Evidence for Thiol/Disulfide Exchange Reactions Between Tubulin and Glyceraldehyde-3-Phosphate Dehydrogenase
Evidence for Thiol/Disulfide Exchange Reactions Between Tubulin and Glyceraldehyde-3-Phosphate Dehydrogenase
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DOI：
10.1002/cm.21204
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发表时间：
2014-12-01
期刊：
影响因子：
2.9
通讯作者：
Kennett, Kelly L.
中科院分区：
文献类型：
--
作者：
Landino, Lisa M.;Hagedorn, Tara D.;Kennett, Kelly L.
虽然硫醇氧化还原反应是调节蛋白质结构和功能的常见机制，但蛋白质二硫键的形成是氧化应激的标志，与神经退化有关。微管蛋白和甘油醛-3-磷酸脱氢酶都含有多个半胱氨酸，这些半胱氨酸被确定为氧化成二硫化物、S亚硝化和S谷胱甘肽基化的靶标。我们发现，GAPDH是除了MAP-2和tau之外的三个重要的脑微管相关蛋白(MAP)之一，具有反应性半胱氨酸。我们通过5-碘代乙酰氨基荧光素标记和免疫印迹检测高分子量链间微管蛋白二硫化物，检测到在兔肌肉GAPDH存在下，过氧化氢对微管蛋白半胱氨酸的氧化作用增加了三到四倍。在硫醇/二硫键交换实验中，微管蛋白恢复到50%的氧化GAPDH半胱氨酸水平，平衡状态有利于还原GAPDH值。此外，我们还报道了氧化的GAPDH被硫氧还蛋白还原酶系统(TRS)修复。被微管蛋白和TRS还原后，GAPDH活性的恢复是时间依赖的，这表明活性部位半胱氨酸149附近的构象发生了变化。脑MAP与氧化微管蛋白的添加减少，微管蛋白二硫键减少，MAP-2和GAPDH的标记减少。由于氧化的GAPDH的微管蛋白修复程度依赖于缓冲强度，因此我们得出结论，静电影响两种蛋白质之间的硫醇/二硫键交换。本文提出的新的相互作用可能保护GAPDH在氧化应激条件下免受抑制。(C)2014年威利期刊公司。
While thiol redox reactions are a common mechanism to regulate protein structure and function, protein disulfide bond formation is a marker of oxidative stress that has been linked to neurodegeneration. Both tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contain multiple cysteines that have been identified as targets for oxidation to disulfides, S-nitrosation and S-glutathionylation. We show that GAPDH is one of three prominent brain microtubule-associated proteins (MAPs), in addition to MAP-2 and tau, with reactive cysteines. We detected a threefold to fourfold increase in tubulin cysteine oxidation by hydrogen peroxide in the presence of rabbit muscle GAPDH by 5-iodoacetamidofluorescein labeling and by Western blot detection of higher molecular weight inter-chain tubulin disulfides. In thiol/disulfide exchange experiments, tubulin restored similar to 50% of oxidized GAPDH cysteines and the equilibrium favored reduced GAPDH. Further, we report that oxidized GAPDH is repaired by the thioredoxin reductase system (TRS). Restoration of GAPDH activity after reduction by both tubulin and the TRS was time-dependent suggesting conformational changes near the active site cysteine149. The addition of brain MAPs to oxidized tubulin reduced tubulin disulfides and labeling of MAP-2 and of GAPDH decreased. Because the extent of tubulin repair of oxidized GAPDH was dependent on buffer strength, we conclude that electrostatics influence thiol/disulfide exchange between the two proteins. The novel interactions presented herein may protect GAPDH from inhibition under oxidative stress conditions. (C) 2014 Wiley Periodicals, Inc.