Role of Ubiquitin in Cardiovascular System

描述（由申请人提供）：n端规则途径是一种泛素蛋白水解途径，将蛋白质的体内半衰期与其n端残基的身份联系起来。精氨酸（Arg）从Arg- trnaarg偶联到n端天冬氨酸（Asp）、谷氨酸（Glu）或半胱氨酸（Cys）是该蛋白水解途径的一部分，因为它可以导致所得到的精氨酸偶联蛋白的泛素化。我们之前已经确定了哺乳动物Ate1基因编码精氨酸转移酶，负责所有已知的蛋白质精氨酸化活性，并表明Ate1-/-胚胎由于各种心血管缺陷而死亡，包括心室发育不全、室间隔缺损和晚期血管生成。这些结果表明，未知底物的ate1依赖性蛋白水解是心肌生长和血管完整性/成熟的重要调节机制。然而，心血管缺陷的确切性质和潜在的分子机制仍然难以捉摸。全基因组功能蛋白质组学方法使我们确定了一组心血管调节因子（Rgs4， Rgs5和Rgs16）作为ate1依赖性精氨酸化的底物，这可能至少部分地支持ate1依赖性心血管稳态。值得注意的是，Rgs4和Rgs5是gtpase激活蛋白（GAP），作为gpcr偶联Ga亚基的负调节因子，并分别被认为是心肌生长和血管成熟/完整的Gq/ gi激活信号的重要调节因子。生化分析表明，这些底物的降解依赖于Cys2残基作为降解决定因素，通过Met氨基肽酶裂解n端Met暴露于n端。在足够的氧气（O2）和一氧化氮（NO）的存在下，n端Cys2似乎被氧化为CysO2，从而产生一种类似于Asp的结构，这是一种允许精氨酸化的残基。精氨酸化RGS蛋白的n端精氨酸残基随后被特异性的E3连接酶结合，其身份尚不清楚。利用基于亲和的蛋白质组学方法，我们分离了一组E3家族（命名为Ubr1至Ubr7），并证明了Ubr1， Ubr2和Ubr4是蛋白精氨酸化特异性的主要E3s，并且Ubr1-/-Ubr2-/-和Ubr4-/-胚胎死于心血管缺陷。基于这些结果，我们假设Rgs4、Rgs5和Rgs16的功能是通过MetAPs-O2/NO-Ate1-Ubr蛋白水解级联调控的。在Aim 1中，我们将利用组织特异性Ate1敲除小鼠与心脏中过表达Gq的转基因小鼠联合，表征Ate1依赖性精氨酸化在心血管发育和信号传导中的生理功能。在Aim 2中，为了了解ate1依赖性心血管发展的分子原理，我们将表征Rgs4和Rgs5的精氨酸化依赖性转换和Cys2的共翻译修饰。在Aim 3中，作为我们表征精氨酸化底物特异性泛素连接酶的长期努力的一部分，我们将表征缺乏Ubr4的小鼠的心血管发育，Ubr4是蛋白质精氨酸化下游新发现的识别成分。1

DESCRIPTION (provided by applicant): The N-end rule pathway is one ubiquitin 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-tRNAArg to N-terminal aspartate (Asp), glutamate (Glu), or cysteine (Cys) is part of this proteolytic pathway in that it can lead to ubiquitination of the resulting Arg-conjugated proteins. We have previously identified the mammalian Ate1 gene encoding Arg-transferases responsible for all known protein arginylation activities and have shown that Ate1-/- embryos die owing to various cardiovascular defects including ventricular hypoplasia, ventricular septal defect, and late angiogenesis. These results suggest that Ate1-dependent proteolysis of unknown substrate(s) is a crucial regulatory mechanism for myocardial growth and blood vessel integrity/maturation. However, the exact nature of the cardiovascular defects and the underlying molecular mechanisms remain elusive. Genomewide functional proteomic approach led us to identify a set of cardiovascular regulators (Rgs4, Rgs5, and Rgs16) as substrates of Ate1-dependent arginylation that may underlie, at least partially, Ate1-dependent cardiovascular homeostasis. Notably, Rgs4 and Rgs5 are GTPase-activating proteins (GAP) that act as negative regulators of GPCR-coupled Ga subunits and have been implicated as important regulators of Gq/Gi-activated signaling for myocardial growth and vascular maturation/integrity, respectively. Biochemical analyses showed that degradation of these substrates depends on the Cys2 residue as a degradation determinant, which is exposed to the N-terminus through cleavage of N-terminal Met by Met aminopeptidases. In the presence of sufficient oxygen (O2) and nitric oxide (NO), N-terminal Cys2 appears to be oxidized to CysO2 to create a structural homolog of Asp, an arginylation-permissive residue. The N-terminal Arg residue of arginylated RGS proteins is subsequently bound by specific E3 ligases whose identities remain unclear. Using an affinity-based proteomic approach, we isolated a set of E3 family (named Ubr1 through Ubr7) and demonstrated that Ubr1, Ubr2, and Ubr4 are the major E3s specific for protein arginylation and that Ubr1-/-Ubr2-/- and Ubr4-/- embryos die of cardiovascular defects. Based on these results, we hypothesize that the functions of Rgs4, Rgs5, and Rgs16 are modulated through the MetAPs-O2/NO-Ate1-Ubr proteolytic cascade. In Aim 1, we will characterize the physiological function of Ate1-dependent arginylation in cardiovascular development and signaling using tissue-specific Ate1 knockout mice in combination with transgenic mice overexpressing Gq in the heart. In Aim 2, to understand the molecular principles underlying Ate1-dependent cardiovascular development, we will characterize arginylation-dependent turnover and cotranslational modifications of Cys2 of Rgs4 and Rgs5. In Aim 3, as part of our long-term efforts to characterize ubiquitin ligases specific of arginylated substrates, we will characterize cardiovascular development of mice lacking Ubr4, a newly identified recognition component downstream of protein arginylation. 1