Arabidopsis 2010: Investigating Coiled-coil Proteins in the Arabidopsis ORFeome

在动物和酵母中发现含有长螺旋结构域的蛋白质参与将信号分子附着到细胞的大型固态成分上，如膜系统、着丝粒、中心体或核支架。越来越多的证据表明，它们在细胞中蛋白质定位的时空调节中起着重要作用。相比之下，这组蛋白质在植物中几乎没有被研究过。这个拟南芥2010项目的目标是鉴定所有含有长螺旋结构域的拟南芥蛋白，并对一个与植物核膜靶向有关的亚群进行功能表征。初步研究已经在拟南芥基因组中确定了1000多个预测的卷曲蛋白，其中近70%是“未知蛋白”。已经确定了在植物细胞的所有主要隔室中具有分选信号的卷曲卷曲蛋白的例子。为了开始对拟南芥中这一大型且未表征的蛋白质类的功能研究，将采用两种方法：(1)将在多处理器集群上建立一个自动计算平台，允许：(a)通过结构预测算法识别拟南芥中所有长的（50个氨基酸或更长）卷曲卷曲蛋白；(b)将输出结果与一系列序列和结构分析程序相结合，以建立预测的核、细胞器和膜定位、家族关系、功能域和其他相关特征；(c)创建一个公开可用的、可搜索的拟南芥卷曲蛋白数据库，其中将添加亚细胞定位、蛋白质-蛋白质相互作用和突变表型等实验数据。(2)对拟南芥WPP-domain蛋白结合的16个功能未知的coil -coil蛋白进行功能研究。WPP结构域是植物特有的核膜靶向结构域，它参与了Ran GTPase激活蛋白（RanGAP）在核边缘的定位。通过实验鉴定出一组能特异性识别WPP结构域的卷曲蛋白。假设是，一些鉴定的蛋白质是特定的亚细胞锚定wpp结构域蛋白。这将通过研究：(a)全长蛋白的体外和体内结合特异性来检验；(b)它们的亚细胞定位；(c)基因敲除或RNAi表型；(d)螺旋蛋白敲除对wpp结构域蛋白- gfp融合体亚细胞定位的影响。第一种方法将提供有关特征的大量信息，这些信息可以帮助建立关于单个蛋白质功能的知情假设。第二种方法将直接在分子、细胞和整个植物水平上研究16种螺旋状“未知蛋白质”的功能。在这个项目中要分析的基因的加入号列表可以在http://www.arabidopsis.org/info/2010_projects/index.html上找到。实验数据将通过出版和随后纳入可从http://www.biosci.ohio-state.edu/~plantbio/Faculty/meier.html作为链接访问的公开数据库提供。拟南芥品系和质粒等资源将通过ABRC提供。本项目将为研究生和本科生提供分子细胞生物学和生物信息学交叉领域的培训机会。在更广泛的范围内，对植物细胞信号空间组织的研究可能会为作物发育工程提供新的途径。

Proteins containing long coiled-coil domains have been found in animals and yeast to be involved in attaching signaling molecules to the large, solid-state components of the cell, such as membrane systems, centromeres, centrosomes, or the nuclear scaffold. There is growing evidence that they play an important role in the spatial and temporal regulation of protein positioning in the cell. In contrast, this group of proteins has been barely investigated in plants.The goal of this Arabidopsis 2010 project is to identify all Arabidopsis proteins that contain long stretches of coiled-coil domains, and to functionally characterize a subgroup that has been implicated in plant nuclear envelope targeting. Preliminary research has identified over 1000 predicted coiled-coil proteins in the Arabidopsis genome, almost 70% of which are "unknown proteins". Examples of coiled-coil proteins with sorting signals for all major compartments of the plant cell have been identified.To begin a functional investigation of this large and uncharacterized protein class in Arabidopsis, a twofold approach will be taken:(1) An automated computational platform will be established on a multi-processor cluster that will allow: (a) to identify all long (50 amino acids or longer) coiled-coil proteins in Arabidopsis by structural prediction algorithms; (b) to integrate the output with a battery of sequence and structure analysis programs to establish predicted nuclear, organellar, and membrane localization, family relationships, functional domains, and other relevant features; and (c) to create a publicly available, searchable database of Arabidopsis coiled-coil proteins, to which experimental data such as subcellular location, protein-protein interactions, and mutant phenotypes will be added.(2) A group of 16 coiled-coil proteins of unknown function, which bind to Arabidopsis WPP-domain proteins, will be functionally investigated. The WPP domain is a nuclear envelope-targeting domain unique to plants, which is involved in positioning Ran GTPase activating protein (RanGAP) at the nuclear rim. A group of coiled-coil proteins has been experimentally identified, which specifically recognize the WPP domain. The hypothesis is that some of the identified proteins are specific subcellular anchors for WPP-domain proteins. This will be tested by investigating: (a) the in vitro and in vivo binding specificity of the full-length proteins; (b) their subcellular localization; (c) knockout or RNAi phenotypes; and (d) the effect of coiled-coil protein knockouts on the subcellular positioning of WPP-domain protein-GFP fusions.The first approach will provide an accessible bounty of information about features that can help build informed hypotheses about the function of individual proteins. The second approach will directly investigate the function of 16 coiled-coil "unknown proteins" on a molecular, cellular, and whole-plant level.A list of the accession numbers of the genes to be analyzed during this project can be found at http://www.arabidopsis.org/info/2010_projects/index.html. Experimental data will be made available by publication and subsequently by incorporation into a publicly available database accessible as link from http://www.biosci.ohio-state.edu/~plantbio/Faculty/meier.html. Resources such as Arabidopsis lines and plasmids will be made available through the ABRC. This project will provide training opportunities for graduate and undergraduate students at the interface of molecular cell biology and bioinformatics. On a broader scope, the investigation of spatial organization of signaling in plant cells might lead to future approaches in developmental engineering of crop plants.