Protein oxidation and proteolysis by the nonradical oxidants singlet oxygen or peroxynitrite.
Exposure of proteins to oxidants leads to increased oxidation followed by preferential degradation by the proteasomal system. The role of the biologically occurring oxidants singlet oxygen and peroxynitrite in oxidation of proteins in living cells and enhanced degradation of these proteins was examined in this study. Subsequent to treatment of an isolated model protein, ferritin, with singlet oxygen or peroxynitrite, there was enhanced degradation by the isolated 20S proteasome. Treatment of clone 9 liver cells (normal liver epithelia) with two different singlet oxygen-generating systems or peroxynitrite leads to a concentration-dependent increase in cellular protein turnover. At high concentrations of these oxidants, the protein turnover decreases without significant loss of cell viability and proteasome activity. To compare the increase of intracellular protein turnover with that obtained with other oxidants, cells were exposed to hydrogen peroxide or xanthine/xanthine oxidase. The maximal increase in protein turnover was similar with the various oxidants. The oxidized protein moieties were removed by enhanced protein turnover. Removal of singlet oxygen- or peroxynitrite-damaged proteins is dependent on the proteasomal system, as suggested by the sensitivity to lactacystin. Our results provide evidence that the proteasomal system is able to selectively recognize and degrade proteins modified by singlet oxygen or peroxynitrite in vitro as well as in living cells.
Grune
T
T
Neurowissenschaftliches Forschungszentrum, Medizinische Fakultät, Charité, Humboldt-Universität zu Berlin, Berlin, Germany. tilman.grune@charite.de
Klotz
L O
LO
Gieche
J
J
Rudeck
M
M
Sies
H
H
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005293:Ferritins / Q000378:metabolism*; D005609:Free Radicals / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009284:Naphthols / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism; D051381:Rats; D026082:Singlet Oxygen / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D005609:Free Radicals; D009097:Multienzyme Complexes; D009284:Naphthols; D011506:Proteins; C067713:lactacystin; D030421:Peroxynitrous Acid; D026082:Singlet Oxygen; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D005293:Ferritins; C058008:3,3'-(1,4-naphthylidene)diproprionate; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine