title: Participation of covalent modification of Keap1 in the activation of Nrf2 by tert-butylbenzoquinone, an electrophilic metabolite of butylated hydroxyanisole.
abstract: Butylated hydroxyanisole (BHA) is an antioxidant and class-2B carcinogen. It is biotransformed to tert-butylhydroquinone (TBHQ), which readily auto-oxidizes to the electrophilic metabolite tert-butylbenzoquinone (TBQ). BHA and TBHQ activate Nrf2, a transcription factor that is negatively regulated by Keap1 and plays a role in the initial response to chemicals causing oxidative or electrophilic stress, although, the exact mechanism of Nrf2 activation remains unclear. Here, we examined the role of TBQ in Nrf2 activation. Exposure of RAW264.7 cells to TBQ activated Nrf2 and up-regulated its downstream proteins; under these conditions, TBQ produced cellular reactive oxygen species (ROS). However, while pretreatment with catalase conjugated with polyethylene glycol (PEG-CAT) did not affect the TBQ-induced activation of Nrf2, the ROS generation caused by TBQ was entirely abolished by PEG-CAT, suggesting that ROS is not the dominant factor for TBQ-dependent Nrf2 activation. A click chemistry technique indicated that TBQ chemically modifies Keap1. Furthermore, ultrahigh performance liquid chromatography-tandem mass spectrometry analysis with purified Keap1 revealed that TBQ covalently binds to Keap1 through Cys23, Cys151, Cys226, and Cys368. These results suggest that TBQ derived from BHA activates Nrf2 through electrophilic modification of Keap1 rather than ROS formation.
authors.affiliation: Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Abiko
authors.forename: Takashi
authors.initials: T
authors.lastname: Miura
authors.forename: Bui Hoang
authors.initials: BH
authors.lastname: Phuc
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shinkai
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kumagai
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology*; D002083:Butylated Hydroxyanisole / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D016227:Benzoquinones; D003598:Cytoskeletal Proteins; D006873:Hydroquinones; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D017382:Reactive Oxygen Species; D002083:Butylated Hydroxyanisole; C018855:2-tert-butylhydroquinone; D003545:Cysteine