The proteasome inhibitors lactacystin, clastro lactacystin beta -lactone, o
r tri-leucine vinyl sulfone (NLVS), in the presence of [S-35]cysteine/methi
onine, caused increased incorporation of S-35 into cellular proteins, even
when protein synthesis was inhibited by cycloheximide, This effect was bloc
ked by incubation with the glutathione synthesis inhibitor buthionine sulfo
ximine. Proteasome inhibitors also enhanced total glutathione levels, incre
ased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma -g
lutamylcysteine synthetase (rate-limiting in glutathione synthesis). Microm
olar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-l
ike activity of purified 20S proteasome, but millimolar GSH or GSSG was inh
ibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like ac
tivity. Enhanced proteasome glutathiolation was verified when purified prep
arations of the 20S core enzyme complex were incubated with [S-35]GSH after
pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro la
ctacystin beta -lactone may promote structural modification of the 20S core
proteasome, with increased exposure of cysteine residues, which are prone
to S-thiolation, Three main conclusions can be drawn from the present work.
First, proteasome inhibitors alter cellular glutathione metabolism. Second
, proteasome glutathiolation is enhanced by inhibitors but still occurs in
their absence, sat physiological GSH and GSSG levels. Third, proteasome glu
tathiolation seems to be a previously unknown mechanism of proteasome regul
ation in vivo, (C) 2001 Academic Press.