Abstract
Purpose: :
To establish whether oxidative stress up–regulates ubiquitin proteassome pathway (UPP) in retinal endothelial cells leading to increased protein degradation under pathologies associated with increased production of reactive oxygen species (ROS)
Methods: :
Retinal endothelial cells were exposed to a continuous flux of hydrogen peroxide produced by glucose oxidase. Endogenous ubiquitin conjugates were detected by Western Blotting. The ubiquitin conjugating activity was determined using radiolabeled ubiquitin or α–lactalbumin. The turnover of ubiquitin conjugates was determined by pulse–chase experiments, using radiolabeled ubiquitin. Levels of mRNA were determined by radioactive Northern Blot.
Results: :
The exposure of retinal endothelial cells to hydrogen peroxide leads to an increase in ubiquitin conjugating activity to both endogenous and exogenous substrates. The levels of endogenous ubiquitin conjugates do not change in response to oxidative stress presumably because the turnover of conjugates is also increased as revealed by pulse–chase experiments with radiolabeled ubiquitin. Exposure to oxidative stress further results in an increase in the levels of mRNA that encode specific ubiquitin fusion proteins.
Conclusions: :
Oxidative stress has been implicated in a variety of retinal diseases including diabetic retinopathy. Data shows that oxidative stress up–regulates ubiquitin proteasome pathway (UPP) activating specific ubiquitin genes and by increasing turnover of ubiquitin conjugates. Up–regulation of UPP may account for cell response to stress in conditions where oxidative stress is overexpressed.
Keywords: oxidation/oxidative or free radical damage • diabetic retinopathy • proteolysis