Nitric oxide-dependent generation of reactive species in sickle cell disease - Actin tyrosine nitration induces defective cytoskeletal polymerization
Nitric oxide-dependent generation of reactive species in sickle cell disease - Actin tyrosine nitration induces defective cytoskeletal polymerization
复制标题
DOI：
10.1074/jbc.m208916200
复制
发表时间：
2003-02-07
影响因子：
4.8
通讯作者：
Freeman, BA
中科院分区：
文献类型：
--
作者：
Aslan, M;Ryan, TM;Freeman, BA
镰状细胞病（SCD）中发生的间歇性血管闭塞导致缺血-再灌注损伤和炎症过程的活化，包括活性氧的产生增强和诱导型一氧化氮合酶（NOS 2）的表达增加。认识到受损的一氧化氮依赖性血管功能以及伴随的氧化和硝化物质的形成与组织活性氧物质产生的速率增加一致，在人SCD组织和SCD小鼠模型中评价了肝脏和肾脏NOS 2表达、组织3-硝基酪氨酸（NO（2）Tyr）形成和细胞凋亡。SCD小鼠和人的肝脏和肾脏NOS 2表达和NO（2）Tyr免疫反应性显著增加，但在非病变组织中没有增加。TdT介导的缺口末端标记（TUNEL）染色显示所有SCD组织中NOS 2和NO（2）Tyr表达水平升高的区域中存在凋亡细胞。气相色谱质谱分析显示，SCD小鼠血浆蛋白NO（2）Tyr含量增加，肝脏和肾脏蛋白NO（2）Tyr衍生物水平增加（分别为21.4 +/- 2.6和37.5 +/- 7.8 ng/mg），野生型小鼠分别为8.2 +/- 2.2和10 +/- 1.2 ng/mg。免疫印迹分析和免疫沉淀SCD小鼠肝脏和肾脏蛋白揭示了一个主要的NO（2）酪氨酸蛋白的42 kDa，与对照组相比。酶的凝胶内消化和MALDI-TOF质谱鉴定这种硝化蛋白肌动蛋白。电喷雾离子化和串联质谱片段分析显示，15个肌动蛋白酪氨酸残基中有3个被硝化（Tyr（91），Tyr（198）和Tyr（240）），其位置显著改变肌动蛋白组装。SCD人类和小鼠组织的共聚焦显微镜显示，硝化导致形态上不同的丝状肌动蛋白的解体。总之，我们已经观察到介导血红蛋白聚合缺陷的镰状细胞病的血红蛋白点突变通过炎症氧化反应被翻译成有缺陷的细胞骨架聚合。
The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.