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 (NO^- ₂)-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 mostOA-NO ₂-reactive Cys residues in Keap1 were Cys³⁸, Cys ²²⁶, Cys²⁵⁷, Cys²⁷³, Cys²⁸⁸, and Cys⁴⁸⁹. Of these, Cys²⁷³ and Cys²⁸⁸ accounted for ∼ 50% of OA-NO₂ reactions in a cellular milieu. Notably, Cys¹⁵¹ was among the least OA-NO₂reactive of the Keap1 Cys residues, with mutation of Cys¹⁵¹ having no effect on net OA-NO ₂ 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 can therefore be categorized as a Cys¹⁵¹- independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.