Role of Ubiquitin in Cardiovascular System

描述(申请人提供)：N-末端规则途径是一种泛素蛋白分解途径，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-tRNAArg到N-末端天冬氨酸(Asp)、谷氨酸(Glu)或半胱氨酸(Cys)的结合是这种蛋白分解途径的一部分，因为它可以导致Arg结合蛋白的泛素化。我们以前已经鉴定了编码精氨酸转移酶的哺乳动物ATE1基因，该基因负责所有已知的蛋白质精氨酸化活性，并表明ATE1-/-胚胎死于各种心血管缺陷，包括室发育不全、室间隔缺陷和晚期血管生成。这些结果表明，未知底物(S)的ATE1依赖的蛋白分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不清楚。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依赖的精氨酸化的底物，该底物可能至少部分地支持ATE1依赖的心血管动态平衡。值得注意的是，RGS4和RGS5是GTP酶激活蛋白(GAP)，它们作为GPCR偶联的GA亚基的负调节因子，分别被认为是GQ/GI激活的心肌生长和血管成熟/完整性信号的重要调节因子。生化分析表明，这些底物的降解依赖于作为降解决定因素的Cys2残基，该残基通过Met氨基肽酶对N-末端Met的切割而暴露在N端。在充足的氧气(O2)和一氧化氮(NO)存在下，N端的Cys2似乎被氧化成CysO2，从而创建了Asp的结构同系物，Asp是一种允许精氨酸化的残基。精氨酸化的RGS蛋白的N-末端Arg残基随后被特定的E3连接酶结合，这些连接酶的身份尚不清楚。利用基于亲和力的蛋白质组学方法，我们分离了一组E3家族(命名为Ubr1到Ubr7)，并证明了Ubr1、Ubr2和Ubr4是蛋白质精氨酸化的主要E3，Ubr1-/-Ubr2-/-和Ubr4-/-胚胎死于心血管缺陷。根据这些结果，我们假设RGS4、RGS5和RGS16的功能是通过MetAPs-O2/NO-ATE1-UBR蛋白水解级联来调节的。在目标1中，我们将利用组织特异性的Ate1基因敲除小鼠与心脏中高表达GQ的转基因小鼠相结合，表征依赖于Ate1的精氨酸化在心血管发育和信号转导中的生理功能。在目标2中，为了了解ATE1依赖的心血管发育的分子机制，我们将表征RGS4和RGS5的Cys2的精氨酸化依赖的周转和共翻译修饰。在目标3中，作为我们长期努力的一部分，我们将表征精氨酸化底物的特定泛素连接酶，我们将表征缺乏Ubr4的小鼠的心血管发育，Ubr4是一种新发现的蛋白质精氨酸化下游识别成分。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