Noncatalytic Interactions between Glutathione S-Transferases and Nitroalkene Fatty Acids Modulate Nitroalkene-Mediated Activation of Peroxisomal Proliferator-Activated Receptor γ

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Abstract
The naturally occurring nitroalkenes, nitrolinoleic (NO2-LA) and nitrooleic (NO2-OA) acids, are among the most potent endogenous ligand activators of PPARγ-dependent transcription. In order to understand mechanisms that regulate cellular response to these nitroalkenes, we previously demonstrated that glutathione conjugation of NO2-LA and MRP1-mediated efflux of the conjugates were associated with significant attenuation of PPARγ activation by this nitroalkene [(2006) Biochemistry 45, 7889−7896]. Here we show that NO2-OA activation of PPARγ is similarly affected by nonenzymatic conjugation and MRP1-mediated efflux. Moreover, the roles of glutathione S-transferases (GSTs) in the glutathione conjugation and bioactivities of NO2-LA and NO2-OA were investigated. While none of the GST isozymes tested (GSTA1-1, A4-4, M1a-1a, and P1a-1a) enhanced the rate of glutathione conjugation, expression of GSTA1-1, M1a-1a, or P1a-1a in MCF7 cells significantly reduced the magnitude of PPARγ-dependent reporter gene transcription in response to NO2-LA and NO2-OA treatment, with GSTP1a-1a expression mediating the most potent inhibition of PPARγ. Although these GSTs failed to catalyze nitroalkene conjugation with glutathione, the nitroalkenes were found to associate avidly with all four GST isozymes as indicated by their ability to inhibit GST activity with Ki’s in the nanomolar range. Treatment of purified GSTP1a-1a with excess NO2-LA and NO2-OA resulted in the formation of covalent adducts between GSTP1a monomers and nitroalkenes, although separate experiments indicated that such covalent bond formation was not necessary for avid GST−nitroalkene interactions. These results suggest that GSTs can inhibit the activation of transcription by nitroalkenes via noncatalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARγ.

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