Modulation of the chymotrypsin-like activity of the 20S proteasome by intracellular redox status: effects of glutathione peroxidase-1 overexpression and antioxidant drugs.
ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.
Kretz-Remy
Carole
C
Stress Oxydant, Chaperon et Apoptose, CNRS-UMR 5534, Université Claude Bernard Lyon-I, 16 rue Dubois, F-69622 Villeurbanne Cédex, France.
Arrigo
André-Patrick
AP
D000975:Antioxidants / Q000494:pharmacology*; D002918:Chymotrypsin / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005979:Glutathione Peroxidase / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D017382:Reactive Oxygen Species / Q000378:metabolism; D000094765:Glutathione Peroxidase GPX1
D000975:Antioxidants; D003374:Coumarins; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D009842:Oligopeptides; D017382:Reactive Oxygen Species; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D005979:Glutathione Peroxidase; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000094765:Glutathione Peroxidase GPX1; C000722067:GPX1 protein, human