Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation |
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| Affiliation: | 1. Department of Cell and Molecular Pharmacology and Experimental Therapeutics;2. Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA;1. Programa de Pós-Graduação em Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Campus Universitário, Camobi, 97105-900 Santa Maria, RS, Brazil;2. Setor de Hematologia/Oncologia, Hospital Universitário de Santa Maria, Universidade Federal de Santa Maria, Campus Universitário, Camobi, 97105-900 Santa Maria, RS, Brazil;1. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA;2. Department of Medicine, Division of Hematology/Oncology, University of California San Diego, San Diego, CA, USA;3. Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, USA;4. Department of Neurosurgery, University of Southern California, Keck School of Medicine, Los Angeles, CA;5. Department of Neurosurgery, Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA;1. Department of Biomedical Sciences, Ohio University, Athens, OH 45701, USA;2. Program in Molecular and Cellular Biology, Ohio University, Athens, OH 45701, USA;3. The Diabetes Institute at Ohio University, Ohio University, Athens, OH 45701, USA;4. OUHCOM Office of Research & Grants, Ohio University, Athens, OH 45701, USA;5. College of Natural Sciences, School of Biological Sciences, University of Texas at Austin, TX 78713, USA |
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| 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 GSTP1-1 A (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 GSTP1-1 A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1 A-, 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 (KD=51.0 vs 57.0 nM), 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. |
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