Arabidopsis 2010: Functional Analysis of Ubiquitin-Protein Ligase (E3) Families in Arabidopis

拟南芥 2010：拟南芥泛素蛋白连接酶 (E3) 家族的功能分析

• 批准号: 0929100
• 负责人: Richard Vierstra
• 金额: $ 467.54万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0929100&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Functional; Analysis; Ubiquitin

Intellectual Merit:The goal of this project is to define the network of Arabidopsis proteins responsible for covalent attachment of the small protein ubiquitin (Ub) to specific cellular targets. The attachment of Ub to other proteins is highly selective and plays an integral role in numerous developmental and metabolic processes in plants. Although addition of Ub was originally discovered as a mechanism to promote protein degradation, recently other functions have become apparent, including roles in DNA repair, lysosomal catabolism, intracellular trafficking, and the regulation of chromatin structure and transcription. Several enzymes are needed for attachment of Ub to targets, but among these the Ub-protein ligases (or E3s) are the crucial enzymes that control both target selectivity and the nature of the Ub linkage. Genome analysis now shows that Arabidopsis may express over 1,500 E3 components, making E3s one of the largest functional protein groups in plants (~5% of the proteome). The diversity of E3s is unique to the plant kingdom, and the large size of this family indicates it presents a versatile mechanism for controlling cellular processes. This versatility makes it of significant interest relative to plant evolution and adaptation to the environment, as well as holding potential for future plant genetic engineering. This project will expand successful collaborative efforts to define E3 proteins families and their targets in Arabidopsis, using the large battery of methods, reagents and mutants, as well as a variety of new genomic and structure-based approaches. The project will continue to identify and annotate the Arabidopsis E3 protein families and define their biochemical activities. Their importance to Arabidopsis physiology, development and survival will be analyzed by reverse genetic analyses and further studied by expression profiling, localization using GFP-E3 fusions, and strategies to identify partners or substrates (yeast-two-hybrid and co-immunoprecipitation). The project will continue to develop protein microarrays as a high-throughput method to unravel the network of proteins involved in E3 action. Further substrates of ubiquitylation will be identified and then confirmed genetically and by global protein stability assays. The relationships between E3s and their partner E2s will be assessed by substrate-specific in vitro assays and genetic analyses. 3-D models for how targets are recognized by representative E3s will be developed by X-ray crystallographic analyses. Finally, the project will transfer knowledge gained from Arabidopsis to other plant species to help define E3 evolution, to understand why plants are so reliant on this post-translational modification, and to identify E3 orthologs in crops, which can then be exploited to improve agricultural productivity. Broader Impact: The program will provide high level training for postdoctoral researchers and graduate students. A mentoring plan is in place to involve trainees specifically in design of the research, and to develop career opportunities. Students and postdocs will present their work at annual project meetings of the investigators, exposing them to the interdisciplinary aspects of the work. In addition to training graduate students and postdoctoral fellows, under-represented minority undergraduates will also be part of the discovery process through a summer research program partnering with the University of West Alabama. This project aims to train, in a highly interdisciplinary context, the next generation of professionals to continue these efforts, and to expose under-represented minorities to plant research.

智力优势：这个项目的目标是定义负责小蛋白泛素（Ub）与特定细胞靶标共价连接的拟南芥蛋白网络。Ub与其他蛋白质的附着具有高度选择性，在植物的许多发育和代谢过程中起着不可或缺的作用。虽然Ub的加入最初被发现是促进蛋白质降解的机制，但最近其他功能已经变得明显，包括DNA修复，溶酶体分解代谢，细胞内运输以及染色质结构和转录的调节。Ub与靶标的连接需要几种酶，但其中Ub蛋白连接酶（或E3s）是控制靶标选择性和Ub连锁性质的关键酶。基因组分析表明，拟南芥可能表达超过1500种E3成分，使E3成为植物中最大的功能蛋白群之一（约占蛋白质组的5%）。E3s的多样性是植物界所独有的，这个家族的庞大规模表明它具有控制细胞过程的多种机制。这种多功能性使其在植物进化和适应环境方面具有重要意义，并在未来的植物基因工程中具有潜力。该项目将利用大量的方法、试剂和突变体，以及各种新的基因组和基于结构的方法，扩大成功的合作努力，以确定拟南芥中的E3蛋白家族及其靶点。该项目将继续鉴定和注释拟南芥E3蛋白家族并确定其生化活性。它们对拟南芥生理、发育和生存的重要性将通过反向遗传分析进行分析，并通过表达谱、GFP-E3融合物定位以及识别合作伙伴或底物的策略（酵母-双杂交和共免疫沉淀）进一步研究。该项目将继续开发蛋白质微阵列作为一种高通量方法来解开参与E3作用的蛋白质网络。进一步的泛素化底物将被鉴定，然后通过遗传和全局蛋白质稳定性测定来确认。E3s及其伴侣E2s之间的关系将通过底物特异性体外测定和遗传分析来评估。具有代表性的E3s如何识别目标的三维模型将通过x射线晶体学分析来开发。最后，该项目将把从拟南芥中获得的知识转移到其他植物物种中，以帮助定义E3进化，理解为什么植物如此依赖于这种翻译后修饰，并确定作物中的E3同源物，然后可以利用它来提高农业生产力。更广泛的影响：该项目将为博士后研究人员和研究生提供高水平的培训。制定了一项指导计划，使受训者专门参与研究的设计，并发展职业机会。学生和博士后将在研究人员的年度项目会议上展示他们的工作，使他们接触到工作的跨学科方面。除了培训研究生和博士后，少数族裔本科生也将通过与西阿拉巴马大学合作的暑期研究项目，成为发现过程的一部分。该项目旨在在高度跨学科的背景下培训下一代专业人员继续这些努力，并使代表性不足的少数民族接触植物研究。