A convenient method to assess chemical modification of protein thiols by electrophilic metals

Abstract

-Although covalent modification of protein thiols by electrophilic metals is implicated in disruption of protein functions associated with toxicity, there are limited methods available to detect such modifications. In the present study, we established a convenient method to assess modification of protein thiols by electrophiles, referred to as a biotin-PEAC 5 -maleimide (BPM)-labeling assay. In this assay, protein S-modification by electrophiles can be estimated by a decrease in protein modification by BPM, a thiol reactive probe. Using methylmercury (MeHg) as a model electrophilic metal, thiol modification of cellular proteins was detected by the BPM-labeling assay in SH-SY5Y cell lysates and primary mouse hepatocytes. The sensitivity and reliability of the assay was confirmed by atomic absorption spectrometry with recombinant Keap1 as a model thiol protein. This assay was applied to not only MeHg but also to other metals such as cadmium and lead. We also established a BPM-precipitation assay with avidin-agarose beads to separate BPM-modified cellular proteins followed by detection with the individual antibodies. This assay was available for detecting MeHg-induced S-modification of cellular Keap1 in SH-SY5Y cells. Taken together, we have developed reliable simple methods to estimate protein S-modification by electrophilic metals.

Cadmium-mediated activation of the HSP90/HSF1 pathway regulated by reactive persulfides/polysulfides

“…The BPM-labeling assay and BPM-precipitation assay were performed as previously described (Toyama et al, 2013). Briefly, recombinant human HSP90 (1 mM) was reacted with cadmium chloride (1, 5, 25, or 100 mM) in 50 mM Tris-HCl (pH 7.5) at 25 C for 30 min, then the mixture was incubated with BPM (25 mM) at 25 C for 30 min.…”

“…Since HSF1 released from HSPs is reported to translocate into the nucleus to bind HSEs, thereby upregulating the downstream HSP genes (Akerfelt et al, 2010; Bjork and Sistonen, 2010; Voellmy, 2004), it seems likely that cadmium-mediated activation of HSF1 is attributable to disruption of the interaction between HSF1 and HSP90. In this context, the HSP90 modification caused by cadmium was evaluated using the BPM-precipitation assay that we previously developed (Toyama et al, 2013). Exposure of the cells to cadmium significantly decreased the HSP90 band intensity (Figs.…”

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

“…As shown in Fig. 1, 2-hydroxy-1,4-NQ (ω = 6.15 eV), 2,3,5,6-tetrametyl-1,4-BQ (ω = 6.36 eV), 2-methyl-1,4-NQ (ω = 6.39 eV), and 2-hydroxy-3-(3-methyl-2-butenyl)-1-,4-NQ (ω = 6.52 eV) had negligible chemical modification capacities as evaluated by the BPM assay (Toyama et al, 2013;Abiko et al, 2015), whereas other quinones arylated proteins in 9000 × g supernatant of mouse liver (Fig. 1).…”

“…The BPM precipitation assay was performed according to procedures described previously, with a minor modification (Abiko et al, 2015;Toyama et al, 2013). A homogenate of liver from C57BL/6J male mice was centrifuged at 600 × g for 10 min, the supernatant was centrifuged at 9000 × g for 20 min, and then the lysate obtained was stored frozen under liquid nitrogen and kept at -80°C before use.…”

“…The BPM precipitation assay was performed according to procedures described previously (Toyama et al, 2013). Briefly, A549 cells were exposed to 25 or 50 μM of TBQ or 1 μM MC after pretreatment of MG132.…”