A method for detecting covalent modification of sensor proteins associated with 1,4-naphthoquinone-induced activation of electrophilic signal transduction pathways

Abstract

While metabolic activation of naphthalene, yielding 1,2-naphthoquinone (1,2-NQ) and 1,4-NQ that can covalently bind to cellular proteins, has been recognized to be associated with its toxicity, the current consensus is that such electrophile-mediated covalent modification of sensor proteins with thiolate ions is also involved in activation of cellular signal transduction pathways for cellular protection against reactive materials. In the present study, we developed an immunochemical assay to detect cellular proteins adducted by 1,4-NQ. Dot blot analysis indicated that the antibody prepared against 1,4-NQ recognized the naphthalene moiety with the para-dicarbonyl group, rather than with the ortho-dicarbonyl group. Furthermore, little cross-reactivity of para-quinones with either a different number of aromatic rings (n = 1) or substituent groups was observed. With this specific antibody against 1,4-NQ, we identified nine target proteins of 1,4-NQ following exposure of human epithelial carcinoma cell line A431 to 1,4-NQ. Among them, heat shock protein 90 (HSP90) and HSP70 are of interest because covalent modification of these chaperones causes activation of heat shock factor-1, which plays a role in the cellular response against electrophiles such as 1,4-NQ. Thus, our method, which does not use radiolabeled compounds, would be applicable for exploring activation of electrophilic signal transduction pathways coupled to covalent modification of sensor proteins during exposure to naphthalene as well as 1,4-NQ.