NITRIC OXIDE-MEDIATED PROTEIN MODIFICATION IN THE LUNG

描述（根据申请人的摘要和具体目标改编）： 该项目是一个多学科的方法重叠化学， 生物化学和细胞生物学，研究内源性和 外源形成的一氧化氮（NO）和其它活性氮物质 (RNS)肺中的蛋白质改变。 总的假设是 谷胱甘肽S-转移酶是酪氨酸硝化的优先目标 和氯化在发炎的肺，因此，可以作为适当的 用于在体内产生活性物质的剂量计。 此外，本发明还提供了一种方法， GST中功能性酪氨酸残基的硝化和/或氯化可以 导致酶失活和细胞防御能力减弱。 具体 目的是：1）完善现有的分析方法， 多个蛋白质修饰（酪氨酸）位点特异性测量 硝化和氯化）在谷胱甘肽S-转移酶中的应用 离子化串联质谱; 2）确定是否化学和 嗜中性粒细胞介导的保守活性位点的硝化和氯化 纯化的大鼠和人谷胱甘肽S-转移酶中的酪氨酰残基 在体外抑制其活性，并确定哪些同工酶最 容易被修改和失活; 3）确定是否 活性氮类或刺激的人中性粒细胞诱导硝化 和/或谷胱甘肽S-转移酶中活性位点酪氨酸的氯化 培养的人气管支气管上皮细胞（HTBECs）中的同工酶，和 以确定这是否会导致这种细胞的活性降低， 防御系统;以及4）确定是否硝化和氯化 谷胱甘肽S-转移酶存在于吸烟者的肺组织中 和患有炎性肺病的患者，并将程度与 这些改变损害了这个关键细胞的功能， 防御系统

DESCRIPTION (Adapted from applicant's abstract and specific aims): This project represents a multidisciplinary approach overlapping chemistry, biochemistry, and cell biology, to study the effect of endogenously and exogenously formed nitric oxide (NO) and other reactive nitrogen species (RNS) upon protein modification in the lung. The overall hypothesis is the glutathione S-transferases are preferential targets for tyrosine nitration and chlorination in the inflamed lung, and hence, may serve as appropriate dosimeters for the production of reactive species in vivo. In addition, nitration and/or chlorination of functional tyrosine residues in GSTs may result in enzyme inactivation and weakened cellular defenses. The specific aims are: 1) to refine existing analytical methods for the simultaneous and site-specific measurement of multiple protein modifications (tyrosine nitration and chlorination) in glutathione S-transferases using electrospray ionization tandem mass spectrometry; 2) to ascertain whether chemical- and neutrophil-mediated nitration and chlorination of conserved active-site tyrosyl residues in purified rat and human glutathione S-transferases inhibits their activity in vitro, and to determine which isozymes are most susceptible to modification and inactivation; 3) to determine whether reactive nitrogen species or stimulated human neutrophils induce nitration and/or chlorination of active-site tyrosines in glutathione S-transferase isozymes in cultured human tracheobronchial epithelial cells (HTBECs), and to determine whether this results in decreased activity of this cellular defense system; and 4) to determine whether nitrated and chlorinated glutathione S-transferases are present in lung tissue from cigarette smokers and patients with inflammatory lung diseases, and to correlate the extent to which these modifications impair the function of this critical cellular defense system.