ATP-Ubiquitin Dependent Proteolysis

描述(由申请人提供)：泛素/26S蛋白酶体途径是真核细胞调控的基本策略，在该途径中，蛋白质通过组装由26S蛋白酶体的19S调控复合体识别的特定的多泛素降解信号而被破坏。经过平行但不同的结合反应的泛素样蛋白的超级家族已经共同进化，以服务于不同于靶向降解的调节功能。这些翻译后修饰的功能特异性需要在连接机制的组件之间精确定向的蛋白质相互作用。在目前的供资期间，已利用常规的生化和遗传方法部分地以高分辨率确定蛋白质相互作用。为下一个供资阶段提出的研究包括三个具体目标，旨在以这些进展为基础。具体目标1将使用生化和遗传学方法研究E1催化的1类泛素样蛋白的激活，并将研究羧基末端2-GRAP结构域作为同源E2/UBC识别的特异性过滤器的作用，2-GRAP结构域与Ubc12氨基末端肽结合在Nedd8激活期间在过渡态稳定中的作用，并表征选定的E1残基在结合与催化作用中的作用。具体目标2将研究一组新的双功能连接酶的机制，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泛素样蛋白来阻止降解。动力学研究将量化UbcH7(泛素特异性)和UbcH8(ISG15特异性)在调节乳腺癌和前列腺癌中涉及的抗有丝分裂14-3-3C蛋白水平方面支持EPF/Trim25连接酶的能力。其他研究将检验泛素和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作为泛素连接酶的同源和异源二聚体的酶学。动力学研究将探讨MDMX刺激的MDM2依赖的泛素化的机制，测试MDM2形成多泛素链的两位点近邻指标化模型，并检测MDMX与该连接酶的真正底物Ku70和Ku80的体外结合活性。这些研究旨在扩大我们对26S蛋白酶体介导的降解的泛素依赖靶向机制的理解，并表征一种新的调控策略，该策略涉及一小部分双功能连接酶，可以为高等真核生物中ISG15连接途径的进化提供功能背景。后者的目标是确定干扰素依赖调节以前未知的作用的机制。用泛素和相关的泛素样蛋白修饰细胞蛋白质构成生物体的基本调节策略，其紊乱与大量的遗传性和获得性疾病有关。这笔资金支持理解这一调节机制的基本原理的工作，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，该途径可以在不产生细胞因子的衰弱副作用的情况下用于治疗。

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.