title: Transthiocarbamoylation of proteins by thiolated isothiocyanates.
abstract: 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.
authors: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors: Takahiro
authors: T
authors: Shibata
authors: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors: Yuuki
authors: Y
authors: Kimura
authors: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors: Akihiro
authors: A
authors: Mukai
authors: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors: Hitoshi
authors: H
authors: Mori
authors: School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
authors: Sohei
authors: S
authors: Ito
authors: Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8552, Japan.
authors: Yukio
authors: Y
authors: Asaka
authors: Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8552, Japan.
authors: Sho
authors: S
authors: Oe
authors: Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8552, Japan.
authors: Hiroshi
authors: H
authors: Tanaka
authors: Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8552, Japan.
authors: Takashi
authors: T
authors: Takahashi
authors: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Electronic address: uchidak@agr.nagoya-u.ac.jp.
authors: Koji
authors: K
authors: Uchida
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D004926:Escherichia coli / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D017879:Isothiocyanates / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry; D019596:Wolves
chemical_list: C111984:6-methylsulfinylhexyl isothiocyanate; D018841:HSP90 Heat-Shock Proteins; C493165:HSP90AB1 protein, human; D017879:Isothiocyanates; D011506:Proteins; D034741:RNA, Small Interfering; D013438:Sulfhydryl Compounds