Seres T, Ravichandran V, Moriguchi T, Rokutan K, Thomas J A, Johnston R B
Department of Pediatrics (Immunology), Yale University School of Medicine, New Haven, CT 06520, USA.
J Immunol. 1996 Mar 1;156(5):1973-80.
Stimulation of the respiratory burst in mouse macrophages or human neutrophils results in the formation of disulfide bonds between low m.w. thiols and sulfhydryl groups on specific cytosolic proteins (S-thiolation). S-thiolation is reversible in certain chemical systems. The aim of the present study was to analyze the dynamic nature of this process in human monocytes under physiologic conditions. We report here that the extent of S-thiolation and the rate of respiratory burst stimulated by opsonized zymosan or phorbol diester increased for 10 to 20 min and then declined (dethiolation) in close association. Individual proteins underwent S-thiolation and dethiolation at different rates. H2O--appeared particularly effective in mediating S-thiolation, based on inhibition of S-thiolation by added catalase and accentuation by azide, which inhibits cellular catalase. S-thiolation did not occur in stimulated monocytes from patients with chronic granulomatous disease. The addition of H2O2 to monocytes or lymphocytes induced rapid S-thiolation (1 to 3 min); a subsequent dethiolation returned most of the proteins to baseline by 15 to 30 min. At 0 degrees C and after addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea, there was effective S-thiolation on exposure to H2O2, but dethiolation was inhibited, suggesting a possible role for glutathione (GSH)/thioredoxin reductase systems in this process. GSH was determined to be the most abundant low m.w. thiol bound to S-thiolated proteins, but gamma-glutamylcysteine and cysteine were also bound. The time of maximal reduction in cytosolic GSH during the respiratory burst (10 min) coincided with the time at which protein-bound GSH was highest. S-thiolation-dethiolation represents a reversible post-translational modification that could protect cellular proteins from irreversible oxidative damage.
刺激小鼠巨噬细胞或人中性粒细胞的呼吸爆发会导致低分子量硫醇与特定胞质蛋白上的巯基之间形成二硫键（S-硫醇化）。在某些化学体系中，S-硫醇化是可逆的。本研究的目的是分析生理条件下人单核细胞中这一过程的动态特性。我们在此报告，经调理的酵母聚糖或佛波酯刺激后，S-硫醇化程度和呼吸爆发速率在10至20分钟内升高，然后密切相关地下降（脱硫醇化）。个别蛋白质经历S-硫醇化和脱硫醇化的速率不同。基于添加过氧化氢酶对S-硫醇化的抑制作用以及叠氮化物（抑制细胞过氧化氢酶）的增强作用，H₂O₂似乎在介导S-硫醇化方面特别有效。慢性肉芽肿病患者受刺激的单核细胞中未发生S-硫醇化。向单核细胞或淋巴细胞中添加H₂O₂会诱导快速的S-硫醇化（1至3分钟）；随后的脱硫醇化在15至30分钟内使大多数蛋白质恢复到基线水平。在0℃以及添加1,3-双（2-氯乙基）-1-亚硝基脲后，暴露于H₂O₂时会发生有效的S-硫醇化，但脱硫醇化受到抑制，这表明谷胱甘肽（GSH）/硫氧还蛋白还原酶系统在这一过程中可能发挥作用。GSH被确定为与S-硫醇化蛋白结合的最丰富的低分子量硫醇，但γ-谷氨酰半胱氨酸和半胱氨酸也会结合。呼吸爆发期间胞质GSH最大程度减少的时间（10分钟）与蛋白结合GSH最高的时间一致。S-硫醇化-脱硫醇化代表一种可逆的翻译后修饰，可保护细胞蛋白质免受不可逆的氧化损伤。
