Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates

Takahiro Shibata,Yuuki Kimura,Akihiro Mukai,Hitoshi Mori,Sohei Ito,Yukio Asaka,Sho Oe,Hiroshi Tanaka,Takashi Takahashi,Koji Uchida

doi:10.1074/jbc.m111.308049

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.

Highlights

Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits
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)
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

Summary

Background

Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Further study on the thiocarbamoylation of Hsp90␤ suggested that the formation of 6-HITC-Hsp90␤ 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. When plant cells are damaged, glucosinolates are hydrolyzed by myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) to produce isothiocyanates They are among the most appreciated nutraceutical components of what have become known as “functional foods” and have been suggested to have important medical benefits. Transthiocarbamoylation of cellular proteins in living cells after exposure to a thiolated isothiocyanate was studied using click chemistry. Based on the identification of a heat shock protein as the major cellular target of transthiocarbamoylation, we studied its functional consequences

EXPERIMENTAL PROCEDURES

The abbreviations used are

RESULTS

DISCUSSION