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

拟南芥 2010：拟南芥中泛素蛋白连接酶 (E3) 家族的功能分析

• 批准号: 0519970
• 负责人: Richard Vierstra
• 金额: $ 406.2万
• 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: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0519970&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Functional; Analysis; Ubiquitin

The goal of this project is to describe the network of Arabidopsis proteins responsible for the covalent attachment of ubiquitin (Ub) and to develop research tools for their analysis. The post-translational conjugation of one or more Ubs to selected intracellular proteins plays an integral role in numerous growth, developmental, and metabolic processes in plants via its ability to modify the function and/or half-life of its targets. Whereas its main function is to commit proteins for degradation by the 26S proteasome, other functions have become apparent more recently, including roles in DNA repair, lysosomal catabolism, intracellular trafficking, and the regulation of transcription. Among the enzymes responsible for Ub conjugation, the Ub-protein ligases (or E3s) are the crucial elements that control both target selectivity and the nature of the Ub linkage. Arabidopsis appears to contain over 1,300 E3 components, making this collection one of the largest functional groups in this plant (~5% of the proteome). This project is directed toward defining the E3 families and their targets in Arabidopsis, using the large battery of methods, reagents and mutants assembled during a previous award. The Arabidopsis E3 protein families will be further annotated and their biochemical activities will be defined. Potential functions will be explored by expression studies with DNA microarrays and by localization using GFP-E3 fusions. Interaction strategies (yeast-two-hybrid and co-immunoprecipitation) using individual E3s as bait will be exploited to identify potential targets and various accessory factors. High-throughput protein microarrays will identify targets, using individual E3s or their target-recognition modules as probes. The processes controlled by ubiquitination will be explored by the phenotypic analysis in bulk of large collections of E2 and E3 mutants. Affinity approaches coupled with tagged Ubs will be employed to isolate ubiquitinated proteins from Arabidopsis. These conjugates will then be identified by various mass spectrometric techniques to provide an extensive library of Ub targets. And finally, the web-accessible PlantsUBQ database (http://plantsubq.genomics.purdue.edu) of genes, mutants, targets, and other information pertinent to ubiquitination will be expanded for broad scientific exposure. Broader Impact: The results generated by this study will form an essential framework for understanding E3 diversity, help reveal specific functions for each E3 type, and likely assign function to a number of uncharacterized Arabidoposis genes, thus helping define the genome of this plant. Ultimately, the results will provide comprehensive information on the role of this post-translational modification in plants that ultimately can be used to devise new strategies to improve crop productivity. In addition to the training of graduate students and postdoctoral fellows, a main educational goal will be the inclusion of minority undergraduates in the discovery process as part of a summer research program.

本项目的目标是描述负责泛素（Ub）共价连接的拟南芥蛋白质网络，并开发用于其分析的研究工具。 一种或多种Ub与所选细胞内蛋白的翻译后缀合通过其改变其靶标的功能和/或半衰期的能力在植物的许多生长、发育和代谢过程中起着不可或缺的作用。 虽然它的主要功能是提交蛋白质降解的26 S蛋白酶体，其他功能已变得明显，最近，包括在DNA修复，溶酶体catalysts，细胞内运输和转录的调节作用。 在负责Ub缀合的酶中，Ub-蛋白连接酶（或E3）是控制靶标选择性和Ub连接的性质的关键元件。 拟南芥似乎含有超过1，300个E3组分，使其成为该植物中最大的功能组之一（约占蛋白质组的5%）。 该项目是针对定义E3家族及其在拟南芥中的目标，使用的方法，试剂和突变体组装在以前的奖励大电池。 拟南芥E3蛋白家族将被进一步注释，并定义其生化活性。 潜在的功能将探索通过DNA微阵列的表达研究和使用GFP-E3融合定位。 相互作用策略（酵母双杂交和免疫共沉淀）使用个别E3作为诱饵将被利用，以确定潜在的目标和各种辅助因素。 高通量蛋白质微阵列将使用单个E3或其靶识别模块作为探针来识别靶。 泛素化控制的过程将通过大量E2和E3突变体的表型分析来探索。 结合标记的Ubs的亲和方法将被用来从拟南芥中分离泛素化蛋白。 然后通过各种质谱技术鉴定这些缀合物，以提供广泛的Ub靶标文库。 最后，可通过网络访问的PlantsUBQ数据库（http：plantsubq.genomics.purdue.edu）的基因、突变体、靶点和其他与泛素化相关的信息将被扩大，以供广泛的科学研究。 更广泛的影响：这项研究产生的结果将形成一个理解E3多样性的基本框架，有助于揭示每种E3类型的特定功能，并可能将功能分配给一些未表征的Arabidoposis基因，从而帮助定义这种植物的基因组。 最终，这些结果将提供有关这种翻译后修饰在植物中的作用的全面信息，最终可用于制定提高作物产量的新策略。 除了研究生和博士后研究员的培训，一个主要的教育目标将是在发现过程中纳入少数民族大学生作为夏季研究计划的一部分。