Peroxiredoxins are a class of abundant antioxidant proteins found in both prokaryotic and eukaryotic cells. Hydrogen peroxide (H2O2) specifically oxidizes these proteins at a catalytic cysteine residue near the N-terminus of the protein; oxidation of this residue causes a conformational change that results in formation of a disulfide bond between a nearby cysteine residue on the same or on a different peroxiredoxin monomer. While once thought to act purely as H2O2 scavengers in cells, several recent studies have suggested that peroxiredoxins also have key roles in signaling pathways in mammalian cells, and there is now considerable interest in the role that the oxidation state of these proteins plays in these different signaling pathways. To date, determining the oxidation state of these proteins in living cells in real time has not been possible. In order to address this problem, we developed a genetically-encoded FRET probe composed of two peroxiredoxin-2 monomers linked by a long flexible linker and flanked by two fluorescent proteins capable of FRET to monitor the oxidation-dependent dimerization of peroxiredoxin-2 in live cells. We characterized this probe in terms of selectivity towards H2O2 as well as sensitivity to different concentrations of external H2O2. The probe responded specifically to hydrogen peroxide, and we were able to detect external peroxide concentrations in the nanomolar range in our experiments, corresponding to picomolar changes in the intracellular concentration. In addition, we were able to use this probe to detect an increase in peroxiredoxin-2 oxidation in response to the cancer therapeutic piperlongumine in a culture of HeLa cells expressing the probe in the cytoplasm. Unlike current methods to detect the oxidation state of peroxiredoxins, which require lysis of cells, this probe allows measurement of the peroxiredoxin-2 oxidation state in living cells, opening up the possibility of observing dynamic signaling processes.

Imaging the oxidation state of peroxiredoxin-2 in living cells

251st National Meeting (2016)

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