Specificity and dynamics of HO detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution.

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]/[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 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 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.