Purpose: Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study was to investigate the effect of oxidative stress on the UPP in cultured human retina pigment epithelial cells.
Methods: To mimic physiological oxidative stress, ARPE-19 cells were exposed to continuously generated H2O(2) or A2E-mediated photooxidation. Proteasome activity was monitored using fluorogenic peptides as substrates. The ubiquitin conjugation activity and activities of E1 and E2 were determined by the thiolester assays. Levels of ubiquitin and ubiquitin conjugates were determined by Western blotting.
Results: Exposure of ARPE-19 cells to 40 to 50 microM H2O(2) for 4 hours resulted in a 30% to 50% reduction in all three peptidase activities of the proteasome. Similarly, exposure of A2E-loaded ARPE-19 cells to blue light resulted in a 40% to 60% reduction in proteasome activity. Loading of A2E or exposure to blue light alone had little effect on proteasome activity. In contrast, exposure of ARPE-19 to low levels of H2O(2) (10 microM) stimulated ubiquitin conjugation activity. Loading of A2E, with or without exposure to blue light, upregulated the levels of ubiquitin-activating enzyme and increased conjugation activity. Exposure to H2O(2) or A2E-mediated photooxidation also resulted in a twofold to threefold increase in levels of endogenous ubiquitin conjugates.
Conclusions: These data show that the proteasome in ARPE-19 is susceptible to oxidative inactivation, whereas activities of the ubiquitin-conjugating enzymes are more resistant to oxidative stress. Oxidative inactivation of the proteasome appears to be one of the mechanisms underlying stress-induced accumulation of ubiquitin conjugates in the cells.