title: Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation.
abstract: 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 (~300 s) 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 36 h) 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 (K(D)=51.0 vs 57.0 nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (K(D)=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.
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA. manevich@musc.edu
authors.forename: Y
authors.initials: Y
authors.lastname: Manevich
authors.forename: S
authors.initials: S
authors.lastname: Hutchens
authors.forename: K D
authors.initials: KD
authors.lastname: Tew
authors.forename: D M
authors.initials: DM
authors.lastname: Townsend
mesh_terms: D000483:Alleles; D017209:Apoptosis / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000235:genetics; D019610:Cytoprotection; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015227:Lipid Peroxidation / Q000235:genetics; D061986:MCF-7 Cells; D009154:Mutation / Q000235:genetics; D018384:Oxidative Stress; D054465:Peroxiredoxin VI / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011485:Protein Binding / Q000235:genetics; D019076:Transgenes / Q000235:genetics
chemical_list: D054465:Peroxiredoxin VI; D005982:Glutathione Transferase