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Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution
Redox Biology ( IF 11.4 ) Pub Date : 2024-04-03 , DOI: 10.1016/j.redox.2024.103141
Lara Vogelsang , Jürgen Eirich , Iris Finkemeier , Karl-Josef Dietz

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 cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]/[GSSG] redox potential, cellular HO 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 from fifteen recombinant proteins at 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 HO 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 HO, 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.

中文翻译:


体外重建揭示胞质氧化还原调节网络 H2O2 解毒的特异性和动态



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。浆细胞区室维持着与谷胱甘肽/谷胱甘肽二硫化物对 (GSH/GSSG) 和 NAD(P)H 系统相连的基于硫醇的氧化还原调节网络。基本网络成分是已知的,并且使用基因编码探针的细胞成像揭示了对 [GSH]/[GSSG] 氧化还原电位、细胞 H2O 和 NAD(P)H+H 量依赖于代谢和环境线索的动态的深入了解。人们对网络组成部分的贡献和相互作用知之甚少,这也是因为遗传方法中的补偿反应。重建十五种浓度重组蛋白的胞质网络,即谷胱甘肽过氧化物酶样 (GPXL)、过氧化还原蛋白 (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,可监测对单个 HO 脉冲的响应。通过基于质谱的蛋白质组学定量,硫醇氧化的主要变化发生在 GPXL 的相关肽中,PRX 的程度较小,而其他含 Cys 的肽仅在氧化还原状态和保护方面显示出较小的变化。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可以降低网络中荧光传感器的氧化,但无法抑制它。结果证明了网络解毒 H2O 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,以及 APX 在调节信号传导而不抑制信号传导并将负担转移到谷胱甘肽氧化方面的重要性。
更新日期:2024-04-03
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