UWY COBRE: MECHANISMS OF HYPOXIA SENSING FROM RHODOBACTER TO HUMANS

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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.