Light-induced changes in protein nitration in photoreceptor rod outer segments.
Light-induced changes in protein nitration in photoreceptor rod outer segments.
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发表时间：
2006-12
期刊：
影响因子：
2.2
通讯作者：
Vikram Palamalai;R. Darrow;D. Organisciak;M. Miyagi
中科院分区：
文献类型：
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作者：
Vikram Palamalai;R. Darrow;D. Organisciak;M. Miyagi
研究目的光对大鼠视网膜蛋白质硝化的调节作用。为了更好地理解蛋白质硝化在强光诱导的感光细胞死亡中的作用，我们检测了视网膜蛋白质硝化并鉴定了视杆细胞外节（ROS）中的靶蛋白。方法将周期性光照饲养的大鼠暴露于强绿色光下8 h，观察抗氧化剂二甲基硫脲（DMTU）的作用。这些大鼠中的一个子集在8小时的光照后在黑暗中保持24小时。用抗硝基酪氨酸抗体对ROS蛋白进行Western分析，以检查蛋白质硝化的变化。用抗硝基酪氨酸抗体的二维免疫印迹，然后用液相色谱串联质谱法来鉴定ROS中的硝化蛋白。三个一氧化氮合酶（NOS）亚型，诱导型，神经元型和内皮型NOS的表达水平进行了半定量免疫印迹分析。结果Western分析显示，DMTU处理组和未处理组大鼠在光处理8 h后的黑暗恢复期，ROS蛋白硝化水平均升高。然而，DMTU有效地减少了蛋白质硝化活性氧在光暴露期间，并在随后的黑暗恢复期。使用二维免疫印迹，然后液相色谱串联质谱分析，我们确定了十个ROS蛋白硝化目标。这些蛋白质大多是糖酵解酶。视网膜中诱导型NOS的水平随着光照的增加而增加。结论：DMTU降低光诱导过程中和光诱导后ROS蛋白质硝化的作用提示蛋白质硝化参与光诱导的感光细胞死亡。糖酵解酶的硝化作用可以改变其活性。在强光照射期间和之后iNOS水平的增加表明，该同种型负责在黑暗恢复期间强光诱导的ROS中的蛋白质硝化。在光暴露期间在ROS中看到的有限硝化反应可能反映了内源性抗氧化剂对活性氧和氮物种产生的淬灭作用。
PURPOSE Light has been shown to modulate protein nitration in rat retinas. To better understand the role of protein nitration in photoreceptor cell death induced by intense light, we examined retinal protein nitration and identified target proteins in rod outer segments (ROS). METHODS Cyclic light-reared rats, treated or not with the antioxidant, dimethylthiourea (DMTU), were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after 8 h of light exposure. Western analysis of ROS proteins with an anti-nitrotyrosine antibody was performed to examine changes in protein nitration. 2D-immunoblots with anti-nitrotyrosine antibody followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins in ROS. The expression levels of three nitric oxide synthase (NOS) isoforms, inducible, neuronal-, and endothelial-NOS were semi-quantified by immunoblot analysis. RESULTS Western analysis revealed that the level of ROS protein nitration increased during the dark recovery period after 8 h of light treatment in both DMTU treated and untreated rats. However, DMTU effectively reduced protein nitration in ROS during light exposure and during the subsequent dark recovery period. Using 2D-immunoblotting followed by liquid chromatography tandem mass spectrometry analysis, we identified ten ROS proteins as nitration targets. Most of these proteins were glycolytic enzymes. The level of inducible-NOS in the retina was increased by light exposure. CONCLUSIONS The effect of DMTU in reducing ROS protein nitration during and after light suggests the involvement of protein nitration during light-induced photoreceptor cell death. Nitration of glycolytic enzymes specifically may alter their activities. Increased levels of iNOS during and after intense light exposure suggest that this isoform is responsible for intense light induced protein nitration in ROS during the dark recovery period. The limited nitration seen in ROS during light exposure may reflect a quenching effect by endogenous antioxidants on the generation of reactive oxygen and nitrogen species.