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Structural and functional characterisation of Dickkopf4 (Dkk4), a key inhibitor of Wnt signalling

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posted on 2016-03-01, 15:14 authored by Alice Mary Barkell
A growing weight of experimental evidence indicates that all four secreted protein members of the cysteine-rich Dkk family, Dkk1, Dkk2, Dkk3 and Dkk4, can act as inhibitors of the Wnt pathway. To date, no structural and little functional information has been reported for Dkk4, which has been the focus of this thesis. Full length human Dkk4, including a C-terminal His6 tag, was successfully expressed in E.coli. An effective refolding and purification protocol was developed giving a final yield of 3.0 – 5.0 mg/L of homogenous, correctly folded protein. The activity of Dkk4 was assessed by investigating both its ability to bind the Wnt co-receptor LRP6, and to inhibit Wnt signalling using a variety of experiments including immunoprecipitation, FACS based cell surface binding and Wnt activated luciferase activity. Refolded Dkk4 showed well-dispersed 3D NMR spectra, which allowed the essentially complete, sequence specific assignment of the backbone atoms. The NMR data clearly indicated that both cysteine-rich domains adopt folded structures and strongly suggests a well-defined interface between the two. In addition, a novel interaction between Dkk4 and heparin was demonstrated using chemical shift perturbation experiments. The generation of models of the N-terminal region and C-terminal domain of Dkk4 allowed the perturbation data to be mapped, and revealed that a conformational change is likely to occur upon heparin binding. It is proposed that cell membrane proteins displaying heparan sulphate, a closely related molecule to heparin, may sequester Dkk4 yielding a high local concentration at the cell surface in order the control the Wnt cascade. Interestingly, Dkk4 may be able to interact with both heparin and LRP6 simultaneously rather than competitively.

History

Supervisor(s)

Carr, Mark; Robinson, Martyn

Date of award

2013-03-01

Author affiliation

Department of Biochemistry

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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