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Title: Structural and functional studies of Wnt signalling pathway antagonists
Author: Malinauskas, Tomas
ISNI:       0000 0004 2724 702X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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Wnt morphogens control embryonic development and adult tissue homeostasis. Deregulation of the Wnt signalling pathway leads to human diseases. This thesis covers work on two antagonists of the Wnt signalling pathway, Wnt inhibitory factor 1 (WIF-1) (Chapters 1 to 5) and secreted Frizzled-related protein 1 (sFRP-1) (Chapters 1 and 6). In vertebrates the N-terminal WIF domain of the six-domain WIF-1 and the cysteine-rich domain of the two-domain sFRP-1 bind to Wnt proteins and inhibit signal transduction. My human WIF domain crystal structure reveals a novel binding site for phospholipid. Two acyl chains of the phospholipid extend deep into the domain while the lipid head group is surface exposed. Biophysical and cellular assays, combined with structure-guided mutagenesis, indicate a WIF domain Wnt-binding surface proximal to the lipid head group, but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The crystal structure of the six-domain WIF-1 reveals EGFs I-V wrapped-back to interface with the WIF domain at EGF III. Binding studies locate a heparan sulfate proteoglycan-binding site in EGFs II-V, consistent with highly conserved, positively charged residues on EGF IV. My human sFRP-1 cysteine-rich domain crystal structures reveal a conserved hydrophobic homodimer interface, conformationally variant N- and C-termini, and a conformationally invariant, putative Wnt-binding site. The site is located proximal to a positively charged surface region, which is consistent with the observed sFRP-1 cysteine-rich domain binding to heparin and heparan sulfate. The combination of heparan sulfate proteoglycan- and Wnt-binding properties suggests a model for WIF-1 and sFRP-1 localisation, and signalling inhibition, within Wnt morphogen gradients. The findings described in this thesis provide insights into the medically-important mechanisms of Wnt signalling pathway.
Supervisor: Jones, Edith Yvonne Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; Biology (medical sciences) ; Molecular biophysics (biochemistry) ; Oncology ; Biology