Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms
Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms
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DOI：
10.1073/pnas.062604199
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发表时间：
2002-03-19
影响因子：
11.1
通讯作者：
Wink, DA
中科院分区：
文献类型：
--
作者：
Espey, MG;Xavier, S;Wink, DA
蛋白质中的 3-硝基酪氨酰加合物已在多种疾病中检测到。通过使用独特的模型系统检查活性氮氧化物物质可能通过硝化阻碍蛋白质功能的机制，该系统利用重组绿色荧光蛋白（GFP）的荧光团中的关键酪氨酰残基。将悬浮在磷酸盐缓冲液中的纯化 GFP 以 0.5 或 5 muM 的步骤暴露于合成过氧亚硝酸盐，导致 3-硝基酪氨酰免疫反应性逐渐增加，同时内在荧光消失（IC50 接近 20 muM）。完整 MCF-7 肿瘤细胞内表达的等量 GFP 发出的荧光很大程度上对这种推注治疗具有抵抗力 (IC50 > 250 muM)。检查了过氧亚硝酸盐输注（1 μM/min）或通过同时等摩尔生成一氧化氮（NO）和超氧化物（例如，3-吗啉代亚胺；NONOates加黄嘌呤氧化酶/次黄嘌呤、甲萘醌或丝裂霉素C）从头形成的更生理相关的条件。尽管二氢罗丹明在这些条件下均发生强烈氧化，但无论二氧化碳是否存在，纯化的和细胞内的 GFP 的荧光降低并不明显，这表明氧化和硝化不一定是耦合的。另外，细胞外和细胞内 GFP 荧光都对血红素过氧化物酶/过氧化氢催化的亚硝酸盐氧化产生的硝化极其敏感。 NO 与硝基氧 2-苯基-4,4,5,5-四甲基咪唑-1-氧基 3-氧化物反应期间形成二氧化氮 (NO2)，表明 NO2 可以进入细胞并通过酪氨酰硝化改变肽功能。总而言之，这些发现证明血红素过氧化物酶催化的 NO2 形成可能在炎症和慢性疾病中发挥关键作用，同时对过氧亚硝酸盐硝化的重要性提出质疑。
3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 muM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC50 approximate to 20 muM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC50 > 250 muM). The more physiologically relevant conditions of either peroxynitrite infusion (1 muM/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxide-catalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO2) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO2 can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidase-catalyzed formation of NO2 may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.