Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

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.