Glutathiolation of the proteasome is enhanced by proteolytic inhibitors.
The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin 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, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.
Demasi
M
M
Ethel Percy Andrus Gerontology Center, Division of Molecular Biology, University of Southern California, 3715 McClintock Avenue, Room 306, Los Angeles, California 90089-0191, USA.
Shringarpure
R
R
Davies
K J
KJ
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002999:Clone Cells; D003545:Cysteine / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007783:Lactones / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013450:Sulfones / Q000494:pharmacology
D017931:DNA Primers; D007783:Lactones; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011480:Protease Inhibitors; D012333:RNA, Messenger; D013450:Sulfones; C423405:tri-leucine-vinyl-sulfone; C067713:lactacystin; C098869:clasto-lactacystin beta-lactone; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide; D000111:Acetylcysteine