Abstract
Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.
Highlights
Encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events
We have demonstrated that the diffusion of H2O2 across fission yeast membranes is limited, and that scavenging mainly by Tpx[1] enhances the gradients up to 300:111
We have demonstrated that steady-state H2O2 levels, enhanced in Δtpx[1], can change basal oxidation of the roGFP2Tpx[1].C169S probe, OxD0 (Fig. 3b)
Summary
Encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. We use a new probe, roGFP2-Tpx[1].C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. The probe detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. Aerobic organisms inevitably produce reactive oxygen species as a side effect of sequential one-electron reduction of oxygen during respiration, among other processes One of these species, hydrogen peroxide (H2O2), has been traditionally linked to the toxicity associated to aerobic metabolism. Very few Cys in proteins fulfill the prerequisites for fast and efficient oxidation by moderate fluctuations of peroxides. Among the proteins unambiguously responding to pre-toxic doses of
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