Role of heat shock proteins in tumor necrosis factor-induced nuclear factor-kappaB signaling pathway
Arslan, Alper Mehmet (2005) Role of heat shock proteins in tumor necrosis factor-induced nuclear factor-kappaB signaling pathway. [Thesis]
Heat shock proteins are a group of proteins that play key roles in thermoregulation, cell cycle control, development and transcriptional regulation through their diverse functions in protein folding/refolding, protein translocation/shuttling, protein activity via stabilization and/or maturation to functionally-competent conformation and protein turnover. In this study, the role of heat shock proteins in tumor necrosis factor-induced nuclear factor-kappaB signaling pathway was investigated. Endogenous heat shock protein 90 levels were found to increase significantly in a time-dependent manner starting from 15 minutes of tumor necrosis factor treatment in HeLa cell line, as shown by immunoblotting. Endogenous heat shock protein 70 levels were found to be unaffected by tumor necrosis factor in these cells. Accordingly, electrophoretic mobility shift assay results showed significantly impaired nuclear factor-kappaB activation in presence of the specific heat shock protein 90 plays an important role in this signaling pathway. To further search for any possible protein-protein inteactions between components of this pathway and heat shock protein 90, transfection of HeLa cells was optimized with green fluorescent protein with 95% efficiency. Immunoprecipitation of endegenous heat shock protein 90 and transfected inhibitor-kappaB kinase alpha were also optimized with high efficiency as a prerequisite to co-immunoprecipitation assay. Work is in process to show the interaction between these proteins by co-immunoprecipitation and to make use of this assay to searh for any possible croos-talk between divirgent downstream pathways of tumor necrosis factor signaling that might occur via heat shock protein 90.
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