A Novel Regulator of Oxygen Homeostasis

描述（由申请人提供）：缺氧是许多人类疾病的中心特征，包括心肌梗死、中风和癌症。尽管对细胞对缺氧的反应进行了大量的研究，但我们对低氧张力引发的途径的理解充其量是不完整的。脯氨酸羟化酶结构域蛋白（PHD）、缺氧诱导因子（HIF）、von Hippel Lindau蛋白（VHL）通路的发现是这方面的最新进展。在该途径中，脯氨酸羟化酶PHD（也称为HIF脯氨酸羟化酶或EGLN）在两个特定的脯氨酸残基上修饰HIF，进而靶向后者，通过依赖于VHL E3泛素连接酶复合物的泛素-蛋白酶体途径降解。ph在羟基化反应中必须使用分子氧，这立即提供了一个令人信服的模型，通过该模型，氧张力可以耦合到蛋白质降解。因此，在常压条件下，HIF被本构羟基化和降解。在缺氧条件下，HIF被低羟基化，逃避VHL介导的降解，然后与参与缺氧适应的基因增强子结合。事实上，PHD:HIF:VHL模型甚至可能提高PHD作为通用氧传感器的可能性。PHD由三种异构体组成，目前的证据表明PHD2是PHD的关键异构体，负责在常氧条件下将HIF维持在基本上无法检测到的水平。在目前的应用中，我们确定了一个因子，我们称之为IOP1，它挑战了PHD:HIF:VHL通路足以解释氧传感的概念。IOP1与PHD2结合，并与氢化酶同源，氢化酶是厌氧细菌中的含铁酶。细菌氢化酶在维持厌氧条件下的氧化还原平衡中起着核心作用。我们提供的证据表明，在哺乳动物细胞中，IOP1可以调节常氧和缺氧条件下的HIF活性，并且这是通过调节HIF蛋白水平来实现的。此外，细菌含铁氢化酶众所周知是氧不稳定的，这增加了IOP1本身可能作为氧传感器的有趣可能性。为了更充分地表征这种缺氧信号通路的新成分，我们将追求以下具体目标。首先，我们将确定IOP1是否调控HIF通路。其次，我们将确定发生这种情况的机制。第三，我们将确定IOP1本身是否作为氧传感器。第四，我们将产生具有IOP1种系缺失的小鼠，然后表征小鼠和来自这些小鼠的细胞的缺氧反应。我们期望这些研究将定义细胞对缺氧反应的新途径。

DESCRIPTION (provided by applicant): Hypoxia is a central feature of many human diseases, including myocardial infarction, stroke, and cancer. Despite substantial research on cellular responses to hypoxia, our understanding of pathways initiated by low oxygen tension remains, at best, incomplete. A major recent advance in this regard is the discovery of the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF):von Hippel Lindau protein (VHL) pathway. In this pathway, the prolyl hydroxylase PHD (also known as HIF prolyl hydroxylase or EGLN) modifies HIF at two specific prolyl residues, which in turn targets the latter for degradation by the ubiquitin-proteasome pathway in a manner dependent of the VHL E3 ubiquitin ligase complex. The obligatory use of molecular oxygen by PHD in the hydroxylation reaction immediately provides a compelling model by which oxygen tension can be coupled to protein degradation. Thus, under normoxic conditions, HIF is constitutively hydroxylated and degraded. Under hypoxic conditions, HIF is hypohydroxylated, escapes VHL mediated degradation, and then binds to the enhancers of genes involved in adaptation to hypoxia. Indeed, the PHD:HIF:VHL model may even raise the possibility of PHD as a universal oxygen sensor. PHD consist of three isoforms, and current evidence indicates that PHD2 is the key PHD isoform responsible for maintaining HIF at essentially undetectable levels under normoxia. In the present application, we identify a factor, which we call IOP1, that challenges the notion that the PHD:HIF:VHL pathway is sufficient to account for oxygen sensing. IOP1 binds to PHD2 and is homologous to hydrogenases, iron-containing enzymes with ancestral origins in anaerobic bacteria. Bacterial hydrogenases play central roles in maintaining redox balance under anaerobic conditions. We provide evidence that in mammalian cells, IOP1 can regulate the HIF activity under both normoxic and hypoxic conditions, and that this occurs through the modulation of HIF protein levels. In addition, bacterial iron-containing hydrogenases are well known to be oxygen-labile, raising the intriguing possibility that IOP1 itself may serve as an oxygen sensor. To more fully characterize this novel component of the hypoxia signaling pathway, we will pursue the following specific aims. First, we will determine whether IOP1 regulates the HIF pathway. Second, we will determine the mechanism by which this occurs. Third, we will determine whether IOP1 itself serves as an oxygen sensor. Fourth, we will generate mice with a germline deletion of IOP1 and then characterize both the mice and hypoxic responses in cells derived from these mice. We anticipate that these studies will define a new pathway by which cells respond to hypoxia.