Professor David Sheehan Research Group
Our laboratory uses redox proteomics to detect oxidative damage to proteins, the key agents of biological change. Earth has existed for some 4,500 million years and the earliest fossils of living cells date to about 3,500 million years ago. Some of these earliest life-forms were photosynthetic using Sun’s energy to drive production of molecular oxygen (O2) from water (H2O) and carbon dioxide (CO2). At first, molecular oxygen was mainly absorbed by iron in rocks. However, about 2,000 million years ago, molecular oxygen began to accumulate in the Earth’s atmosphere giving us the oxidising atmosphere we have today. This had profound effects on biological evolution leading to larger cells, multicellular organisms and an explosion of new phyla. An enduring legacy is that the interior of living cells are still reducing environments in contrast to their oxidising surroundings. This also led to adoption of aerobic metabolism – the burning of glucose in the presence of molecular oxygen – which dominates biology today. In comparison with anaerobic metabolism, far more available energy results from aerobic than anaerobic metabolism per gram of glucose and, today, more than 90% of a cell’s energy comes from the electron transport chain located in the cell’s powerhouses, the mitochondria. This is because of oxygen’s unusually high potential energy which means that, when oxygen is reduced to water (which happens in the electron transport chain of mitochondria), much more available energy is released than for reduction of almost any other element. But there is a problem.
As oxygen is being reduced, intermediate forms of oxygen species (collectively termed reactive oxygen species; ROS) are produced and these often leak from mitochondria and are also formed in some other cellular processes. These are highly toxic chemicals which can even kill cells. Fortunately, cells are well-adapted to this situation and have evolved extensive antioxidant defences which maintain a state of redox homeostasis. But, if these defences are reduced (as happens in ageing) or if supply of ROS increases (a common feature of toxic materials such as environmental pollutants), then important biochemical components of cells can become chemically modified and damaged. This is a phenomenon known as oxidative stress. Our lab is interested especially in oxidative stress as an index of environmental pollution, but this phenomenon also occurs in important pathologies such as cancer, Alzheimer’s and kidney disease. Quantitatively, about 70% of ROS are absorbed by proteins with smaller amounts interacting with DNA and cell membranes. For this reason, our focus is on the cell’s protein complement, the proteome. Unlike the cell’s genome, the proteome is a dynamic quantity which changes under conditions of stress, pathology or stage of cell cycle. Thus proteomics (profiling of the proteome with high-throughput methods) offers a means of getting insights to biochemical processes affected by toxic chemicals. In addition, it is increasingly recognised that ROS and other redox-active species are also crucial to normal cell functioning in processes such as redox signalling.
- Glutathione transferases
- Redox proteomics in environmental monitoring
- Redox proteomics in pathology
- Toxicology of nanoparticles
Glutathione transferases (GSTs) are key Phase II detoxification enzymes which also defend against oxidative stress. We have pioneered their study in fungi and have discovered that unique GST-like genes are widespread in fungal genomes. 60% of these are elongation factors involved in protein synthesis. We are also interested in mining new fungal genomes such as that of Trichosporon asahii for clues to protein-based metal-resistance.
Allocati, N., Masulli, M., Del Boccio, P., Pieragostino, D., D’Antonio, D., Sheehan, D., Di Ilio C. (2013) Identification of an elongation factor 1Bgprotein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus. Folia Microbiologica IN PRESS.
We are interested in effects of oxidative stress on protein side-chains. Two dimensional SDS PAGE (2-D SDS PAGE), coupled with appropriate selection and identification methodologies, allows the identification of proteins specifically targeted by ROS. We develop these methodologies in yeast and bacterial systems and apply them to bioindicator organisms (clams and mussels) to reveal oxidative stress arising from environmental pollution in marine estuaries. In particular, we label oxidised amino acid side-chains with fluors and scan gels for fluorescence. By cutting spots from gels, tryptic digesting and mass spectrometry we can obtain identifications of proteins targeted by ROS.
Pedriali A., Riva C, Parolini M, Cristoni S., Sheehan D., Binelli A. (2013) A redox proteomic investigation of oxidative stress caused by benzoylecgonine in the freshwater bivalve Dreissena polymorpha. Drug Testing and Analysis 5, 646-645.
Sheehan D. (2013) Next-generation genome sequencing makes non-model organisms increasingly accessible for proteomic studies. J. Proteomics Bioinform. 6, e-21. Do1: 10.4172/jpb.10000e21.
McDonagh B., Martinez-Acedo P., Vázquez J., Padilla A., Sheehan D., Bárcena J.A. (2012) Application of iTRAQ Reagents to relatively quantify the reversible redox state of cysteine residues. International Journal of Proteomics Pages; 514847 DOI: 10.1155/2012/514847
Company, R., Torreblanco, A., Cajaraville, M., Bebianno, M.B., Sheehan D. (2012) Comparison of thiol subproteomes from different Mid-Atlantic ridge vent sites. Science of the Total Environment 437, 413-421.
In collaboration with colleagues in UCC’s Dept of Physiology we can apply our redox proteomics toolkit to important pathologies such as hypertension, and muscle hypoxia.
Tyther, R., McDonagh, B. and Sheehan D. (2011) Proteomics in investigation of protein nitration in kidney disease: Technical challenges and perspectives from the spontaneously hypertensive rat. Mass Spectrometry Reviews30, 121-141.
Tyther, R., Ahmeda, A., Johns, E., McDonagh B. and Sheehan D. (2010) Proteomic profiling of perturbed protein sulfenation in renal medulla of the spontaneously hypertensive rat J. Proteome Res. 9, 2678-2687.
Nanoparticles have at least one dimension <100nm and seem able to cross key biobarriers such as blood-brain, skin and lungs. They are being used for ever-more applications and represent an important category of emerging environmental threat. We use redox proteomics to screen panels of nanoparticles to assess their relative toxicity to a variety of organisms.
Madadlou, A., Iacopino, D., Sheehan, D., Emam-Djomeh, Z., and Mousavi M.E. (2010) Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres. Biochimica et Biophysica Acta – General Subjects 1800,459-465.
Tedesco, S., Doyle, H., Blasco, J., Redmond, G. and Sheehan, D. (2010) Oxidative stress and toxicity of gold nanoparticles in Mytilus edulis. Aquatic Toxicology 100, 178-186.
Past lab members
Tahirah Al J’Afar PhD
Sultan Alharbi MSc
- Catherine Cole, PhD candidate, National Oceanography Centre, University of Southampton, UK.
- Sidra Ilyas, PhD candidate, University of the Punjab, Lahore, Pakistan.
- Rafael Trevisan, PhD candidate, Universidade Federal de Santa Catarina, Florianapolis, Brazil. He is in Ireland on the Brazilian Government’s Science Without Frontiers Programme.
- Badreddine Sellami, PhD candidate at the University of Carthage, Bizerte, Tunisia. He has spent three training-periods in our laboratory.
- Ricardo Fernandez Cisnal, PhD candidate at the University of Cordoba, Spain.