REGULATION OF NF-KAPPA B BY NITRIC OXIDE IN THE LUNG

NF-κ B是一种转录因子，调节许多炎症反应基因。 在这种炎症状态下，一氧化氮（NO）的细胞产生增加。 根据细胞类型、刺激、浓度和递送形式，已经发现NO对NF-κ B具有刺激性或抑制性。 本研究旨在检测NO或NO相关分子对呼吸道上皮细胞NF-κ B活性的影响。 我们的初步研究表明，NO对呼吸道上皮细胞NF-κ B B有抑制作用。NO发挥这种作用的机制尚不清楚。 我们推测NO通过在p50-p65异源二聚体或活化途径中的另一种蛋白上形成亚硝基硫醇来调节NF-κ B B。 为了研究这个假设，我们计划：1。确定外源性和内源性NO或NO相关分子对呼吸道上皮细胞中NF-κ B活性的影响，并确定这些影响是否对细胞内巯基水平敏感。 2.在体外分析重组NF-κ B蛋白，以确定它们是否可以被亚硝基化以及在分子上的哪个或哪些位点发生亚硝基化。 3.确定这些S-亚硝基化NF-κ B蛋白是否存在于细胞内。 4.确定这些蛋白质的S-亚硝基化改变其细胞功能的机制。申请人的长期目标是成为研究肺中基因转录调节的独立研究者，特别是当其涉及氧化和亚硝化应激时。 有了这个奖项，申请人将获得必要的培训和经验，在分子生物学技术，以实现这一目标。

NF-Kappa B is a transcription factor which regulates numerous inflammatory response genes. Cellular production of nitric oxide (NO) is increased in such inflammatory states. Depending upon the cell type, stimulus, concentration, and form of delivery, NO has been found to be either stimulatory or inhibitory to NF-Kappa B. This proposed research is designed to test the effects of NO or NO-related molecules on NF-Kappa B activity in the respiratory epithelium. Our preliminary studies indicate that NO has an inhibitory effect on NF-Kappa B in respiratory epithelial cells. The mechanism(s) by which NO exerts this effect is not understood. We hypothesize that NO regulates NF-Kappa B via the formation of a nitrosothiol on the p50-p65 heterodimer or another protein in the activation pathway. To investigate this hypothesis we plan to: 1. Determine the effects of exogenous and endogenous NO or NO-related molecules on NF-Kappa B activity in respiratory epithelial cells and determine whether these effects are sensitive to intracellular thiol levels. 2. Analyze recombinant NF-Kappa B proteins in vitro to determine whether they can be nitrosylated and at which site(s) this occurs on the molecule. 3. Determine whether these S-nitrosylated NF-Kappa B proteins are present intracellularly. 4. Determine the mechanism by which S-nitrosylation of these proteins alters their cellular function. The applicants long term goals are to become an independent investigator studying the regulation of gene transcription in the lung, in particular, as it relates to oxidative and nitrosative stress. With this award, the applicant will obtain the necessary training and experience in molecular biological techniques to achieve this goal.