THE PROTEASOME IS A TARGET OF OXIDATIVE DAMAGE IN CULTURED HUMAN RETINA PIGMENT EPITHELIAL CELLS
Xinyu Zhang 1,
Jilin Zhou 2,
Alexandre F. Fernandes 1,
Janet R. Sparrow 2,
Paulo Pereira 3,
Allen Taylor 1,
and
Fu Shang 4*
1 HNRC, Tufts University, Boston, Massachusetts, United States
2 Department of Ophthalmology, Columbia University, New York, New York, United States
3 Center of Ophthalmology, IBILI - University of Coimbra, Coimbra, Portugal
4 HNRC, Tufts University, 711 Washington Street, Boston, Massachusetts, 02111, United States
* To whom correspondence should be addressed. E-mail: fu.shang{at}tufts.edu.
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Abstract |
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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 cultured human 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 and activities of E1 and E2s were determined by the thiolester assays. Levels of ubiquitin and ubiquitin conjugates were determined by Western blotting.
Results: Exposure of ARPE-19 cells to 40-50 µM 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 (10 µM) 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.
Key Words:
retinal pigment epithelium, oxidative damage, proteinases, lipid peroxidation, photooxidation
