Functional Anatomy of the Cul3 Ubiquitin Ligase

Cul3 泛素连接酶的功能解剖

• 批准号: 6919560
• 负责人: JEFFREY W HARPER
• 金额: $ 30.51万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919560
• 关键词: RNA interference; SDS polyacrylamide gel electrophoresis; cell cycle; enzyme activity; functional /structural genomics; green fluorescent proteins; human tissue; ligase; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; ubiquitin

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.

描述（由申请人提供）：SCF泛素连接酶途径采用Cullin蛋白来组装底物特异性模块和泛素结合酶，从而促进底物泛素化。我们最近的工作表明，Cullin-3（CUL3）以类似于结合CULL的SKPL/F-box复合物的方式结合了大型BTB域蛋白。我们假设BTB蛋白充当CUL3的底物特定适配器。这一假设的证据来自我们的发现，即秀丽隐杆线虫BTB蛋白MEL-26通过其数学领域与其遗传定义的靶标MEI相互作用。消除从减数分裂退出的MEI-1蛋白取决于MEL-26，CUL-3和CULLIN激活剂NEDD8，这表明CUL3/MEL-26复合物作为泛素连接酶起作用，可在此细胞周期过渡过程中促进MEI-1销毁。此外，我们最近发现，通过其BTB结构域与CUL3相互作用，并通过其Kelch域与转录因子NRF2相互作用。在这里，我们寻求进一步确定BTB蛋白在靶向泛素倾斜中的作用。在AIM 1中，我们将阐明两个基于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蛋白二聚化在体内泛素化活性中的作用。在AIM 2中，我们将通过晶体学和生化研究来检查CUL-3/MEL-26/MEI-1复合物的结构，扩展E3的结构分析。在AIM 3中，我们将采用针对BTB蛋白的新开发的RNAi文库来定义CUL3/BTB复合物在细胞分裂和单体环蛋白E周转中的作用。此外，在BTB/CUL3途径的背景下，将开发出识别采用功能丧失遗传学并适用于全基因组筛查的泛素化靶标的新型方法。这些实验将：确认并扩展了BTB蛋白充当CUL3的底物特定衔接子的假设，将揭示MEL-26 BTB蛋白的CUL-3和底物识别的结构基础，并将应用功能基因组方法的功能，以使泛素依比克蛋白 - 依比克蛋白 - 依比克蛋白 - 依比克蛋白 - 依比克蛋白的功能和靶标在人类细胞中的问题。