title: 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.
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, FIN-70211 Kuopio, Finland.
authors.forename: Emilia
authors.initials: E
authors.lastname: Kansanen
authors.forename: Gustavo
authors.initials: G
authors.lastname: Bonacci
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Schopfer
authors.forename: Suvi M
authors.initials: SM
authors.lastname: Kuosmanen
authors.forename: Kit I
authors.initials: KI
authors.lastname: Tong
authors.forename: Hanna
authors.initials: H
authors.lastname: Leinonen
authors.forename: Steven R
authors.initials: SR
authors.lastname: Woodcock
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Carsten
authors.initials: C
authors.lastname: Carlberg
authors.forename: Seppo
authors.initials: S
authors.lastname: Ylä-Herttuala
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Freeman
authors.forename: Anna-Liisa
authors.initials: AL
authors.lastname: Levonen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D005227:Fatty Acids / Q000737:chemistry*; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D000072019:Kelch-Like ECH-Associated Protein 1; D008041:Linoleic Acids / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000737:chemistry*; D009574:Nitro Compounds / Q000737:chemistry; D009829:Oleic Acids / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry
chemical_list: C558925:9-nitrooctadec-9-enoic acid; D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D005227:Fatty Acids; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D008041:Linoleic Acids; D051267:NF-E2-Related Factor 2; D009574:Nitro Compounds; D009829:Oleic Acids; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D003545:Cysteine