Regulation and Catalysis of Human Insulin Degrading Enzyme

描述（由申请人提供）：金属蛋白酶是人类五种蛋白酶中最丰富的。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（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。越来越多的遗传学证据有力地表明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- 2.6 A分辨率解析了与胰岛素B链、A β、胰淀素和胰高血糖素复合的人IDE的X射线晶体结构。我们的结构揭示了一种新的机制，底物识别和控制的催化IDE。具体地，我们发现IDE由两个56 kDa的功能性N-和C-末端结构域（分别为IDE-N和IDE-C）组成，并且它们形成一个封闭的笼，其大小刚好足以包封小肽如胰岛素。IDE-N和IDE-C之间的广泛接触使IDE的降解室无法接近衬底。IDE通常保持在这种闭合构象中，并且IDE结构域的重新定位是允许基底进入催化室的关键控制步骤。封闭的基板经历构象变化，以与IDE的两个离散区域相互作用，以使其降解。在这个应用程序中，我们建议更好地理解这个有趣的规则。我们将进行致突变分析，以开始解决开放过程以及确定IDE催化循环的两个关键步骤的结构，无底物IDE闭合和开放构象。我们还将获得IDE如何识别含二硫键的IDE底物和高亲和力拟肽异羟肟酸盐的结构基础，这些物质可以有效地抑制IDE活性。此外，我们建议构建过度活跃的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。这些目标的成功将不仅拓宽我们的知识，在蛋白酶如何识别其底物和控制其蛋白水解活性，但也提供了有价值的信息，在未来的IDE为基础的治疗设计。

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.