Noncatalytic interactions between glutathione S-transferases and nitroalkene fatty acids modulate nitroalkene-mediated activation of peroxisomal proliferator-activated receptor gamma.

The naturally occurring nitroalkenes, including nitrolinoleic (NO2-LA) and nitrooleic (NO2-OA)1 acids, are implicated in the modulation of multiple signaling pathways resulting in several important bioactivities including vasodilation, inhibition of inflammation, and inhibition of platelet activation and function (1-4). In addition, these nitrated unsaturated fatty acids are among the most potent endogenous ligand activators of PPARγ—a nuclear receptor involved in the transcription of genes associated with carbohydrate and lipid homeostasis, cellular differentiation, and inflammation (5-8). The physiological importance of these nitroalkenes is indicated by their relative abundance in vivo with aggregate levels reported to be around 1-2 μM in normal human plasma and erythrocytes (9)—levels in excess of those necessary to significantly activate PPARγ-dependent transcription in vitro (10, 11). In order to understand how nitroalkene activities are regulated at the cellular level, we previously demonstrated that NO2-LA forms conjugates with glutathione, rapidly and non-enzymatically, under physiological conditions (12). These conjugates are efficiently effluxed by MRP1, a ubiquitous ATP-dependent glutathione conjugate transporter, and combined conjugation plus MRP1-mediated efflux profoundly attenuates PPARγ-dependent transcription activation in response to NO2-LA (12). Here we demonstrate that activation of PPARγ by NO2-OA is similarly affected by non-enzymatic conjugation and efflux. Moreover, we investigated whether expression of representative isozymes of the soluble forms of glutathione S-transferase (GST) would influence NO2-LA and NO2-OA bioactivities either by catalyzing their conjugation with glutathione or by an alternative mechanism. Indeed, we show that while expression of GSTA1-1, GSTM1a-1a, or GSTP1a-1a significantly attenuates the activation of PPARγ-dependent transcription by NO2-LA and NO2-OA, none of these isozymes, or GSTA4-4, is able to catalyze glutathione conjugation of the nitroalkenes. Rather, these GSTs appear to influence transcription via avid covalent and non-covalent interactions with the nitroalkenes and their glutathione conjugates thereby sequestering them from their nuclear target, PPARγ.