The effect of Peroxiredoxin 6 (Prdx6) mutation at its active site on its interaction with piGST

Abstract

Prdx6, a 1‐Cys member of the selenium‐independent peroxiredoxin superfamily, has both peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of recombinant Prdx6 mediated by πGST is required for the peroxidatic catalytic cycle (PNAS 101: 3780, 2004). In a previous abstract, we reported that transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increases the lipid peroxidase activity in the cells. A dramatic increase in the interaction between Prdx6 and πGST in cells upon their treatment with tert‐butyl hydroperoxide was shown by the Duolink Proximity Ligation Assay (DPLA). To examine the role of the peroxidase or PLA2 active site amino acids of Prdx6 on interactions with πGST, plasmids expressing mutated Prdx6 (C47S or S32A) were transfected into Prdx6 null mouse pulmonary microvascular endothelial cells. Interaction between the 2 proteins as shown by DPLA was abolished by mutation at the active site for peroxidase activity (C47), but mutation of the active site for PLA2 activity (S32) had no effect. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST.