Investigating the molecular function of UbcH10

Al Awadh, Ahmed Abdullah (2021). Investigating the molecular function of UbcH10. University of Birmingham. Ph.D.

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UbcH10 is an E2-conjugating enzyme specific for the Anaphase-Promoting Complex/Cyclosome (APC/C) and a proto-oncogene product that is overexpressed in many human cancers. The molecular basis of UbcH10 function is not, however, completely understood. To investigate the molecular function of UbcH10 in more detail and identify novel UbcH10-interacting proteins, our laboratory had previously performed UbcH10 interactomic studies. As such, it was hypothesised that novel UbcH10-associated proteins might be novel APC/C substrates or APC/C regulators. To this end, we investigated the function of the novel UbcH10-interacting protein and RhoA-specific RhoGEF, PDZ-RhoGEF in more detail. It was determined that PDZ-RhoGEF associates with UbcH10 both in vitro and in vivo and is targeted for proteasomal degradation in early mitosis. In this regard, it was determined that PDZ-RhoGEF associates with Cdc20, at least in part, through a highly conserved KEN box present in PDZ-RhoGEF and that ablation of the KEN box stabilises PDZ-RhoGEF protein levels. Moreover, it was determined that PDZ-RhoGEF is phosphorylated upon a number of S/TP sites in mitosis by Cdk1 and that pharmacological inhibition of Cdk1 in combination with proteasomal inhibitors limits the degradation of PDZ-RhoGEF in mitosis. These data suggest that PDZ-RhoGEF is targeted for APC/C-Cdc20-mediated degradation in a Cdk1- dependent manner.

Previous studies have suggested that UbcH10 might possess intrinsic E3 ubiquitin ligase activity. As such it was hypothesized that UbcH10 autoubiquitylation activity might affect UbcH10 function and that UbcH10 E3 ligase activity represented a potential therapeutic target. To this end, it was shown that UbcH10 possesses intrinsic E3 ligase activity and is able to undergo extensive autopolyubiquitylation independent of the APC/C, on one or more lysine acceptor residues. Mutational analyses determined that K119 is the major lysine acceptor residue for polyubiquitin chain formation both in vitro and in vivo. In this regard, it was also determined that UbcH10 can assemble polyubiquitin chains with K6, K11 and K48 homotypic ubiquitin linkages. A high-throughput quantitative UbcH10 ubiquitylation assay for drug-screening was also developed and confirmed the requirement for lysine acceptor residues in UbcH10 autopolyubiquitylation activity. These data indicate that UbcH10 can potentially modify its own activity through self-polyubiquitylation and that UbcH10 E3 ligase activity could potentially be targeted pharmacologically in order to inhibit UbcH10 function.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Medical & Dental Sciences
School or Department: Institute of Cancer and Genomic Sciences
Funders: Other
Other Funders: Saudi Arabia Cultural Bureau
Subjects: Q Science > Q Science (General)
Q Science > QP Physiology
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)


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