Validation of the multiple sensor mechanism of the Keap1-Nrf2 system.
The Keap1-Nrf2 system plays a critical role in cellular defense against electrophiles and reactive oxygen species. Keap1 possesses a number of cysteine residues, some of which are highly reactive and serves as sensors for these insults. Indeed, point mutation of Cys151 abrogates the response to certain electrophiles. However, this mutation does not affect the other set of electrophiles, suggesting that multiple sensor systems reside within the cysteine residues of Keap1. The precise contribution of each reactive cysteine to the sensor function of Keap1 remains to be clarified. To elucidate the contribution of Cys151 in vivo, in this study we adopted transgenic complementation rescue assays. Embryonic fibroblasts and primary peritoneal macrophages were prepared from mice expressing the Keap1-C151S mutant. These cells were challenged with various Nrf2 inducers. We found that some of the inducers triggered only marginal responses in Keap1-C151S-expressing cells, while others evoked responses in a comparable magnitude to those observed in the wild-type cells. We found that tert-butyl hydroquinone, diethylmaleate, sulforaphane, and dimethylfumarate were Cys151 preferable, whereas 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PG-J(2)), 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im), ebselen, nitro-oleic acid, and cadmium chloride were Cys151 independent. Experiments with embryonic fibroblasts and primary macrophages yielded consistent results. Experiments testing protective effects against the cytotoxicity of 1-chloro-2,4-dinitrobenzene of sulforaphane and 15d-PG-J(2) in Keap1-C151S-expressing macrophages revealed that the former inducer was effective, while the latter was not. These results thus indicate that there exists distinct utilization of Keap1 cysteine residues by different chemicals that trigger the response of the Keap1-Nrf2 system, and further substantiate the notion that there are multiple sensing mechanisms within Keap1 cysteine residues.
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
Kai
K
Takaya
Takafumi
T
Suzuki
Hozumi
H
Motohashi
Ko
K
Onodera
Susumu
S
Satomi
Thomas W
TW
Kensler
Masayuki
M
Yamamoto
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D000069462:Dimethyl Fumarate; D005650:Fumarates / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D054833:Isoindoles; D017879:Isothiocyanates / Q000494:pharmacology; D000072019:Kelch-Like ECH-Associated Protein 1; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism; D008298:Maleates / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D009828:Oleanolic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D016566:Organoselenium Compounds / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D013454:Sulfoxides; D015533:Transcriptional Activation / Q000187:drug effects
C472829:1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole; D048868:Adaptor Proteins, Signal Transducing; D000975:Antioxidants; D001393:Azoles; D003598:Cytoskeletal Proteins; D005650:Fumarates; D006873:Hydroquinones; D007093:Imidazoles; D054833:Isoindoles; D017879:Isothiocyanates; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D008298:Maleates; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D016566:Organoselenium Compounds; D016877:Oxidants; D013454:Sulfoxides; C042986:ebselen; D009828:Oleanolic Acid; C018855:2-tert-butylhydroquinone; D016660:NAD(P)H Dehydrogenase (Quinone); C491732:Nqo1 protein, mouse; D005721:Glutamate-Cysteine Ligase; D000069462:Dimethyl Fumarate; C014476:diethyl maleate; C016766:sulforaphane