Purpose. The two main routes that cells used for degrading intracellular proteins are the ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway. The objective of this study was to investigate the relationship between UPP and autophagy-lysosome pathway in protein turnover in cultured human retina pigment epithelial cells.
Methods. We inhibited the UPP with proteasome-specific inhibitor and determined the activation of autophagosome by LC3, which level was determined by western blotting. Vice versa, we inhibited the autophagy-lysosome pathway with bafilomycin A1. Proteasome activity was monitored using fluorogenic peptides as substrates. Levels of p62, ubiquitin and ubiquitin conjugates were determined by western blotting.
Results. Autophagy inhibition increased levels of ubiquitin conjugates associated with P62 accumulates after long time inhibition. On the other hand, the autophagosome activity was significant increased by the inhibition of UPP. The level of LC3 was increased by 2-fold after 24 hours inhibition.
Conclusions. These data show that autophagy compromise dose not only affect long-lived proteins degradation, but also the ubiquitin-proteasome system. Either impairment of the UPP and autophagy-lysosome pathway could lead to the development of AMD and AMD-related phenotypes.