Regulation and Catalysis of Human Insulin Degrading Enzyme

描述(申请人提供)：金属蛋白酶是人类五类蛋白酶中含量最丰富的。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，这两种蛋白分别与糖尿病和阿尔茨海默病的发生有关。越来越多的遗传证据有力地表明，IDE是治疗2型糖尿病和阿尔茨海默病的潜在药物靶点。为了开发开发工具来探索IDE的治疗潜力，我们最近以2.1-2.6A的分辨率解决了人的IDE与胰岛素B链、AP、胰淀素和胰高血糖素的络合物的X射线晶体结构。我们的结构揭示了底物识别和IDE催化控制的新机制。具体来说，我们发现IDE由两个56 kDa的功能性N-末端和C-末端结构域(分别是IDE-N和IDE-C)组成，它们形成了一个封闭的笼子，大到足以包裹胰岛素等小肽。IDE-N和IDE-C之间的广泛接触使底物无法接触到IDE的降解室。IDE通常保持这种封闭的构象，而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，导致其降解。在本申请中，我们建议更好地理解这一耐人寻味的规定。我们将进行突变分析，以开始研究IDE的开放过程，并确定IDE催化循环的两个关键步骤的结构，即无底物的IDE封闭构象和开放构象。我们还将获得IDE如何识别含有二硫键的IDE底物和能够有效钝化IDE活性的高亲和力仿肽羟胺类化合物的结构基础。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和控制其蛋白分解活性的认识，而且还将为未来基于IDE的疗法的设计提供有价值的信息。

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.