Shinkai Yasuhiro, Abiko Yumi, Ida Tomoaki, Miura Takashi, Kakehashi Hidenao, Ishii Isao, Nishida Motohiro, Sawa Tomohiro, Akaike Takaaki, Kumagai Yoshito
†Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
¶Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
Chem Res Toxicol. 2015 May 18;28(5):838-47. doi: 10.1021/tx500416y. Epub 2015 Apr 9.
Sulfhydration by a hydrogen sulfide anion and electrophile thiolation by reactive sulfur species (RSS) such as persulfides/polysulfides (e.g., R-S-SH/R-S-Sn-H(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH2-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH2-SH) and a generator of reactive oxygen species (ROS), we demonstrate that protein thiols can be modified by a reactive sulfenic acid to form disulfide adducts that undergo rapid cleavage in the presence of glutathione (GSH). As expected, 1,2-NQH2-SAc is rapidly hydrolyzed and partially oxidized to yield 1,2-NQ-SH, resulting in a redox cycling reaction that produces ROS through a chemical disproportionation reaction. The sulfenic acid forms of 1,2-NQ-SH and 1,2-NQH2-SH were detected by derivatization experiments with dimedone. 1,2-NQH2-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH2 adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH2-SAc caused an extensive chemical modification of cellular proteins in both cases. Protein adduction by 1,2-NQ through a thio ether (C-S-C) bond slowly declined through a GSH-dependent S-transarylation reaction, whereas that originating from 1,2-NQH2-SAc through a disulfide (C-S-S-C) bond was rapidly restored to the free protein thiol in the cells. Under these conditions, 1,2-NQH2-SAc activated Nrf2 and upregulated its target genes, which were enhanced by pretreatment with buthionine sulfoximine (BSO), to deplete cellular GSH. Pretreatment of catalase conjugated with poly(ethylene glycol) suppressed Nrf2 activation by 1,2-NQH2-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress.
硫化氢阴离子介导的巯基化以及诸如过硫化物/多硫化物（例如，R-S-SH/R-S-Sn-H(R)）等活性硫物种（RSS）介导的亲电硫醇化是亲电信号传导中的独特反应。使用1,2-二羟基萘-4-硫代乙酸酯（1,2-NQH2-SAc）作为1,2-二羟基萘-4-硫醇（1,2-NQH2-SH）的前体和活性氧（ROS）的生成剂，我们证明蛋白质巯基可被活性亚磺酸修饰形成二硫键加合物，该加合物在谷胱甘肽（GSH）存在下会迅速裂解。正如预期的那样，1,2-NQH2-SAc迅速水解并部分氧化生成1,2-NQ-SH，导致通过化学歧化反应产生活性氧的氧化还原循环反应。通过与达米酮的衍生化实验检测到1,2-NQ-SH和1,2-NQH2-SH的亚磺酸形式。1,2-NQH2-SOH在半胱氨酸171处修饰Keap1，产生Keap1-S-S-1,2-NQH2加合物。随后将A431细胞暴露于1,2-NQ或1,2-NQH2-SAc在两种情况下均导致细胞蛋白质的广泛化学修饰。1,2-NQ通过硫醚（C-S-C）键进行的蛋白质加合通过依赖GSH的S-转芳基化反应缓慢下降，而源自1,2-NQH2-SAc通过二硫键（C-S-S-C）键进行的加合在细胞中迅速恢复为游离蛋白质巯基。在这些条件下，1,2-NQH2-SAc激活Nrf2并上调其靶基因，用丁硫氨酸亚砜胺（BSO）预处理可增强这种上调，以耗尽细胞内的GSH。聚乙二醇偶联的过氧化氢酶预处理可抑制1,2-NQH2-SAc对Nrf2的激活。这些结果表明，在氧化应激下，RSS介导的可逆亲电信号传导通过亚磺酸的形成而发生。
