OXYGEN SENSING AND SIGNAL TRANSDUCTION

氧传感和信号传导

基本信息

批准号：
6177831
负责人：
H. Franklin Bunn
金额：
$ 38.23万
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.

对缺氧的适应取决于许多生理上重要基因的诱导，例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于控制通气至关重要的酪氨酸羟化酶由颈动脉体。这些基因的缺氧上调取决于氧传感和信号转导的常见模式，从而导致转录因子 HIF-1 的激活。有推定证据表明氧传感器是一种黄素血红素蛋白，具有 NAD(P)H 氧化酶的功能。在该提案的具体目标 1 中，将采用遗传策略来克隆和表征表达具有共有 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还原酶具有很强同源性的新基因。这些基因之一特别令人感兴趣，因为它编码488个残基的融合蛋白，该融合蛋白在N端侧与细胞色素b5具有同源性，在C端侧与细胞色素b5还原酶具有同源性。我们将这种蛋白质命名为 b5b5R3，广泛表达于细胞和组织中。该提案最具挑战性的方面是确定 b5b5R3 或其他两个新基因 b5R1 和 b5R2 是否充当氧传感器。表达将通过反义实验而消除，对于 b5b5R3，通过小鼠 ES 细胞中的靶向敲除来消除。相反，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度表达来测试。彻底的光谱分析，包括紫外共振拉曼，将伴随着酶活性和底物特异性的研究。具体目标 2 描述了一种用于表征从质膜分离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）和 240 kDa 血红素蛋白。如果蛋白质测序表明任一蛋白质是新的，则将对其进行分子克隆和表达，并如上所述评估其功能。具体目标 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。这些实验应该提供有关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的途径中的分子事件的全面信息。