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Regulation of Cellular Metabolism by LAR Phosphatase

Principal Supervisor: Dr Debbie Cunningham - School of Biosciences

Co-supervisors: Dr Andy Philp, Dr Neil Hotchin

PhD project title: Regulation of Cellular Metabolism by LAR Phosphatase

University of Registration: Birmingham

Project outline:

Generation of reactive oxygen species (ROS) as products of normal metabolic functions of the cell such as mitochondrial oxidative metabolism are counteracted by their elimination via antioxidant defences of the cell in order to maintain redox homeostasis. Excessive production of ROS or an ineffective antioxidant response can lead to oxidative stress, resulting in damaging oxidation of proteins, lipids and DNA. It is also becoming increasingly evident that oxidative stress impacts directly on cell signalling pathways by altering the oxidative state of specific cysteine residues on proteins. Oxidative damage to biomolecules and aberrant regulation of cell signalling pathways can contribute to a range of diseases, including cancer, degenerative disorders, atherosclerosis, diabetes and also the ageing process itself. In response to oxidative stress cells use a variety of enzymatic and non-enzymatic anti-oxidant systems to eliminate ROS and maintain redox homeostasis. Regulation of protein expression at the transcriptional level and regulation of protein degradation in order to fine tune relevant protein turnover are important mechanisms that cells rely on to combat oxidative stress.

We have recent evidence that a protein tyrosine phosphatase known as leucocyte-antigen related (LAR) plays a role in regulating turnover of proteins involved in maintenance of redox balance, including a number of mitochondrial proteins1. The mechanism by which LAR is specifically regulating the levels of these proteins is as yet unknown. LAR phosphatase itself is reversibly oxidised and inactivated through oxidation of the catalytic cysteine. This inactivation will lead to increased tyrosine phosphorylation of LAR substrates and result in modulation of specific downstream pathways. This regulation could be a feedback mechanism in order to regulate the redox status of the cell following ‘sensing’ of the redox status of the cell by LAR.

The aim of this project is to characterise the mechanism by which LAR phosphatase regulates protein turnover in response to oxidative stress, and understand how LAR impacts on mitochondrial function. Mass spectrometry based proteomics methods will be used to identify LAR substrates that play a role in regulating levels of proteins involved in cellular metabolism. In parallel, mitochondrial function will be investigated via measurement of mitochondrial respiration, and complimentary enzyme assays and immunoblotting. By analysing both the function and content of mitochondrial proteins, our aim is to directly link LAR to mitochondrial regulation.

References:

  1. Sarhan, A.R., Patel, T.R., Creese, A.J., Tomlinson, M.G., Hellberg, C., Heath, J.K., Hotchin, N.A., Cunningham, D.L. (2016) Regulation of Platelet Derived Growth Factor Signalling by LAR Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study Mol Cell Proteomics 15, 1823-1836

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Molecular cell biology techniques including cell culture, western blotting, immunoprecipitation and protein knock down studies using siRNA
  • Cell metabolic profiling (mitochondrial respiration, substrate utilisation)
  • Quantitative proteomics (SILAC: Stable Isotope Labelling of Amino Acids in Cell Culture)
  • Large dataset handling
  • Bioinformatics

Contact: Dr Debbie Cunningham, University of Birmingham