Regulation and Catalysis of Human Insulin Degrading Enzyme

描述（由申请人提供）：胰岛素降解酶（IDE）是一种进化上保守的110 kDa金属蛋白酶，参与胰岛素和淀粉样蛋白的清除。(?)。在啮齿动物和人类中积累的遗传证据有力地支持IDE在2型糖尿病和阿尔茨海默病进展中的作用。因此，从分子角度了解IDE的功能、催化机制和调控对于制定可行的IDE治疗策略至关重要。我们已经解决了人类IDE与胰岛素，A？以及其他功能相关的底物，如利钠肽和促炎趋化因子CCL3/CCL4。IDE有两个55 kDa结构域，形成一个封闭的催化室以捕获其底物。我们的结构和生化分析揭示了IDE如何使用一个封闭的催化室来选择性地识别其底物的全局特征。本研究的长期目标是：(1)阐明IDE的功能及其调控机制；(2)阐明IDE在人类疾病中的作用。本应用程序的目的是了解(1)在催化过程中IDE的开闭构象开关和动力学的分子基础；(2)IDE调节的分子机制。此外，我们将开发IDE的有效化学调节剂，作为潜在的治疗剂和工具，以解决该酶的生物学功能。核心假设是IDE的开闭构象开关是IDE的关键调控步骤，受二聚化、翻译后修饰、细胞因子和化学调节剂的变构调节。这项拟议研究的基本原理是，了解IDE的调节和功能，并开发IDE的小化学调节剂，最终将使我们能够更好地设计基于IDE的治疗策略，具体针对某些人类疾病，如糖尿病、阿尔茨海默病和炎症。在初步数据的指导下，我们将在三个具体目标中研究IDE的调节和功能：在Aim 1中，我们将使用单分子福斯特共振能量转移分析来解决IDE催化过程中发生的构象开关和动力学，并确定IDE的催化是如何被调节的。目的2是使用两种不同的筛选方法来开发有效的小分子化合物，这些化合物可以调节IDE的活性，并使用这些化合物来解决IDE的生物学功能。目的3是结合结构、生化和突变研究，通过二聚化、生理相关细胞因子（如中间丝蛋白、巢蛋白和静脉蛋白）和翻译后修饰（如磷酸化和乙酰化）来解决IDE调节的分子基础。这项拟议中的研究意义重大，因为它将对糖尿病和阿尔茨海默病中涉及的一种关键酶的动力学和调节产生新的见解，因为它还将导致发现新的化学线索，可以有效地调节这种酶。拟议的研究具有创新性，因为它采用生物物理、生化、细胞和药物化学方法来研究IDE的调节和功能。

DESCRIPTION (provided by applicant): Insulin degrading enzyme (IDE) is an evolutionarily conserved, 110 kDa metalloprotease that is involved in the clearance of insulin and amyloid ? (A?). Accumulating genetic evidence in rodents and humans strongly support the role of IDE in the progression of type 2 diabetes mellitus and Alzheimer's disease. Thus, it is vital to understand the functions, catalytic mechanism, and regulation of IDE from a molecular perspective to develop viable IDE-based therapeutic strategies. We have solved the structures of human IDE in complex with insulin, A?, and other functionally relevant substrates such as natriuretic peptides and proinflammatory chemokines, CCL3/CCL4. IDE has two 55 kDa domains, which form an enclosed catalytic chamber to entrap its substrates. Our structural and biochemical analyses reveal how IDE uses an enclosed catalytic chamber to selectively recognize the global features of its substrates. The long-term goal of this research is (1) to delineate the function(s) of IDE and the mechanism(s) of its regulation and (2) to elucidate the role of IDE in human diseases. The objectives of this application are to understand (1) the molecular basis for the open-closed conformational switch and dynamics of IDE during catalysis and (2) the molecular mechanism for the regulation of IDE. In addition, we will develop potent chemical modulators of IDE to be used as potential therapeutic agents and as tools to address the biological functions of this enzyme. The central hypothesis is that the open-closed conformational switch of IDE is the key regulatory step of IDE that is subject to allosteric regulation by dimerization, posttranslational modifications, cellular factors, and chemical modulators. The rationale for the proposed research is that understanding the regulation and functions of IDE and developing small chemical modulators of IDE will ultimately allow us to better design IDE-based therapeutic strategies specific to certain human diseases such as diabetes, Alzheimer's disease, and inflammation. Guided by our preliminary data, we will study the regulation and functions of IDE in three specific aims: in Aim 1, we will use single molecule Forster resonance energy transfer analyses to address the conformational switches and dynamics that occur during catalysis of IDE and to determine how the catalysis of IDE is regulated. Aim 2 is to use two distinct screening methods to develop potent small molecule compounds that can modulate the activity of IDE and use such compounds to address the biological functions of IDE. Aim 3 is to combine structural, biochemical, and mutational studies to address the molecular basis for the regulation of IDE by dimerization, physiologically relevant cellular factors such as intermediate filament proteins, nestin and vimentin, and by posttranslational modifications such as phosphorylation and acetylation. The proposed research is significant because it will generate new insights in the dynamics and regulation of a key enzyme involved in diabetes and Alzheimer's disease and because it will also lead to the discovery of new chemical leads that can potently modulate this enzyme. The proposed research is innovative because it employs biophysical, biochemical, cellular, and medicinal chemical approaches to investigate the regulation and functions of IDE.