Functional Anatomy of the Cul3 Ubiquitin Ligase

描述(申请人提供)：SCF泛素连接酶途径利用cullin蛋白组装底物特异性模块和泛素结合酶，从而促进底物泛素化。我们最近的工作揭示了cullin-3(CUL3)以类似于Skp1/F-box复合体的方式结合BTB结构域蛋白大家族，Skp1/F-box复合体与cull结合。我们假设BTB蛋白作为底物特异的CUL3适配子发挥作用。这一假说的证据来自我们的发现，线虫BTB蛋白Mel-26通过其数学结构域与其基因定义的靶标Mei-1相互作用。伴随着减数分裂退出的Mei-1蛋白的消除依赖于Mel-26、Cul-3和cullin激活子Nedd8，这表明CUL3/Mel-26复合体在这一细胞周期转变中作为泛素连接酶促进Mei-1的破坏。此外，我们最近发现，Keap1通过其BTB结构域与CUL3相互作用，并通过其kelch结构域与转录因子Nrf2相互作用，在体内降解Nrf2和在体外泛化Nrf2是必需的。在这里，我们试图进一步确定BTB蛋白在靶向泛素化中的作用。在目标1中，我们将阐明两种基于CUL3的泛素连接酶Mel-26/CUL3和Keap1/CUL3对靶泛素化的生化要求，并将研究BTB-蛋白二聚在体外泛素化活性和体内蛋白质转换中的作用。在目标2中，我们将通过结晶学和生化研究来研究CuL-3/MEL-26/MEI-1络合物的结构，从而扩展我们对E3的结构分析。在目标3中，我们将利用新开发的针对BTB蛋白的RNAi文库来确定CUL3/BTB复合体在细胞分裂和单体Cyclin E周转中的作用。此外，将在BTB/CUL3途径的背景下开发利用功能丧失遗传学识别泛素化靶标并适用于全基因组筛选的新方法。这些实验将：证实和推广BTB蛋白作为CUL3底物特异性适配子的假设，揭示CuL-3和Mel-26 BTB蛋白识别底物的结构基础，并将功能基因组方法的力量应用于人类细胞中泛素连接酶的功能和靶标识别问题。

DESCRIPTION (provided by applicant): The SCF ubiquitin ligase pathway employs cullin proteins to assemble substrate specificity modules and ubiquitin conjugating enzymes, thereby promoting substrate ubiquitination. Our recent work has revealed that cullin-3 (Cul3) binds a large family of BTB-domain proteins in a manner analogous to the Skpl/F-box complex, which binds Cull. We hypothesize that BTB proteins function as substrate specific adaptors for Cul3. Evidence for this hypothesis comes from our finding that the C. elegans BTB protein Mel-26 interacts with its genetically defined target Mei-1 through its MATH domain. Elimination of Mei-1 protein that accompanies exit from meiosis depends upon Mel-26, Cul-3, and the cullin activator Nedd8, suggesting that the Cul3/Mel-26 complex functions as a ubiquitin ligase to facilitate Mei-1 destruction during this cell cycle transition. In addition, we have recently found that Keap1, which interacts through its BTB domain with Cul3 and through its kelch domain with the transcription factor Nrf2, is required for degradation of Nrf2 in vivo and ubiqutination of Nrf2 in vitro. Here, we seek to further establish the role of BTB proteins in targeted ubiquitintation. In Aim 1, we will elucidate the biochemical requirements for target ubiquitination by two Cul3-based ubiquitin ligases, Mel-26/Cul3 and Keap1/Cul3, and will examine the role of BTB-protein dimerization in ubiquitination activity in vitro and protein turnover in vivo. In aim 2, we will extend our structural analysis of E3s by examining the architecture of the Cul-3/Mel-26/Mei-1 complex through crystallographic and biochemical studies. In aim 3, we will employ newly developed RNAi libraries against BTB proteins to define the role of Cul3/BTB complexes in cell division and monomeric cyclin E turnover. In addition, novel approaches for the identification of ubiquitination targets employing loss-of-function genetics and applicable to genome-wide screens will be developed in the context of the BTB/Cul3 pathway. These experiments will: confirm and extend the hypothesis that BTB proteins function as substrate specific adaptors of Cul3, will reveal the structural basis for Cul-3 and substrate recognition by the Mel-26 BTB protein, and will apply the power of functional genomic approaches to the problem of ubiquitin ligase function and target identification in human cells.