Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. James (Wing-Yiu) Choy

Abstract

The Wnt/b-catenin signaling pathway is critical to embryonic development and adult tissue homeostasis. Mutations to Wnt signaling components can cause dysregulation of the pathway, leading to various human diseases such as cancer. The partially disordered protein Chibby (Cby) is a conserved nuclear protein that acts as an antagonist in the Wnt/b-catenin signaling pathway. Cby’s antagonism is accomplished via two mechanisms. First, by competing with the Tcf/Lef family of transcription factors, Cby abrogates the b-catenin-mediated transcription of Wnt signaling genes. Moreover, upon phosphorylation on serine 20 by the kinase Akt, Cby forms a complex with the protein 14-3-3 to facilitate the nuclear export of b-catenin. Structurally, Cby is composed of an unstructured N-terminal half, while its C-terminal half harbours a coiled-coil domain. Cby’s N-terminal half comprises a 14-3-3 binding motif, while its C-terminal half mediates the interaction with b-catenin, as well as TC-1, an antagonist of Cby. In this thesis, the molecular details of Cby’s structural elements and its interactions with the Wnt signaling components 14-3-3, b-catenin and TC-1 were investigated. The Cby/14-3-3 interaction was studied by using a combined approach of nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC) and X-ray crystallography. While the solved crystal structure revealed a canonical binding mode, NMR spectroscopy and ITC revealed that residues flanking Cby’s 14-3-3 binding motif are involved in the interaction. Next, hydrogen-deuterium exchange mass spectrometry revealed that in addition to Cby’s disordered N-terminus, Cby contains a disordered C-terminal extension. ITC and NMR experiments demonstrate that the disordered N-terminus negatively regulates target binding between TC-1 and Cby’s coiled-coil domain. Lastly, mutagenesis studies suggest that Cby’s coiled-coil domain utilizes differing binding modes when interacting with b-catenin and TC-1, with Cby binding as a monomer to b-catenin and as a dimer to TC-1. In conclusion, this thesis demonstrates how Cby’s structural elements collectively mediate protein-protein interactions in the Wnt signaling pathway.


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