Regulation and Catalysis of Human Insulin Degrading Enzyme

人胰岛素降解酶的调控与催化

• 批准号: 7637276
• 负责人: WEI-JEN TANG
• 金额: $ 28.32万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7637276
• 关键词: Address; Affinity; Alanine; Alzheimer' s Disease; Amyloid; Animals; Atrial Natriuretic Factor; B-insulin; Binding; Biochemical; Brain; C-terminal; Catalysis; Catalytic Domain; Cell Culture Techniques; Cell Line; Cells; Cerebrum; Charge; Complex; Crystallization; Culture Media; Cultured Cells; Cysteine; Data; Development; Diabetes Mellitus; Dimerization; Disulfides; Drug Delivery Systems; Effectiveness; Encapsulated; Engineering; Enzyme Gene; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Failure; Family; Florida; Frequencies; Future; Genetic; Glucagon; Human; In Vitro; Insulin; Insulinase; Ions; Kinetics; Knowledge; Lead; Metalloproteases; Modeling; Molecular; Molecular Conformation; Mus; Mutation; Neuroblastoma; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Peptide Hydrolases; Peptides; Point Mutation; Process; Property; Proteins; Proteolysis; Recombinants; Reducing Agents; Regulation; Research Personnel; Resolution; Rodent; Role; Scanning; Structural Models; Structure; Subfamily lentivirinae; Testing; Therapeutic; Transfection; Universities; Work; Zinc; base; design; dimer; disulfide bond; enzyme activity; enzyme structure; enzyme substrate; enzyme substrate complex; hydroxamate; improved; inhibitor/antagonist; insight; islet amyloid polypeptide; loss of function mutation; mouse model; mutant; novel; overexpression; oxidation; peptidomimetics; preference; prevent; programs; protein structure; research study; success; therapeutic target; tool; tris(2-carboxyethyl)phosphine

DESCRIPTION (provided by applicant): Metalloprotease is the most abundant within the five protease classes in humans. Insulin degrading enzyme (IDE) is a zinc-metalloprotease that is involved in the clearance of insulin and amyloid (3 (A3), two key proteins for the development of diabetes and Alzheimer's disease, respectively. Accumulating genetic evidence strongly suggests that IDE is a potential drug target for type 2 diabetes and Alzheimer's disease. In order to develop tools to explore the therapeutic potential of IDE, we have recently solved the x-ray crystal structures of human IDE in complex with insulin B chain, Ap, amylin, and glucagon at 2.1-2.6A resolution. Our structures reveal a novel mechanism for substrate recognition and control of catalysis of IDE. Specifically, we found that IDE consists of two 56kDa functional N- and C-terminal domains (IDE-N and IDE- C, respectively) and they form an enclosed cage just large enough to encapsulate small peptides such as insulin. The extensive contacts between IDE-N and IDE-C keep the degradation chamber of IDE inaccessible to substrates. IDE stays in this closed conformation normally and the repositioning of IDE domains is the key control step in allowing substrate access to the catalytic chamber. The enclosed substrate undergoes conformational changes to interact with two discrete regions of IDE for its degradation. In this application, we propose to better understand this intriguing regulation. We will perform mutagenic analysis to begin to address the opening process as well as determine the structures of two key steps for the catalytic cycle of IDE, substrate-free IDE closed and open conformations. We will also obtain the structural basis in how IDE recognizes disulfide-bond containing IDE substrates and high affinity peptidomimetic hydroxamates that can potently inactivate IDE activity. Furthermore, we propose to construct hyperactive IDE mutants and test their ability to degrade Ap in cultured neuronal cells. Success of these aims will not only broaden our knowledge in how proteases recognize their substrates and control their proteolytic activity but also provide valuable information in the future design of IDE-based therapeutics.

描述（由申请人提供）：金属蛋白酶是人类五种蛋白酶类别中最丰富的。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（3（A3）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。不断积累的遗传证据强烈表明，IDE是2型糖尿病和阿尔茨海默病的潜在药物靶点。为了开发工具来探索 IDE 的治疗潜力，我们最近以 2.1-2.6A 分辨率解析了人 IDE 与胰岛素 B 链、Ap、胰岛淀粉样多肽和胰高血糖素复合物的 X 射线晶体结构。我们的结构揭示了一种底物识别和 IDE 催化控制的新机制。具体来说，我们发现 IDE 由两个 56kDa 功能性 N 端和 C 端结构域（IDE-N 和 IDE-C， 它们形成一个封闭的笼子，大小刚好足以封装胰岛素等小肽。 IDE-N 和 IDE-C 之间的广泛接触使基材无法接近 IDE 的降解室。 IDE 通常保持这种闭合构象，IDE 结构域的重新定位是允许底物进入催化室的关键控制步骤。封闭的底物发生构象变化 与 IDE 的两个离散区域相互作用以降低其性能。在此应用中，我们建议更好地理解这一有趣的法规。我们将进行诱变分析，开始解决开放过程，并确定 IDE 催化循环的两个关键步骤的结构，即无底物 IDE 闭合构象和开放构象。我们还将获得 IDE 如何识别含有二硫键的 IDE 底物和高亲和力的结构基础 拟肽异羟肟酸盐，可有效灭活 IDE 活性。此外，我们建议构建高活性的 IDE 突变体并测试它们在培养的神经元细胞中降解 Ap 的能力。这些目标的成功不仅将拓宽我们对蛋白酶如何识别其底物并控制其蛋白水解活性的知识，而且还为未来基于 IDE 的疗法的设计提供有价值的信息。