Baty James W, Hampton Mark B, Winterbourn Christine C
Free Radical Research Group, Department of Pathology, Christchurch School of Medicine and Health Sciences, PO Box 4345, Christchurch, New Zealand.
Biochem J. 2005 Aug 1;389(Pt 3):785-95. doi: 10.1042/BJ20050337.
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 microM 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 BUB1beta, HSP90beta (heat-shock protein 90beta) and proteosome components. At 20 microM 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.
硫醇蛋白在细胞抗氧化防御和氧化还原信号传导中起着重要作用。据推测，活性氧化剂会导致选择性硫醇氧化，但不同细胞蛋白和关键靶点的相对敏感性尚未得到很好的表征。我们将Jurkat细胞暴露于过氧化氢中10分钟，并通过用碘乙酰胺荧光素标记和二维电泳来测量可逆氧化蛋白的变化。在200微摩尔过氧化氢浓度下，该浓度会导致丝裂原活化蛋白（MAP）激酶细胞外信号调节激酶（ERK）的激活、生长停滞和凋亡，观察到的变化相对较少。共有28个斑点发生可逆氧化（标记强度增加），24个斑点标记强度降低。后者包括过氧化物还原酶1和2的亚型，它们发生了不可逆氧化。甘油醛-3-磷酸脱氢酶（GAPDH）的氧化很显著，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BUB1β、热休克蛋白90β（HSP90β）和蛋白酶体成分。在20微摩尔过氧化氢浓度下，变化较少，但GAPDH和过氧化物还原酶2仍被修饰。二硝基氯苯处理抑制了细胞硫氧还蛋白还原酶并部分耗尽了谷胱甘肽，导致几种蛋白质发生可逆氧化，包括硫氧还蛋白1以及过氧化物还原酶1和2。大多数变化与过氧化氢处理的不同，二硝基氯苯几乎没有增强过氧化氢处理所引起的变化。相对较少的蛋白质，包括脱氧胞苷激酶、核苷二磷酸激酶和一种蛋白酶体激活亚基，仅对联合处理有反应。因此，过氧化氢的大多数作用与硫氧还蛋白氧化无关。我们的研究已确定过氧化物还原酶2和GAPDH是两种对氧化剂最敏感的细胞蛋白，并突出了过氧化物还原酶多么容易发生不可逆氧化。
