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Experimental and Theoretical Modelling of the MAPK Pathway

Maddison, Louise Elizabeth

[Thesis]. Manchester, UK: The University of Manchester; 2012.

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Abstract

The MAPK pathway plays a crucial role in regulating cellular response to external stimuli. Binding of growth factors and other mitogenic signals to cell surface receptors initiates a phosphorylation-dependent relay of protein activation, resulting in altered transcription, ultimately regulating cell proliferation and differentiation. Signalling through this pathway is regulated by the coordinated function of specific protein kinases and protein phosphatases. As perturbation of this signalling system is often associated with diseases such as cancer, modelling is a useful means to help understand the outcomes that may result following changes in component levels or activity. The determination of absolute quantification data, in copies per cell, for proteins of the MAPK pathway will allow the expansion of and improved accuracy within predictive models.The strategy used within this thesis is based on the established technique of stable isotope dilution, generating isotopically labelled peptides using the QconCAT methodology. Recombinant DNA techniques were used to generate artificial concatamers of large numbers of tryptic peptides as quantification standards. A QconCAT, LM1, of 49 KDa (29 tryptic peptides), corresponding to the scaffold proteins was designed and built to encode two peptides per protein. A second QconCAT, LM2, of 58 KDa (34 tryptic peptides), encoded peptides from the dual-specificity phosphatases (DUSPs) and substrates. Quantification was performed using ultra performance liquid chromatography coupled to mass spectrometry. A selected reaction monitoring (SRM) approach was employed where the most intense y-ions per peptide were selected either from experimental data or predictions in silico. Using the ratio of the signal for the light:heavy isotopologues, the amount of light isotopologue can be inferred, allowing copies per cell quantifications to be established. Native peptides were present below the lower limit of quantification, and therefore the upper bounds of copies per cell were obtained for the three cell lines; colon cancer cells HCT 116 (K-Ras mutant) and HT-29 (B-Raf mutant) and a control cell line of HEK-293. Finally, mathematical modelling was undertaken to explore the mass-action kinetics of a three component scaffold signalling molecule. It was found that the optimal scaffold concentration is between the lowest and second lowest concentration of signalling protein.

Layman's Abstract

An emerging research field, known as Systems Biology, has the potential to provide a vital link to new drug discoveries to combat cancer. Systems Biology combines laboratory results and specialised software to simulate human cells in the computer. By working with these virtual cells, new insights into the disease can be revealed. Cells communicate to each other across a complex network of protein to protein interactions. When a small molecule known as a growth factor attaches to the outside of the cell, a relay of signals is stimulated. These signals travel through the cell to reach the nucleus. The nucleus holds the genetic instructions for making new cells and the instructions are acted upon. During cancer these signalling pathways between proteins become confused. For instance one protein called Ras can become permanently switched on and so the cells grow uncontrollably creating a tumour. The concentrations of proteins in healthy and cancerous human cells can be measured using mass spectrometry, a type of molecular weighing scales. The computer software contains a map of all the interactions and concentrations of each protein to build a virtual model of a cell. Experiments which would be too time consuming and complex to carry out in a laboratory can be investigated in a virtual environment. Through these techniques, we can improve our understanding of diseased cells, and realise which proteins are susceptible to intervention. This provides new drug targets to the pharmaceutical industry in the fight against cancer.