治疗策略的工具，以保留 IDE 活性，以有效清除 Ap 和胰岛素等肽。公共健康相关性：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目标。拟议的研究可能会提供一种治疗这些问题的方法 通过操纵这种关键酶的活性来治疗疾病。