Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism

Abstract

Nitro-fatty acids (NO(2)-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-NO(2)) 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-nitro-octadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO(2)-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 ∼50% of OA-NO(2) reactions in a cellular milieu. Notably, Cys(151) was among the least OA-NO(2)-reactive of the Keap1 Cys residues, with mutation of Cys(151) having no effect on net OA-NO(2) reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO(2) enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO(2) can therefore be categorized as a Cys(151)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.

FIGURE 1.

Functional importance of OA-NO₂-reactive Cys residues. A and B, HEK-293T cells were co-transfected with ARE-luciferase reporter vector, Nrf2-overexpressing vector, and vector expressing wild type Keap1 or Cys to Ser mutations of the indicated amino acids. 24 h after transfection, cells were treated with OA-NO₂, 15d-PGJ₂, or SFN for 16 h, and luciferase activity was measured. Results are normalized to β-galactosidase and represented as -fold change versus pGL3-SV40-control vector for each treatment. Values are represented as mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01 when compared with ARE-luc + Nrf2 + Keap1-wt.

FIGURE 2.

OA-NO₂ reacts with Keap1 in a cellular milieu. HEK-293T cells were transfected with FLAG-CMV (empty vector) or FLAG-Keap1-overexpressing vector and treated with the indicated concentrations of OA-NO₂. A, Keap1-adducted OA-NO₂ was exchanged to β-ME from immunoprecipitated Keap1 in the presence of a [¹³C₁₈]OA-NO₂ internal standard and quantified by LC-MS/MS as β-ME-OA-NO₂. The lower panels show transfection efficiency of FLAG-Keap1 constructs. B, β-ME-OA-NO₂ levels captured upon exchange to β-ME in immunoprecipitated WT Keap1 and the following Cys to Ser mutant Keap1: C273S, C288S, C273S/C288S, or C151S/C273S/C288S. β-ME exchange reactions were conducted in the presence of [¹³C₁₈]OA-NO₂ and quantified by LC-MS/MS. Values are represented as mean ± S.D. (error bars) *, p < 0.05; **, p < 0.01; ***, p < 0.001 when compared with respective control. ns, not significant.

FIGURE 3.

Nitroalkene regioisomer-specific activation of ARE. A, HEK-293T cells were transfected with the ARE luciferase reporter and β-galactosidase control vector. 24 h after transfection, the cells were incubated with 9-OA-NO₂, 10-OA-NO₂, or a 1:1 mix of both isomers for 16 h. The data are represented as -fold change from control (vehicle) ± S.D. (error bars); n = 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001 when compared with a mix of both isomers. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 when compared with 10-OA-NO₂. B, human umbilical vein endothelial cells treated with vehicle, 5 μm 1:1 mix of 9-OA-NO₂ and 10-OA-NO₂, 9-OA-NO₂, or 10-OA-NO₂ for 2 h. ChIP assays were performed with chromatin extracts using an anti-Nrf2 antibody. Real-time quantitative PCR was performed using primers specific for a distal enhancer region of the HMOX1 gene containing multiple ARE elements. Non-precipitated input chromatin served as a reference, and IgG-precipitated template served as specificity control. -Fold induction of Nrf2 association was calculated. Values are represented as mean ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 when compared with vehicle.

FIGURE 4.

Exposure to OA-NO₂ or 15d-PGJ₂ does not cause dissociation of Keap1 and Cul3. HEK-293T cells were co-transfected with FLAG-Keap1 and HA-Cul3 and treated with 15d-PGJ₂, OA-NO₂, or SFN. Cell lysates were immunoprecipitated (IP) with FLAG or HA antibodies (top), and the amount of bound Keap1 or Cul3 was detected with Western blot using FLAG or HA antibodies. The bottom shows the transfection efficiency of total cell lysates (input control). Western blots are representative of three independent experiments.