Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2011; 286(49):42150-61. DOI: 10.1074/jbc.M111.308049
Source: PubMed

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.

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    • "It has been speculated that, in addition to competing with ATP for binding to the N-terminal domain of HSP90, inhibitors such as GA and 17-AAG, because of the electrophilic properties of their benzoquinone moiety, may have the ability to react with cysteine residues of HSP90 and cause conformational changes that could further hinder the function of the chaperone (Beck et al, 2011). Modifications of cysteine residues of HSP90 by sulphhydryl-reactive small molecules have been shown to occur both in vitro and in vivo (Nardai et al, 2000; Carbone et al, 2005; Martinez-Ruiz et al, 2005; Shibata et al, 2011). HSP90 is among the proteins that are modified by the electrophilic lipid peroxidation product 4-hydroxy-2-nonenal (Jacobs and Marnett, 2007, 2010; Smathers et al, 2011) and by its azido-and alkynyl-tagged derivatives (Vila et al, 2008). "
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    • "Considering that most, if not all, of these chemical structures are simple and small, it is reasonable to assume that they largely interact with proteins in a non-specific manner. In support of this notion, several ITCs including SFN were shown to bind to unidentified multiple proteins in cultured cells[10], [11], though the authors did not highlight those findings in their studies. Such non-selective bindings to proteins by xenobiotic chemicals are presumed to be potentially proteotoxic. "
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