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 - 绝素蛋白酶，参与清除胰岛素和淀粉样蛋白P的清除，这是两种用于糖尿病和阿尔茨海默氏病的关键蛋白。另外，IDE可以结合某些主要在疼痛神经生理学中起作用的阿片类肽。蛋白酶抑制剂可用于治疗疼痛，因为它们可能会增加体内阿片类肽的寿命，并且成瘾的发展风险降低。此外，在阿片类肽结合后，IDE被选择性地激活淀粉样蛋白-P降解，但被抑制为胰岛素水解。但是，不 晶体结构与阿片类肽家族的任何成员的复合物中存在；因此，我建议用代表每类阿片类肽，enkephalins，dynorphins和内啡肽的代表来解决IDE的晶体结构。我还计划通过IDE表征每个阿片类肽的动力学参数和裂解位点。此外，我建议检查IDE是否可以影响阿片类肽的代谢，以及它是否参与模型培养细胞中阿片类药物介导的信号传导。阿片类肽与IDE的相互作用的表征可能揭示有关IDE经历构象变化的线索，这在阿片类肽结合上经历了促进对AP清除的选择性激活，并可能为开发新的IDE抑制剂提供基础，旨在独家分解某些肽抑制剂。积累的证据强烈表明，氧化和亚硝基化是糖尿病，阿尔茨海默氏病和心血管疾病涉及的因素，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。我将检查是否可以在培养的神经母细胞瘤细胞N2a（3-淀粉样蛋白的前体蛋白。位置定位的诱变）中发生氧化/硝化抑制IDE，用于识别X射线晶体学的影响，并通过氧化的影响来确定X射线晶体的影响。定义IDE的氧化和亚硝基化的分子基础，并可以作为一种治疗策略来保留IDE活性的工具，以有效清除AP和胰岛素等胰岛素：胰岛素降解的酶是一种令人兴奋的酶。通过操纵这种关键酶的活性来进行疾病。