Proteome-wide Light/Dark Modulation of Thiol Oxidation in Cyanobacteria Revealed by Quantitative Site-specific Redox Proteomics
Proteome-wide Light/Dark Modulation of Thiol Oxidation in Cyanobacteria Revealed by Quantitative Site-specific Redox Proteomics
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DOI：
10.1074/mcp.m114.041160
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发表时间：
2014-12-01
影响因子：
7
通讯作者：
Qian, Wei-Jun
中科院分区：
文献类型：
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作者：
Guo, Jia;Nguyen, Amelia Y.;Qian, Wei-Jun
蛋白质巯基氧化是光合生物光合作用、代谢和基因表达的重要调控机制。在此，我们提出了蛋白质组范围内的定量和位点特异性的分析在体内巯基氧化调制的光/暗的蓝藻集胞藻属PCC 6803，产氧光合原核生物，使用树脂辅助巯基富集的方法。我们的蛋白质组学方法集成了树脂辅助富集与同量异位串联质量标签标记，使可逆氧化硫醇的位点特异性和定量测量。在光、暗和3-（3，4-二氯苯基）1，1-二甲基脲（一种光系统II抑制剂）条件下，对来自1，060种蛋白质的类似于2，100个Cys位点的氧化还原动力学进行了定量。除相对定量外，还定量了类似于1，350个Cys位点的氧化的化学计量或百分比（可逆氧化/总硫醇）。总体结果显示，在许多关键的生物过程，包括光合电子传递，碳固定，糖酵解硫醇氧化的广泛变化。此外，氧化还原灵敏度沿着化学计量数据使得能够预测感兴趣的蛋白质的潜在功能性Cys位点。氧化还原敏感的Cysites在NADP依赖的甘油醛-3-磷酸脱氢酶，过氧化物氧还蛋白（AhpC/TSA家族蛋白Sll 1621），和葡萄糖6-磷酸脱氢酶的功能意义，进一步证实了定点诱变和生化研究。总之，我们的研究结果提供了重要的见解光合生物的广泛的氧化还原调节。
Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of similar to 2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for similar to 1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover, the redox sensitivity along with the stoichiometric data enabled prediction of potential functional Cys-sites for proteins of interest. The functional significance of redox-sensitive Cyssites in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin (AhpC/TSA family protein Sll1621), and glucose 6-phosphate dehydrogenase was further confirmed with site-specific mutagenesis and biochemical studies. Together, our findings provide significant insights into the broad redox regulation of photosynthetic organisms.