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Title: Investigating the role of the Abruptex region within the Notch receptor
Author: Rowntree, Thomas
ISNI:       0000 0004 7966 3041
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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The aim of this project was to investigate the structure and function of the Abruptex (Ax) region within the Notch receptor. Previous data from D. melanogaster shows that missense mutations in this region (EGF24-29) lead to a phenotype indicative of increased signalling. A proposed mechanism for this involves an interaction between the Abruptex region and the ligand binding region of the receptor to negatively regulate signalling. In this project, hNotch1 protein constructs were expressed from prokaryotic and eukaryotic systems to investigate the structure of the region, and interactions with the ligand binding region. After establishing a pipeline for characterizing Ax EGF-like domain constructs by small angle X-ray scattering, structural analysis revealed an extended structure for human Notch1 and Notch2 EGF23-27, with a kink at the EGF25-26 interface inferred from bioinformatics analysis. This suggests that additional non-canonical cysteines in human Notch1 EGF25 and EGF27 do not have a structural role. Binding assays suggested an interaction between EGF12 and EGF24, with binding to EGF12 dissimilar to ligand binding and requiring both O-Fuc and O-Gluc modifications on opposite sides of the domain. A decrease in binding was observed for the EGF24 Abruptex mutant D909V suggesting involvement of the N-terminal of this domain. In addition, a cell-based transactivation assay utilizing hJagged1 as the ligand was established to investigate the impact of Abruptex variants on activation of the mammalian Notch1 receptor. This showed a decrease in activation associated with a variant containing Abruptex missense mutations in EGF24 and EGF25, and a lack of activation in an EGF24-25 deletion variant. These results suggest that in the case of Jagged1, the Abruptex region has a role in optimizing transactivation. The enhanced signalling phenotype indicative of Abruptex mutants may be mediated by other means such as modulating cis-inhibition.
Supervisor: Handford, Penny Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available