ATP-Ubiquitin Dependent Proteolysis

描述（由申请人提供）：泛素/26 S蛋白酶体途径是真核细胞调控的基本策略，其中通过在蛋白质上组装特异性多聚泛素降解信号（由26 S蛋白酶体的19 S调控复合物识别），靶向蛋白质进行破坏。一个超家族的泛素样蛋白进行平行的，但不同的共轭反应已共同进化，以提供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连接机制的组分之间精确定向的蛋白质相互作用。在目前的资助期间，传统的生物化学和遗传学方法已被部分利用，以确定高分辨率的蛋白质相互作用。为下一个资助期提出的研究包括三个具体目标，旨在巩固这些进展。具体目标1将使用生物化学和遗传学方法来研究E1催化的1类泛素样蛋白的活化，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Ubc 12氨基末端肽结合在Nedd 8活化期间的过渡态稳定中的作用，并表征所选E1残基的结合作用与催化作用。具体目标2将研究一种新的双功能连接酶亚组的机制，该亚组能够用泛素修饰其同源蛋白底物以靶向降解或干扰素诱导的ISG 15泛素样蛋白以阻断降解。动力学研究将量化UbcH 7（泛素特异性）和UbcH 8（ISG 15特异性）支持Epf/Trim 25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。其他研究将检查泛素与ISG 15修饰对14-3-3C半衰期的影响。具体目标3将继续研究Mdm 2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Mdm 2依赖性泛素化的机制，测试Mdm 2形成聚泛素链的双位点近端索引模型，并检查MdmX与Ku 70和Ku 80（该连接酶的真正底物）的体外缀合活性。这些研究旨在扩展我们对26 S蛋白酶体介导的降解的泛素依赖性靶向机制的理解，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ISG 15连接途径的进化提供功能背景。后一个目标是预期定义一个以前未知的作用，干扰素依赖regulation.Modification的细胞蛋白与泛素和相关的泛素样蛋白构成了一个基本的监管策略的生物体，紊乱，这是牵连在一个大的队列的遗传性和获得性疾病的机制。该基金支持理解这种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，这种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。

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.