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Conflict of interest
Peroxiredoxin VI (Prdx6) is a dual-functioning antioxidant enzyme that acts to protect biological membranes against damage caused by lipid peroxidation , . The enzyme displays glutathione peroxidase and calcium-independent PLA, activities with specificity for phospholipid hydroperoxides providing physiological protection after cell exposure to oxidative stress. The glutathione peroxidase activity of Prdx6 is regulated by initial heterodimerization with, and subsequent S-glutathionylation by, glutathione-loaded glutathione -transferase Pi (GSTP) , , . One distinctive function of GSTP is its ability to form a redox complex with a target protein with subsequent delivery of bound and activated glutathione (thiolate anion) for S-glutathionylation of target proteins . In the case of Prdx6, the catalytic Cys47 is buried in the hydrophobic globular core region of the protein. After reduction of peroxides, this cysteine becomes oxidized to a sulfenate and GSTP participates in overcoming the accessibility barrier for glutathione (GSH) to reach this hydrophobic region and reactivate Prdx6. Glutathione -transferases are multifunctional isoenzymes that can detoxify xenobiotics and endogenous metabolites primarily by catalyzing their conjugation with GSH , . GSTP1-1 is overexpressed in tumors and in cell lines selected for resistance to certain anticancer agents and has been shown to be polymorphic , a characteristic that imbues potential selective catalytic properties on the individual isozymes. The genetic polymorphisms in the GSTP1 gene arise from nucleotide transitions that change cgrp antagonist 105 from Ile to Val and codon 114 from Ala to Val, generating four GSTP1 alleles: wild-type GSTP1-1A (Ile105/Ala114), GSTP1-1B (Val105/Ala114), GSTP1-1C (Val105/Val114), and GSTP1-1D (Ile105/Val114) , . The Ile105→Val105 and Ala114→Val114 substitutions do not alter glutathione-binding affinity, but cause a steric change at the substrate-binding site of the enzyme , . The GSTP variants have different catalytic rates for the formation of thioether conjugates between GSH and some small-molecule electrophiles , . Altered conformation of the substrate-binding site(s) may contribute to final substrate specificity. The hydrophobicity and size of residue 114 could serve as an important determinant of the substrate specificity of each of the GSTP1 isozymes . On the other hand, because GSTP1-1D, bearing Ile105 and Val114, has enzyme activity toward CDNB (1-Chloro-2,4-dinitrobenzene) comparable to that of GSTP1-1A, Val105 may circumvent the influence of Val114 . There are some examples in which distinct polymorphisms influence response to specific anticancer drugs. For example, GSTP1-1A has a role in the acquisition of cisplatin resistance reportedly through enhancing the formation of platinum–glutathione conjugates . There are also some indications of epidemiological correlations for GSTP isotype expression with etiological aspects of endometrial , bladder, and testicular cancers . In this report we sought to determine if polymorphic variants of GSTP1 have a general impact on the response to oxidative stress by directly influencing the peroxidase functions of Prdx6. Materials and methods
Results Unlike most tumor cell lines, MCF-7 cells have intrinsically low expression levels of GSTP1-1 . Prdx6 expression in these cells was measureable and was unaffected by forced expression of various GSTP1-1 allelic variants (Figs. 1A, 2C, and 2D). Our data confirm both barely detectable levels of GSTP1 and appropriate expression levels of GSTP1-1 after selection of pertinent clones of transfectants (WT transfected with empty vector (V), Y7F mutant, and allelic variants; Figs. 1B, 2A, and 2B). GSTM was detected in MCF-7 cells, but was unaffected by expression of GSTP1-1 allelic variants (Fig. 2B). In contrast, levels of PhGPx (GPx4) expression were decreased by expression of GSTP1-1 allelic variants (Fig. 2D). The redox state of Prdx6 is regulated through its heterodimerization with GSTP1, which serves to deliver activated GSH (GS−) as a source of electrons for reduction of its catalytic cysteine sulfenate. Consequently, disulfide-based homo/heterodimerization of Prdx6 correlates with protein reduction (activation). We used TCEP to reduce the disulfide of Prdx6 homo/heterodimerization (Fig. 1A, lower and upper arrows, respectively). Quantification of Prdx6 monomer/dimer patterns (from Fig. 1A) was influenced by the GSTP1-1 variant expression. For example, either GSTP1-1A or GSTP1-1C was more effective at forming the heterodimer with Prdx6 than either GSTP1-1B or GSTP1-1D (Figs. 1C and 1D). This would be expected to result in better reduction (activation) of Prdx6 by either GSTP1-1A or GSTP1-1C. Expression and purification of His-tagged GSTP variants in E. coli provided a source of essentially homogeneous proteins, which were used for subsequent in vitro experiments (Fig. 1E).