Regulation and Catalysis of Human Insulin Degrading Enzyme

人胰岛素降解酶的调控与催化

• 批准号: 7898366
• 负责人: WEI-JEN TANG
• 金额: $ 24.02万
• 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: 2009-08-31 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7898366
• 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

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.

金属蛋白酶是人类五个蛋白酶类中最丰富的。胰岛素降解酶 （IDE）是一种与清除胰岛素和淀粉样蛋白清除有关（3（A3），两个键 分别用于糖尿病和阿尔茨海默氏病的蛋白质。积累的遗传 有证据表明，IDE是2型糖尿病和阿尔茨海默氏病的潜在药物靶标。在 为了开发探索IDE治疗潜力的工具，我们最近解决了X射线晶体 人类IDE与胰岛素B链，AP，淀粉纤维和胰高血糖素在2.1-2.6a分辨率下的结构。 我们的结构揭示了底物识别和控制IDE催化的新机制。 具体而言，我们发现IDE由两个56KDA功能N-和C末端结构域（IDE-N和IDE- c分别）和它们形成一个封闭的笼子，刚好足以封装小肽，例如 胰岛素。 IDE-N和IDE-C之间的广泛接触保持IDE的降解室 底物无法接近。 IDE通常保持在这种封闭的构象中，并重新定位IDE 域是允许底物进入催化室的关键控制步骤。封闭的 底物经历构象变化，以与IDE的两个离散区域相互作用以降解。 在此应用程序中，我们建议更好地理解这一有趣的法规。我们将执行诱变 分析开始解决开放过程，并确定两个关键步骤的结构 IDE的催化循环，无基质IDE闭合和开放构象。我们还将获得结构 IDE如何识别含有IDE底物和高亲和力肽型的二硫键键的基础 可以有效灭活IDE活性的羟胺。此外，我们建议构建多动态 IDE突变体并测试其在培养的神经元细胞中降解AP的能力。这些目标的成功不会 只扩大我们在蛋白酶识别其底物并控制其蛋白水解活性方面的知识 但也提供基于IDE的治疗剂的未来设计中的宝贵信息。