Genetic Analysis of Neuronal Hypoxic Stress Resistance

神经元耐缺氧应激的遗传分析

• 批准号: 9979647
• 负责人: Christopher G Rongo
• 金额: $ 32.55万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-15 至 2021-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979647
• 关键词: Address; Affinity; Animal Model; Animals; Anoxia; Behavior; Behavioral; Binding; Biological Models; Caenorhabditis elegans; Cell Nucleus; Cerebral Palsy; Consumption; Cyclic GMP-Dependent Protein Kinases; Cysteine; Development; Disease; Endosomes; Environment; Enzymes; Esthesia; Etiology; Foundations; Gene Expression; Generations; Genes; Genetic; Genetic Models; Genetic Transcription; Glutamate Receptor; Guanylate Cyclase; Hydroxylation; Hypoxia; Hypoxia Inducible Factor; Ischemic Stroke; Lobular Neoplasia; MAP Kinase Gene; Malignant Neoplasms; Mammals; Measures; Mediating; Mediator of activation protein; Mitochondria; Molecular Chaperones; Mutation; Myocardial Infarction; Neuronal Hypoxia; Neurons; Organism; Orthologous Gene; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Pattern; Phosphorylation; Physiology; Play; Procollagen-Proline Dioxygenase; Proline; Proteins; Proteolysis; Proteomics; Pulmonary Hypertension; Receptor Inhibition; Recycling; Regulation; Reproducibility; Resistance; Role; Scaffolding Protein; Side; Spinal cord injury; Stress; Synapses; Synaptic Receptors; Testing; Tissues; Transcriptional Regulation; Traumatic Brain Injury; Ubiquitination; Work; deprivation; disorder prevention; factor A; genetic analysis; human disease; hypoxia inducible factor 1; in vivo; intestinal epithelium; mutant; novel; p38 Mitogen Activated Protein Kinase; parkin gene/protein; phosphoric diester hydrolase; receptor recycling; recruit; response; sensor; stem; synaptic function; tissue culture; ubiquitin ligase

PROJECT SUMMARY Hypoxia (low O2) plays a central role in a diverse array of human diseases. O2 is sensed by the hypoxia response pathway comprising a prolyl hydroxylase (PHD) enzyme, which uses O2 to hydroxylate specific prolines on the Hypoxia Inducible Factor α (HIFα). Once hydroxylated, HIFα is ubiquitinated by the Von Hippel-Lindau (VHL) ubiquitin ligase, resulting in its proteolysis. When hypoxia ensues, PHD enzymes lack the O2 to hydroxylate HIFα, resulting in HIFα stabilization, entry into the nucleus, and the transcriptional regulation of multiple target genes. We currently do not know all of the proteins that regulate this pathway, how this pathway is modulated in different tissue types, or how it uses a single O2 sensor with a low affinity for O2 to respond to a broad dynamic range of O2 concentration. Because of the essential requirement of pathway components in early development and viability in mammals, we also know little about how the pathway actually works in vivo in an intact animal. To address these questions, this proposal takes advantage of genetics and an intact, isogenic model organism (C. elegans) that can thrive under hypoxia and whose environment and genetics can be controlled with fidelity and reproducibility. C. elegans possess single genes for the PHD (EGL-9), the VHL (VHL-1), and the HIFα (HIF-1). We recently identified four new regulators/mediators of this pathway. First, the PMK-1 ortholog of p38 MAPK promotes EGL-9 function under normoxia. Second, the EGL-4 ortholog of Protein Kinase G (PKG) is a substrate of PMK-1 that is required for PMK-1 to regulate EGL-9 activity. Third, the PDR- 1 ortholog of the ubiquitin ligase Parkin inhibits HIF-1 in neurons. Fourth, the CHN-1 ortholog of the ubiquitin ligase and chaperone CHIP, a factor known to work with Parkin, inhibits HIF-1 in neurons. We hypothesize that PMK-1 regulates the pathway by activating EGL-4 via phosphorylation. We believe that they allow for an additional layer of regulation, expanding the dynamic range of O2 sensation and modulating the timing of the response. We also hypothesize that PDR-1 and CHN-1 form an ubiquitin ligase pair that regulates HIF-1 independently of (and in different tissues from) regulation by VHL-1, thereby allowing context-specific and tissue-specific patterns of hypoxia response. Here we will characterize the mechanism by which CHN-1, PDR-1, EGL-4, and PMK-1 regulate the pathway in vivo. We will measure HIF-1 ubiquitination and turnover, target gene expression, EGL-9 activity and subcellular localization, hypoxia survival, O2 consumption and ATP generation, oxidative stress, and mitochondrial dynamics in mutants for these factors. We will directly test whether PDR-1 and CHN-1 regulate the pathway through HIF-1. We will test whether PMK-1 regulates the pathway by phosphorylating EGL-4. We will use a proteomics approach to identify downstream substrates of EGL-4 that operate as part of the pathway. At its conclusion, these studies will have provided the foundation for examining whether the orthologs of these factors conduct similar roles in mammals.

项目总结