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 incre ased the intracellular degradation of both ''short-lived'' and ''long- lived,'' metabolically radiolabeled, cell proteins in cultures of Clon e 9 liver cells (normal liver epithelia), The oxidative stresses emplo yed 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 gen eral, exposure to more severe oxidative stress produced a concentratio n-dependent decline in intracellular proteolysis, in some cases to bel ow baseline levels, Oxidatively modified ''foreign'' proteins (superox ide dismutase and hemoglobin) were also selectively degraded, in compa rison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of f oreign proteins added to cell lysates was greatly increased by mild ox idative modification, but depressed by more severe oxidative modificat ion, 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 fo r most of the selective degradation observed with mild oxidative stres s; up to approximately 95% for intracellular proteolysis and 65-80% fo r degradation of foreign modified proteins, Seven days of daily treatm ent with an antisense oligodeoxynucleotide, directed against the initi ation codon region of the proteasome C2 subunit gene, severely depress ed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced incre ase in intracellular proteolysis by approximately 95%, without signifi cantly affecting baseline proteolytic rates. Extensive studies reveale d only small or no increases in the overall capacity of oxidatively st ressed cells to degrade oxidatively modified protein substrates; a fin ding supported by both Western blot and Northern blot analyses which r evealed no significant increase in the levels of proteasome subunit po lypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, t hus increasing their proteolytic susceptibility, In contrast, severe o xidative stress diminishes intracellular proteolysis, probably by gene rating severely damaged cell proteins that cannot be easily degraded ( e.g, cross-linked/aggregated proteins), and by damaging proteolytic en zymes, We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective de gradation of oxidatively modified proteins in Clone 9 Liver cells.