Protein tyrosine nitration in cytokine-activated murine macrophages - Involvement of a peroxidase/nitrite pathway rather than peroxynitrite
Protein tyrosine nitration in cytokine-activated murine macrophages - Involvement of a peroxidase/nitrite pathway rather than peroxynitrite
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DOI：
10.1074/jbc.m100585200
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发表时间：
2001-09-07
影响因子：
4.8
通讯作者：
Mayer, B
中科院分区：
文献类型：
--
作者：
Pfeiffer, S;Lass, A;Mayer, B
过氧亚硝酸盐是由一氧化氮（NO）和超氧阴离子自由基(O)快速反应形成的。（-）(2))被认为在各种炎症和感染性疾病中介导蛋白酪氨酸硝化。然而，最近的一项体外研究表明，过氧亚硝酸盐在生物相关的稳态浓度下表现出较差的硝化效率（Pfeiffer， S., Schmidt， K., and Mayer， B. (2000) J.生物化学，275,6346-6352）。为了研究完整细胞中蛋白酪氨酸硝化作用的分子机制，在免疫刺激下激活小鼠RAW 264.7巨噬细胞，诱导NO合成酶表达（干扰素- γ与脂多糖或酶聚糖A联合），随后测定蛋白结合的3-硝基酪氨酸水平，并释放硝化作用的潜在触发因子(NO， O。（-）(2) H2O2，过氧亚硝酸盐，亚硝酸盐)。3-硝基酪氨酸水平在刺激后16-18 h开始升高，在刺激后20-24 h达到最大值。O的形成。（-）(2)在刺激后1-5 h达到最大值，刺激后5 h降至基线。NO的释放分别在干扰素- γ /脂多糖和干扰素- γ /酶聚糖A刺激后的6小时和9小时达到峰值，随后在接下来的4小时内迅速下降到基线。NO的形成导致亚硝酸盐的积累，在刺激后15小时左右达到50 μ m的水平。只有在细胞因子激活的细胞用12-肉豆酸酯-13-乙酸佛波处理后，过氧亚硝酸盐才会有明显的释放，这导致二氢膦胺氧化增加2.2倍，而3-硝基酪氨酸的水平却没有显著增加。叠氮化物和过氧化氢酶能抑制酪氨酸的硝化作用，亚硝酸盐能模拟酪氨酸的硝化作用。与此同时，NO/O的时间进程也存在显著差异。（-）(2)释放与3-硝基酪氨酸形成的对比，这些结果表明激活的巨噬细胞中的蛋白酪氨酸硝化是由亚硝酸盐依赖的过氧化物酶反应引起的，而不是由过氧亚硝酸盐引起的。
Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O . (-)(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O . (-)(2), H2O2, peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O . (-)(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at similar to6 and similar to9 h after stimulation with interferon-gamma /lipopolysaccharide and interferon-gamma /zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 mum 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O . (-)(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.