E2 selectivity and activation by CHIP

描述(申请人提供)：泛素化是多种细胞信号的一种常见途径，包括通过蛋白酶体中的降解实现蛋白质周转。信号系统的缺陷直接与一系列疾病有关，包括帕金森氏症等神经退行性疾病，以及包括乳腺癌和卵巢癌在内的一系列人类癌症。泛素化过程涉及一系列酶来激活泛素(Ub)并将其转移到底物上。在最后一步中，E3泛素连接酶作用于底物和泛素结合的E2。最近的数据支持这一理论，即在大多数情况下，特定的E2/E3对将使底物单泛素化，或使先前泛素化的底物多泛素化。CHIP(Hsc70相互作用蛋白的羧基末端)是一个含有E3泛素连接酶的U-box，它与伴侣蛋白相互作用，参与细胞对应激的反应。用于研究特定E3的E2结合伙伴的酵母-双杂交筛选已经建立，并将用于研究与CHIP的相互作用(AIM 1)。然后将使用核磁共振来确认和绘制相互作用图，并将通过体外泛素化实验来测试功能。目标2着重于泛素结合的E2被激活的机制 通过与E3连接酶相互作用进行泛素转移。功能分析支持一种机制，即E2~Ub需要与E3结合才能在泛素化转移中发挥作用；然而，目前还没有关于任何E2~Ub/E3复合体的结构信息。活性位点突变体E2将被用来稳定Ub带电形式，在三元络合物的分析中将进行三种不同的滴定：在每一次实验中都将对一种蛋白质进行同位素标记。了解E2/E3复合体的功能将极大地促进对参与特定信号通路的E2和E3蛋白的分配。这些研究将有助于确定CHIP与E2机制在E2特异性方面的相互作用，以及参与刺激泛素转移的机制。公共卫生报告：泛素化是一种常见的细胞信号通路，与一系列疾病直接相关。这项确定泛素化机制组件之间特定功能相互作用的工作是监测和治疗导致疾病的系统故障的必要步骤。

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.