Electrophilic Nitro-fatty Acids Activate NRF2 by a KEAP1 Cysteine 151-independent Mechanism

Nitro-fatty acids (NO2-FAs) are electrophilic signaling mediators formed in vivo via nitric oxide (NO)- and nitrite (NO2-)-dependent reactions. Nitro-fatty acids modulate signaling cascades via reversible covalent post-translational modification of nucleophilic amino acids in regulatory proteins and enzymes, thus altering downstream signaling events, such as Keap1-Nrf2-antioxidant response element (ARE)-regulated gene expression. In this study, we investigate the molecular mechanisms by which 9- and 10-nitro-octadec-9-enoic acid (OA-NO2) activate the transcription factor Nrf2, focusing on the post-translational modifications of cysteines in the Nrf2 inhibitor Keap1 by nitroalkylation and its downstream responses. Of the two regioisomers, 9-nitrooctadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO2-reactive Cys residues in Keap1 were Cys(38), Cys(226), Cys(257), Cys(273), Cys(288), and Cys(489). Of these, Cys(273) and Cys(288) accounted for similar to 50% of OA-NO2 reactions in a cellular milieu. Notably, Cys(151) was among the least OA-NO2-reactive of the Keap1 Cys residues, with mutation of Cys(151) having no effect on net OA-NO2 reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO2 enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO2 canthereforebe categorized as a Cys(151)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.