Genetic Analysis of Neuronal Hypoxic Stress Resistance

项目总结 低氧(低氧)在多种人类疾病中起着核心作用。氧气是通过缺氧而感觉到的 反应途径包括一个脯氨酸羟基酶(PHD)，它使用O2来羟化特定的 缺氧诱导因子α(HIFα)上的脯氨酸。一旦羟化，低氧诱导因子α被冯泛素化 Hippel-Lindau(VHL)泛素连接酶，导致其蛋白分解。当缺氧接踵而至时，PHD酶缺乏 O2羟化缺氧诱导因子α，使缺氧诱导因子α稳定，进入细胞核，并参与转录调控 多个靶标基因。我们目前还不知道调节这一途径的所有蛋白质，这是如何 途径在不同的组织类型中被调制，或者它如何使用对O2具有低亲和力的单个O2传感器来 对较宽的氧气浓度动态范围作出反应。因为路径的本质要求 在哺乳动物的早期发育和生存能力中，我们也对该途径实际上是如何进行的知之甚少 在一个完整的动物体内起作用。 为了解决这些问题，这项提议利用了遗传学和完整的等基因模型。 能在低氧条件下茁壮成长，其环境和遗传可控的生物体(线虫) 具有保真度和可重复性。线虫具有PHD(EGL-9)、VHL(VHL-1)和 低氧诱导因子α(HIF-1)。我们最近发现了这一途径的四个新的调节/调节因子。首先，PMK-1 P38MAPK同源基因在常氧条件下促进EGL-9功能。第二，蛋白质的EGL-4同源基因 蛋白激酶G(PKG)是PMK-1的底物，是PMK-1调节EGL-9活性所必需的。第三，人民民主共和国-- 泛素连接酶Parkin的1个同系物抑制神经元中的HIF-1。第四，泛素的CHN-1同源基因 连接酶和伴侣芯片，一个已知对帕金有效的因素，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 它们允许额外的一层调节，扩大氧气感觉的动态范围 调整响应的时序。我们还假设PDR-1和CHN-1形成泛素连接酶 在不同组织中独立于VHL-1的调节而调节HIF-1的对，从而允许 特定背景和特定组织的缺氧反应模式。在这里，我们将通过以下几点来描述该机制 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 以及周转、靶基因表达、EGL-9活性和亚细胞定位、低氧存活、O2 这些因子在突变体中的消耗和ATP生成、氧化应激和线粒体动力学。 我们将直接检测PDR-1和CHN-1是否通过HIF-1调节该通路。我们将测试一下 PMK-1通过磷酸化EGL-4来调节该通路。我们将使用蛋白质组学的方法来鉴定 EGL-4的下游底物，作为途径的一部分。在其结论中，这些研究将有 为研究这些因子的同源基因是否在哺乳动物中扮演类似的角色提供了基础。

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.