The immune system and its regulation in health and disease. (360G-Wellcome-087798_Z_08_Z)

The project aims to provide an understanding of the membrane localization and regulatory signalling mechanisms of PRL3, a protein phosphatase which has been shown to have roles in regulation of cell migration, proliferation and invasion and been implicated in colorectal cancer metastasis; A1.We will establish the structural basis for PRL3's substrate selectivity by solving structures of bound ligands and complexes, pinpointing the specificity determinants. We will focus on interactions with phosphorylated ezrin and integrin 1b sequences by NMR (or X-ray crystallographic) methods. Key catalytic residues will be mutated to trap bound substrates, allowing us to identify interactions. This information will be used to initiate fragment screening for compounds that bind to pockets bordering the active site. Features of these fragments will be incorporated into the design of inhibitors and peptidomimetics that bind to the active states of PRL3. Selectivity will be assessed by comparing affinities with PRL1 and PRL2. A2. In order to reconcile PRL3's low basal activity and the upregulated activity that contributes to metastatic progression, we will characterize ligands and allosteric sites that could influence PRL3's enzymatic function. Novel ligands will be identified by thermal shift assays and validated by SPR and NMR, and respective ligand interactions of the three PRL enzymes will be compared. Interactions with both the integrin a1 cytoplasmic tail, which helps recruit PRL3 to its receptor substrate and soluble lipid headgroups will be investigated. Binding modes and structures will be determined by NMR (or crystallography). Novel ligand interactions will be validated using binding site mutants and assaying PRL signalling activities. The effects of novel ligands on PRL3's kinetic activity will be measured. The activated PRL3 conformation which is responsible for metastatic signalling will be used to initiate virtual and fragment screening in order to identify hits and develop lead molecules. A3: The predicted membrane insertion interface of PRL enzymes is unprecedented in its size and complexity. We hypothesize that its interactions alter the enzyme's conformation and oligomeric state, thus influencing its intrinsic activity and proximity to receptor substrates and ligands. We will characterize the PRL3's interactions with mixed micelles in order to solve structures of the membrane complex. The influence of micelles on the oligomeric state will be determined by AUC and NMR-based translational diffusion measurements. Due to the large membrane interactive surface predicted by the computer modelling program MODA, we will also test interactions with bicelles, vesicles and novel membrane discs we are developing, thus minimizing curvature effects. Key residues that contact membrane lipids will be mutated for assays of bilayer affinity, lipid specificity and subcellular localization of GFP fusions.