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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。氧化胁迫诱导的细胞程序性死亡（PCD）在人类和植物疾病中起着重要作用。斯通和威尔逊实验室正在采取非常不同的实验方法来理解控制植物和人类神经元组织中PCD的分子机制。Stone实验室开发了一种筛选以鉴定对诱导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年）。Wilson实验室正在研究与家族性帕金森病（PD）及其在其他生物体中的亲属有关的人DJ-1蛋白的结构-功能关系（Wilson et al.，2003年; Canet-Aviles等人，2004; Wilson等人，2004年）。DJ-1被认为可以保护细胞免受ROS介导的凋亡，但其作用方式仍不清楚。我们假设，我们可以通过研究在遗传上易处理的真核生物A. thaliana.以.在拟南芥中，DJ-1样蛋白由三个基因编码，其编码两个单独的DJ-1样结构域的融合串联阵列。总之，我们有专业知识来全面剖析这三个未定性的功能。拟南芥DJ-1样蛋白，这可能揭示了人类DJ-1在预防PD中的作用。 本研究的具体目标是：1）亚克隆全长cDNA克隆，选择A.将拟南芥DJ-1样蛋白转化到合适的载体中用于过表达和X射线晶体结构测定。2)确定A.拟南芥DJ-1样蛋白，产生双重和三重敲除突变体，并表征突变体对各种氧化还原相关的非生物胁迫的敏感性（例如，H2 O2、百草枯、甲萘醌、强光、UV-C）。确定人类、酵母或细菌DJ-1同源物是否可以补充拟南芥突变体。3)将GFP融合到A.拟南芥DJ-1样蛋白来检测亚细胞定位，并产生启动子：：GUS融合体来检测组织定位。4)鉴定与A.通过酵母双杂交筛选和免疫共沉淀实验，获得了拟南芥DJ-1样蛋白。

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.