title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.
abstract: The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Saito
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan taka23@med.tohoku.ac.jp masiyamamoto@med.tohoku.ac.jp.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Hiramoto
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Soichiro
authors.initials: S
authors.lastname: Asami
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Naganuma
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Suda
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Iso
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Morita
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Liam
authors.initials: L
authors.lastname: Baird
authors.affiliation: Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Furusawa
authors.affiliation: Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Negishi
authors.affiliation: Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ichinose
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan taka23@med.tohoku.ac.jp masiyamamoto@med.tohoku.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D018384:Oxidative Stress; D016415:Sequence Alignment; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; D003545:Cysteine