STRUCTURAL STUDIES OF DJ-1 FROM MULTIPLE EUKARYOTES

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。 人类DJ-1在细胞对氧化应激的反应中起重要作用，DJ-1的某些点突变与罕见的常染色体隐性遗传早发性帕金森？S病有关。此外，DJ-1被独立发现是一种依赖ras的癌基因，并与某些类型的癌症有关。我们和其他人的研究表明，DJ-1是一种同源二聚体，含有高度保守的半胱氨酸残基，具有低的pKa值，这是DJ-1 S保护活性所必需的。基于之前人DJ-1的原子分辨结构，我们已经制造并结晶了一些点突变，这些点突变改变了DJ-1中这个功能关键的半胱氨酸残基的反应性。我们建议确定这些突变体的X射线晶体结构，以便为我们完成的DJ-1半胱氨酸反应性的生化研究提供明确的结构解释。这项拟议工作的主要目标是确定人类DJ-1半胱氨酸反应性的结构决定因素。 此外，我们正在从植物中鉴定DJ-1超家族的一个新成员的结构。与DJ-1超家族的所有其他特征成员不同，植物DJ-1被预测为具有独特结构特征的伪二聚体单体。我们将确定一个具有代表性的植物DJ-1蛋白的X射线晶体结构，并利用这些结果形成关于植物DJ-1蛋白功能的可测试假设。这些实验是正在进行的一项努力的一部分，目的是从几种不同的模式生物中全面描述真核生物DJ-1的功能。

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. Human DJ-1 plays an important role in the cellular response to oxidative stress, and certain point mutations in DJ-1 have been associated with rare forms of autosomal recessive early-onset Parkinson?s disease. In addition, DJ-1 was independently discovered as a ras-dependent oncogene and has been implicated in certain types of cancer. Studies by us and others have revealed that DJ-1 is a homodimer that contains a highly conserved cysteine residue with a depressed pKa value that is essential for DJ-1?s protective activity. Based on a previous atomic resolution structure of human DJ-1, we have made and crystallized a number of point mutants that alter the reactivity of this functionally critical cysteine residue in DJ-1. We propose to determine the X-ray crystal structures of these mutants in order to provide a clear structural explanation for our completed biochemical study of cysteine reactivity in DJ-1. The principal goal of the proposed work is to identify the structural determinants of cysteine reactivity in human DJ-1. In addition, we are undertaking the structural characterization of a new member of the DJ-1 superfamily from plants. Unlike all other characterized members of the DJ-1 superfamily, plant DJ-1 is predicted to be a pseudo-dimeric monomer with unique structural features. We will determine the X-ray crystal structure of a representative plant DJ-1 protein and use these results to form testable hypotheses about the function of plant DJ-1 proteins. These experiments are part of an ongoing effort to comprehensively characterize the function of eukaryotic DJ-1 from several different model organisms.