OXYGEN SENSING AND SIGNAL TRANSDUCTION

氧传感和信号传导

• 批准号: 6646463
• 负责人: H. Franklin Bunn
• 金额: $ 40.06万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6646463
• 关键词: NAD(P)H oxidoreductase; SDS polyacrylamide gel electrophoresis; antisense nucleic acid; binding proteins; biological signal transduction; carbon monoxide; cell membrane; chimeric proteins; cytochrome b; cytochrome b5 reductase; embryonic stem cell; erythropoietin; flavoproteins; free radical oxygen; gene expression; genetic transcription; hemoprotein; hypoxia; iron; mitochondria; nitric oxide; protein structure function; radiotracer; spectrometry; transcription factor

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.

对低氧的适应依赖于许多重要生理基因的诱导，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和控制颈动脉小体通气性的酪氨酸羟基酶。这些基因的低氧上调依赖于一种共同的氧气感知和信号转导模式，导致转录因子HIF-1的激活。有推定证据表明，氧感应器是一种黄色素蛋白，其功能是NAD(P)H氧化酶。在本提案的具体目标1中，将采用遗传策略来克隆和表征表达具有共同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还原酶有很强同源性的新基因。其中一个基因特别令人感兴趣，因为它编码一个488个残基的融合蛋白，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的蛋白质在细胞和组织中广泛表达。这项提议最具挑战性的方面将是确定b5b5R3或另外两个新基因b5r1和b5r2是否起到氧气传感器的作用。反义实验将取消表达，对于b5b5R3，将通过在小鼠ES细胞中定向敲除来取消表达。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UV共振拉曼，将伴随着酶活性和底物专一性的研究。特定目的2描述了一种从质膜中分离的两种蛋白质的生化方法：一种是与b5b5R3相同的50 kDa NADPH和黄素结合的血红素蛋白，另一种是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的，将对其进行分子克隆和表达，并对其功能进行如上所述的评估。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物种的贡献。这些实验应该提供有关该途径中分子事件的全面信息，这些分子事件将细胞内氧分压的降低与生物重要基因的转录激活联系起来。