Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
J Biol Chem. 2011 Dec 9;286(49):42150-42161. doi: 10.1074/jbc.M111.308049. Epub 2011 Oct 13.
Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examined the effect of sulfhydryl molecules on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we observed significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addition, the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chemical insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl molecules by 6-HITC-ME and observed that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide containing Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins.
异硫氰酸酯是一种具有膜通透性的亲电试剂，可与巯基形成加合物，据认为具有重要的医学益处。在这里，我们研究了异硫氰酸酯-巯基缀合物，并研究了它们将异硫氰酸酯部分转移到细胞蛋白上的亲电潜力。当我们研究巯基分子对 6-甲基亚磺酰基己基异硫氰酸酯（6-HITC）诱导的细胞反应的影响时，我们观察到即使存在 N-乙酰-L-半胱氨酸或谷胱甘肽（GSH），6-HITC 也能显著诱导血红素加氧酶-1 的表达。此外，真正的 6-HITC-β-巯基乙醇（6-HITC-ME）缀合物显著上调了酶的表达，表明巯基异硫氰酸酯具有亲电性。为了深入了解巯基异硫氰酸酯诱导的细胞反应，我们研究了 6-HITC-ME 对巯基分子的转硫代氨基甲酰化作用，并观察到 6-HITC-ME 与 GSH 孵育时，生成了一个对应于 6-HITC 与 GSH 缀合物的单一产物。为了测试巯基异硫氰酸酯在活细胞中硫代氨基甲酰化蛋白质的功能能力，我们设计了一种新的探针，结合了异硫氰酸酯反应基团和炔基官能团，并揭示了 6-HITC-ME 暴露于细胞后蛋白质的转硫代氨基甲酰化作用。硫代氨基甲酰化的靶标包括热休克蛋白 90β（Hsp90β），它是 Hsp90 家族中的一种伴侣 ATP 酶，参与蛋白质成熟和靶向。为了鉴定 Hsp90β 修饰的位点，我们利用纳升 LC/MALDI-TOF MS/MS 并提出，肽中含有 Cys-521 的巯基与 6-HITC 反应，导致体外重组 Hsp90β 中 6-HITC 处理后的共价加合物。Cys-521 位选择性结合得到了计算机建模的支持。对 Hsp90β 的硫代氨基甲酰化的进一步研究表明，6-HITC-Hsp90β 缀合物的形成可能导致热休克因子-1 的激活，迅速发出潜在的热休克反应信号。这些数据表明，巯基异硫氰酸酯是一种活性代谢物，可通过细胞蛋白的转硫代氨基甲酰化作用，为细胞反应做出贡献。
