UWY COBRE: MECHANISMS OF HYPOXIA SENSING FROM RHODOBACTER TO HUMANS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。对大多数生物来说，感知和响应缺氧是至关重要的。在西方社会，心脏病、中风和癌症等主要死亡原因中，缺氧是一种病理生理因素。从单细胞原核生物到人类，许多缺氧感知的分子机制都是保守的。此外，介导缺氧反应的蛋白质包含类似的结构模块，代表性的例子是PAS结构域。该项目的一个目标是阐明线粒体系球形红杆菌的缺氧感知机制，该机制涉及含有PAS结构域的转录抑制因子PpsR。阐明PpsR感知缺氧的分子机制以及信号转导级联中相关成分的特征，有助于揭示哺乳动物缺氧依赖基因的表达机制。我们通过双杂交筛选、体外结构域-结构域相互作用和突变体分析研究了PpsR不同结构域的作用。我们发现PpsR的PAS结构域参与蛋白质-蛋白质相互作用，即类似于它们在真核生物中的作用。这是在原核生物中首次证明PAS结构域的这种功能。为了阐明缺氧依赖的信号转导级联，我们正在寻找可能与PpsR相互作用的蛋白质，并在PpsR中构建可能影响其氧化还原敏感性的突变。我们与Affymetrix， Inc. （Santa Clara， CA）达成了生产R. sphaeroides基因芯片的协议，该芯片将用于缺氧依赖基因表达的全基因组评估。我们优化了样品制备条件，并利用小鼠基因芯片进行了小鼠基因表达谱的初步实验。我们可以开始在经历人工诱导缺血的小鼠中描述全局基因表达。

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. Sensing and responding to hypoxia are of critical importance to most organisms. Hypoxia is a pathophysiological component of the major causes of mortality in Western societies: heart attack, stroke, and cancer. Many molecular mechanisms of hypoxia sensing are conserved from unicellular prokaryotes to humans. Furthermore, proteins mediating hypoxic responses contain similar structural modules, a representative example being the PAS domains. One goal of this project is to elucidate a mechanism of hypoxia sensing in a mitochondria lineage bacterium Rhodobacter sphaeroides that involves a PAS domain-containing transcriptional repressor PpsR. Elucidation of the molecular mechanism of hypoxia sensing by PpsR as well as characterization of the components involved in the signal transduction cascade could shed light on the mechanisms of hypoxia dependent gene expression in mammals. We studied the roles of various domains of PpsR by using two-hybrid screens, domain-domain interactions in vitr o and mutant analysis. We found that the PAS domains of PpsR are involved in protein-protein interactions, i.e. similar to their role in the eukaryotes. It is the first demonstration of this function of the PAS domains in prokaryotes. To elucidate the hypoxia dependent signal transduction cascade, we are searching for proteins that might interact with PpsR and constructing mutations in PpsR that might affect its redox sensitivity. We reached an Agreement with Affymetrix, Inc. (Santa Clara, CA) for production of the R. sphaeroides GeneChips that will be used for whole genome assessment of hypoxia dependent gene expression. We optimized conditions for sample preparation and performed initial experiments on gene expression profiling in mice using mouse GeneChips. We are in a position to start characterizing global gene expression in mice experiencing artificially induced ischemia.