Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Author "Barry, Christopher James"

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    Modelling the glucocorticoid receptor dimerisation cycle
    (Stellenbosch : Stellenbosch University, 2017-03) Barry, Christopher James; Rohwer, J. M.; Louw, A.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The Nobel prize winning discovery of the human glucocorticoid (GC), cortisol, was instrumental in steroidal anti-inflammatory medication development. GCs are employed to combat diseases caused by malfunctions in the immune response such as rheumatoid arthritis, allergies, asthma, sepsis, acute transplant rejection and graft-versus-host disease. However, as with many members of the steroid class, GCs regulate a plethora of biological processes and consequently therapeutic use is associated with a number of side effects. The majority of GC effects are mediated through activation of their cognate steroid receptor, the glucocorticoid receptor (GR). In the inactive form, the GR resides in the cytoplasm as a monomer. Upon ligand binding the receptor-ligand complex translocates into the nucleus. Once inside the nucleus, the GR can either remain a monomer and act as a trans-acting transcriptional repressor, which is associated with the positive effects of GC treatments. Alternatively, the GR can dimerise and act as a cis-acting transcriptional activator, associated with the side effects of GC treatments. Therefore, ligand binding and dimerisation are major factors that determine GC signal transduction and subsequent induction or repression of transcription. Ligand activation of GR can follow two pathways, which occur simultaneously: either via the "classical pathway", which consists of ligand binding to monomeric GR, which subsequently dimerises, or via the "alternative pathway", where GR dimerises independently of ligand and ligand subsequently binds to the dimer. Being hydrophobic, GCs are able to pass through the cell membrane without transporters, hence, at any given time, their cellular concentration is roughly equal in most tissues. Conversely, GRs are present throughout the body at different concentrations depending on tissue type, inter individual variation, physiological conditions and disease state. Taken together, GR level is likely a primary cause of variations in GC activity. Until recently, the influence of GR concentration on GC activity had not been quantified nor had the molecular mechanism been elucidated. In 2013, Robertson et al. showed that the Hill coeficient and potency of GR-Dexamethasone (Dex) binding increased with an increase in GR concentration. The shifts in Hill coeficient and potency were abolished when dimerisation was abrogated using a dimerisation deficient mutant. The same study showed that high levels of wild type GR displayed ligand-independent dimerisation, which is a prerequisite for cooperative ligand binding. A major outcome of this project was the formulation of a mathematical model of the GR dimerisation cycle and Dex binding. Significantly, this model captured GR concentration-dependent shifts in potency and Hill coefficient when simulating GRDex saturation binding experiments, albeit not to the same extent as experimental data from literature. This correlates with the increase in potency and Hill coefficient with an increase in GR concentration shown by Robertson et al.. Furthermore, this model is capable of simultaneously predicting GR-GC binding in cells with different GR concentrations, which more closely resembles a transiently transfected cell population. Using a method developed in this study, the specific binding of a population of cells can be scaled to the relative distribution of GR within that cell population. The kinetic basis for the increase in potency was determined in this study as a GR concentration-dependent decrease in koff as kon remained constant. This decrease in koff was eliminated when dimerisation was abrogated and therefore the concentration-dependent shift in potency is most likely attributed to the dimerisation reactions present in both the classical and alternate pathways of GR activation. This project comprised a novel approach of simulating GR-GC binding, considered a requisite step of GR activation. The findings demonstrate that the GC signal transduction system is more sensitive to GR concentration than has been previously anticipated. This has implications for GC signal transduction research, steroid research in general, as well as for therapeutic regimes and the development of GC resistance.