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的N-末端修饰的特异性和机制。N-末端修饰酶（NTMEs），其中包括28个肽酶和11个转移酶，将进行研究。NTME控制蛋白质成熟、活化或调节、降解以及潜在的植物基因调节的重要基本方面。来自植物遗传学，细胞生物学以及基础和高通量生物化学的最先进方法被整合到该计划中，以将NTME领域从“同源活性”过渡到固体生物化学。本项目将解决以下问题：（1）拟南芥NTME蛋白是否具有酶活性？(2)通过组合肽库确定的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蛋白是协同作用还是后协同作用？(5)NTME T-DNA敲除突变体的影响是什么？总的来说，这些数据将建立每个NTME的特异性和位置，从而建立一套蛋白质组的规则，这将通过其亚细胞定位和其N-末端的一级序列预测蛋白质的命运。更广泛的影响：该项目将：（1）生产和分发针对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的扩展特异性的新型组合肽文库，以及（4）生产NTME的纯合单突变体和双突变体的集合。交互式NTME网站（http：//www.cepceb.ucr.edu/members/walling/2010_NTMEs）将提供拟南芥NTME基因的列表、NTME测定、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）将分发本项目产生的突变体。除了建立第一个生化规则来预测蛋白质在亚细胞区室中的命运外，该计划还提供了一个独特的机会，可以在植物N-末端修饰酶的新兴领域培养本科生，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.