Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells
Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells
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DOI：
10.1042/bj20050337
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发表时间：
2005-08-01
影响因子：
4.1
通讯作者：
Winterbourn, CC
中科院分区：
文献类型：
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作者：
Baty, JW;Hampton, MB;Winterbourn, CC
巯基蛋白在细胞抗氧化防御和氧化还原信号传导中是重要的。据推测，反应性氧化剂导致选择性巯基氧化，但不同的细胞蛋白质和关键目标的相对敏感性没有得到很好的表征。我们将Jurkat细胞暴露于H2 O2 10分钟，并通过碘乙酰胺荧光素标记和二维电泳测量可逆氧化蛋白的变化。在200 μ M的H2 O2，这导致激活的MAP（丝裂原活化蛋白）激酶ERK（细胞外信号调节激酶），生长停滞和凋亡，相对较少的变化。共有28个斑点可逆氧化（标记强度增加），24个斑点减少。后者包括过氧化物酶1和2的亚型，它们被不可逆地氧化。GAPDH（甘油醛-3-磷酸脱氢酶）的氧化是惊人的，其他受影响的蛋白质包括谷胱甘肽S-转移酶P1-1，烯醇化酶，蛋白激酶A的调节亚基，膜联蛋白VI，有丝分裂检查点丝氨酸/苏氨酸蛋白激酶BUB 1 β，HSP 90 β（热休克蛋白90 β）和蛋白体成分。在20 μ M H2 O2时，变化较少，但GAPDH和peroxiredoxin 2仍被修饰。二硝基氯苯治疗，抑制细胞硫氧还蛋白还原酶和部分耗尽GSH，引起可逆氧化的几种蛋白质，包括硫氧还蛋白1和过氧化物酶1和2。大多数变化与H2 O2不同，与H2 O2的变化几乎没有增强二硝基氯苯。相对较少的蛋白质，包括脱氧胞苷激酶，核苷二磷酸激酶和蛋白酶体激活剂亚基，仅对联合治疗有反应。因此，H2 O2的大部分作用与硫氧还蛋白氧化无关。我们的研究已经确定peroxiredoxin 2和GAPDH作为两个最氧化剂敏感的细胞蛋白，并强调如何容易peroxiredoxins进行不可逆氧化。
Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 mu M H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3 -phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1 beta, HSP90 beta (heat-shock protein 90 beta) and proteosome components. At 20 mu M H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.