PURPOSE: 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.
PURPOSE: 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.