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R. Sihvola, K. Kaarniranta, T. Ryhänen, R. Miettinen, M. Teräsvirta, A. Salminen, H. Uusitalo; Proteasome Inhibitor–Induced Cytoplasmic Protein Aggregation Is Modified With Geldanamycin in Human ARPE–19 Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5133.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Pathogenesis of age–related macular degeneration (AMD) involves to impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in RPE cells. In addition to lysosomal protein degradation, many cellular proteins are degraded in proteasomes. Proteasomes are large multicatalytic proteinase complexes that are found in the cytosol and in the nucleus of eukaryotic cells having a central role in cellular protein turnover. The ubiquitin–proteasome system selectively degrades intracellular proteins. The proteins that are modulated by the proteasome are involved in the control of inflammatory processes, cell cycle regulation, and gene expression. In response to various stresses, cells increase the expression of heat shock proteins (Hsps). They function as a molecular chaperones, in order to prevent the accumulation of cellular cytotoxic protein aggregates. Role of Hsp70, Hsp90 and proteasome inhibition were evaluated in cellular aggregation in human RPE cells (ARPE–19). Methods: Accumulation Hsp70 and ubiquitinated proteins were analyzed by Western blotting. Transmission electron microscopy was used to detect cellular organelles in ARPE–19 cells. Interleukin–6 (IL–6) and nitric oxide (NO) levels were measured by ELISA assays to study inflammation process. Results: MG–132 proteasome inhibitor caused robust accumulation of Hsp70 protein and ubiquitinated proteins in ARPE–19 cells. Hsp90 inhibitor geldanamycin potentiated, influence of the proteasome inhibitor, when analyzed ubiquitinated proteins. Electron microscopy analyses showed highly, in size and context, varied cytoplasmic protein aggregates in response to proteasome inhibition. Interestingly, the largest cytoplasmic aggregates disappeared with geldanamycin treatment. Levels of inflammation markers (IL–6 and NO) were not changed, when exposed to proteasome inhibitor. Conclusions: This study reveals that ubiquitin–proteasome pathway is an important way to control protein turnover in the RPE cells. In addition, Hsp70 and Hsp90 are closely related to regulation of cytoplasmic protein aggregation together with proteasomes.
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