Oxidative stress upregulates the ubiquitin proteasome pathway inretinal endothelial cells

[摘要] Purpose: To establish whether oxidative stress in retinalendothelial cells upregulates the ubiquitin proteasome pathway (UPP)leading to increased protein degradation in diabetes.Methods: Retinal endothelial cells were exposed to a continuous fluxof hydrogen peroxide produced by glucose oxidase. Endogenous ubiquitinconjugates were detected by western blotting. The ubiquitin conjugatingactivity was determined using radiolabeled ubiquitin orα-lactalbumin. The turnover of ubiquitin conjugates was determinedby pulse-chase experiments, using radiolabeled ubiquitin. Levels of mRNAwere determined by radioactive northern blot and by real time PCR.Results: The exposure of endothelial cells to physiologicalconcentrations of hydrogen peroxide led to an increase in ubiquitinconjugating activity to both endogenous and exogenous substrates.Remarkably, the endogenous ubiquitin conjugates did not change inresponse to oxidative stress presumably because the turnover ofconjugates was also increased as revealed by pulse-chase experimentswith radiolabeled ubiquitin. Exposure of retinal endothelial cells tooxidative stress further resulted in an increase in the levels of mRNAthat encode for polyubiquitin chains or ubiquitin fused to carboxylextension proteins.Conclusions: Oxidative stress upregulated UPP and increased turnoverof ubiquitin conjugates. Upregulation of UPP may account for cellresponse to stress in conditions where oxidative stress isoverexpressed.

[学科分类] 生物化学/生物物理