Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins
Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins
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DOI：
10.1002/jnr.20771
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发表时间：
2006-03-01
影响因子：
4.2
通讯作者：
Bolognani, F
中科院分区：
文献类型：
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作者：
Bizzozero, OA;Ziegler, JL;Bolognani, F
本研究旨在确定脑谷胱甘肽（GSH）急性耗竭诱导的氧化应激是否足以产生蛋白羰基（PCOs）。为此，将大鼠脑切片分别与GSH耗尽剂1，3-双[2-氯乙基]-1-亚硝基脲（BCNU）和马来酸二乙酯（DEM）孵育，并在用二硝基苯肼衍生后在Western印迹上评估蛋白质羰基化。用1 mM BCNU或10 mM DEM孵育2小时可使GSH水平降低> 70%。在这些条件下，几种蛋白质（40-120 kDa）的羰基化增加了2-3倍。羰基化蛋白质的分离表明，增加的PCO代表氧化蛋白质的量的上升。铁螯合剂去铁胺，超氧化物清除剂芦丁和H2 O2淬灭剂二甲基硫脲都防止DEM诱导的蛋白质羰基化和脂质过氧化（TBARS），表明潜在的机制涉及铁催化产生的羟基自由基从H2 O2（芬顿反应）。用叠氮化钠和氨基三唑抑制过氧化氢酶活性，用巯基琥珀酸抑制谷胱甘肽过氧化物酶活性并没有增加PCOS或TBARS，这表明活性氧（ROS）的产生增加而不是细胞抗氧化防御受损是GSH耗尽后H2 O2积累的原因。PCO的形成不受黄嘌呤氧化酶抑制剂oxypurinol的影响，但SKF-525 A和羰基氰3-氯苯腙可减少PCO的形成，表明微粒体单加氧酶系统和线粒体电子传递系统是ROS的主要来源。与这些发现一致，亚细胞分级分离研究表明，线粒体和突触体是主要的含PCO的细胞器。这些结果也得到了大脑中PCO的解剖分布的支持。我们的观察可能是重要的背景下，多发性硬化症，减少谷胱甘肽，线粒体功能障碍，过量生产的活性氧，增加蛋白质羰基化都有报道。(C)2006 Wiley-Liss，Inc.
This study was aimed at establishing whether oxidative stress induced by acute depletion of brain glutathione (GSH) is sufficient to generate protein carbonyls (PCOs). To this end, rat brain slices were incubated separately with the GSH depletors 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU) and diethyl maleate (DEM), and protein carbonylation was assessed on Western blots after derivatization with dinitrophenyl hydrazine. Incubation with 1 mM BCNU or 10 mM DEM for 2 hr decreased GSH levels by >70%. Under these conditions the carbonylation of several proteins (40-120 kDa) increased by 2-3 fold. Isolation of carbonylated proteins showed that augmented PCOs represents a rise in the amount of oxidized protein. The iron chelator deferoxamine, the superoxide scavenger rutin and the H2O2 quencher dimethylthiourea all prevented DEM-induced protein carbonylation and lipid peroxidation (TBARS), indicating that the underlying mechanism involves the iron-catalyzed generation of hydroxyl radicals from H2O2 (Fenton reaction). Inhibition of catalase activity with sodium azide and aminotriazole, and glutathione peroxidase activity with mercaptosuccinic acid did not increase PCOs or TBARS, suggesting that increased production of reactive oxygen species (ROS) rather than compromised cellular antioxidant defenses is the cause for the accumulation of H2O2 after GSH depletion. PCO formation was not affected by the xanthine oxidase inhibitor oxypurinol but it was reduced by SKF-525A and carbonyl cyanide 3-chlorophenylhydrazone, indicating that the microsomal monooxygenase system and the mitochondrial electron transport system are the major sources of ROS. Consistent with these findings, subcellular fractionation studies showed that mitochondria and synaptosomes are the major PCO-containing organelles. These results were also supported by the anatomic distribution of PCOs in brain. Our observations may be important in the context of multiple sclerosis where decreased GSH, mitochondrial dysfunction, excessive production of ROS, and increased protein carbonylation have all been reported. (C) 2006 Wiley-Liss, Inc.