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

Chaperone binding motifs in disordered WNT signalling proteins

Early stage researcher 9 (ESR9) project

Supervision: Stefan Rüdiger

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

Aim:
The aim of this project is to compare the binding specifity of the signalling chaperone Hsp90 with that of the Hsp70 machine.

Methodology:
ESR9 will use peptide arrays to screen to binding sites of the molecular chaperones in cytosolic factors of the Wnt cascade (collaboration Friedler, private sector secondment: Pepscan). ESR9 will systematically vary binding and non-binding sites to extract specific chaperone binding sequence properties, and compare this to known chaperone binding motifs (collaboration Attana). ESR9 will determine kinetics for association and dissociation, in comparison to fluorescent labelled substrate proteins, which is established in the laboratory. ESR9 will collaborate with ESR8 to localise the interaction sites in the protein structures. ESR9 will design mutants that modulate chaperone affinity and test them in vivo (international secondment: Koo).

Collaborators:
Friedler, Koo, Attana, Pepscan, UPE

Project goals:
ESR9 will determine for the first time binding motifs of Hsp90 in substrates, in particular in disordered proteins. ESR9 will determine rules how to modulate chaperone binding, differentiate between Hsp90 and Hsp70 and interfere with chaperoning in vivo.

Risk assessment and contingency plans:
Chaperone binding to peptide arrays is established in the lab. Peptide mapping of chaperone specificity was pioneered by the Rüdiger and Friedler labs. Two companies will ensure the availability of latest technology. Protein purification of chaperones is established in the lab. Design of mutations may affect protein function, but disordered regions are typically more tolerant and we can assay for eventual functional effects.

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.