THE PROTEASOME IS A TARGET OF OXIDATIVE DAMAGE IN HUMAN RETINA PIGMENT EPITHELIAL CELLS

Authors: ZHANG, XINYU - JM USDA HNRCA @ TUFTS; ZHOU, JILIA - COLUMBIA UNIV, NY; FERNANDES, ALEXANDRE - UNIV OF COIMBRA, PORTUGAL; SPARROW, JANET - COLUMBIA UNIV, NY; PEREIRA, PAULO - UNIV OF COIMBRA, PORTUGAL; Taylor, Allen; Shang, Fu

Submitted to: Investigative Ophthalmology and Visual Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2008
Publication Date: 4/11/2008
Citation: Zhang, X., Zhou, J., Fernandes, A.F., Sparrow, J., Pereira, P., Taylor, A., Shang, F. 2008. The proteasome is a target of oxidative damage in human retina pigment epithelial cells. Investigative Ophthalmology and Visual Science. DOI: 10.1167/iovs.07-1559.

Interpretive Summary: Several age-related degenerative diseases are known to be associated with the dysfunction of the ubiquitin-proteasome pathway (UPP). This study investigates the effect of oxidative stress on the UPP in retina pigment epithelial cells. To achieve this, ARPE-19 cells were exposed to H2O2 or A2E-mediated photo-oxidation while proteasome activity was monitored using fluorogenic peptides. Levels of ubiquitin conjugates were determined by Western blotting. Results indicated that 40-50 uM H2O2 exposure for 4 hours led to 30-50% reduction in peptidase activity. Exposure of A2E loaded ARPE cells to blue light led to a 40-60% reduction. Loading A2E cells and exposure to blue light alone had little effect in proteasome activity, while exposure of ARPE cells to low levels of H2O2 and A2E loading stimulated ubiquitin conjugation activity. A2E-mediated photo-oxidation and H2O2 also resulted in a 2-3 fold increase in endogenous ubiquitin conjugates levels. In summary, data shows that proteasome in ARPE-19 is prone to oxidative inactivation whereas activities of the ubiquitin conjugating enzymes are more resistant to oxidative stress. Proteasome oxidative inactivation may be one of the mechanisms underlying stress-induced accumulation of ubiquitin conjugates in the cells.

Technical Abstract: Purpose: Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study is to investigate the effect of oxidative stress on the UPP in retina pigment epithelial cells. Methods: To mimic physiological oxidative stress, ARPE-19 cells were exposed to continuously generated H2O2 or A2E-mediated photo-oxidation. The proteasome activity was monitored using fluorogenic peptides as substrates. The ubiquitin conjugation activity was determined by the thioester assay. Levels of ubiquitin and ubiquitin conjugates were determined by Western blotting. Results: Exposure of ARPE-19 cells to 40-50 uM H2O2 for 4 h resulted in a 30-50% reduction in all the three peptidase activities of the proteasome. Similarly, exposure of A2E loaded ARPE-19 cells to blue light resulted in a 40-60% 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 H2O2 (3-20 uM) stimulated ubiquitin conjugation activity. Loading of A2E, with or without blue light exposure, up-regulated the levels of ubiquitin activating enzyme and increased conjugation activity. Exposure to H2O2 or A2E-mediated photo-oxidation also resulted in a 2-3 fold increase in levels of endogenous ubiquitin conjugates. Conclusion: 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.