G protein regulation by ubiquitination

摘要：异三聚体G蛋白是细胞外信号进入细胞内所必需的 因此，它对多细胞生物体的生存至关重要。大多数神经递质和 荷尔蒙与细胞表面受体结合，激活G蛋白，从而引发细胞反应。作为调解人 在细胞表面受体和细胞内效应器之间，G蛋白被很好地定位为靶点 翻译后监管。新出现的证据表明，G蛋白受单泛素化调节， 它在膜蛋白的内化、运输和降解过程中发挥着重要的作用。无处不在的 连接酶(Rsp5，人类Nedd4的同源基因)已被发现是连接的必要条件和充分条件 体内和体外G-α亚基(Gpa1，人G-α-I的同源基因)的单素化。这个 这项提议的直接目标是确定Rsp5催化的G蛋白单偶联素化是如何 以及它如何影响G蛋白激活和信号传递的基本特性。长期的职业生涯 目标是成为G蛋白研究领域的首席研究员，专注于了解 翻译后修饰调节G蛋白的机制及其在G蛋白中的作用 信号转导。在目标1(K99相)中，杂三聚体G对G-α单素化的调节 将对蛋白质组装进行研究。假说是Gpa1单素化和异三聚体 复杂的装配是相互排斥的。这一假说预测G-β/伽马结合抑制Gpa1 Rsp5的单泛素化，反过来，Gpa1单泛素化抑制其与G- 贝塔/伽马。在目标2(K99和R00阶段)，磷酸化在G-α单泛素化中的作用将 要下定决心。Gpa1被多个位置的多个激酶磷酸化 泛素化。该假说认为Gpa1单泛素化需要磷酸化。在AIM 3(R00 阶段)，将研究去泛素化对G-α的调节。内在化的去泛素化 蛋白质和再循环回到质膜被证明可以重新敏化G蛋白信号 小路。泛素酶Ubp12被认为是一种特异性的Gpa1脱泛素酶。这个 假设是Ubp12使Gpa1去泛素化并促进其循环。在K99阶段的 获奖后，将获得应用生物物理蛋白质分析方面的重要培训(包括结构、 功能和蛋白质结合分析)，通过北方大学结构生物学中心 北卡罗来纳州教堂山(北卡罗来纳州)。通过课程作业，将获得基础课程的额外培训 药理学原理。这项研究将在生物化学系和 北卡罗来纳大学的药理学，它拥有非常强大的研究基础设施，包括世界著名的G 蛋白质研究人员，一流的研究核心设施和优越的教育机会。一种能力 研究G蛋白的体外泛素化以及体内泛素化是酵母实验的独特优势 系统。这不仅使深入研究G蛋白的调节机制成为可能 泛素化，还要研究泛素化对G蛋白结构基本性质的影响， 功能和蛋白质的相互作用。重要的是，酵母系统之间存在许多强烈的相似之处 在这里学习过，还有人类。因此，如果这些研究的结果将对人类G蛋白产生广泛的影响 研究。

Abstract: 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.