Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome.
Exposure to various forms of mild oxidative stress significantly increased the intracellular degradation of both "short-lived" and "long-lived," metabolically radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal liver epithelia). The oxidative stresses employed were bolus H2O2 addition; continuous H2O2 flux; the redox cycling quinones, menadione and paraquat; and the aldehydic products of lipid peroxidation, 4-hydroxynonenal, malonyldialdehyde, and hexenal. In general, exposure to more severe oxidative stress produced a concentration-dependent decline in intracellular proteolysis, in some cases to below baseline levels. Oxidatively modified "foreign" proteins (superoxide dismutase and hemoglobin) were also selectively degraded, in comparison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of foreign proteins added to cell lysates was greatly increased by mild oxidative modification, but depressed by more severe oxidative modification. The proteinase activity was recovered in > 300-kDa cell fractions, and inhibitor profiles and immunoprecipitation studies indicated that the multicatalytic proteinase complex, proteasome, was responsible for most of the selective degradation observed with mild oxidative stress; up to approximately 95% for intracellular proteolysis and 65-80% for degradation of foreign modified proteins. Seven days of daily treatment with an antisense oligodeoxynucleotide, directed against the initiation codon region of the proteasome C2 subunit gene, severely depressed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced increase in intracellular proteolysis by approximately 95%, without significantly affecting baseline proteolytic rates. Extensive studies revealed only small or no increases in the overall capacity of oxidatively stressed cells to degrade oxidatively modified protein substrates; a finding supported by both Western blot and Northern blot analyses which revealed no significant increase in the levels of proteasome subunit polypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility. In contrast, severe oxidative stress diminishes intracellular proteolysis, probably by generating severely damaged cell proteins that cannot be easily degraded (e.g. cross-linked/aggregated proteins), and by damaging proteolytic enzymes. We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective degradation of oxidatively modified proteins in Clone 9 liver cells.
Grune
T
T
Department of Biochemistry and Molecular Biology, Albany Medical College, New York 12208.
Reinheckel
T
T
Joshi
M
M
Davies
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D016373:DNA, Antisense; D004848:Epithelium / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008054:Lipid Peroxides / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013312:Stress, Physiological / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology
D016373:DNA, Antisense; D006454:Hemoglobins; D008054:Lipid Peroxides; D009097:Multienzyme Complexes; D010455:Peptides; D011506:Proteins; D014812:Vitamin K; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D010269:Paraquat