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.

项目摘要 缺氧（低O2）在人类疾病的潜水员阵列中起着核心作用。 O2被缺氧感测 完成丙酰羟化酶（PHD）酶的响应途径，该酶使用O2羟基特异性 缺氧诱导因子α（HIFα）的脯氨酸。一旦羟基化，HIFα被VON泛素化 嬉皮 - 林达（VHL）泛素连接酶，导致其蛋白水解。当出现缺氧时，博士酶缺乏 O2至羟基HIFα，导致HIFα稳定，进入核和转录调节 多个靶基因。我们目前不知道所有调节该途径的蛋白质，如何 途径在不同的组织类型中进行调制，或者它如何使用对O2的亲和力低的单个O2传感器 对O2浓度的广泛动态范围响应。由于路径的必要要求 早期发展和哺乳动物生存能力的组成部分，我们对路径的实际方式知之甚少 在完整动物中在体内工作。 为了解决这些问题，该提案利用了遗传学和完整的等源模型 可以在缺氧下壮成长的生物（秀丽隐杆线虫），并且可以控制其环境和遗传学 具有保真度和可重复性。秀丽隐杆线虫具有博士学位（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/调解人。首先，PMK-1 p38 MAPK的直系同源物促进了常氧化的EGL-9功能。其次，蛋白质的EGL-4直系同源 激酶G（PKG）是PMK-1的底物，它是PMK-1调节EGL-9活性所必需的。第三，pdr- 1泛素连接酶Parkin的直系同源物抑制神经元中的HIF-1。第四，泛素的CHN-1直系同源 连接酶和伴侣芯片是已知与帕金一起使用的因素，它抑制神经元中的HIF-1。 我们假设PMK-1通过通过磷酸化激活EGL-4来调节途径。我们相信 它们允许额外的调节层，扩大了O2感觉的动态范围和 调节响应的时机。我们还假设PDR-1和CHN-1形成泛素连接酶 通过VHL-1独立于（和不同组织中）调节HIF-1的对，从而允许 缺氧反应的上下文特异性和组织特异性模式。在这里，我们将通过 哪个CHN-1，PDR-1，EGL-4和PMK-1在体内调节途径。我们将测量HIF-1泛素化 和营业额，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产生，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调节途径。我们将测试是否 PMK-1通过磷酸化EGL-4来调节途径。我们将使用蛋白质组学方法来识别 EGL-4的下游基板作为途径的一部分运行。在结论中，这些研究将有 为检查这些因素的直系同源物是否在哺乳动物中起着相似的作用，为基础提供了基础。