My research interests are in three main areas.
- Molecular basis of galactosemia and other inherited metabolic diseases
- Characterisation of liver fluke proteins
- Human drug target characterisation
1. Molecular basis of galactosemia and other inherited metabolic diseases
My research group seek to understand the molecular basis of galactosemia, an inherited metabolic disease in which patients are unable to metabolise the monosaccharide galactose. The disease arises from mutations in the genes encoding the enzymes of the Leloir pathway of galactose metabolism. Using recombinant expression systems we have recapitulated many of the disease-associated variant proteins and used biochemical and biophysical methods to characterise them. Our work has demonstrated that protein misfolding is a key, fundamental cause of galactose 1-phosphate uridylyltransferase deficiency (type I or classical galactosemia). This misfolding results in lower protein stability, greater protease susceptibility and, often, reduced enzymatic activity.
Reduced enzyme activity is also a cause of galactokinase deficiency (type II galactosemia). We are also interested in engineering this enzyme and the related N-acetylgalactosamine kinase (GALK2) to expand their substrate range. In collaboration with Dr Meilan Huang (Chemistry & Chemical Engineering, Queens University Belfast) we are investigating the details of the catalytic mechanism of human galactokinase. We collaborated with Prof Hazel Holden (University of Wisconsin, WI, USA) who determined the structure of human galactokinase (and also the yeast galactokinase, Gal1p and human galactose mutarotase, GALM).
Protein misfolding and, sometimes, loss of a tightly bound NAD+ cofactor play a role in the loss of catalytic turnover seen in UDP-galactose 4’-epimerase deficiency (type III galactosemia). We worked with Judy Fridovich-Keil (Emory University, GA, USA) who has developed yeast models for types I and III galactosemia. We have also worked with Dr Steffen Lindert (UCSD, CA, USA) who used computational chemistry to investigate the structural consequences of disease-associated variants of these proteins and we have applied bioinformatics approaches to predict the severity of uncharacterised variants.
Our current work aims to deepen our understanding of the molecular details of these events and seeks to work towards possible therapies based on stabilisation of the proteins or novel dietary components.
We are interested in applying the techniques and approaches which have been successful in understanding galactosemia to other inherited metabolic diseases – and are open to collaboration in this area.
2. Characterisation of liver fluke proteins
We also characterise enzymes and calcium binding proteins from the common liver fluke Fasciola hepatica. This parasite infects many millions of humans, mostly in the developing world and is also an important parasite of livestock. Recently we have discovered three calmodulin like proteins (FhCaM1, FhCaM2 and FhCaM3) and two unusual calcium binding proteins (FhCaBP3 and FhCaBP4). For all these proteins we have built molecular models, characterised their ability to interact with divalent cations and assessed their binding to known calmodulin antagonists.
We discovered and characterised the plasma membrane calcium ATPase (PMCA) from F. hepatica – again assessing its ability to interact with several drug-like molecules. In this case we developed a yeast “model” in which the yeast’s PMCA was “swapped” with the liver fluke’s. This enabled us to extract cell membranes from the yeast cells and characterise this membrane protein in a suitable environment.
Several metabolic enzymes are also being characterised, for example citrate synthase, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase (TPI). Current work is focussing on extending our knowledge of the biochemical properties of these proteins, including trying to understand their roles in the fluke (where this is not already known). This work is of value because it may result in the identification of viable, novel drug targets and for the fundamental science which we may discover about these proteins from an invertebrate.
We have also studied tubulins from the liver fluke. The organism expresses at least 11 different types of α- and β-tubulin, which are localised to different cells and tissues. We demonstrated that β-tubulin isotype 2 interacts with albendazole, an anthelmintic drug and may be a one of its targets.
We are interested in identifying inhibitors of these enzymes and also expanding our work into other helminth species. For example, we recently collaborated with scientists at the University of Cambridge (Dr Ed Farnell and Prof David Dunne) to characterise the TPI from Schistosoma mansoni.
3. Human drug target characterisation
We are also interested in the fundamental characterisation of human drug targets. Recently, we have been working in the human NAD(P)H- and NRH-quinone oxidoreductases (NQO1 and NQO2). We have studied the enzymology of these proteins (and also of the related yeast and bacterial enzymes Lot6p and MdaB). Much of this work has been in collaboration with Prof Ian Stratford (University of Manchester).
Research keywords: Protein biochemistry, enzymology, calcium binding proteins, galactose-metabolising enzymes, galactokinase, UDP-galactose 4‘-epimerase, inherited metabolic disease, galactosemia, liver fluke enzymes, triose phosphate isomerase, pharmacological chaperones, SDR family enzymes, GHMP family enzymes, recombinant expression in Escherichia coli.