title: The proteasome and intracellular redox status: implications for apoptotic regulation in lens epithelial cells.
abstract: OBJECTIVE
  This study aimed to investigate redox regulation of the proteasome as well as the effect of proteasome inhibition on intracellular oxidative status and apoptosis.
  
  
  METHODS
  Oxidative stress was induced in cultured human lens epithelial cells (HLECs) and intact mouse lenses by 100 microM H2O2. HLECs were also exposed to the reduced and the oxidized forms of glutathione (GSH/GSSG) and the reducing agent dithiotreitol (DTT). The chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured using synthetic fluorogenic substrates. Superoxide as well as peroxide production, mitochondrial membrane potential, and the level of GSH was measured in HLECs after proteasome inhibition by MG-132 or lactacystin. Apoptosis was determined by measuring caspase-3 activation and by studying apoptotic nuclei after staining with Hoechst 33342.
  
  
  RESULTS
  All three peptidase activities of the proteasome were inhibited by 100 microM H2O2 and by the oxidized form of glutathione (GSSG), whereas the reduced form (GSH) stimulated chymotrypsin-like and peptidylglutamyl peptidase activities in HLECs lysates. Intact mouse lenses exposed to 100 microM H2O2 exhibited loss of transparency and trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH levels. Inhibition of the proteasome in cultured HLECs caused significant increase in apoptosis and disturbed intracellular redox balance. Simultaneous addition of exogenous GSH completely abolished the increased apoptosis seen after MG-132 treatment.
  
  
  CONCLUSIONS
  This study supports the hypothesis that intracellular proteolytic and oxidative regulatory systems are tightly coupled. The current data also indicate that apoptosis by proteasome inhibition is mediated through oxidative mechanisms.
authors.affiliation: Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Göteborg, Sweden. Anne.Petersen@gu.se
authors.forename: A
authors.initials: A
authors.lastname: Petersen
authors.forename: T
authors.initials: T
authors.lastname: Carlsson
authors.forename: J-O
authors.initials: JO
authors.lastname: Karlsson
authors.forename: M
authors.initials: M
authors.lastname: Zetterberg
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000502:physiology; D005978:Glutathione / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D007908:Lens, Crystalline / Q000166:cytology / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D016877:Oxidants; C067713:lactacystin; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; D005978:Glutathione; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D019803:Glutathione Disulfide; D000111:Acetylcysteine