Role of Ubiquitin in Cardiovascular System

泛素在心血管系统中的作用

• 批准号: 7141372
• 负责人: YONG TAE KWON
• 金额: $ 36.14万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7141372
• 关键词: G protein; arginine; cardiogenesis; developmental genetics; embryo /fetus tissue /cell culture; guanosinetriphosphatase activating protein; laboratory mouse; organ culture; posttranslational modifications; protein binding; protein isoforms; protein structure function; proteolysis; proteomics; ubiquitin; ubiquitin protein ligase

DESCRIPTION (provided by applicant): The N-end rule pathway is one ubiquitin (Ub) proteolytic pathway that relates the in vivo half-life of a protein to the identity of its N terminal residue. Conjugation of arginine (Arg) from Arg-tRNA(Arg) to N-terminal aspartate (Asp), glutamate (Glu), or cysteine (Cys) is part of this proteolytic pathway in that it leads to ubiquitylation of the resulting, Arg-conjugated proteins. We previously observed that ATE1-/- embryos die due to various cardiovascular defects. However, the exact nature of the cardiovascular defects and the underlying molecular mechanisms are unknown. The overall goal of this project is to determine the physiological role of ATE1-dependent arginylation in the myocardial growth and to gain insights into the cardiovascular pathways/molecules in which the ATE1-dependent proteolytic pathway participates. Functional proteomic approach to identify the substrates of the N-end rule pathway and subsequent preliminary characterization of the selected candidate substrates led to the hypothesis that RGS4, RGS5, and RGS16 are ATE1 substrates that may underlie the ATE1-dependent cardiovascular system. RGS proteins are GTPase-activating proteins (GAP) for G(alpha) subunits and negatively regulate the G protein- coupled receptor (GPCR) signaling. RGS4, RGS5, and/or RGS16 have been implicated as important negative regulators of the Gq/Gi-activated signaling for myocardial growth and vascular maturation/integrity. We then asked the mechanism by which these RGS proteins are ubiquitylated for proteolysis by ubiquitin ligases (E3s). Using an affinity-based proteomic approach, we identified a novel protein family, named UBR1-UBR7, that potentially acts for ubiquitylation of RGS4, RGS5, and RGS16. Our overall hypothesis is that the cellular concentrations of RGS4, RGS5, and RGS16, and thus their functions as regulators of the cardiovascular G protein pathways, are regulated through the MetAPs-O2-ATE1-UBR proteolytic cascade. To address this hypothesis and related issues, we propose the following Aims. Aim 1: To test whether ATE1 has an in vivo role in intrinsic myocardial growth. Aim 2: To determine the role of ATE 1-dependent arginylation for turnover of RGS4, RGS5, and RGS16. Aim 3: To elucidate the molecular mechanism by which RGS4, RGS5, and RGS16 are ubiquitylated for proteolysis. Aim 4: To examine the physiological consequences of ATE1 knockout on the G protein pathways.

描述（由申请人提供）：N-末端规则途径是一种泛素（Ub）蛋白水解途径，其将蛋白质的体内半衰期与其N末端残基的身份联系起来。精氨酸（Arg）从Arg-tRNA（Arg）到N-末端天冬氨酸（Asp）、谷氨酸（Glu）或半胱氨酸（Cys）的缀合是该蛋白水解途径的一部分，因为它导致所得Arg缀合蛋白的泛素化。我们先前观察到ATE 1-/-胚胎由于各种心血管缺陷而死亡。然而，心血管缺陷的确切性质和潜在的分子机制尚不清楚。本项目的总体目标是确定ATE 1依赖性腺苷酸化在心肌生长中的生理作用，并深入了解ATE 1依赖性蛋白水解途径参与的心血管途径/分子。功能性蛋白质组学方法鉴定N-末端规则途径的底物和随后对所选候选底物的初步表征导致了以下假设：RGS 4、RGS 5和RGS 16是ATE 1底物，可能是ATE 1依赖性心血管系统的基础。RGS蛋白是G（α）亚基的GTP酶激活蛋白（GAP），并且负调节G蛋白偶联受体（GPCR）信号传导。RGS 4、RGS 5和/或RGS 16已被认为是心肌生长和血管成熟/完整性的Gq/Gi激活信号传导的重要负调节剂。然后，我们问的机制，这些RGS蛋白被泛素连接酶（E3）的蛋白水解泛素化。使用基于亲和力的蛋白质组学方法，我们鉴定了一个新的蛋白质家族，命名为UBR 1-UBR 7，其可能作用于RGS 4、RGS 5和RGS 16的泛素化。我们的总体假设是，RGS 4、RGS 5和RGS 16的细胞浓度，以及它们作为心血管G蛋白通路调节剂的功能，通过MetAPs-O2-ATE 1-UBR蛋白水解级联反应进行调节。为了解决这一假设和相关问题，我们提出了以下目标。目的1：检测ATE 1是否在体内内在心肌生长中发挥作用。目的2：确定ATE 1依赖性腺苷酸化对RGS 4、RGS 5和RGS 16周转的作用。目的3：阐明RGS 4、RGS 5和RGS 16泛素化蛋白水解的分子机制。目的4：研究ATE 1基因敲除对G蛋白通路的生理影响。