Inhibition of NF-kappaB-DNA binding by mercuric ion: utility of the non-thiol reductant, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), on detection of impaired NF-kappaB-DNA binding by thiol-directed agents.
Mercuric ion (Hg(2+)), a potent thiol inhibitor, prevents expression of nuclear factor kappaB (NF-kappaB) by mercaptide bond formation with a critical cysteine moiety (cys(62)) on the p50 subunit required for DNA binding. NF-kappaB-DNA binding is typically measured in reaction mixtures in which dithiothreitol (DTT) or other thiol reductants are used to maintain cys(62) in the reduced state. However, the presence of thiol reductants prevents accurate assessment of the Hg(2+) concentration required to prevent NF-kappaB-DNA binding because of competitive mercaptide bond formation. In the present studies we evaluated the efficacy of tris(2-carboxyethyl)phosphine-HCl (TCEP), a non-thiol reducing agent which does not bind Hg(2+), on NF-kappaB-DNA binding in vitro, using recombinant p50 protein and a (32)P-labelled kappaB oligonucleotide. We also measured the minimal Hg(2+) concentration required to prevent this interaction in the presence of either reagent. DTT promoted NF-kappaB-DNA binding in concentrations from 0.25 to 2.6mM in binding reactions. However, in the presence of 0.25mM DTT, inhibition of NF-kappaB binding was seen only at Hg(2+) concentrations greater than 100 microM and results were highly variable. In contrast, TCEP promoted NF-kappaB-DNA binding in a dose-related manner in concentrations from 0.25 to 6mM. In the presence of even 6mM TCEP, Hg(2+) prevented NF-kappaB-DNA binding at concentrations as low as 20 microM in binding reactions. Similar findings were observed with regard to the thiol alkylating agent N-ethylmaleimide (NEM). These findings demonstrate the utility of TCEP as reductant in nuclear binding reaction assays involving the interaction of thiol constituents. They also demonstrate inhibition of NF-kappaB-DNA binding at Hg(2+) concentrations comparable to those known to initiate toxicity and apoptotic cell death in vivo.
Dieguez-Acuña
F J
FJ
Department of Environmental Health, 4225 Roosevelt Way NE, Suite 100, University of Washington, Seattle, WA 98105, USA.
Woods
J S
JS
D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D008628:Mercury / Q000494:pharmacology*; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D009841:Oligonucleotides / Q000494:pharmacology; D010720:Phosphines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D019163:Reducing Agents / Q000494:pharmacology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D004247:DNA; D004229:Dithiothreitol; D005033:Ethylmaleimide; D008628:Mercury; D016328:NF-kappa B; D009841:Oligonucleotides; D010720:Phosphines; D011994:Recombinant Proteins; D019163:Reducing Agents; D013439:Sulfhydryl Reagents; C080938:tris(2-carboxyethyl)phosphine