| Popis: |
Aggregation of misfolded proteins in the cell is often indicative of a failing protein quality control system, whose responsibility is to mediate either the refolding or degradation of these aberrant proteins. Heat-shock (HS), which causes proteins to misfold, leads to a marked increase of the ubiquitination of proteins that display poor solubility in the cell. The E3 ubiquitin ligase Rsp5 has previously been shown to be the major ubiquitin ligase that targets cytosolic misfolded proteins upon HS in S. cerevisiae. Its closest mammalian orthologue, Nedd4-1, was also found to be responsible for an increased ubiquitination upon HS in HeLa and MEF cells, however further work is needed to better characterize this response. Such characterization included looking at which proteins get ubiquitinated upon HS to then determine whether they share common characteristics such as cellular localization, and if they are newly synthesized versus pre-existing. Using diGly enrichment coupled with mass spectrometry, we identified over 300 proteins that were further ubiquitinated upon HS. An important portion of these proteins are localized in the nucleus, whereas proteins associated to the mitochondria are markedly under-represented. As well, proteins involved in processes such as pre-mRNA processing, cell cycle and SUMOylation were found to be enriched. Using a pulse-SILAC approach, we found that a large portion of proteins ubiquitinated after HS were pre-existing, whereas some were newly synthesized including ribosomal proteins. We also investigated the potential role of other Nedd4 family members in the HS ubiquitination response in Hek293 cells. Using shRNA, we found that the knockdown of the Itch E3, but not of Nedd4-1, caused a decrease of the HS-induced ubiquitination response in these cells. Accordingly, we also showed a reduced cell viability upon Itch knocked down under HS. |
