Degradation pathways in protein quality control

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

Misfolded protein species bear the constant risk to impair important cellular functions and to form aggregates. To safeguard the proteome, cells have evolved a sophisticated protein quality control (PQC) network, including molecular chaperones and the ubiquitin-proteasome system (UPS). This PhD thesis includes two studies, which focused on different aspects how this system is implemented in the cell. The first study addressed the identification of a cytosolic PQC pathway for the model protein Sec3-913, a mutant version of the essential exocyst subunit Sec3, in fission yeast. The data show that, albeit being functional, Sec3-913 is targeted for degradation by the UPS at elevated temperatures. Different components, including HSP70-type molecular chaperones, the ubiquitinprotein ligase Pib1 and the deubiquitinating enzyme Ubp3 were found to promote Sec3-913 degradation. In addition to the mutant variant, also wild-type Sec3 is subject to Pib1-mediated proteasomal degradation, in particular under stress conditions.The second study includes a systematic screen for PQC degrons, using the well-studied hu man dihydrofolate reductase (DHFR) as a model protein. Two regions in the DHFR sequence, Deg1 and Deg2, could be identified as PQC degrons. When fused to an unrelated reporter protein, both degrons trigger the proteasomal degradation of the fusion partner. By modifying the Deg1 and Deg2 polypeptides, hydrophobic residues emerged to be most essential for degron function. However, sequence context and length also seem to have a crucial impact on degron strength. While Deg2 mediated degradation is clearly dependent on the HSP70s Ssa1 and Ssa2 and the E3 Ubr1, recognition of Deg1 presumably relies on other components of the UPS. The Deg1 and Deg2 regions are partially buried within full length DHFR, suggesting that both degrons only take effect upon a DHFR misfolding event leading to their exposure. Additional investigations of the recessive megaloblastic anemia-linked D153V DHFR variant revealed that this amino acid substitution generates a de novo and HSP70 dependent PQC degron. Due to its localization at the DHFR surface, this degron likely confers degradation of DHFR, leading to the loss-of-function disease phenotype.
ForlagDepartment of Biology, Faculty of Science, University of Copenhagen
Antal sider158
StatusUdgivet - 2019

ID: 248894693