Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, and driven by changes in communication between cells. Heparin-binding proteins (HBPs) of the plasma membrane and extracellular matrix play a central role in cell communication; their interaction with heparan sulfate/heparin regulates many facets of their activities, including bioavailability and signaling in the cell microenvironment (plasma membrane and extracellular matrix). We have developed two approaches for the analysis of the interactions of these proteins:
(i) Purification on a heparin column and label-free relative quantification by mass spectrometry.
(ii) Structural proteomics identification of lysine residues involved in binding the polysaccharide.
The weaknesses of our current data are that we do not know (i) if a protein is actually a HBP or a protein bound to a HBP; (ii) where the heparin binding (or in the tissue, heparan sulfate binding sites) sites are physically on most of these proteins; (iii) which arginine residues are involved in binding. Most critically, in terms of understanding the network of interactions in the cell microenvironment, the identification of binding sites in the proteins has only been demonstrated in vitro, with purified proteins; we have no information on the actual interactions between protein and polysaccharide that occur in a healthy of a diseased tissue.
The aim of this PhD will be to identify directly the interactions of HBPs in mouse pancreas and in a model of acute pancreatitis with the endogenous heparan sulfate. The first aim will be to develop arginine specific chemistry suitable for mass spectrometry identification of these residues. This methodology will then be deployed, alongside the existing lysine selective approach, on pericellular matrix and plasma membrane heparin binding proteins in healthy mouse pancreas and in a mouse model of acute pancreatitis.
A wide range of state-of-the-art techniques will be used. In broad terms these cover the following areas:
Molecular cell biology: recombinant protein production;
Proteomics: analysis of protein-glycosaminoglycan interactions by structural proteomics;
Systems biology: Identification of disease mechanisms in terms of changes of levels of protein, of occupied heparin binding sites and of proteins bound to HBPs, using cytoscape, canonical pathways, gene ontology, and other systems biology tools.
This project is open to applicants who are able to obtain their own funding for tuition fee, consumable laboratory costs and living expenses.
A fees bursary may be available for suitably qualified applicants.
The 2015-16 PhD tuition fees are: UK/EU students £4,052.00 p.a.; international students £17,690.00.
In addition fees of between £1,000 and £12,000 per year are required for research costs depending on the type of project. An estimated maintenance allowance of £820 per month is required to cover accommodation, meals, transport etc.
The above figures are for guidance only, details will be provided when an offer is made.
Ori, A., Wilkinson, M.C. and Fernig, D.G. (2011). A systems biology approach for the investigation of the heparin/heparan sulfate interactome. J. Biol. Chem. 286:19892-19904.
Nunes, Q.M., Mournetas, V., Lane, B., Sutton, R., Fernig, D.G. and Vasieva O. (2013). The heparin-binding protein interactome in pancreatic digestive diseases. Pancreatology 13: 598-604.