Arabidopsis 2010 Project: Collaborative Research: A Functional Genomics Approach to N-terminal Modifying Enzymes

这个2010年的项目将确定拟南芥蛋白质组N末端修饰的特异性和机制。N-末端修饰酶(NTME)包括28个多肽酶和11个转移酶。NTME控制着蛋白质成熟、激活或调节、降解以及潜在的植物基因调控等重要的基本方面。该计划融合了植物遗传学、细胞生物学以及基础和高通量生物化学的最新方法，将NTMe领域从“同源活性”转变为以固体生物化学为基础的领域。该项目将解决以下问题：(1)拟南芥NTMe蛋白是否具有酶活性？(2)由组合多肽文库确定的NTMe酶的特异性是什么？(3)NTMe RNA和蛋白质在何时何地积累？(4)NTMe蛋白是共翻译还是翻译后作用？(5)NTMe T-DNA敲除突变体有什么影响？总的来说，这些数据将确定每个NTMe的特异性和位置，从而为蛋白质组建立一套规则，通过蛋白质的亚细胞定位和N末端的初级序列来预测蛋白质的命运。更广泛的影响：该项目将：(1)生产和分发39种NTMe蛋白的抗血清，(2)开发表达和测定植物NTME的方法，(3)合成用于确定NTME的扩展特异性的新的组合肽库，以及(4)产生NTME的纯合单突变和双突变集合。NTMe互动式网站(http://www.cepceb.ucr.edu/members/walling/2010_NTMEs)将提供拟南芥NTMe基因的清单、NTMe分析、多肽酶和转移酶研究社区的链接以及访问该项目开发的资源的机制。拟南芥生物资源中心(ABRC)将分发该项目产生的突变。除了建立第一个预测蛋白质在亚细胞室中命运的生化规则外，这个项目还提供了一个独特的机会，在一个新出现的植物N末端修饰酶领域培训本科生、研究生和博士后研究员。

This 2010 project will determine the specificity and mechanisms of N-terminal modifications of the Arabidopsis proteome. The N-terminal modification enzymes (NTMEs), which include 28 peptidases and 11 transferases, will be studied. NTMEs control important fundamental aspects of protein maturation, activation or regulation, degradation, and potentially of plant gene regulation. State-of-the-art methods from plant genetics, cell biology and both basic and high throughput biochemistry are integrated in this program to transition the NTME field from "activity by homology" to one grounded in solid biochemistry. The project will address the following questions: (1) Do Arabidopsis NTME proteins have enzymatic activities? (2) What are the specificities of NTME enzymes as determined by combinatorial peptide libraries? (3) When and where do NTME RNAs and proteins accumulate? (4) Do NTME proteins act co- or post-translationally? (5) What are the impacts of NTME T-DNA knock-out mutants? Collectively, these data will establish the specificity and location of each NTME and thereby establish a set of rules for the proteome, which will predict the fate of a protein by its subcellular localization and the primary sequence of its N-terminus. Broader Impacts: This project will: (1) produce and distribute antisera to 39 NTME proteins, (2) develop methods to express and assay plant NTMEs, (3) synthesize novel combinatorial peptide libraries for determining the extended specificities of NTMEs, and (4) produce a collection of homozygous single and double mutants for NTMEs. The interactive NTME web site (http://www.cepceb.ucr.edu/members/walling/2010_NTMEs) will provide a list of the Arabidopsis NTME genes, NTME assays, links to the peptidase and transferase research communities, and mechanisms to access the resources developed by this project. The Arabidopsis Biological Resource Center (ABRC) will distribute mutants generated in this project. In addition to establishing the first biochemical rules to predict the fate of a protein in a subcellular compartment, this program provides a unique opportunity to train undergraduates, graduate students and postdoctoral fellows in a newly emerging field of N-terminal modifying enzymes in plants.