G protein regulation by ubiquitination

描述(由申请人提供)：异源三聚体G蛋白是将细胞外信号转化为细胞内反应所必需的，因此对多细胞生物体的生存至关重要。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，从而引发细胞反应。作为细胞表面受体和细胞内效应分子之间的中介，G蛋白被认为是翻译后调控的靶点。新的证据表明，G蛋白受单泛素化的调节，它在膜蛋白的内化、运输和降解中具有广泛的作用。泛素连接酶(Rsp5，人Nedd4的同源基因)在体内和体外被发现是G-α亚基(Gpa1，人G-α-I的同源基因)单素化的必要条件和充分条件。这项建议的直接目标是确定Rsp5催化的G蛋白单泛素化是如何调节的，以及它如何影响G蛋白激活和信号传递的基本性质。长期的职业目标是成为G蛋白研究领域的首席研究员，专注于了解G蛋白翻译后修饰的调节机制及其参与G蛋白信号转导。在目标1(K99阶段)，将研究异源三聚体G蛋白组装对G-α单泛素化的调节。假设Gpa1单素化和杂三聚体组装是相互排斥的。这一假说预测，G-β/伽马结合抑制Rsp5对Gpa1的单泛素化，反过来，Gpa1单泛素化抑制其与G-β/伽马的重新结合。在目标2(K99和R00阶段)，将确定磷酸化在G-α单泛素化中的作用。Gpa1被泛素化位点周围多个位置的多个激酶磷酸化。该假说认为Gpa1单泛素化需要磷酸化。在目标3(R00阶段)，将研究去泛素化对G-α的调节。内化蛋白的去泛素化和再循环回到质膜可以使G蛋白信号通路重新变得敏感。泛素酶Ubp12被认为是一种特异性的Gpa1脱泛素酶。假设是Ubp12使Gpa1去泛素化并促进其循环。在K99奖项阶段，将通过北卡罗来纳大学教堂山分校(UNC)结构生物学中心在应用生物物理蛋白质分析(包括结构、功能和蛋白质结合分析)方面获得重要培训。通过课程作业，将获得药理学基本原理方面的额外培训。这项研究将在北卡罗来纳大学的生物化学和药理学系进行。北卡罗来纳大学拥有极其强大的研究基础设施，包括世界知名的G蛋白质研究人员、出色的研究核心设施和优越的教育机会。在体外和体内研究G蛋白泛素化的能力是酵母实验系统的独特优势。这不仅可以深入研究G蛋白泛素化的调控机制，而且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，在这里研究的酵母系统与人类之间有许多强烈的相似之处。因此，这些研究的结果将对人类G蛋白的研究产生广泛的影响。 与公共健康相关：G蛋白是重要的分子，使细胞能够感知环境并对其做出反应。糖尿病、失明和抑郁症等疾病都是由G蛋白信号失灵引起的。这项研究解决了G蛋白是如何调节的新发现。

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.