title: Functional interaction of glutathione S-transferase pi and peroxiredoxin 6 in intact cells.
abstract: Peroxiredoxin 6 (Prdx6) is a 1-Cys member of the peroxiredoxin superfamily that plays an important role in antioxidant defense. Glutathionylation of recombinant Prdx6 mediated by π glutathione S-transferase (GST) is required for reduction of the oxidized Cys and completion of the peroxidatic catalytic cycle in vitro. This study investigated the requirement for πGST in intact cells. Transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increased phospholipid peroxidase activity as measured in cell lysates and protected intact cells against a peroxidative stress. siRNA knockdown indicated that this increased peroxidase activity was Prdx6 dependent. Interaction between πGST and Prdx6, evaluated by the Duolink Proximity Ligation Assay, was minimal under basal conditions but increased dramatically following treatment of cells with the oxidant, tert-butyl hydroperoxide. Interaction was abolished by mutation of C47, the active site for Prdx6 peroxidase activity. Depletion of cellular GSH by treatment of cells with buthionine sulfoximine had no effect on the interaction of Prdx6 and πGST. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST and that the interaction plays an important role in regenerating the peroxidase activity of Prdx6.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6068, United States.
authors.forename: Suiping
authors.initials: S
authors.lastname: Zhou
authors.forename: Yu-Chin
authors.initials: YC
authors.lastname: Lien
authors.forename: Tea
authors.initials: T
authors.lastname: Shuvaeva
authors.forename: Kristine
authors.initials: K
authors.lastname: DeBolt
authors.forename: Sheldon I
authors.initials: SI
authors.lastname: Feinstein
authors.forename: Aron B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000166:cytology; D005978:Glutathione / Q000378:metabolism; D051549:Glutathione S-Transferase pi / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000098:blood supply; D061986:MCF-7 Cells; D051379:Mice; D055806:Microvessels / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress; D054465:Peroxiredoxin VI / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005978:Glutathione