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Role of HSP70 and p62 in regulation of autophagy clearance in ARPE‐19 cells
Author(s) -
KAARNIRANTA K
Publication year - 2009
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2009.4333.x
Subject(s) - autophagy , microbiology and biotechnology , proteasome , aggresome , lipofuscin , mg132 , hsp70 , protein aggregation , viability assay , ubiquitin , proteasome inhibitor , protein degradation , lysosome , endosome , cytoplasm , chemistry , biology , heat shock protein , cell , intracellular , biochemistry , apoptosis , gene , enzyme
Purpose A hallmark of RPE cell aging is lysosomal lipofuscin accumulation reflecting a weakened capacity for protein degradation in lysosomes. The presence of lipofuscin increases the misfolding of intracellular proteins, which evokes additional stress in the RPE cells. If the capacity of molecular chaperones to repair protein damages is overwhelmed, then the proteins are mainly cleared in proteasomes or in lysosomes including autophagy. We demonstrate that autophagy is a master clearance mechanism in proteasome inhibitor ‐induced aggregation in ARPE‐19 cells. Methods The HSP70, p62 and ubiquitin expression levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate morphological changes. HSP70 and p62 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results The proteasome inhibitor MG132 evoked the formation of juxtanuclear protein aggregates in ARPE‐19 cells. It also caused a robust accumulation of HSP70 and p62 proteins and ubiquitin‐protein conjugates that all colocalized with the formed protein aggregates. We found that protein aggregation is a temporary process, a cessation of proteasome inhibition led to autophagosome‐mediated removal of cytoplasmic protein aggregates. Interestingly, the p62 rather than the HSP70 regulate autophagy activity. However, both of them have essential function in regulation of cellular viability. Conclusion In addition to classical lysosomal proteolysis, there is the increasing evidence that heat shock proteins, proteasomes and autophagy regulate protein turnover in the RPE cells and thus have important roles in AMD disease.