E2 selectivity and activation by CHIP

描述（由申请人提供）：泛素化是多种细胞信号的常见途径，包括通过蛋白酶体降解的蛋白质周转。信号系统的缺陷直接涉及一系列疾病，包括神经退行性疾病，如帕金森氏症，以及一系列人类癌症，包括乳腺癌和卵巢癌。泛素化过程涉及一系列酶来激活并将泛素（Ub）转移到底物上。在最后一步中，E3泛素连接酶的作用是定位底物和泛素缀合的E2。最近的数据支持的理论，在大多数情况下，一个特定的E2/E3对将monoubiquitinate底物，或polyubiquitinate以前的泛素化底物。CHIP（Hsc 70相互作用蛋白的羧基末端）是一种含有E3遍在蛋白连接酶的U盒，它与伴侣蛋白相互作用，并参与细胞对应激的反应。已经建立了用于研究特定E3的E2结合伴侣的酵母双杂交筛选，并将用于研究与CHIP的相互作用（目的1）。然后将使用NMR来确认和绘制相互作用，并通过体外泛素化测定来测试功能。目的2关注泛素结合的E2被激活的机制， 泛素通过与E3连接酶相互作用转移。功能分析支持这样一种机制，其中E2~Ub需要与E3结合才能在泛素化转移中发挥作用;然而，没有任何E2~Ub/E3复合物的结构信息。活性位点突变体E2将用于稳定不带电形式，并且在三元复合物的分析中将进行三个单独系列的滴定：在每个实验中将对一种蛋白质进行同位素标记。了解E2/E3复合物的功能将极大地促进分配参与特定信号通路的E2和E3蛋白的尝试。这些研究将有助于确定CHIP与E2机制的相互作用，包括E2特异性和刺激泛素转移的机制。公共卫生关系：泛素化是一种常见的细胞信号传导途径，与一系列疾病直接相关。这项工作，以确定特定的功能之间的相互作用的组件的泛素化机制是一个必要的步骤，在监测和治疗系统故障，导致疾病。

DESCRIPTION (provided by applicant): Ubiquitination is a common pathway for multiple cellular signals including protein turnover via degradation in the proteasome. Defects in the signaling system have been directly implicated in a range of diseases including neurodegenerative disorders such as Parkinson's, and a range of human cancers including breast and ovarian. The ubiquitination process involves a cascade of enzymes to activate and transfer ubiquitin (Ub) to a substrate. In the final step, the E3 ubiquitin ligase acts to localize both a substrate and ubiquitin-conjugated E2. Recent data support the theory that in most cases a specific E2/E3 pair will either monoubiquitinate a substrate, or polyubiquitinate a previously ubiquitinated substrate. CHIP (carboxylterminus of Hsc70 interacting protein) is a U-box containing E3 ubiquitin ligase that interacts with chaperone proteins and is involved in the cellular response to stress. A yeast-two hybrid screen for investigating E2 binding partners for a specific E3 has been established, and will be used to investigate interactions with CHIP (Aim 1). NMR will then be used to confirm and map the interactions, and function will be tested by in vitro ubiquitination assay. Aim 2 focuses on the mechanism whereby the ubiquitin-bound E2 is activated for ubiquitin transfer by interaction with an E3 ligase. Functional assays support a mechanism in which binding to an E3 is required for E2~Ub to function in ubiquitination transfer; however there is no structural information available for any E2~Ub/E3 complex. An active site mutant E2 will be used to stabilize the Ubcharged form, and three separate series of titrations will be performed in analysis of the ternary complex: one protein will be isotopically labeled in each experiment. Understanding the function of E2/E3 complexes will greatly facilitate attempts to assign E2 and E3 proteins involved in specific signaling pathways. These studies will assist to define the interactions of CHIP with E2 machinery both in E2 specificity and the mechanism involved in stimulation of ubiquitin transfer. PUBLIC HEALTH RLEVANCE: Ubiquitination is a common cellular signaling pathway which has been directly implicated in a range of diseases. This work to define specific functional interactions between components of the ubiquitination machinery is a necessary step in monitoring and treating system failures which lead to disease.