Esben Poulsen:
Interplay between molecular chaperones and the ubiquitin-proteasome system in targeting misfolded proteins for degradation

Date: 15-10-2014    Supervisor: Rasmus Hartmann-Petersen

The focus of this PhD study has been the ubiquitin-proteasome system (UPS), with a particular emphasis on the recognition and degradation of misfolded proteins. Given the large number of proteins potentially involved in any given degradation pathway, several high-throughput screening techniques have been used, both in the yeast model organism S. pombe and in mammalian tissue culture cells. The articles included in this thesis cover four studies. The first study was focused on finding the specific UPS components involved in degradation of ubiquitin-fusion proteins and misframed ubiquitin. This study was based on the implementation of high-throughput siRNA screening, using a library against more than 600 UPS components. The second study was focused on studying proteins associated with the 26S proteasome in the fission yeast S. pombe. This study includes a proteomics analysis of proteins interacting with purified 26S proteasomes, and the subsequent characterization of two novel proteasome interacting proteins. The third study was aimed at analyzing the chaperone-assisted pathway leading to degradation of misfolded kinetochore proteins in S. pombe. In this study chaperones, E2s, E3s and DUBs involved in the specific degradation pathway were identified. This work had its origin in epistasis mapping of two Hsp70 co-chaperone proteins, which also formed the basis for the fourth study. In this final study, the Hsp70 co-chaperones were overexpressed in S. pombe. This led to a cell growth defect which, by RNA sequencing, was shown to be caused by a broad cellular stress response. In addition to these studies, this thesis contains a review article, covering the protein quality control systems active in the nucleus of yeast model systems and higher eukaryotes. In conjunction with the first study, which relied on siRNA screening, this thesis also contains a thorough methodology paper on using siRNA screening coupled with high-throughput fluorescence microscopy to study the ubiquitin proteasome system in mammalian culture cells.