OXYGEN SENSING AND SIGNAL TRANSDUCTION

对缺氧的适应依赖于一些生理上重要基因的诱导，如调节红细胞质量的促红细胞生成素，促进缺血组织中新血管形成的血管内皮生长因子，以及对控制颈动脉体通气至关重要的酪氨酸羟化酶。这些基因的缺氧上调依赖于氧传感和信号转导的共同模式，导致转录因子HIF-1的激活。有推测的证据表明，氧传感器是一种黄素血红素蛋白，其功能是NAD(P)H氧化酶。在本提案的Specific Aim 1中，将采用遗传策略克隆和表征表达具有一致的NADPH和黄素结合域的蛋白质的基因。我们发现了三个与细胞色素b5还原酶有很强同源性的新基因。其中一个基因是特别有趣的，因为它编码一个488残基融合蛋白，在n端，与细胞色素b5同源，在c端，与细胞色素b5还原酶同源。这种我们命名为b5b5R3的蛋白在细胞和组织中广泛表达。这项提议中最具挑战性的方面将是确定b5b5R3或其他两个新基因b5R1和b5R2是否起氧气传感器的作用。在小鼠胚胎干细胞中，通过反义实验和靶向敲除b5b5R3来消除表达。相反，由这三个候选基因编码的蛋白质的功能特性将通过在哺乳动物细胞和细菌中的过表达来测试。全面的光谱分析，包括紫外共振拉曼，将伴随着酶活性和底物特异性的研究。Specific Aim 2描述了一种生化方法，用于表征从质膜分离的两种蛋白质：一种50 kDa的NADPH和黄素结合血红素蛋白，可能与b5b5R3相同，另一种240 kDa的血红素蛋白。如果蛋白质测序表明其中一种蛋白质是新颖的，则将对其进行分子克隆和表达，并按照上述方法评估其功能。特异性Aim 3侧重于信号转导过程，确定血红素配体一氧化碳和一氧化氮对HIF-1激活的影响，以及由假定的氧化酶传感器产生的活性氧的贡献。这些实验应该提供一个全面的信息，在连接细胞内氧张力的降低和生物学上重要基因的转录激活的途径中的分子事件。

Adaptation to hypoxia depends on the induction of a number of physiologically important genes such as erythropoietin, which regulates red blood cell mass, vascular endothelial growth factor, which promotes new blood vessel formation in ischemic tissue and tyrosine hydroxylase which is essential for the control of ventilation by the carotid body. The hypoxic up-regulation of these genes depends upon a common mode of oxygen sensing and signal transduction, leading to the activation of the transcription factor HIF-1. There is presumptive evidence that the oxygen sensor is a flavo-heme protein that functions as an NAD(P)H oxidase. In Specific Aim 1 of this proposal, a genetic strategy will be employed to clone and characterize genes that express proteins that have consensus NADPH and flavin binding domains. We have discovered three novel genes that have strong homology to cytochrome b5 reductase. One of these genes is of particular interest since it encodes a 488 residue fusion protein which, on the N-terminal side, bears homology to cytochrome b5 and, and on the C-terminal side, to cytochrome b5 reductase. This protein which we designate b5b5R3 is widely expressed in cells and tissues. The most challenging aspect of this proposal will be to determine whether b5b5R3, or the other two novel genes, b5R1 and b5R2, function as the oxygen sensor. Expression will be abolished by antisense experiments and, in the case of b5b5R3, by targeted knockout in mouse ES cells. Conversely, the functional properties of the proteins encoded by these three candidate genes will be tested by overexpression both in mammalian cells and in bacteria. Thorough spectroscopic analysis, including UV resonance Raman, will be accompanied by studies of enzymatic activity and substrate specificity. Specific Aim 2 describes a biochemical approach for characterization of two proteins isolated from the plasma membrane: a 50 kDa NADPH and flavin binding heme protein, that could be identical to b5b5R3, and a 240 kDa heme protein. If protein sequencing indicates that either protein is novel, it will be molecularly cloned and expressed, and its function will be assessed as described above. Specific Aim 3 focuses on the signal transduction process, determining the impact of the heme ligands carbon monoxide and nitric oxide on HIF-1 activation as well as the contribution of reactive oxygen species generated by the putative oxidase sensor. These experiments should provide a comprehensive body of information on the molecular events in the pathway that link a decrease in intracellular oxygen tension to transcriptional activation of biologically important genes.