Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
Free Radic Biol Med. 2012 Aug 15;53(4):817-27. doi: 10.1016/j.freeradbiomed.2012.06.023. Epub 2012 Jun 23.
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.
Keap1-Nrf2 系统在细胞防御亲电试剂和活性氧方面起着至关重要的作用。Keap1 具有多个半胱氨酸残基，其中一些高度反应灵敏，可作为这些应激原的传感器。事实上，Cys151 点突变会使某些亲电试剂的反应丧失。然而，这种突变并不影响另一组亲电试剂，这表明 Keap1 的半胱氨酸残基中存在多种传感器系统。每个反应性半胱氨酸对半胱氨酸残基传感器功能的精确贡献仍有待阐明。为了阐明 Cys151 在体内的贡献，本研究采用了转基因互补挽救测定。从表达 Keap1-C151S 突变体的小鼠中制备胚胎成纤维细胞和原代腹腔巨噬细胞。用各种 Nrf2 诱导剂处理这些细胞。我们发现，一些诱导剂仅在 Keap1-C151S 表达细胞中触发轻微反应，而其他诱导剂则在与野生型细胞观察到的反应相当的程度上引发反应。我们发现，叔丁基对苯二酚、二乙基马来酸酯、萝卜硫素和富马酸二甲酯是 Cys151 优选的，而 15-脱氧-Δ(12,14)-前列腺素 J(2)(15d-PG-J(2))、2-氰基-3,12 二氧代辛烷-1,9 二烯-28-咪唑啉(CDDO-Im)、依布硒啉、硝基油酸和氯化镉则不是 Cys151 优选的。胚胎成纤维细胞和原代巨噬细胞的实验得出了一致的结果。在 Keap1-C151S 表达的巨噬细胞中测试萝卜硫素和 15d-PG-J(2)对 1-氯-2,4-二硝基苯细胞毒性的保护作用的实验表明，前一种诱导剂是有效的，而后者则不是。这些结果表明，不同的化学物质通过不同的方式利用 Keap1 半胱氨酸残基来触发 Keap1-Nrf2 系统的反应，进一步证实了 Keap1 半胱氨酸残基中存在多种感应机制的观点。
