Genetic Analysis of Neuronal Hypoxic Stress Resistance

神经元耐缺氧应激的遗传分析

基本信息

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
项目类别：
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

项目摘要

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，缺氧可感知氧气。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我们目前不知道调节该途径的所有蛋白质，以及这是如何实现的。途径在不同的组织类型中进行调节，或者如何使用对 O2 亲和力较低的单个 O2 传感器来调节由于途径的基本要求，可以响应广泛的动态范围的 O2 浓度。尽管该通路是哺乳动物早期发育和生存能力的组成部分，但我们对该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）具有保真度和重现性。秀丽隐杆线虫拥有 PHD (EGL-9)、VHL (VHL-1) 和单基因。我们最近发现了该通路的四个新调节因子/介导因子，首先是 PMK-1。 p38 MAPK 的直系同源物在常氧条件下促进 EGL-9 功能其次，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。泛素连接酶 Parkin 的 1 直向同源物抑制神经元中的 HIF-1 第四，泛素的 CHN-1 直向同源物。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。我们相信 PMK-1 通过磷酸化激活 EGL-4 来调节该途径。它们允许额外的一层调节，扩大 O2 感觉的动态范围，我们还发现 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），从而允许在这里，我们将通过以下方式来描述缺氧反应的具体机制。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 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。我们将测试 PDR-1 和 CHN-1 是否通过 HIF-1 调节该通路。 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。这些研究得出的结论是，EGL-4 的下游底物作为该途径的一部分发挥作用。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。