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

Abstract

Nitro-fatty acids (NOIn 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-nitrooctadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. (2), the enzyme xanthine oxidoreductase (3), and the transcription factor peroxisome proliferator-activated receptor ␥ (PPAR␥) (4). Moreover, NO 2 -FAs activate heat shock (5) and antioxidant response pathways (5, 6) via mechanisms that remain to be defined. Antioxidant response element (ARE)-regulated genes play an essential role in the protection against endogenous and exogenous stresses (7). The transcription factor nuclear factor E2-related factor-2 (Nrf2) can activate these genes via binding to AREs as a heterodimer with small Maf proteins (7). Under basal conditions, Nrf2 is bound to its inhibitor Kelch-like ECHassociated protein 1 (Keap1), which functions as an adaptor molecule in the Cul3-based E3 ligase complex. Nrf2 is then rapidly ubiquitinated and degraded (8, 9). During periods when cellular concentrations of oxidative or electrophilic species are elevated, the interaction of Nrf2 with the ubiquitin ligase complex is disrupted, enabling the escape of Nrf2 from degradation, its nuclear translocation, and transactivation of target genes.Keap1 is a Cys-rich protein with 27 Cys residues in the human and 25 Cys residues in the murine protein. Keap1 has four functional domains: the Bric-a-Brac, tramtrack, broad complex (BTB) domain, the intervening region (IVR), the Kelch domain (also known as the double glycine repeat), and the C-terminal region. Alkylation or oxidation of Keap1 Cys residues, predominantly within the IVR, leads to the inactivation of Keap1 and is the central mechanism for the activation of Nrf2 (10 -12). A number of studies utilizing mass spectrometry (MS) analysis show that electrophilic inducers of Nrf2 modify several different Cys residues in recombinant Keap1. These data indi-* This work was supported, in whole or in part, by National Institutes of Health BTB, Bric-a-Brac, tramtrack, broad complex; IVR, intervening region; 15d-PGJ 2 , 15-deoxy-⌬12,14-prostaglandin J 2 ; HEK, human embryonic kidney; ␤-ME, ␤-mercaptoethanol; OA-NO 2 , 9-and 10-nitro-octadec-9-enoic acid; 9-OA-NO 2 , 9-nitro-octadec-9-enoic acid; 10-OA-NO 2 , 10-nitro-octadec-9-enoic acid; LNO 2 , 9-, 10-, 12-, or 13-nitro-octadeca-9,12-dienoic acid.

Conjugated Linoleic Acid Is a Preferential Substrate for Fatty Acid Nitration

“…Nitration of CLA by ⅐ NO 2 Gas and the Electrophilic Reactivity of NO 2 -CLA-The electrophilic nitroalkene of NO 2 -FA mediates the signaling actions of these species through the posttranslational protein modifications of cysteine and to a lesser extent histidine (15,34). To probe NO 2 -CLA electrophilicity, reactivity toward ␤-ME was evaluated (26).…”

“…6b) (15,36), was analyzed by immunofluorescence microscopy. There was a significant increase in HO-1 expression in the cytosol of colonic epithelial cells of rats gavaged with CLA and NO 2 Ϫ (Fig.…”

“…In human coronary artery endothelium for example, fatty acid nitroalkenes significantly influence the expression of ϳ400 metabolic and anti-inflammatory-related genes (13). Specific cellular nitroalkylation targets include functionally significant thiol residues in the transcriptional regulatory proteins PPAR␥ (14), Keap1/ Nrf2 (Kelch-like ECH-associated protein 1 (Keap1)/regulator of nuclear factor (erythroid-derived-2)-like 2 (Nrf2)) (15), heat shock factor-1 (HSF-1), and NF-B (4).…”

15-Hydroxyprostaglandin Dehydrogenase Generation of Electrophilic Lipid Signaling Mediators from Hydroxy Ω-3 Fatty Acids

“…Mechanisms of action include induction of the expression of anti-inflammatory genes regulated by the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). Electrophilic fatty acids form Michael adducts with cysteines, for example primarily the Cys 273 and Cys 288 in KEAP1 (Kelch-like ECH-associated protein 1), which is responsible for sequestering Nrf2 in the cytosol and regulating its proteasomal degradation (14). This adduction promotes the release of Nrf2 and its translocation to the nucleus, where it promotes antioxidant response element gene expression (14,15).…”

“…Electrophilic fatty acids form Michael adducts with cysteines, for example primarily the Cys 273 and Cys 288 in KEAP1 (Kelch-like ECH-associated protein 1), which is responsible for sequestering Nrf2 in the cytosol and regulating its proteasomal degradation (14). This adduction promotes the release of Nrf2 and its translocation to the nucleus, where it promotes antioxidant response element gene expression (14,15). Electrophilic fatty acids also induce heat shock factor expression and suppress proinflammatory gene expression through Cys 38 alkylation and inhibition of DNA binding by the p65 subunit of NF-B (14, 16 -18).…”