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.
- SourceAvailable from: Tatiana N Boronina
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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). "
ABSTRACT: Background: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. Methods: Combining chemical synthesis, biological evaluation, and structure–activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. Results: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. Conclusion: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.British Journal of Cancer 12/2013; 110(1). DOI:10.1038/bjc.2013.710 · 4.84 Impact Factor
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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, , 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. "
ABSTRACT: Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.PLoS ONE 03/2013; 8(3):e58641. DOI:10.1371/journal.pone.0058641 · 3.23 Impact Factor
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ABSTRACT: All cells have developed various mechanisms to respond and adapt to a variety of environmental challenges, including stresses that damage cellular proteins. One such response, the heat shock response (HSR), leads to the transcriptional activation of a family of molecular chaperone proteins that promote proper folding or clearance of damaged proteins within the cytosol. In addition to its role in protection against acute insults, the HSR also regulates lifespan and protects against protein misfolding that is associated with degenerative diseases of aging. As a result, identifying pharmacological regulators of the HSR has become an active area of research in recent years. Here, we review progress made in identifying small molecule activators of the HSR, what cellular targets these compounds interact with to drive response activation, and how such molecules may ultimately be employed to delay or reverse protein misfolding events that contribute to a number of diseases.Chemical Research in Toxicology 07/2012; 25(10):2036-53. DOI:10.1021/tx300264x · 3.53 Impact Factor