TY - JOUR
T1 - Validation of the multiple sensor mechanism of the Keap1-Nrf2 system
AU - Takaya, Kai
AU - Suzuki, Takafumi
AU - Motohashi, Hozumi
AU - Onodera, Ko
AU - Satomi, Susumu
AU - Kensler, Thomas W.
AU - Yamamoto, Masayuki
N1 - Funding Information:
We are grateful to Drs. K. Taguchi, H. Kurokawa and F. Katsuoka for critical advice. We thank Bruce Freeman, Ph.D. (University of Pittsburgh), for providing OA-NO 2 . This work was supported in part by Grants-in-Aids for Creative Scientific Research and Scientific Research from JSPS, JST CREST , Target Protein Program from MEXT, Tohoku University Global COE Program for Conquest of Signal Transduction Diseases with “Network Medicine”, the NAITO Foundation, and the Takeda Science Foundation.
PY - 2012/8/15
Y1 - 2012/8/15
N2 - 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-Δ12,14-prostaglandin J2 (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.
AB - 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-Δ12,14-prostaglandin J2 (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.
KW - Keap1
KW - Nrf2
KW - Reactive cysteine
KW - Stress response
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U2 - 10.1016/j.freeradbiomed.2012.06.023
DO - 10.1016/j.freeradbiomed.2012.06.023
M3 - Article
C2 - 22732183
AN - SCOPUS:84864461148
SN - 0891-5849
VL - 53
SP - 817
EP - 827
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 4
ER -