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.
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