REGENTS OF THE UNIVERSITY OF COLORADO, THE
The ability of cells, tissues, and organisms to sense and respond to changes in oxygen levels is often crucial to their survival. Cellular adaptation to hypoxia is a good example of this. Intermittent episodes of hypoxia are associated with a variety of human patho-physiological states (e.g., sleep apnea, central hypoventilation syndrome, and vascular occlusion); these can alter metabolism, induce angiogenesis, and affect inflammatory responses. Several studies have reported that adaptation to hypoxia requires the induction of a large number of nuclear genes and that the mitochondrial respiratory chain plays an important role in the induction of some of these hypoxic genes in mammalian and yeast cells. The overall goal of our studies is to understand the role of the mitochondrion in this process. These studies combine proteomic, genomic, and genetic strategies to analyze hypoxic gene induction in yeast. In addition, parallel studies will be done with mammalian cells in culture. Aim 1 will use gene profiling methods to identify hypoxic nuclear genes that are under the control of the mitochondrial respiratory chain in yeast. Aim 2 will focus on the mitochondrial - dependent hypoxic gene induction pathway itself. Genetic manipulation will be used to identify and order yeast genes that are essential for the induction of mitochondrial-dependent hypoxic nuclear genes. Aim 3 will evaluate the importance of mitochondrially-generated reactive oxygen species and nitric oxide in yeast hypoxic gene induction, with emphasis on understanding the role of protein tyrosine nitration and protein carbonylation in hypoxic signaling. Aim 4 focuses on hypoxic gene induction in mammalian cells. It addresses two questions that are controversial or incompletely explored. First, it will use methodologies that have been successful in yeast to ask if mammalian cells experience transient oxidative or nitrosative stress when exposed to hypoxia or anoxia and, if so, identify proteins carbonylated or tyrosine nitrated under anoxic or hypoxic conditions. Second, it will use in RNAi knock down experiments to assess the importance of these carbonylated or nitrated proteins in hypoxic signaling in mammalian cells. The failure of cells to respond properly to hypoxia can lead to a variety of pathological states (e.g., anemia, myocardial infarction, retinopathy, and the growth of tumors). An understanding of hypoxic signaling pathways may lead to new therapies for these diseases and may help with our understanding of human aging.