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T. Ryhänen, K. Kaarniranta, E. Kuusisto, R. Miettinen, A. Salminen, M. Teräsvirta, H. Uusitalo; Geldanamycin Modulates Ubiquitin–Proteasome Pathway Mediated Protein Aggregation in Human Arpe–19 Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2889.
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Retinal pigment epithelial cells (RPE) maintain survival of photoreceptors. One of the important functions of RPE cells is to break down membranous discs shed from photoreceptor outer segment. In age–related macular degeneration the break down process is impaired. This leads to accumulation of lipofuscin in lysosomes of RPE cells. The majority of non–specific protein break down takes place in lysosomes. However the lysosomal share of all protein degradation in cells is only 20%. Proteasomes degradate the rest 80% of proteins. All proteins that proteasomes break down are tagged with ubiquitin. The role of proteasomes in age–related macular degeneration is not known. In response to various stresses, cells increase the expression of heat shock proteins (Hsps). They function as molecular chaperones, in order to prevent the accumulation of cellular cytotoxic protein aggregates. Role of Hsp90, Hsp70, ubiquitine and proteasome inhibition were evaluated in cellular aggregation in human RPE cells (ARPE–19).
Cellular localization of Hsp90, Hsp70 and ubiquitin were studied by immunofluorescense and phase contrast microscopy. Transmission electron microscopy was used to detect cellular organelles in ARPE–19 cells. Cell viability was analyzed by MTT –assay.
Electron microscopy showed a robust accumulation of juxtanuclear protein aggregates in response to proteasome inhibitor, MG–132. The size and context of protein aggregates varied highly. Hsp70 and ubiquitine but not Hsp90 colocalized with the protein aggragates. When the cells were subjected to Hsp90 inhibitor geldanamycin the amount of protein aggregates was clearly decreased. Proteasome inhibitor increased cell death in ARPE–19 cells.
This study reveals that ubiquitin–proteasome pathway is an important way to control protein turnover in the RPE cells. In addition, Hsp90 inhibitor reflects to the cytoplasmic protein aggregation in ARPE–19 cells.
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