WNT-mediated signaling relay in stem cells and oncogenesis - from basic biology to applications

Control of complex assembly in the WNT cascade

Early stage researcher 8 (ESR8) project

Supervision: Stefan Rüdiger

Lab webpage:  http://www.uu.nl/medewerkers/SGDRudiger

Aim:
The aim of this project is to determine the role of the molecular chaperones in the Wnt signalling cascade.

Methodology:
ESR8 will purify the relevant players of the WNT cascade and the relevant molecular chaperones in the cytosol. ESR8 will use biochemical and biophysical techniques to determine affinity and binding kinetics of the chaperones with all players of the cascade (Fluorescence, Mass Spectrometry (private sector secondment: Attana, collaboration UPE, Bryja) and NMR, profiting from a recent technical breakthrough of the Rüdiger groups to analyse Hsp90 by NMR). ESR8 will collaborate with ESR9, who will map binding sites of chaperones in WNT proteins at the peptide level (international secondment: Friedler). In collaboration with the Maurice group, ESR8 will analyse the functional impact of the chaperones on the WNT proteins in vivo, also profiting from specific inhibitors of both chaperones.

Collaborators:
Maurice, Bryja, Friedler, Attana, UPE

Project goals:
ESR8 will deliver a comprehensive picture of interaction partners of the molecular chaperones Hsp70 and Hsp90 in the WNT signalling cascade and will get mechanistic insights into modulation of complex assemblies mediating signalling.

Risk assessment and contingency plans:
Identification of novel interactions is per se non-predictable. Here, however, also negative results will be meaningful as we can validate them by comparing them to the known interactions of Hsp90 within the WNT kinases. The protein purification risk is limited we already can produce soluble domains of all factors involved.

Key publications:

1. G.E. Karagöz, A.M.S. Duarte, J. Ippel, C. Uetrecht, T. Sinnige, M. van Rosmalen , J. Hausmann, A.J.R. Heck, R. Boelens and S.G.D Rüdiger ‘The N-terminal domain of human Hsp90 triggers binding to the co-chaperone p23’, Proc Natl Acad Sci USA, 108, 580 (2011)

2. D.P. Minde, Z. Anvarian, S.G.D. Rüdiger# and Maurice, MM# (2011) # joint corresponding authors. ‘Messing up disorder: How do missense mutations in the tumor suppressor protein APC lead to cancer?’ Mol Cancer 10, 101 (2011). Marked “highly accessed” by publisher.

3. T. Didenko, R. Boelens and S.G.D. Rüdiger (2011).3D DOSY–TROSY to determine the translational diffusion coefficient of large protein complexes.’ PEDS 24, 99-103.