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Jiao Zhan, Jie-er He, Fu Shang, Mingxing Wu, Xinyu Zhang; Crosstalk between autophagy and the ubiquitin-proteasome pathway in human RPE cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1731.
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The accumulation of damaged or misfolded proteins is hypothesized to contribute to RPE dysfunction in age-related macular degeneration (AMD). The ubiquitin-proteasome pathway (UPP) and the autophagy-lysosome pathway (ALP) are the two major systems for clearance of misfolded or damaged proteins. The aim of the present study was to investigate how these two systems communicate and coordinate with each other in RPE cells to eliminate intracellular toxic misfolded or damaged proteins or their aggregates.
Cultured ARPE-19 cells were treated with proteasome inhibitor MG132 and lysosomotropic agent Chloroquine (CQ) respectively. The levels of ubiquitinated proteins and γ-tubulin were analyzed by western blotting and immunofluorescence to monitor protein aggregation. The activity of the ALP was determined by LC3-II and LAMP1, the hallmarks of autophagy and the lysosome. The fluorescent granules of LC3 and LAMP1 were confirmed under fluorescence microscopy. The levels of HDAC6, p62 were determined with immunofluorescence and Western Blotting.
The levels of ubiquitinated protein aggregations significantly increased and formed juxtanuclear aggresomes after the treatment of MG132 in RPE cells, indicating that inhibition of the UPP caused protein aggregation. The levels of LC3-II increased and aggregated around the ubiquitinated proteins in MG132 treated cells. The levels of HDAC6, p62 also increased in MG132 treated cells, indicating inhibition of the UPP up-regulated autophagy activity. Treatment of cells with CQ induced vacuole formation and secondary increases in protein levels of LC3-II and P62. In addition, high mass ubiquitin conjugates increased but levels of low mass ubiquitin conjugates decreased oppositely upon CQ treatment. In contrast to proteasome inhibition, the levels of HDAC6 decreased after CQ treatment.
Our results indicate that there is a crosstalk between the UPP and the ALP in human RPE cells. When the UPP was impaired, the ALP was induced to compensate for the limited activity of the UPP. When the ALP was compromised, the UPP was also up-regulated. The crosstalk and cooperation between the two proteolytic pathways may play an important role in eliminating misfolded or other forms of damaged proteins.
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