Structural and Functional Analysis of Oxygen Sensor

说明(由申请人提供)本建议的目标是 氧传感器的鉴定及氧传感器的阐述 路径。氧气在许多生理过程和机制中是必不可少的。 因为氧气感应似乎存在于所有需氧生物体中。低氧诱导性 到目前为止，HIF因子和von Hippel-Lindau(VHL)肿瘤抑制蛋白 哺乳动物氧气感应途径中唯一具有良好特性的蛋白质。 常压下产生的活性氧物种已被证明触发了 HIF蛋白的氧化修饰和随后的降解 VHL依赖的泛素化；因此，HIF只有在 低氧条件，从而上调重要的生理靶点 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/b5R)的克隆与鉴定 这似乎是一个很好的候选氧气传感器。B5/b5R基因已经被 在人类、小鼠、大鼠、果蝇和线虫中发现。成绩单 在所有的人类细胞系和组织中都存在。该蛋白质为NAD(P)H 在常氧条件下能产生超氧化物的氧化还原酶。在……里面 将对这一提议、结构和功能进行研究 以阐明其在氧感应和信号转导中的作用 路径。在特定目标1中，结合改变的突变b5/b5R蛋白 氧和NAD(P)H的性质将被表达，动力学 将对超氧化物的产量进行测量。获得和失去功能的突变体将 将其导入人体细胞系，以干扰氧感应。在……里面 特定目标2，电子从NAD(P)H转移到 FAD对血红素和氧气的研究将通过两种X射线衍射分析来进行 以及野生型和突变型b5/b5R蛋白的共振拉曼光谱。在……里面 特异靶3，重组HIF、VHL和VHL之间的物理相互作用 将研究b5/b5R蛋白，以重建亚细胞氧 体外传感途径。小鼠低氧反应的遗传学研究， 果蝇和线虫应该提供更多关于 B5/b5R在氧气传感中的作用和对体外重建设计的帮助 实验。从这些拟议研究中获得的知识将有助于 对于我们对不同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.