Disulfiram is a potent inhibitor of proteases of the caspase family.
We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.
Nobel
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CS
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Kimland
M
M
Nicholson
D W
DW
Orrenius
S
S
Slater
A F
AF
D000427:Alcohol Deterrents / Q000494:pharmacology*; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004221:Disulfiram / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics
D000427:Alcohol Deterrents; D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases; D005978:Glutathione; D004221:Disulfiram; D019803:Glutathione Disulfide