Functional Interaction of piGST and Peroxiredoxin 6 (Prdx6) in Cultured Cells

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Prdx6 is a moonlighting protein that has both PLA2 and peroxidase activities. Partial purification of native enzyme from bovine lung revealed the presence of πGST in an active preparation and we have observed that purified recombinant Prdx6 from E coli generally lacks peroxidase activity unless incubated with GSH‐loaded πGST. We provided direct evidence for the formation of a complex between Prdx6 and πGST in vitro (Biochemistry, 2006, Vol. 45, p360), and showed that delivery of recombinant Prdx6 into cells (MCF7) that lack πGST did not increase the phospholipid hydroperoxide peroxidase activity unless πGST was co‐delivered (PNAS, 2004, Vol. 101, p3780). These data indicate an in vitro interaction between Prdx6 and πGST that is required for the peroxidase activity of Prdx6. To evaluate this functional interaction in vivo, we used the Duolink Proximity Ligation Assay to explore the interaction of πGST and Prdx6 in vivo. There is a dramatic increase in interaction between endogenous πGST and Prdx6 in mouse pulmonary microvascular endothelial cells (MPMVEC) upon their treatment with tert‐butyl hydroperoxide. Furthermore, transfection of MCF7 cells with a vector expressing πGST increases the phospholipid hydroperoxide peroxidase activity in the cells, presumably due to activation of Prdx6 by πGST. These in vivo studies provide evidence that πGST functionally interacts with Prdx6 in the intact cell.