NITRIC OXIDE-MEDIATED PROTEIN MODIFICATION IN THE LUNG

描述(根据申请者的抽象和具体目标改编)：此 该项目代表了交叉化学的多学科方法， 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)和其他活性氮物种 (RNS)在肺中的蛋白质修饰。总体假设是 谷胱甘肽S转移酶是酪氨酸硝化的首选靶标 以及炎症肺部的氯化作用，因此可以适当地起作用 活体内产生活性物质的剂量计。此外, GSTs中功能性酪氨酸残基的硝化和/或氯化可 导致酶失活和细胞防御能力减弱。具体的 目标是：1)改进现有的分析方法，用于同时和 多种蛋白质修饰(酪氨酸)的位点特异性测量 电喷雾法测定谷胱甘肽S转移酶 电离串联质谱仪；2)确定化学和化学物质 中性粒细胞介导的保守活性中心的硝化和氯化 纯化大鼠和人谷胱甘肽S转移酶中酪氨酸残留量的研究 在体外抑制它们的活性，并确定哪些同工酶最 易被修改和失活；3)确定 活性氮类或受刺激的人中性粒细胞诱导硝化 谷胱甘肽S转移酶活性部位酪氨酸的氯化 培养的人气管支气管上皮细胞(HTBECs)的同工酶，以及 以确定这是否会导致该细胞活性降低 防御系统；以及4)确定硝化和氯化 吸烟者肺组织中存在谷胱甘肽S转移酶 和患有炎症性肺部疾病的患者，并将其程度与 这些改变损害了这一关键细胞的功能 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.