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Abstract：
The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.
摘要：
硫醇氧化还原态是细胞生物学中蛋白质的决定性功能特征。浆细胞区室维持与谷胱甘肽/谷胱甘肽二硫化物对(GSH/GSSG)和NAD(P)H系统连接的基于硫醇的氧化还原调节网络。基本网络成分是已知的，使用基因编码探针的体内细胞成像揭示了对[GSH]2/[GSSG]氧化还原电位动力学的了解，细胞H2O2和NAD（P）HH的含量取决于代谢和环境线索。较少理解的是网络组件的贡献和相互作用，也是因为遗传方法中的代偿反应。从15种体内浓度的重组蛋白中重建拟南芥的胞质网络，即谷胱甘肽过氧化物酶样（GPXL），过氧化物酶(PRX)，谷氧还蛋白（GRX），硫氧还蛋白,NADPH依赖性硫氧还蛋白还原酶A和谷胱甘肽还原酶，并应用Grx1-roGFP2或roGFP2-Orp1作为动态传感器，允许监测对单个H2O2脉冲的响应。通过基于质谱的蛋白质组学定量的巯基氧化的主要变化发生在GPXL的相关肽中，在较小程度上的PRX，而其他含Cys的肽仅显示其氧化还原状态和保护作用的微小变化。将抗坏血酸过氧化物酶（APX）与脱氢抗坏血酸还原酶一起滴定到系统中，降低了网络中荧光传感器的氧化，但无法抑制它。结果表明，该网络具有使H2O2解毒的能力，电子流的部分独立分支对特定细胞信号传导具有重要意义，并且APX在不抑制信号传导和将负担转移到谷胱甘肽氧化的情况下调节信号传导的重要性。
