Biological Role of Two Metalloenzymes: insulin-degrading enzyme and neprilysin

描述（由申请人提供）：本奖项的目的是完成我的培训，并在美国学术机构建立一个高度跨学科的实验室，重点研究淀粉样蛋白- (a)和利钠肽降解金属肽酶的生物学功能、催化机制和活性调控。在本提案中，我重点关注胰岛素降解酶（IDE）和NEP (NEP)，这两种含隐窝锌金属蛋白酶与人类疾病高度相关，因为它们能够降解多种肽和激素，包括几种形式的A2，阿尔茨海默病患者大脑中淀粉样斑块的主要成分。因此，IDE和NEP代表了通过操纵这两种酶来控制人类A2水平的令人兴奋的治疗潜力。然而，仅仅控制IDE和NEP的活性可能会导致不可接受的副作用的出现，因为这两种蛋白酶也会改变作为底物的其他生理激素的水平，如调节心血管功能的利钠肽。因此，我的实验室将采取全面、多方面的方法，将原子分辨率结构、蛋白质组学数据和生化结果与从细胞更大背景中收集的信息结合起来，彻底了解这两种金属酶的功能。这里概述的研究计划是高度跨学科的，整合了结构生物学、生物化学、生物物理学和细胞生物学的技术和概念，以回答关于IDE和NEP的三个基本问题：1)IDE和NEP如何影响活细胞中的利钠肽水平和由此产生的信号动力学，当IDE和NEP耗尽或被敲除时，细胞会产生什么后果？2)由于利钠肽家族成员的不同生化特性，它们是否可以作为IDE和NEP表征底物选择性机制的工具？3) IDE和NEP通过氧化和亚硝基化失活的分子基础是什么？这些生理翻译后修饰剂如何影响底物的转换？该奖项的指导阶段（目标1）的重点是建立IDE的基本属性。在独立阶段（目标2），在指导阶段建立的方法将扩展到新经济政策的研究。总之，这两个目标的成功完成可以为合理开发一类新的药物提供基础，这些药物靶向a /利钠肽降解金属蛋白酶，旨在选择性地分解某些肽底物。此外，通过氧化和亚硝基化确定IDE和NEP失活的分子过程将有助于设计在氧化环境中保持IDE和NEP活性的治疗策略。

DESCRIPTION (provided by applicant): The objective of this award is to complete my training and to establish a highly interdisciplinary laboratory at an American academic institution, focused on the study of the biological function, catalytic mechanism and activity regulation of amyloid- (A ) and natriuretic peptide-degrading metallopeptidases. In this proposal, I focus on insulin-degrading enzyme (IDE) and neprilysin (NEP), two crypt-containing zinc metalloproteases highly relevant in human disease due to their ability to degrade multiple peptides and hormones, including several forms of A2, the main constituent of amyloid plaques in the brains of Alzheimer disease patients. Thus, IDE and NEP represent an exciting therapeutic potential toward the control of A2 levels in humans through the manipulation of these two enzymes. However, simply controlling IDE and NEP activity may result in the emergence of unacceptable side effects, since these two proteases could also alter the levels of other physiological hormones recognized as substrates, such as the natriuretic peptides, which function to regulate cardiovascular function. Consequently, my laboratory will take a comprehensive, multifaceted approach to combine atomic resolution structures, proteomic data, and biochemical results with the information gathered from the larger context of the cell to thoroughly understand the function of these two metalloenzymes. The research plan outlined here is highly interdisciplinary, integrating techniques and concepts from structural biology, biochemistry, biophysics, and cell biology to answer three fundamental questions about IDE and NEP: 1) How do IDE and NEP affect natriuretic peptide levels and the resulting signaling dynamics in the living cell and what are the cellular consequences incurred when IDE and NEP are depleted or knocked out? 2) With the different biochemical properties of each member of the natriuretic peptide family, can they be employed as tools to characterize the mechanism of substrate selectivity by IDE and NEP? 3) What is the molecular basis for the inactivation of IDE and NEP by oxidation and nitrosylation and how do these physiological post- translational modifiers affect substrate turnover? The emphasis in the mentored phase of the award (Aim 1) will be to establish the fundamental properties of IDE. In the independent phase (aim 2), the methods established in the mentored phase will be expanded to the study of NEP. Together, the successful completion of both aims can provide a basis for the rational development of a new class of drugs, targeting A /natriuretic peptide- degrading metalloproteases, designed to selectively catabolize certain peptide substrates. Furthermore, defining the molecular process of IDE and NEP inactivation by oxidation and nitrosylation would facilitate the design of therapeutic strategies to preserve IDE and NEP activity in an oxidative environment. PUBLIC HEALTH RELEVANCE: Because of their ability to recognize substrates that can affect a wide range of physiological activities, insulin- degrading enzyme and neprilysin are exciting targets for the development of therapies to treat a range of chronic ailments, including diabetes, Alzheimer disease and cardiovascular disease. The proposed research aims to elucidate the extent of the biological functions of these two proteins at the molecular, cellular, and whole-organism level to facilitate the design of therapeutic strategies that maximize their potential while minimizing side effects.