Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes

Bruce Morgan, Koen Van Laer, Theresa N E Owusu, Daria Ezeriņa, Daniel Pastor-Flores, Prince Saforo Amponsah, Anja Tursch, Tobias P Dick

Onderzoeksoutput: Articlepeer review

182 Citaten (Scopus)

Samenvatting

Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H2O2 probes, roGFP2-Tsa2ΔCR and roGFP2-Tsa2ΔCPΔCR, that afford such sensitivity. These probes are ∼50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H2O2 homeostasis. The roGFP2-Tsa2ΔCR probe revealed real-time interplay between basal H2O2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H2O2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H2O2 levels and cellular growth rate.

Originele taal-2English
Pagina's (van-tot)437-443
Aantal pagina's7
TijdschriftNature Chemical Biology
Volume12
Nummer van het tijdschrift6
DOI's
StatusPublished - jun 2016

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