G protein regulation by ubiquitination

描述（由申请人提供）：异源三聚体G蛋白对于将细胞外信号转导为细胞内反应至关重要，因此对于多细胞生物体的存活至关重要。大多数神经递质和激素与细胞表面受体结合，并激活G蛋白，引起细胞反应。作为细胞表面受体和细胞内效应物之间的介体，G蛋白被很好地定位为翻译后调节的靶点。新出现的证据表明，G蛋白的调节monoubiquitination，这有一个很好的建立在膜蛋白的内化，运输和降解的作用。已经发现泛素连接酶（Rsp 5，人Nedd 4的直系同源物）对于体内和体外G-α亚基（Gpa 1，人G-α-i的直系同源物）的单泛素化是必要的和足够的。该提案的直接目标是确定Rsp 5催化的G蛋白单泛素化是如何调节的，以及它如何影响G蛋白活化和信号传导的基本特性。长期的职业目标是成为G蛋白研究领域的主要研究者，重点是了解G蛋白通过翻译后修饰调控的机制及其参与G蛋白信号转导。在目标1（K99期）中，将研究通过异源三聚体G蛋白组装对G-α单泛素化的调节。假设Gpa 1单泛素化和异源三聚体复合物组装是相互排斥的。该假设预测G-β/γ结合抑制Rsp 5引起的Gpa 1单泛素化，并且推测Gpa 1单泛素化抑制其与G-β/γ的再结合。在目标2（K99和R 00阶段）中，将确定磷酸化对G-α单泛素化的作用。gpa 1在泛素化位点周围的多个位置被多个激酶磷酸化。假设磷酸化是Gpa 1单泛素化所必需的。在目标3（R 00阶段）中，将研究通过去泛素化对G-α的调节。已显示内化蛋白的去泛素化和再循环回到质膜可使G蛋白信号传导途径重新敏感。泛素蛋白酶Ubp 12被认为是一种特异的Gpa 1去泛素化酶。假设Ubp 12去泛素化Gpa 1并促进其再循环。在该奖项的K99阶段，重要的培训将通过查佩尔山的北卡罗来纳州大学结构生物学中心获得应用生物物理蛋白质分析（包括结构，功能和蛋白质结合测定）。通过课程，将获得药理学基本原理的额外培训。该研究将在生物化学和药理学系进行，该系拥有非常强大的研究基础设施，包括世界知名的G蛋白研究教师，优秀的研究核心设施和上级教育机会。能够在体外和体内研究G蛋白泛素化是酵母实验系统的独特优势。这不仅允许深入研究G蛋白泛素化的调控机制，而且还可以深入研究泛素化对G蛋白结构，功能和蛋白质相互作用的基本性质的影响。重要的是，这里研究的酵母系统和人类之间存在许多强烈的相似之处。因此，如果这些研究结果将对人类G蛋白的研究产生广泛的影响。 公共卫生相关性：G蛋白是重要的分子，使细胞能够感知和响应其环境。糖尿病、失明和抑郁症等疾病是由于G蛋白信号传导功能障碍而表现出来的。这项研究解决了G蛋白如何调节的新发现。

DESCRIPTION (provided by applicant): Heterotrimeric G proteins are essential for transduction of extracellular signals into intracellular responses and therefore critical for the survival of multi-cellular organisms. Most neurotransmitters and hormones bind to cell surface receptors and activate G proteins that elicit cellular responses. As mediators between cell surface receptors and intracellular effectors, G proteins are well positioned to serve as targets of post-translational regulation. Emerging evidence indicates that G proteins are regulated by monoubiquitination, which has a well established role in membrane-protein internalization, trafficking and degradation. A ubiquitin ligase (Rsp5, ortholog of human Nedd4) has been discovered to be necessary and sufficient for monoubiquitination of the G-alpha subunit (Gpa1, ortholog of human G-alpha-i) in vivo and in vitro. The immediate objective of this proposal is to determine how Rsp5-catalyzed G protein monoubiquitination is regulated and how it affects fundamental properties of G protein activation and signaling. The long-term career goal is to become a principal investigator in the G protein research field with a focus on understanding the mechanisms of G protein regulation by post-translational modifications and their involvement in G protein signal transduction. In Aim 1 (K99 phase), the regulation of G-alpha monoubiquitination by heterotrimeric G protein assembly will be investigated. The hypothesis is that Gpa1 monoubiquitination and heterotrimeric complex assembly are mutually exclusive. This hypothesis predicts that G-beta/gamma binding inhibits Gpa1 monoubiquitination by Rsp5, and reciprocally, that Gpa1 monoubiquitination inhibits its re-association with G- beta/gamma. In Aim 2 (K99 and R00 phases), the role of phosphorylation on G-alpha monoubiquitination will be determined. Gpa1 is phosphorylated by multiple kinases at multiple positions surrounding the site of ubiquitination. The hypothesis is that phosphorylation is required for Gpa1 monoubiquitination. In Aim 3 (R00 phase), the regulation of G-alpha by de-ubiquitination will be investigated. De-ubiquitination of internalized proteins and recycling back to the plasma membrane has been shown to re-sensitize G protein signaling pathways. The ubiquitin protease Ubp12 has been implicated as a specific Gpa1 de-ubiquitinase. The hypothesis is that Ubp12 de-ubiquitinates Gpa1 and promotes its recycling. During the K99 phase of the award, significant training will be acquired in applied biophysical protein analysis (including structural, functional, and protein binding assays) through the Center for Structural Biology at the University of North Carolina at Chapel Hill (UNC). Through coursework, additional training will be acquired in fundamental principles of pharmacology. The research will be conducted in the departments of Biochemistry and Pharmacology at UNC, which has an exceptionally strong research infrastructure including world renowned G protein research faculty, outstanding research core facilities and superior educational opportunities. The ability to study G protein ubiquitination in vitro as well as in vivo is a unique advantage of the yeast experimental system. Not only does this allow in-depth research into the regulation mechanisms underlying G protein ubiquitination, but also into the effects of ubiquitination on the fundamental properties of G protein structure, function and protein interactions. Importantly, many strong parallels exist between the yeast system being studied here and humans. Therefore, the results if these studies will have a broad impact on human G protein research. PUBLIC HEALTH RELEVANCE: G proteins are important molecules that allow cells to sense and respond to their environment. Diseases such as diabetes, blindness, and depression, among others, manifest due to malfunctions of G protein signaling. This research addresses new discoveries into how G proteins are regulated.