Research interests
Proteins at the Phospholipid Interface
Many enzymes and other proteins interact with the phospholipid (membrane) surface in order to function. We have a particular interest in (a) the types of interactions that are involved between the protein and the phospholipid interface and how do these interactions affect subsequent protein function and (b) the consequences of such interactions in terms of cellular functions. Overall, events at the membrane interface are linked to a number of inflammatory diseases. An underlying theme is the role of anionic phospholipids and also tryptophan residues on the interfacial binding surface of the protein. Project areas under investigation are:
Fatty acid binding proteins
This project is investigating the mechanism by which these proteins interact with anionic interfaces with release of bound ligand - normally fatty acid. As a result of charge reversal mutagenesis and tryptophan insertion mutagenesis we are defining the conformational changes in the protein as a result of ligand and membrane interactions. Such conformational changes may be a prerequisite for targeting of fatty acids or fatty acyl-CoAs within the cell.
Human secreted phospholipases A2
At present our major interest in what is known as the group IIA enzyme. The IIA enzyme is regarded as being an acute phase protein, the serum level of which arises dramatically in infection and after tissue damage. The enzyme may play a prime role in the development of certain cancers and has been linked to atherosclerosis. A number of project areas are under investigation with respect to this enzyme.
A major role of this IIA enzyme is believed to be anti-bacterial and this is one area of research in the laboratory. The project involves a detailed analysis of this property by making use of site directed mutagenesis in order to understand the molecular mechanisms involved in this process. The enzyme is part of the innate immune response and probably acts synergistically with other anti-bacterial peptides and proteins. A key feature in the response is the binding to anionic membrane surfaces characteristic of bacterial membranes and damaged or apototic human cells.
The role of tryptophan in promoting the binding of interfacial enzymes to the membrane interface is of major interest. This research project involves mutagenesis of non-polar amino acid residues on the interfacial binding surface of the group IIA enzyme with the insertion of tryptophan and other aromatic residues in order to define the effect of such residues on interfacial binding and catalysis. We have successfully turned the IIA enzyme into an enzyme with similar properties to the group V enzyme as a result of tryptophan-insertion mutagenesis. Such mutations allow a detailed study of structure and function.
The inability of the human group IIA enzyme to hydrolyse the plasma membrane of mammalian cells is a remarkable characteristic of this phospholipase A2. However it is possible that this protein can bind to receptors on the cell surface with resulting affects on signal transduction and cell function. Such interactions with the cell surface may also result in protein internalisation. Our recent work using fluorescently-labelled enzyme has highlighted the ability of the enzyme to bind to and be internalized by activated macrophages (THP-1 cells). Moreover we have shown that this cationic enzyme is able to aggregate anionic phospholipid vesicles and enhance vesicles uptake into THP-1 cells in a process involving macropinocytosis and cell surface heparan sulphate proteoglycans. Enzyme catalysis is not required for this process. We propose that a physiological role for this enzyme is to facilitate the removal of anionic extracellular debris including anionic microparticles generated as a result of trauma, infection and the inflammatory response. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.
Research group
Molecular and Cellular Biosciences
Professor David C WiltonBiological Sciences Faculty of Natural & Environmental Sciences Life Sciences Building 85 University of Southampton Highfield Campus Southampton SO17 1BJ
