Investigating WASp-Cdc42 interaction (360G-Wellcome-202640_Z_16_Z)

Rho proteins are involved in many cellular processes. Classically, Rho proteins such as Cdc42 are involved in activating proteins that regulate the actin cytoskeleton. Deregulated Rho proteins have been implicated in tumourigenesis, invasion and metastasis of many cancers types. Cdc42 binds its effector WASp with a tight Kd, therefore peptides that mimic the ability of WASp to bind Cdc42 could be the basis for a new generation of potential anti-cancer agents. The aim of the project is to use site-directed mutagenesis to mutagenise the unstructured Cdc42 binding region of the WASp protein and quantify the binding of each mutant WASp to Cdc42. The mutant proteins will be expressed and purified in E.coli as GST-fusion proteins. Mutants will be assayed for their dissociation constant using a scintillation proximity assay. This will identify key residues that contribute to the high affinity binding of WASp and Cdc42. The wider aim of this research is to gather the data needed as a starting point for the generation of therapeutic Cdc42 binding peptides using a biased peptide library-based selection and screening experiment.