Investigating the Post-Translational Modification of Notch1 Intracellular Domain: From Proteolytic Cleavage to Mass Spectrometric Profiling

The Notch signaling pathway is a highly conserved cell-to-cell contact dependent signaling mechanism that plays a pivotal role in cell fate determination, cellular development and its dysregulation has been widely associated with cancer. Thus, post-translational modifications (PTM) further fine-tune Notch activity by modulating its trafficking, stability, and transcriptional dynamics. This thesis investigates the post-translational regulation of the Notch1 intracellular domain (N1ICD), focusing on its potential proteolytic processing, post-translational modification (PTM) landscape, and associated protein interactors. In the first part of the study, the role of NRDc, a zinc-dependent metallopeptidase, in mediating cryptic cleavage of N1ICD, particularly at ~50 kDa, was explored. Pharmacological inhibition with 1,10-phenanthroline did not significantly alter the expression of this cleavage product, suggesting that NRDc may not directly mediate this cleavage. Building upon these findings, the study expanded the characterization of the PTM profile of N1ICD using GFP-based immunoprecipitation from cells ectopically and endogenously expressing Notch1::GFP. This approach revealed various PTMs, including phosphorylation, methylation, oxidation, ubiquitination, and sulfone formation. Notably, MS analysis identified a phosphorylation site that aligns with residues recognized by Pin1 and MAPK, implicating a role in regulating Notch1 stability. Additionally, several potential protein interactors were identified, involved in RNA processing, protein folding, and the structural cytoskeleton. Overall, this thesis offers a powerful approach for future studies into the endogenous regulation of Notch1 and its role in diseases such as cancer.

Name thesis:degree_name

M.Sc. Biological Sciences

Level thesis:degree_level

Master

Discipline thesis:degree_discipline

Faculty of Mathematics and Science

Department dc:contributor.department

Department of Biological Sciences

Grantor dc:publisher

Brock University

Year dc:date.issued

2026

Author dc:creator

• Ubias, Chanille

Advisor dc:contributor.advisor

• Necakov, Aleksandar

• Notch1
• Post-translational modifications
• NRDc
• Proteolytic cleavage

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10464/20056

OAI identifier oai:identifier

oai:brocku.scholaris.ca:10464/20056