The cytoskeletal-signalling interface
We know that cells sense changes in their environment via receptors which activate signalling cascades. Furthermore, we know about some of the changes in cytoskeletal organisation that can occur as a result of this. What we know less about is how information from signalling networks is integrated and passed to the cytoskeleton. It follows that there must be proteins (or protein complexes) which will interact with the final components of signalling cascades and the first proteins involved in cytoskeletal reorganisation. Once such protein is IQGAP, which has the job of collecting information from various signalling pathways, integrating it and passing it on to the actin cytoskeleton. It is known to interact with the signalling proteins CDC42 and calmodulin as well as the cytoskeletal protein actin. If we want to understand how it functions as an integrator protein, then we need to study IQGAP and the interactions it makes with its partner proteins at a fundamental biochemical level and this is what my laboratory aims to do. In addition, I collaborate with John Nelson in his investigations of another cell signalling molecule, the 67 kDa laminin receptor.
The macromolecular organisation of metabolic pathways
Most undergraduate textbooks give the impression that the cytoplasm is just a bag of enzymes in which metabolic reactions take place with substrates being acted upon by one enzyme and the products then drifting around until they encounter (by random diffusion) the next enzyme in the pathway. Increasingly, this view is being challenged. There are many reasons why this "bag of enzymes" model is unlikely to be correct: many intermediates in metabolic pathways are unstable, or toxic. Some are intermediates on more than one pathway and a free-diffusion model makes it difficult to explain how particular pathways are selected to match the metabolic needs of the cell. Furthermore this model is inherently inefficient and slow. In fact the cytoplasm is a highly specialised environment in which many metabolic pathways take place in multi-enzyme clusters or "metabolons". There is tantalising evidence for the existence of such metabolons in a number of pathways. However, there has been very little characterisation of these complexes. My laboratory aims to fill this gap in our knowledge by identifying and characterising interactions between enzymes in key pathways of intermediary metabolism.
Galactose metabolism in yeast and humans
Although galactose differs from glucose at only in the stereochemistry at carbon-4, it is not recognised by many of the enzymes of glucose metabolism. Therefore, the initial metabolism of galactose proceeds through the Leloir pathway. This pathway requires four enzymic activities: galactose mutarotase, galactokinase, galactose-1-phosphate uridyl transferase and UDP-galactose 4-epiemrase. In humans these enzymic activities are present in four separate proteins. In the yeast Saccharomyces cerevisiae, there are only three proteins with the mutarotase and epimerase activities both being found in a single protein, Gal10p. Mutations in all of the enzymes except the mutarotase have been identified which cause the disease galactosemia. We are interested in several facets of Leloir pathway enzymology: the enzymatic mechanism of galactokinase (and the related protein, N-acetylgalactosamine kinase), the effects of disease-causing mutations on the function of the enzymes, the relationship (if any) between the two activities in Gal10p and whether these enzymes are organised into higher order complexes. These projects involve collaborations with Richard Reece (University of Manchester, UK) Judy Fridovich-Keil (Emory University, Atlanta, USA) and Hazel Holden (University of Wisconsin, USA).
Small protease inhibitors as potential therapeutic agents
Proteases are essential for cellular function. They are required to break down unwanted proteins in a regulated fashion. However, failure of regulation, excess production or mislocalisation of the proteases will result in dangerous damage to essential cellular components. There are a number of small proteins (usually < 20 kDa) which act as potent and (usually) specific inhibitors of protease activity. These inhibitors help regulate the action of cellular proteases and some are also involved in innate immune responses to pathogens. We are expressing, purifying and characterising a number of these small protease inhibitors with the long-term goal of using protein engineering to adapt them for therapeutic use. This work is carried out partly in collaboration with Cliff Taggart (School of Medicine, Dentistry & Biomedical Sciences, QUB).
Novel ways to use Green Fluorescent Protein (GFP) to detect protein-protein interactions
The explosion of genomic and proteomic information in the last few years has lead to an increased need for tools to detect and protein-protein interactions - especially in vivo. GFP can be split into two non-fluorescent halves (or hapto-GFPs) and, amazingly, when the two halves are brought into close proximity the ability to fluoresce is restored. One way to bring the two halves close together is to fuse them to proteins which interact with each other. This is the basis of a potentially powerful tool for detecting and simultaneously localising protein-protein interactions in vivo. We are working to develop and enhance this system in a variety of organisms. This is a joint project with Neil McFerran, Angela Mousley and John Nelson (School of Biological Sciences, QUB).
Biochemical characterisation of drug targets in the liver fluke
The liver fluke is a major parasite of livestock in Northern Ireland. It also affects millions of humans, mainly in the developing world. Although there are effective drug therapies (eg the benzimidazole compound, triclabendazole) which are licensed for use both in farm animals and humans, resistance to these drugs is increasing. As far as we know, very little effort is being made in the pharmaceutical industry to develop new drugs. Nor is the molecular mechanism of action of current drugs fully understood. In collaboration with molecular parasitologists based in the School (Alan Trudgett and Liz Hoey), we are pursuing two lines of investigation: determination of the mechanism of action of existing benzimidazole drugs and the investigation of liver fluke proteins with the aim of evaluating their potential as drug targets.
Effects of low energy ions on DNA
The use of ion beams to treat cancerous tumours shows great promise, with considerable advantages over conventional radiotherapy in terms of reduced side-effects and greater success rates. It is presumed that these therapies work by causing irreversible damage to DNA in tumour cells, thus killing them. However, not much is known about the molecular mechanisms of DNA damage by low energy ions (< 1 keV.Da-1). Yet recent work suggests that these ions can cause damage and, furthermore, ions of these energies are likely to be present at the track ends and as a consequence of secondary ionisations. We are studying the effects of low energy ions on model DNA systems using a combination of biochemical and physical techniques. In an exciting new project we have begun to investigate the effects of antiproton beams on biological materials at CERN. These are collaborative projects with various colleagues at QUB in the School of Maths & Physics (including Fred Currell, Tom Field, Jorge Kohanoff, Adam Hunniford and Bob McCullough) and CCRCB (Kevin Prise and Giuseppe Schettino). In addition, I am building a collabration with Peter van der Burgt (NUI Maynooth, Ireland) to investigate the effects of electrons on DNA and its constituents.