Proteolysis in Cultured Liver Epithelial Cells during Oxidative Stress

Abstract

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.

Activation of Chaperone-mediated Autophagy during Oxidative Stress

“…Whereas during severe oxidative stress, protein aggregation and cross-linking are common events, mild oxidative stress has been shown to generate excellent proteolytic substrates (Grune et al, 1995;Gomes-Marcondes and Tisdale, 2002). Our work supports that in the case of CMA, mild oxidation of CMA substrates facilitates not only their degradation by lysosomal proteases but also their binding/ translocation across the lysosomal membrane ( Figure 4A).…”

“…Thus, mild oxidation induces partial protein unfolding and facilitates proteolytic cleavage (Grune et al, 1995. However, persistent or extensive oxidative damage usually promotes protein aggregation, due to the exposure of patches of hydrophobic amino acids.…”

Biochemistry and pathology of radical-mediated protein oxidation

“…Evidence for a crucial role for the proteasome in the degradation of such proteins is unconvincing [184]. The support from studies using antisense RNA inhibition of proteasome function [185] appears to be undermined by gross re-utilization of radioactive amino acids, such that the measured ' degradation ' rates are implausible and more probably reflect protein synthesis rates.…”

The role of the ubiquitin/proteasome system in cellular responses to radiation

“…The response of the Ub/proteasome system following exposure of cells to various oxidative stressors, including hydrogen peroxide, has been examined in some detail (Grune et al, 1995;Grune, 2000). ATP-dependent degradation of fluorogenic substrates through the 26S proteasome is much more sensitive to hydrogen peroxide treatment than the ATP-and Ub-independent 20S degradation pathway (Reinheckel et al, 1998;Reinheckel et al, 2000;Shringarpure and Davies, 2002;Shringarpure et al, 2002).…”

“…ATP-dependent degradation of fluorogenic substrates through the 26S proteasome is much more sensitive to hydrogen peroxide treatment than the ATP-and Ub-independent 20S degradation pathway (Reinheckel et al, 1998;Reinheckel et al, 2000;Shringarpure and Davies, 2002;Shringarpure et al, 2002). Indeed, modest levels of oxidative stress increase the degradation rate of modified proteins (Grune et al, 1995), as oxidized proteins are preferentially removed by the more resistant 20S proteasome pathway (Shringarpure et al, 2002). Current evidence therefore suggests a division of labor between the 20S and 26S proteasome in response to oxidative stress that allows the 26S proteasome to slow down degradation of ubiquitinylated proteins and activate pathways leading to appropriate cellular responses without compromising the need to remove potentially cytotoxic nonfunctional proteins, which is performed independently.…”