Pace Paul E, Fu Ling, Hampton Mark B, Winterbourn Christine C
Mātai Hāora - Centre for Redox Biology & Medicine, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Lifeomics, Beijing, 102206, China.
Free Radic Biol Med. 2025 Feb 1;227:221-232. doi: 10.1016/j.freeradbiomed.2024.10.314. Epub 2024 Nov 1.
Oxidation of thiol proteins and redox signaling occur in cells exposed to HO but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of HO with CO/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with HO were measured by isotopically labeling reduced thiols and analysis by mass spectrometry. The effects of bicarbonate and of knocking out either Prdx1 or Prdx2 were examined. Approximately 14,000 Cys-peptides were detected, of which ∼1 % underwent 2-10 fold loss in thiol content with HO. Those showing the most oxidation were not affected by the presence of bicarbonate or knockout of either Prdx. Consistent with previous evidence that bicarbonate potentiates inactivation of glyceraldehyde-3-phosphate dehydrogenase, the GAPDH active site Cys residues were significantly more sensitive to HO when bicarbonate was present. Several other proteins were identified as promising candidates for further investigation. Although we identified some potential protein candidates for Prdx-dependent oxidation, most of the significant differences between KO and WT cells were seen in proteins for which HO unexpectedly increased their CysSH content over untreated cells. We conclude that facilitation of protein oxidation by bicarbonate or Prdx-mediated relays is restricted to a small number of proteins and is insufficient to explain the majority of the oxidation of the cell thiols that occured in response to HO.
暴露于羟基自由基（HO）的细胞中会发生硫醇蛋白的氧化和氧化还原信号传导，但具体机制尚不清楚。我们运用氧化还原蛋白质组学，来探寻特定蛋白质氧化的证据，其机制要么涉及HO与CO/碳酸氢盐反应生成活性更强的过氧一碳酸酯，要么通过过氧化物还原酶（Prdxs）介导的反应。通过对还原型硫醇进行同位素标记并用质谱分析，来测定用HO处理Jurkat T淋巴瘤细胞时特定半胱氨酸巯基（Cys-SH）残基氧化态的变化。研究了碳酸氢盐以及敲除Prdx1或Prdx2的影响。检测到约14,000个半胱氨酸肽段，其中约1%的肽段在HO处理后硫醇含量下降了2至10倍。那些显示出最大氧化程度的肽段不受碳酸氢盐存在与否或敲除任一Prdx的影响。与先前关于碳酸氢盐增强甘油醛-3-磷酸脱氢酶失活的证据一致，当存在碳酸氢盐时，甘油醛-3-磷酸脱氢酶活性位点的半胱氨酸残基对HO更为敏感。还鉴定出了其他几种有望进一步研究的蛋白质。尽管我们确定了一些可能通过Prdx依赖机制发生氧化的蛋白质候选物，但敲除组（KO）和野生型（WT）细胞之间的大多数显著差异出现在HO处理后其半胱氨酸巯基（CysSH）含量比未处理细胞意外增加的蛋白质中。我们得出结论，碳酸氢盐或Prdx介导的反应促进蛋白质氧化仅限于少数蛋白质，不足以解释细胞中大多数因HO而发生的硫醇氧化。
