Regulation and Catalysis of Human Insulin Degrading Enzyme

金属蛋白酶是人类五种蛋白酶中含量最丰富的一种。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的清除(3(A3)，两个关键 分别与糖尿病和阿尔茨海默病的发生有关的蛋白质。积累遗传 强有力的证据表明，IDE是治疗2型糖尿病和阿尔茨海默病的潜在药物靶点。在……里面 为了开发工具来探索IDE的治疗潜力，我们最近解决了x射线晶体 人IDE与胰岛素B链、AP、胰淀素和胰升糖素的复合体的结构，分辨率为2.1-2.6A。 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(IDE-N和IDE-N-)组成。 C)，它们形成一个封闭的笼子，大到足以包裹小肽，如 胰岛素。IDE-N和IDE-C之间的广泛接触保持了IDE的降解室 底物无法接触到的。IDE通常保持这种封闭的构象，并且IDE的重新定位 结构域是允许底物进入催化室的关键控制步骤。随函附上的 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。我们将进行诱变 分析开始处理开放过程，并确定两个关键步骤的结构 IDE的催化循环，无底物的IDE封闭和开放构象。我们还将获得结构上的 IDE识别含二硫键的IDE底物和高亲和力多肽的基础 能有效钝化IDE活性的异羟甲酸酯。此外，我们建议构建过度活跃的 在培养的神经细胞中筛选突变体并测试其降解AP的能力。这些目标的成功不会 只是拓宽了我们对蛋白酶如何识别其底物并控制其蛋白分解活性的认识 也为基于IDE的疗法的未来设计提供了有价值的信息。

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.