Abstract
dc:description.abstractAll cells contain numerous multi-subunit protein complexes of exceptional abundance and physiological importance. Prominent examples include the ribosome, proteasome, chaperonin, tubulin, haemoglobin, and many others. A major challenge for the cell is to synthesise individual subunits in the correct stoichiometry. Any imbalance in subunit synthesis results in excess unassembled “orphan” subunits lacking their interaction partner(s). Orphans are accentuated in disease states such as cancer where altered gene expression caused by aneuploidy is common. Acute aneuploidy is known to cause proteotoxicity, probably as a consequence of high orphan burden. How orphan subunits are targeted for degradation to maintain protein homeostasis remains an unanswered question for most of the cell’s major protein complexes. The work described in this thesis investigated how the seven beta subunits of the 20S core proteasome (named PSMB1-7) are targeted for degradation when produced in excess. I used a mammalian *in vitro* translation system to identify candidate interactors of nascent PSMB subunits and found several proteins related to the ubiquitin-proteasome system. Using fluorescent reporters of PSMB subunit degradation in cells depleted of individual candidates, I found that the E3 ubiquitin ligases KCMF1 and UBR4 are required for efficient degradation of unassembled PSMB subunits. Biochemical assays showed that KCMF1 and UBR4 form a stable complex that physically interacts with nascent PSMB subunits. Moreover, using a cell-based flow cytometry assay, I determined which domains of KCMF1 and UBR4 are required for effective degradation of PSMB subunits. Analysis of orphans from other protein complexes showed that the KCMF1-UBR4 complex was also required for efficient degradation of PSMC5 (a 19S proteasomal subunit) and CCT4 (a chaperonin subunit). This was unexpected because orphaned PSMC5 and CCT4 were recently shown to be multiply mono-ubiquitinated by the E3 ubiquitin ligases HERC1 and HERC2, respectively. This led to the hypothesis that the KCMF1-UBR4 complex might build ubiquitin chains on pre-ubiquitinated orphans, thereby improving their recognition by the proteasome. *In vitro* reconstitution of orphan ubiquitination by the KCMF1-UBR4 complex showed that substrate pre-ubiquitination is both necessary and sufficient to create a KCMF1-UBR4 substrate. This reconstitution system revealed that the KCMF1-UBR4 complex builds K48-linked polyubiquitin chains and helped to define the key domains on both ubiquitin and UBR4 involved in substrate recognition. UBR4 mutants impaired in substrate recognition *in vitro* were also impaired in substrate degradation in cell-based assays. These results support a model in which the KCMF1-UBR4 E3 ligase complex acts as a chain elongator for orphaned subunits that are mono-ubiquitinated by a priming ligase. Elongation by the KCMF1-UBR4 complex generates K48 polyubiquitin chains that are ultimately required for efficient degradation by the proteasome. The KCMF1-UBR4 complex may therefore represent a general quality control pathway for the degradation of many types of orphan subunits. Consistent with this model, a wide range of cancer cells are observed to be strongly reliant on KCMF1 and UBR4 for their fitness.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Carrillo Roas, Sara
- Advisor dc:contributor.advisor
-
- Hegde, Ramanujan Shankar
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.106511
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/365090