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

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 (Biochemistry 45: 7889-7896, 2006). Here we show that NO2-OA activation of PPARγ is similarly affected by non-enzymatic 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 non-catalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARγ.

Lactacidosis modulates glutathione metabolism and oxidative glutamate toxicity

“…Net loss of intracellular GSH occurs when GSH is converted to GSSG by glutathione peroxidase and then exported (Dringen and Hirrlinger 2003), through the action of GSH S ‐transferases (GSTs) (Rinaldi et al. 2002) that couple GSH to various organic substrates thereby leading to their export (Lo and Ali‐Osman 2007), and via the non‐enzymatic coupling of GSH to organic molecules (Bates et al. 2009).…”

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

“…These products are preferentially formed intracellularly via nonenzymatic conjugation in a compartment where GSH concentrations are ‫ف‬ 6 mM ( 15,35 ). In this regard, glutathione-S-transferases (GSTA1-1, A4-4, M1a-1a, and P1a-1a) do not participate in GSH conjugation of nitrated LA and OA ( 35 ).…”

“…These products are preferentially formed intracellularly via nonenzymatic conjugation in a compartment where GSH concentrations are ‫ف‬ 6 mM ( 15,35 ). In this regard, glutathione-S-transferases (GSTA1-1, A4-4, M1a-1a, and P1a-1a) do not participate in GSH conjugation of nitrated LA and OA ( 35 ). Inactive GSH conjugates are then exported to the extracellular milieu through MRPs to enter the circulation ( 15 ) and processed by hepatic ␥ -glutamyl transpeptidases and renal dipeptidases to yield cysteine conjugates analogous to those of leukotriene metabolism (36)(37)(38).…”

Nitro-fatty acid pharmacokinetics in the adipose tissue compartment

“…The time-dependent enrichment of nitro-alkene metabolites in the extracellular compartment could be a consequence of NO 2 -FA-GSH adduct export via multidrug resistance protein-1. This, in the presence of low extracellular GSH concentrations, can more readily dissociate to regenerate free nitro-alkenes or be passively transported across the cellular membrane, a pathway also shared by nitro-alkane metabolites (49)(50)(51). Furthermore, the extracellular time-dependent decrease Fig.…”