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.

该项目的目的是描述拟南芥蛋白质的网络，负责泛素（UB）的共价依恋并开发研究工具进行分析。 将一个或多个UB与选定的细胞内蛋白的翻译后结合在植物中通过修改其靶标的功能和/或半寿命的能力在植物中的许多生长，发育和代谢过程中起着不可或缺的作用。 尽管其主要功能是将26S蛋白酶体降解的蛋白质提交蛋白质，但其他功能最近变得显而易见，包括在DNA修复中的作用，溶酶体分解代谢，细胞内运输和转录调节。 在负责UB偶联的酶中，UB蛋白连接酶（或E3S）是控制靶向选择性和UB链接性质的关键要素。 拟南芥似乎包含超过1300个E3组件，使该系列成为该工厂中最大的功能组之一（占蛋白质组的5％）。 该项目是针对拟南芥中定义E3家族及其目标的，使用了先前奖励期间组装的大量方法，试剂和突变体。 拟南芥E3蛋白家族将进一步注释，并将定义其生化活性。 通过使用DNA微阵列的表达研究和使用GFP-E3融合的定位，将探索潜在功能。 将利用使用单个E3作为诱饵的互动策略（酵母 - 两种杂种和共免疫沉淀），以识别潜在的靶标和各种辅助因素。 高通量蛋白微阵列将使用单个E3或其目标识别模块作为探针识别目标。 由泛素化控制的过程将通过大部分E2和E3突变体集合中的表型分析来探索。 结合带标记的瑞银的亲和力方法将采用将泛素化蛋白与拟南芥分离。 然后，这些结合物将通过各种质谱技术来识别，以提供广泛的UB目标库。 最后，基因，突变体，靶标和其他与泛素化有关的基因，突变体和其他信息的基因，突变体，靶标和其他信息将扩大以进行广泛的科学暴露，将扩大基因，突变体和其他信息的基因，突变体和其他信息。 更广泛的影响：本研究产生的结果将构成理解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许多未表征的拟南芥基因，从而有助于定义该植物的基因组。 最终，结果将提供有关这种翻译后修饰在植物中作用的全面信息，这些信息最终可用于设计新策略以提高作物生产率。 除了对研究生和博士后研究员的培训外，作为夏季研究计划的一部分，主要的教育目标还包括在发现过程中纳入少数群体本科生。