OXYGEN SENSING AND SIGNAL TRANSDUCTION

氧传感和信号传导

基本信息

批准号：
2883915
负责人：
H. Franklin Bunn
金额：
$ 30.71万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-01 至 2004-08-31
项目状态：
已结题

项目摘要

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.

对低氧的适应取决于诱导许多具有生理上重要的基因，例如促红细胞生成素，这些基因调节红色血细胞肿块，血管内皮生长因子，促进缺血性组织中的新血管形成和酪氨酸羟化酶中的新血管形成，这对于甲壳体身体的通风控制至关重要。这些基因的低氧上调取决于氧气传感和信号转导的常见模式，导致转录因子HIF-1的激活。有推论性的证据表明，氧气传感器是一种起作用的Flavo-heme蛋白，可作为NAD（p）H氧化酶起作用。在本提案的特定目的1中，将采用遗传策略来克隆并表征表达具有共识NADPH和黄素结合结构域的蛋白质的基因。我们发现了三个与细胞色素B5还原酶具有强大同源性的新型基因。这些基因之一特别令人感兴趣，因为它编码了488个残基融合蛋白，该蛋白在N端侧与细胞色素B5以及C-末端的CytoChrome B5还原酶具有同源性。我们指定B5B5R3的这种蛋白质在细胞和组织中广泛表达。该提案最具挑战性的方面是确定B5B5R3或其他两个新型基因B5R1和B5R2是否充当氧气传感器。反义实验将消除表达，而在B5B5R3的情况下，将通过小鼠ES细胞中的靶向敲除。相反，这三个候选基因编码的蛋白质的功能特性将通过哺乳动物细胞和细菌中的过表达来测试。包括紫外线共振拉曼在内的彻底光谱分析将伴随着酶促活性和底物特异性的研究。具体目标2描述了一种从质膜分离出的两种蛋白质的生化方法：50 kDa NADPH和黄素结合血红素蛋白，可能与B5B5R3相同，以及240 kDa的血红素蛋白。如果蛋白质测序表明任何一种蛋白质都是新颖的，则将被分子克隆和表达，并且将如上所述评估其功能。具体目标3的重点是信号转导过程，确定了血红素一氧化碳和一氧化氮对HIF-1激活的影响，以及假定氧化酶传感器产生的活性氧的贡献。这些实验应提供有关途径中分子事件的全面信息，这些信息将细胞内氧张力的降低与生物学重要基因的转录激活联系起来。