Role of Ubiquitin in Cardiovascular System

描述（由申请人提供）：N-末端规则途径是一种泛素蛋白水解途径，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-tRNAArg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，因为它可以导致所得Arg缀合蛋白的泛素化。我们以前已经确定了哺乳动物Ate 1基因编码的精氨酸转移酶负责所有已知的蛋白质腺苷酸化活动，并已表明，Ate 1-/-胚胎死亡，由于各种心血管缺陷，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，Ate 1依赖的未知底物的蛋白水解是心肌生长和血管完整性/成熟的重要调控机制。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（Rgs 4，Rgs 5和Rgs 16）作为Ate 1依赖性腺苷酸化的底物，这可能是Ate 1依赖性心血管稳态的至少部分基础。值得注意的是，Rgs 4和Rgs 5是GTP酶激活蛋白（GAP），其充当GPCR偶联的Ga亚基的负调节剂，并且已经被认为分别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调节剂。生化分析表明，这些底物的降解依赖于Cys 2残基作为降解决定簇，其通过由Met氨基肽酶切割N-末端Met而暴露于N-末端。在足够的氧气（O2）和一氧化氮（NO）的存在下，N-末端Cys 2似乎被氧化为CysO 2，以创建一个结构同源物的天冬氨酸，一个允许的残基。N-末端精氨酸残基的乙酰化RGS蛋白随后结合的特定E3连接酶的身份仍然不清楚。利用基于亲和性的蛋白质组学方法，我们分离了一组E3家族（命名为Ubr 1至Ubr 7），并证明Ubr 1，Ubr 2和Ubr 4是蛋白质乙酰化的主要E3，Ubr 1-/-Ubr 2-/-和Ubr 4-/-胚胎死于心血管缺陷。基于这些结果，我们假设Rgs 4、Rgs 5和Rgs 16的功能通过MetAPs-O2/NO-Ate 1-Ubr蛋白水解级联反应来调节。在目标1中，我们将使用组织特异性Ate 1基因敲除小鼠与心脏中过表达Gq的转基因小鼠组合来表征Ate 1依赖性腺苷酸化在心血管发育和信号传导中的生理功能。在目标2中，为了了解Ate 1依赖性心血管发展的分子原理，我们将表征Rgs 4和Rgs 5的Cys 2的磷酸化依赖性周转和共翻译修饰。在目标3中，作为我们长期努力的一部分，以表征泛素连接酶特异性的β-淀粉酰化底物，我们将表征心血管发育的小鼠缺乏Ubr 4，一个新发现的识别组件下游的蛋白β-淀粉酰化。1 公共卫生相关性：心血管过程涉及许多蛋白质，这些蛋白质的功能可以通过与不用作蛋白质结构单元的化合物（例如，磷酸盐和GTP）。我们以前已经证明，精氨酸，一个主要的氨基酸，蛋白质N-末端的翻译后共轭在心脏发育和血管生成中起着重要的作用。在一项独立的研究中，我们已经证明，Ate 1编码的R-转移酶介导的蛋白酶体降解的一组心血管调节剂（Rgs 4，Rgs 5和Rgs 16）通过共轭精氨酸的N-末端Cys 2残基作为一个关键的降解决定因素。因此，Rgs蛋白的乙酰化是控制心脏和血管中Rgs偶联的G蛋白信号传导的潜在药物靶标。有趣的是，我们最近的研究结果表明，Cys 2依赖的蛋白酶体降解RGS蛋白不仅需要乙酰化，但也氧化，后者是另一个独特的修饰作为不可逆的蛋白酶体降解前的许可步骤。Rgs蛋白的Cys 2残基的氧化反过来又受到以下因素的严格控制： 分子如O2的生物利用度。哺乳动物的心脏消耗的氧气是大脑的3-20倍，因此需要持续供应氧气来维持其功能。在心脏病、癌症、脑血管疾病和慢性阻塞性肺病中，O2稳态被破坏，这些疾病是最常见的死亡原因，占美国所有死亡人数的三分之二。关于其在心血管信号传导中的作用以及心脏感知其浓度以调节细胞内过程的机制知之甚少。这项研究的成功结果可能提供一种以前未知的机制，传感O2和相关分子，以响应循环血液环境的变化。最后，我们的研究结果表明，一组E3家族（命名为Ubr 1，Ubr 2，和Ubr 4），通过结构保守的锌指结构域，称为Ubr盒识别N-末端精氨酸调控的Rgs蛋白的营业额。这些Ubr E3连接酶的表征不仅将确定的乙酰化依赖性蛋白水解的生理功能，但也可能揭示一个新的治疗目标，以控制心血管过程中的病理生理条件。

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.