Structural and Functional Analysis of Oxygen Sensor

描述（由申请人提供）本提案旨在 氧传感器的识别和氧传感的说明 通路氧气在许多生理过程和机制中是必不可少的 似乎存在于所有需氧生物中。缺氧诱导 HIF因子和VHL肿瘤抑制蛋白的研究进展 是哺乳动物氧传感通路中唯一特征明确的蛋白质。 在常氧下产生的活性氧物质已被证明可以触发 HIF蛋白的氧化修饰和随后的降解， VHL依赖性泛素化;因此，HIF仅在 低氧条件，从而上调生理学上重要靶点 基因，如促红细胞生成素和血管内皮生长因子。我们有 一种新的细胞色素b型NAD（P）H氧化酶（b5/b5 R）的克隆和鉴定 这看起来是一个很好的候选氧传感器。b5/b5 R基因已经被 在人、小鼠、大鼠、果蝇和蠕虫中鉴定。笔录 存在于所有人类细胞系和组织中。该蛋白质是NAD（P）H 在常氧条件下能够产生超氧化物的氧化还原酶。在 这一建议，结构和功能研究将进行这一点， 候选人，以阐明其在氧传感和信号传导中的作用 通路在特异性目的1中，具有改变的结合的突变b5/b5 R蛋白 氧和NAD（P）H的性质将被表达， 测量超氧化物的产生。获得和丧失功能的突变体将 转染到人类细胞系中，以干扰氧传感。在 具体目标2，电子从NAD（P）H转移到 FAD血红素和氧将研究这两个X射线衍射分析 以及通过共振拉曼光谱法对野生型和突变型b5/b5 R蛋白的研究。在 具体目标3，重组HIF、VHL和 b5/b5 R蛋白将被研究，以重建亚细胞氧 体外感受途径。小鼠缺氧反应的遗传学研究， 果蝇和C. elegans应该提供额外的功能信息， b5/b5 R在氧传感中的作用，并有助于体外重建的设计 实验从这些拟议的研究中获得的知识将有助于 我们对各种生理和病理生理学的理解 过程，包括肿瘤发生和对缺血的适应。

DESCRIPTION (provided by applicant) This proposal is aimed at the identification of the oxygen sensor and the elucidation of the oxygen sensing pathway. Oxygen is essential in many physiological processes, and mechanisms for oxygen sensing seem to exist in all aerobic organisms. Hypoxic inducible factors (HIF) and von Hippel-Lindau (VHL) tumor suppressor proteins are so far the only well-characterized proteins in the mammalian oxygen sensing pathway. Reactive oxygen species generated under normoxia have been shown to trigger the oxidative modification and the subsequent degradation of HIF proteins through VHL-dependent ubiquitylation; consequently, HIF is stabilized only under hypoxic conditions, thereby up-regulating physiologically important target genes, such as erythropoietin and vascular endothelial growth factor. We have cloned and characterized a novel cytochrome b-type NAD(P)H oxidase (b5/b5R) which appears to be a good candidate oxygen sensor. The b5/b5R gene has been identified in human, mouse, rat, fruit flies and nematode worms. The transcript is found in all human cell-lines and tissues. The protein is a NAD(P)H oxidoreductase capable of generating superoxide under normoxic conditions. In this proposal, structural and functional studies will be performed on this candidate in order to elucidate its role in the oxygen sensing and signaling pathway. In Specific Aim 1, mutant b5/b5R proteins with altered binding properties for oxygen and NAD(P)H will be expressed and the kinetics of superoxide production will be measured. Gain- and loss-of-function mutants will be transfected into human cell-lines in order to perturb oxygen sensing. In Specific Aim 2, the structural basis for electron transfer from NAD(P)H through FAD to heme and to oxygen will be studied by both X-ray diffraction analyses and by resonance Raman spectroscopy on wild-type and mutant b5/b5R proteins. In Specific Aim 3, the physical interaction between recombinant HIF, VHL and b5/b5R proteins will be studied in order to reconstitute the subcellular oxygen sensing pathway in vitro. Genetic studies on hypoxic responses in mouse, Drosophila and C. elegans should provide additional functional information on b5/b5R in oxygen sensing and help in the design of the in vitro reconstitution experiments. The knowledge gained from these proposed studies will contribute to our understanding of diverse physiological and pathophysilological processes, including tumorigenesis and adaptation to ischemia.