Mechanisms of Oxygen Sensing

描述(由申请人提供)：氧气平衡对生命和健康至关重要，包括肺和心血管健康。慢性缺氧与多种病理因素有关，包括慢性阻塞性肺疾病、肺癌、肺高压、纤维化和炎症等。对氧敏感通路的研究已经揭示了氧稳态的重要介体，然而，对氧敏感的分子机制的详细了解仍不清楚。更充分地了解细胞氧感应通路可能会导致确定治疗肺部和心血管疾病的新靶点。在细胞水平上，低氧激活了低氧诱导转录因子(HIF)。HIF与大量靶基因启动子/增强区域的低氧反应元件结合，从而激活包括糖酵解、血管生成和红细胞生成在内的遗传程序。在常氧条件下，HIF蛋白亚基被脯氨酸羟基酶结构域蛋白2(PHD2)羟化，目的是使其被von Hippel-Lindau(VHL)泛素连接酶泛素化和蛋白酶体降解。在低氧条件下，PHD2的活性被抑制，允许HIF蛋白的积累，随后与HIF亚基结合以诱导转录反应。氧水平降低PHD2活性的潜在机制尚不完全清楚。我们先前已经证明，缺氧增加了线粒体复合体III产生的ROS，从而抑制了HIF蛋白的羟化，从而抑制了HIF蛋白的稳定。在这个提案中，我们将利用蛋白质组学的方法来测试缺氧诱导的线粒体ROS是否通过结合未知蛋白、PHD2的翻译后修饰和/或氧化PHD2中的半胱氨酸残基来抑制PHD2的活性。目前，临床上还没有成功的靶向抑制HIF的药物。这项探索性拨款将提供详细的理解 了解PHD2在缺氧过程中是如何调节的，从而为HIFs的治疗靶向提供分子基础。

DESCRIPTION (provided by applicant): Oxygen homeostasis is essential for life and health, including pulmonary and cardiovascular health. Chronic hypoxia is associated with a myriad of pathologies including pulmonary related diseases such as COPD, lung cancer, pulmonary hypertension, fibrosis and inflammation. Research on oxygen sensing pathways has revealed important mediators of oxygen homeostasis, however, detailed knowledge of the molecular mechanisms of oxygen sensing remain uncovered. Fuller understanding of the cellular oxygen sensing pathways may lead to identification of novel therapeutic targets for treatment of pulmonary and cardiovascular disease. At the cellular level, hypoxia activates the Hypoxia Inducible transcription Factors (HIFs). HIFs bind to hypoxia-response elements in the promoter/enhance regions of a large number of target genes resulting in activation of a genetic program that includes upregulation of glycolysis, angiogenesis, and erythropoiesis. Under normoxic conditions, the HIF¿ protein subunit is hydroxylated by Prolyl Hydroxylase Domain protein 2 (PHD2), targeting it for ubiquitination by the von Hippel-Lindau (VHL) ubiquitin ligase, and proteasomal degradation. The activity of PHD2 is inhibited under hypoxic conditions, allowing the accumulation of HIF¿ protein and the subsequent binding to HIF¿ subunit to induce the transcriptional response. The underlying mechanism by which oxygen levels diminish PHD2 activity is not fully understood. We have previously demonstrated that hypoxia increases the generation of ROS from mitochondrial complex III resulting in inhibition of HIF¿ protein hydroxylation and subsequent HIF¿ protein stabilization. In this proposal we will utilize a proteomic approach to test whether hypoxia-induced mitochondrial ROS lead to inhibition of PHD2 activity by binding of unidentified proteins, post-translational modification of PHD2, and/or oxidizing of cysteine residues within PHD2. Currently, there have been no successful drugs in the clinic targeting inhibition of HIFs. This exploratory grant will provide detailed understanding of how PHD2 is regulated during hypoxia leading to rationale molecular basis for therapeutic targeting of HIFs.