Genetic Analysis of Neuronal Hypoxic Stress Resistance

神经元耐缺氧应激的遗传分析

• 批准号: 9753252
• 负责人: Christopher G Rongo
• 金额: $ 32.24万
• 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 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753252
• 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; MAPK14 gene; Malignant Neoplasms; Mammals; Measures; Mediating; Mediator of activation protein; Mitochondria; Molecular Chaperones; Mutation; Myocardial Infarction; 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; 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.

项目概要 缺氧（低氧气）在多种人类疾病中起着核心作用。 O2 因缺氧而被感知 响应途径包含脯氨酰羟化酶 (PHD)，该酶使用 O2 羟化特定的 缺氧诱导因子 α (HIFα) 上的脯氨酸。一旦羟基化，HIFα 就会被 Von 泛素化 Hippel-Lindau (VHL) 泛素连接酶，导致其蛋白水解。当缺氧发生时，PHD 酶缺乏 O2 羟基化 HIFα，导致 HIFα 稳定、进入细胞核和转录调控 多个靶基因。我们目前还不知道调节该途径的所有蛋白质，这如何 途径在不同的组织类型中受到调节，或者如何使用对 O2 亲和力较低的单个 O2 传感器来调节 响应宽动态范围的 O2 浓度。由于途径的基本要求 尽管该通路是哺乳动物早期发育和生存能力的组成部分，但我们对这条通路实际上是如何运作的也知之甚少。 在完整动物体内发挥作用。 为了解决这些问题，该提案利用了遗传学和完整的等基因模型 可以在缺氧下茁壮成长并且其环境和遗传可以控制的生物体（秀丽隐杆线虫） 具有保真度和再现性。线虫拥有 PHD (EGL-9)、VHL (VHL-1) 和 HIFα (HIF-1)。我们最近确定了该途径的四个新调节器/调节器。首先，PMK-1 p38 MAPK 的直系同源物在常氧条件下促进 EGL-9 功能。二、蛋白质的EGL-4直系同源物 激酶 G (PKG) 是 PMK-1 的底物，是 PMK-1 调节 EGL-9 活性所必需的。第三，PDR- 泛素连接酶 Parkin 的 1 种直系同源物可抑制神经元中的 HIF-1。四、泛素的CHN-1直系同源物 连接酶和伴侣 CHIP 是已知与 Parkin 一起作用的因子，可抑制神经元中的 HIF-1。 我们假设 PMK-1 通过磷酸化激活 EGL-4 来调节该通路。我们相信 它们允许额外的调节层，扩大 O2 感觉的动态范围， 调整响应的时间。我们还假设 PDR-1 和 CHN-1 形成泛素连接酶 对 HIF-1 的调节独立于 VHL-1 的调节（并且在不同的组织中），从而允许 缺氧反应的环境特异性和组织特异性模式。在这里，我们将通过以下方式来描述该机制： 其中 CHN-1、PDR-1、EGL-4 和 PMK-1 在体内调节该途径。我们将测量 HIF-1 泛素化 和周转、靶基因表达、EGL-9 活性和亚细胞定位、缺氧存活、O2 这些因素的突变体中的消耗和 ATP 生成、氧化应激和线粒体动力学。 我们将直接测试PDR-1和CHN-1是否通过HIF-1调节该通路。我们将测试是否 PMK-1 通过磷酸化 EGL-4 来调节该途径。我们将使用蛋白质组学方法来识别 作为该途径一部分起作用的 EGL-4 下游底物。得出结论时，这些研究将 为检查这些因子的直系同源物是否在哺乳动物中发挥相似的作用提供了基础。