STRUCTURE/FUNCTION RELATIONSHIPS OF EUKARYOTIC DJ-1-LIKE PROTEINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 氧化应激诱导的细胞程序性死亡(PCD)在人类和植物疾病中都发挥着重要作用。斯通和威尔逊的实验室正在采取截然不同的实验方法，分别了解植物和人类神经组织中控制PCD的分子机制。Stone实验室开发了一种筛选方法来鉴定对一种诱导PCD的真菌毒素具有抗性的拟南芥突变体(Asai等人，2000；Stone等人，2000；Stone等人，2005)。威尔逊实验室正在研究与家族性帕金森氏病(PD)及其亲属在其他生物中有关的人类DJ-1蛋白的结构-功能关系(Wilson等人，2003；Canet-Aviles等人，2004；Wilson等人，2004)。DJ-1被认为可以保护细胞免受ROS诱导的细胞凋亡，但其作用机制尚不清楚。我们假设，通过研究人类DJ-1蛋白同源物在遗传上易受控制的真核生物A.thaliana中的结构-功能关系，我们可以了解更多关于人类DJ-1蛋白的功能。在A.thaliana中，DJ-1样蛋白由三个基因编码，这三个基因编码两个单独的DJ-1样结构域的融合串联阵列。总之，我们拥有专业知识来全面剖析这三个未鉴定的拟南风DJ-1样蛋白的功能，这可能有助于揭示人类DJ-1在预防帕金森病中的作用。 这项建议的具体目的是： 1)克隆拟南芥DJ-1类蛋白的全长cDNA克隆，筛选定点突变体，构建高效表达载体，并进行X射线晶体结构测定。 2)鉴定拟南芥DJ-1类蛋白的敲除突变，产生双重和三重敲除突变体，并鉴定突变体对各种与氧化还原相关的非生物胁迫(如H202、百草枯、甲萘二酮、强光、UV-C)的敏感性。确定人、酵母或细菌DJ-1同源物是否可以补充拟南芥突变体。 3)构建与拟南芥DJ-1类似蛋白的GFP融合以研究亚细胞定位，并创建启动子：：GUS融合以研究组织定位。 4)通过酵母双杂交筛选和免疫共沉淀实验鉴定与拟南芥DJ-1类蛋白相互作用的蛋白质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Oxidative stress-induced programmed cell death (PCD) plays important roles in both human and plant disease. The Stone and Wilson labs are taking very different experimental approaches to understanding the molecular mechanisms controlling PCD in plants and human neuronal tissues, respectively. The Stone lab developed a screen to identify Arabidopsis thaliana mutants resistant to a fungal toxin that induces PCD (Asai et al., 2000; Stone et al., 2000; Stone et al., 2005). The Wilson lab is examining the structure-function relationships of the human DJ-1 protein implicated in familial Parkinson's disease (PD) and its relatives in other organisms (Wilson et al., 2003; Canet-Aviles et al., 2004; Wilson et al., 2004). DJ-1 is proposed to protect cells from ROS-mediated apoptosis, but its mode of action remains unclear. We hypothesize that we can learn more about the function of the human DJ-1 protein by studying the structure-function relationships of its homologs in a genetically tractable eukaryote, A. thaliana. In A. thaliana, DJ-1-like proteins are encoded by three genes, which encode fused tandem arrays of two individual DJ-1-like domains. Together, we have the expertise to comprehensively dissect the functions of these three uncharacterized A. thaliana DJ-1-like proteins, which may shed light on the role of human DJ-1 in protecting against PD. The specific aims of this proposal are to: 1) Subclone full-length cDNA clones and select site-directed mutants of the A. thaliana DJ-1-like proteins into appropriate vectors for overexpression and X-ray crystal structure determination. 2) Identify knock-out mutations in the A. thaliana DJ-1-like proteins, generate double and triple knock-out mutants, and characterize mutant sensitivity to various redox-related abiotic stresses (e.g., H202, paraquat, menadione, high light, UV-C). Determine whether human, yeast or bacterial DJ-1 homologs can complement the Arabidopsis mutants. 3) Create GFP fusions to the A. thaliana DJ-1-like proteins to examine subcellular localization, and create promoter::GUS fusions to examine tissue localization. 4) Identify proteins that interact with A. thaliana DJ-1-like proteins by yeast two-hybrid interaction screens and co-immunoprecipitation experiments.