Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation

• Published in: Free radical biology & medicine 2013-01, Vol.54, p.62-70
• Main Authors: Manevich, Y., Hutchens, S., Tew, K.D., Townsend, D.M.
• Format: Article
• Language: English
• Subjects: Alleles; Allelic variants; antioxidants; Apoptosis - genetics; cell death; Cell Membrane - metabolism; Cell Membrane Permeability - genetics; Cytoprotection; fluorescence; Free radicals; genetic polymorphism; Glutathione; Glutathione S-transferase; glutathione transferase; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; human population; Humans; image analysis; Lipid peroxidation; Lipid Peroxidation - genetics; MCF-7 Cells; mutants; Mutation - genetics; oxidants; Oxidative Stress; peroxidase; Peroxidase function; peroxides; peroxiredoxin; Peroxiredoxin VI; Peroxiredoxin VI - metabolism; phospholipids; plasma membrane; Polymorphism, Genetic; Protein Binding - genetics; proteins; Protein–protein interactions; Transgenes - genetics
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2012.10.556

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (∼300s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1A-, GSTP1-1B-, and GST1-1D-expressing cells OH generation resulted (after 36h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (KD=51.0 vs 57.0nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (KD=101.0 (94.0) nM). In silico modeling of the GSTP1-1–Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress. [Display omitted] ► GSTP1-1 allelic variants control plasma membrane lipid peroxidation in MCF-7 cells. ► GSTP1-1A (and -1C) activates peroxidase function of Prdx6 in MCF-7 cells and in vitro. ► GSTP1-1A (and -1C) protein has higher affinity for Prdx6 than GSTP1-1B (and -1D).