ATP-Ubiquitin Dependent Proteolysis

ATP-泛素依赖性蛋白水解

• 批准号: 7372097
• 负责人: ARTHUR L HAAS
• 金额: $ 32.38万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7372097
• 关键词: 26S proteasome; Active Sites; Address; Adverse effects; Affinity; Binding; Binding Proteins; Biochemical Genetics; Breast; Class; Disease; Enzymatic Biochemistry; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Family; Funding; G22P1 gene; Genetic; Goals; Half-Life; Immunosuppressive Agents; In Vitro; Inherited; Interferons; Kinetics; Ligase; Ligation; Malignant neoplasm of prostate; Maps; Mediating; Messenger RNA; Methods; Mitotic; Modeling; Modification; Organism; Pathway interactions; Peptides; Polyubiquitin; Positioning Attribute; Post-Translational Protein Processing; Property; Protein Isoforms; Proteins; Proteolysis; Reaction; Regulation; Research Design; Resolution; Role; Signal Transduction; Sirolimus; Site; Specificity; Structure; Testing; Ubiquitin; Ubiquitin Like Proteins; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitination; Viral; Work; XRCC5 gene; adenylate; base; cohort; cytokine; design; genetic manipulation; grasp; human SFN protein; inhibitor/antagonist; neoplastic; novel; paralogous gene; research study; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The ubiquitin/26S proteasome pathway is a fundamental strategy for eukaryotic cell regulation in which proteins are targeted for destruction by assembly on them of specific polyubiquitin degradation signals that are recognized by the 19S regulatory complex of the 26S proteasome. A super family of ubiquitin-like proteins that undergo parallel but distinct conjugation reactions have co-evolved to serve regulatory functions distinct from targeted degradation. The functional specificities of these post-translational modifications require precisely directed protein interactions among components of the ligation machinery. During the current funding period conventional biochemical and genetic approaches have been exploited in part to define the protein interactions at high resolution. Studies proposed for the next funding period comprise of three Specific Aims designed to build upon these advances. Specific Aim 1 will use biochemical and genetic approaches to study E1-catalyzed activation of Class 1 ubiquitin-like proteins and will examine the role of the carboxyl terminal 2-grasp domain as a specificity filter for cognate E2/Ubc recognition, the role of 2-grasp domain versus Ubc12 amino-terminal peptide binding in transition state stabilization during Nedd8 activation, and characterize selected E1 residues in binding versus catalytic roles. Specific Aim 2 will examine the mechanism of a novel subset of Bi-functional Ligases capable of modifying their cognate protein substrates with either ubiquitin to target degradation or the interferon- induced ISG15ubiquitin-like protein to block degradation. Kinetic studies will quantify the ability of UbcH7 (ubiquitin specific) and UbcH8 (ISG15 specific) to support Epf/Trim25 ligase in regulating levels of the anti-mitotic 14-3-3C protein implicated in breast and prostate cancers. Other studies will examine the consequences of ubiquitin versus ISG15 modification on the half life of 14-3-3C. Specific Aim 3 will continue studies on the enzymology of Mdm2 and MdmX homo- and heterodimers as ubiquitin ligases. Kinetic studies will examine the mechanism of MdmX-stimulated Mdm2-dependent ubiquitination, test a two-site Proximal Indexation model for polyubiquitin chain formation by Mdm2, and examine the in vitro conjugating activity of MdmX with Ku70 and Ku80, bona fide substrates of this ligase. These studies are designed to extend our understanding of the mechanism of ubiquitin-dependent targeting for 26S proteasome-mediated degradation and to characterize a new regulatory strategy involving a small subset of Bi-functional Ligases that can provide a functional context for the evolution of the ISG15 ligation pathway in higher eukaryotes. The latter goal is anticipated to define the mechanism for a previously unknown role for interferon-dependent regulation.Modification of cellular proteins with ubiquitin and related ubiquitin-like proteins constitutes a fundamental regulatory strategy for organisms, derangement of which is implicated in a large cohort of inherited and acquired diseases. This funding supports work to understand the underlying principles of this regulatory mechanism and seeks to explicate one pathway by which interferon exerts its anti-viral and immunomodulatory effects that can be exploited therapeutically without the debilitating side effects of the cytokine.

描述（由申请人提供）：泛素/26S蛋白酶体途径是真核细胞调节的基本策略，其中通过在蛋白质上组装由26S蛋白酶体的19S调节复合物识别的特定多泛素降解信号来靶向破坏蛋白质。经历平行但不同的缀合反应的泛素样蛋白超级家族共同进化，以提供与靶向降解不同的调节功能。这些翻译后修饰的功能特异性需要连接机器组件之间精确定向的蛋白质相互作用。在当前的资助期间，传统的生化和遗传学方法已部分用于以高分辨率定义蛋白质相互作用。为下一个资助期提出的研究包括旨在以这些进展为基础的三个具体目标。具体目标 1 将使用生化和遗传学方法来研究 E1 催化的 1 类泛素样蛋白的激活，并将检查羧基末端 2-grasp 结构域作为同源 E2/Ubc 识别的特异性过滤器的作用、2-grasp 结构域与 Ubc12 氨基末端肽结合在 Nedd8 激活过程中过渡态稳定中的作用，并表征选定的 E1 残基的结合作用与催化作用。具体目标 2 将检查双功能连接酶的新子集的机制，该连接酶能够用泛素修饰其同源蛋白底物以靶向降解，或用干扰素诱导的 ISG15 泛素样蛋白修饰其同源蛋白底物以阻止降解。动力学研究将量化 UbcH7（泛素特异性）和 UbcH8（ISG15 特异性）支持 Epf/Trim25 连接酶调节乳腺癌和前列腺癌中涉及的抗有丝分裂 14-3-3C 蛋白水平的能力。其他研究将检验泛素与 ISG15 修饰对 14-3-3C 半衰期的影响。具体目标 3 将继续研究 Mdm2 和 MdmX 同二聚体和异二聚体作为泛素连接酶的酶学。动力学研究将检查 MdmX 刺激的 Mdm2 依赖性泛素化机制，测试 Mdm2 多聚泛素链形成的两位点近端标记模型，并检查 MdmX 与该连接酶的真正底物 Ku70 和 Ku80 的体外缀合活性。这些研究旨在扩展我们对泛素依赖性靶向 26S 蛋白酶体介导的降解机制的理解，并表征涉及一小部分双功能连接酶的新调控策略，该策略可以为高等真核生物中 ISG15 连接途径的进化提供功能背景。预计后一个目标将定义干扰素依赖性调节的先前未知作用的机制。用泛素和相关泛素样蛋白修饰细胞蛋白构成了生物体的基本调节策略，其紊乱与大量遗传性和获得性疾病有关。这笔资金支持了解这种调节机制的基本原理，并试图阐明干扰素发挥其抗病毒和免疫调节作用的一种途径，该途径可以在治疗上利用，而不会产生细胞因子的衰弱副作用。