Role of Ubiquitin in Cardiovascular System

泛素在心血管系统中的作用

• 批准号: 7873043
• 负责人: YONG TAE KWON
• 金额: $ 35.04万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873043
• 关键词: Address; Affinity; Arginine; Aspartate; Binding; Blood Vessels; Boxing; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Culture Techniques; Cell Proliferation; Cells; Congenital Heart Defects; Cysteine; Defect; Embryo; Embryonic Heart; Figs - dietary; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; GTPase-Activating Proteins; Gases; Genes; Glutamates; Goals; Growth; Half-Life; Heart; Homeostasis; In Vitro; Individual; Knock-out; Mediating; Molecular; Myocardial; N-terminal; Names; Nature; Oxygen; Pathway interactions; Physiological; Protein Family; Protein Isoforms; Proteins; Proteolysis; Proteomics; RGS Proteins; Role; Screening procedure; Signal Transduction; Specificity; Testing; Transferase; Ubiquitin; Ventricular; Ventricular Septal Defects; angiogenesis; base; cofactor; in vivo; inhibitor/antagonist; insight; novel; small molecule; tRNA-arginine complex; ubiquitin 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(申请人提供)：N-末端规则途径是一种泛素(Ub)蛋白分解途径，它将蛋白质在体内的半衰期与其N末端残基的特性联系起来。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)的结合是这一蛋白分解途径的一部分，因为它导致了Arg结合蛋白的泛素化。我们先前观察到ATE1-/-胚胎死于各种心血管缺陷。然而，心血管缺陷的确切性质和潜在的分子机制尚不清楚。该项目的总体目标是确定ATE1依赖的精氨酸化在心肌生长中的生理作用，并深入了解ATE1依赖的蛋白分解途径参与的心血管途径/分子。用功能蛋白质组学方法确定N-端规则通路的底物，并随后对选定的候选底物进行初步表征，导致假设RGS4、RGS5和RGS16是ATE1底物，可能是ATE1依赖的心血管系统的基础。RGS蛋白是G(α)亚基的GTP酶激活蛋白(GAP)，负向调节G蛋白偶联受体(GPCR)信号转导。RGS4、RGS5和/或RGS16被认为是GQ/GI激活的心肌生长和血管成熟/完整性信号的重要负调控因子。然后，我们询问了这些RGS蛋白被泛素连接酶(E3s)泛素化以进行蛋白分解的机制。利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UBR1-UBR7，它可能对RGS4、RGS5和RGS16的泛素化起作用。我们的总体假设是，RGS4、RGS5和RGS16的细胞浓度，从而它们作为心血管G蛋白通路的调节功能，是通过MetAPs-O2-ATE1-UBR蛋白水解级联反应来调节的。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：检测血管紧张素转换酶1(ATE1)是否在体内对内源性心肌生长起作用。目的：研究依赖AT1的精氨酸化对RGS4、RGS5和RGS16周转的影响。目的：阐明RGS4、RGS5和RGS16泛素化降解蛋白质的分子机制。目的4：研究ATE1基因敲除对G蛋白通路的生理影响。