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 PUBLIC HEALTH RELEVANCE: Cardiovascular processes involve many proteins whose functions can be turned on and off by posttranslational conjugation with compounds that are not used as a building block in protein (e.g., phosphate and GTP). We have previously shown that posttranslational conjugation of arginine, one of principal amino acids, to the protein N-terminus plays an important role in cardiac development and angiogenesis. In an independent study, we have demonstrated that Ate1-encoded R-transferases mediate proteasomal degradation of a set of cardiovascular regulators (Rgs4, Rgs5, and Rgs16) via the conjugation of arginine to the N-terminal Cys2 residue as a critical degradation determinant. Therefore, the arginylation of the Rgs proteins is a potential drug target to control Rgs-coupled G- protein signaling in the heart and blood vessels. Intriguingly, our more recent results suggest that Cys2- dependent proteasomal degradation of Rgs proteins requires not only arginylation but also oxidation, the latter being yet another unique modification as a licensing step prior to irreversible proteasomal degradation. The oxidation of the Cys2 residue of the Rgs proteins is in turn tightly controlled by bioavailability of molecules such as O2. The mammalian heart consumes O2 3-20 times more than the brain, and thus requires a constant supply of O2 for its function. O2 homeostasis is disrupted in heart disease, cancer, cerebrovascular disease, and chronic obstructive pulmonary disease, which represent the most common causes of mortality and accounts for two-thirds of all deaths in the U.S. Although O2 is a major determinant of cardiac gene expression and numerous cellular processes, extremely little is known about its role in the cardiovascular signaling and the mechanism by which the heart senses its concentration to modulate intracellular processes. Successful results from this study may provide a previously unknown mechanism sensing O2 and related molecules in response to a changing environment of circulating blood. Finally, our results suggest that the turnover of arginylated Rgs proteins is regulated by a set of E3 family (named Ubr1, Ubr2, and Ubr4) that recognize the N-terminal Arg through a structurally conserved zinc-finger domain, termed the Ubr box. The characterization of these Ubr E3 ligases will not only identify the physiological function of the arginylation-dependent proteolysis but also may reveal a new therapeutic target to control pathophysiological conditions in cardiovascular processes.