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

项目描述（由申请人提供）：该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，专注于淀粉样蛋白（A）和利钠肽降解金属肽酶的生物学功能，催化机制和活性调节的研究。在这项提案中，我专注于胰岛素降解酶（IDE）和脑啡肽酶（NEP），这两种含隐窝的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控制人类A2水平的令人兴奋的治疗潜力。然而，简单地控制IDE和NEP活性可能会导致出现不可接受的副作用，因为这两种蛋白酶也可能改变被认为是底物的其他生理激素的水平，例如用于调节心血管功能的利钠肽。因此，我的实验室将采取一种全面的、多方面的方法，将联合收割机的原子分辨率结构、蛋白质组学数据和生化结果与从细胞的更大背景中收集的信息结合起来，以彻底了解这两种金属酶的功能。本文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如何影响利钠肽水平和活细胞中产生的信号动力学，以及IDE和NEP耗尽或敲除时产生的细胞后果是什么？2)由于利钠肽家族的每个成员具有不同的生化特性，它们能否作为工具来表征IDE和NEP的底物选择性机制？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周转？该奖项的指导阶段（目标1）的重点将是建立IDE的基本属性。在独立阶段（目标2），将把在指导阶段建立的方法扩大到对非经济政策的研究。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，靶向A /利钠肽降解金属蛋白酶，旨在选择性分解代谢某些肽底物。此外，通过氧化和亚硝基化来定义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。 公共卫生相关性：由于其识别可影响广泛生理活性的底物的能力，胰岛素降解酶和脑啡肽酶是开发治疗一系列慢性疾病（包括糖尿病、阿尔茨海默病和心血管疾病）的疗法的令人兴奋的靶标。拟议的研究旨在阐明这两种蛋白质在分子，细胞和整个生物体水平上的生物学功能的程度，以促进治疗策略的设计，最大限度地发挥其潜力，同时最大限度地减少副作用。

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.