GSTP catalyzed reduction of Prdx enhances its
GSTP1-1 catalyzed reduction of Prdx6 enhances its peroxidase activity. Using standard NADPH/GR/GSH-coupled assays we used cell lysates from the transiently transfected Retapamulin to measure the influence of GSTP1 allelic variation on peroxidase activity. Specificity for detection of Prdx6 activity was provided by selection of PLPCOOH as a substrate. The catalytically inactive purified Y7F mutant of GSTP1-1A was the negative control and did not facilitate PLPCOOH reduction (Fig. 3A). For the variants, the hierarchy for reduction of PLPCOOH was GSTP1-1A>GSTP1-1C>GSTP1-1B>GSTP1-1D (all were preloaded with GSH for all in vitro experiments). When PLPCOOH was used as a substrate, preincubation of the cell lysates (or in vitro proteins) with MJ33 (a PLPCOOH binding to Prdx6 inhibitor at 100μM) abolished peroxidase activity (Table 1). For general Prdx6-mediated peroxidase activity H2O2 was also used as a substrate (Fig. 3B). To eliminate the possible effects of catalase, cell lysates were preincubated with 100μM 3-AT. Our results mirrored those for PLPCOOH: GSTP1-1A>GSTP1-1C>GSTP1-1B>GSTP1-1D (with no activity for GSTP1-1A Y7F mutant). Of note, the addition of MJ33 as a specific Prdx6 inhibitor influenced only the reduction of PLPCOOH and not H2O2. All data for peroxidase activities of Prdx6 for the cell lysates and in vitro experiments are summarized in Table 1. For quantification of plasma membrane-associated lipid peroxidation, MCF-7 cells transfected with GSTP1-1 allelic variants were labeled with DPPP, which specifically accumulates in the lipid bilayers of plasma membranes and becomes fluorescent after oxidation by lipid hydroperoxides . After extracellular chemical generation of OH (Cu2+–ASC reaction, ∼100μM), kinetic analyses (Fig. 4A) showed that expression of GSTP1-1A and GSTP1-1C quantitatively suppressed lipid peroxidation in the plasma membranes of MCF-7 cells, whereas expression of the Y7F mutant of GSTP1-1A, GSTP1-1B, and GSTP1-1D showed a rapid (∼300s) accumulation of lipid hydroperoxides. In addition, 36h incubation of MCF-7 cells transfected with GSTP1-1 allelic variants after OH exposure in fresh medium compromised the viability of cells transfected with GSTP1-1B, GSTP1-1D, and the Y7F mutant of GSTP1-1A compared to cells transfected with GSTP1-1A or GSTP1-1C (Fig. 4B). Confocal imaging of WT and transiently transfected MCF-7 cells (labeled with DPPP) showed substantial DPPP-associated fluorescence localized primarily (10min after Cu2+–ASC addition) in plasma membranes of GSTP1-1B-, GSTP1-1D-, and Y7F mutant of GSTP1-1-expressing cells (Fig. 5). Consistent with the kinetic results (Fig. 4A) only minimal DPPP fluorescence was detected in the plasma membranes of GSTP1-1A- and GSTP1-1C-transfected cells (Fig. 5). Activation of Prdx6 by heterodimerization with GSTP requires effective binding interactions of the participating proteins. To determine the affinity of binding for the proteins forming the heterodimer, purified recombinant Prdx6 and GSTP1-1 (A, B, C, D, or Y7F mutant) were used for fluorescent labeling and FRET-based analyses. Our data showed similar high affinities of GSTP1-1A and GSTP1-1C for Prdx6 (Fig. 6, Table 2). Binding affinities for GSTP1-1B and GSTP1-1D for Prdx6 were statistically significantly lower (Fig. 6C). Fig. 6D presents actual volume (amino acids 105 and 114) changes in the vicinity of the allelic variant GSTP1-1 close contact site with Prdx6 (Fig. 7). Our data show small changes for GST1-1C compared to those for GSTP1-1B or GSTP1-1D. Preincubation of Prdx6 with GSTP1-1A before titration with GSTP1-1B (or GSTP1-1C or GSTP1-1D) indicated essentially the same binding sites for each of the GTTP1-1 allelic variants. Only maximal numbers of binding sites (Bmax) and not binding affinities (KD) were altered. The absence of GSH in the binding mixture produced extensive decreases in binding affinity values, indicating that GSH loading of GSTP1-1 plays a crucial role in stabilizing the Prdx6–GSTP1 complex (Table 2).