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）在各种人类疾病中起着核心作用。氧气通过缺氧被感知 包括脯氨酰羟化酶（PHD）酶的反应途径，其使用O2将特异性脯氨酰羟化， 脯氨酸对缺氧诱导因子α（HIFα）的影响。一旦羟基化，HIFα被Von Hippel-Lindau（VHL）泛素连接酶，导致其蛋白水解。当缺氧加剧时，PHD酶缺乏 O2使HIFα羟基化，导致HIFα稳定化，进入细胞核，转录调节 多个靶基因。我们目前还不知道所有调节这一途径的蛋白质， 在不同的组织类型中，或者它如何使用对O2具有低亲和力的单个O2传感器， 响应宽动态范围的O2浓度。由于路径的基本要求 尽管我们对哺乳动物早期发育和生存能力的组成部分知之甚少，但我们对该途径实际上是如何运作的也知之甚少 在完整的动物体内起作用。 为了解决这些问题，该提案利用遗传学和完整的等基因模型 有机体（C.它们可以在缺氧条件下茁壮成长，其环境和遗传学可以被控制 具有保真度和可重复性。C.线虫具有PHD（EGL-9）、VHL（VHL-1）和 HIFα（HIF-1）。我们最近确定了这一途径的四个新的调节剂/介质。第一，PMK-1 p38 MAPK的直系同源物在常氧下促进EGL-9的功能。第二，蛋白质的EGL-4直系同源物 激酶G（PKG）是PMK-1调节EGL-9活性所需的PMK-1的底物。第三，人民民主共和国- 泛素连接酶Parkin的1直系同源物抑制神经元中的HIF-1。第四，泛素的CHN-1直系同源物 连接酶和伴侣CHIP，一种已知与帕金一起工作的因子，抑制神经元中的HIF-1。 我们推测PMK-1通过磷酸化激活EGL-4来调节该通路。我们认为 它们允许额外的调节层，扩大O2感觉的动态范围， 调节响应的时间。我们还假设PDR-1和CHN-1形成泛素连接酶 一对独立于VHL-1调节HIF-1（并且在不同组织中），从而允许 环境特异性和组织特异性缺氧反应模式。在这里，我们将通过以下方式描述该机制： 其中CHN-1、PDR-1、EGL-4和PMK-1在体内调节该通路。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 消耗和ATP的产生，氧化应激和这些因素的突变体中的线粒体动力学。 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 PMK-1通过磷酸化EGL-4调节该途径。我们将使用蛋白质组学方法来识别 EGL-4的下游底物作为途径的一部分起作用。在其结束时，这些研究将有 为研究这些因子的直系同源物是否在哺乳动物中发挥类似作用提供了基础。