Modelling the glucocorticoid receptor dimerisation cycle

Barry, Christopher James (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Die ontdekking van die menslike glukokortikoïed (GC) kortisol, wat met die Nobelprys bekroon is, het 'n belangrike bydrae gelewer tot die ontwikkeling van steroïedale anti-inflammatoriese medikasie. GCs word gebruik om siektes wat deur gebreke in die immuunstelsel veroorsaak word, soos rumatoïede artritis, allergieë, asma, sepsis, akute oorplanting verwerping en ent-versus-gasheer siekte, te behandel. Soos met baie lede van die steroïed-klas van molekule, reguleer GCs egter 'n wye reeks van biologiese prosesse, en om hierdie rede gaan terapeutiese gebruik dikwels gepaard met 'n aantal newe-effekte. Die meerderheid van die GC-effekte word bemiddel deur aktivering van die glukokortikoïed-reseptor (GR). Die onaktiewe vorm van GR kom in die sitoplasma voor as 'n monomeer en beweeg na die selkern wanneer dit aan 'n gepaste steroïed bind. Sodra die GR binne die selkern is kan dit as monomeer as 'n transkripsionele onderdrukker optree, wat geassosieer word met die positiewe effekte van GC behandeling. Alternatiewelik kan die GR optree as `n dimeer, in hierdie geval as 'n transkripsiefaktor deur te bind aan GC responselemente om gene te aktiveer. Hierdie meganisme van werking word geassosieer met die newe-effekte van GC behandeling. Dus bepaal ligandbinding en reseptor-dimerisering die seintransduksie van GCs en die daaropvolgende induksie of repressie van transkripsie. Die aktivering van GR deur ligandbinding kan twee weë volg wat gelyktydig plaasvind: in die "klassieke pad" bind die ligand eers aan monomeriese GR wat later dimeriseer, terwyl GR in die "alternatiewe pad" 'n dimeer vorm in die afwesigheid van ligand, waaraan die ligand daarna bind. Omdat GCs hidrofobies is, is dit in staat om sonder transportproteïene deur die selmembraan te beweeg, dus is hul sellulêre konsentrasie min of meer gelyk in die meeste weefsels. Aan die ander kant is GRs teenwoordig in verskillende konsentrasies in die liggaam, afhangende van die tipe weefsel, variasie tussen individue, fisiologiese toestande en siektes. Met hierdie punte in gedagte kan daar afgelei word dat die GR waarskynlik die belangrikste bydraende faktor is wat differensiële GC aktiwiteit bepaal. Tot onlangs was die invloed van GR konsentrasie op GC aktiwiteit, asook die molekulêre meganisme hiervan, nie bepaal nie. In 2013 het Robertson et al. getoon dat die Hill koëffisiënt van GR-dexametasoon binding asook die potensie toegeneem het wanneer die totale GR konsentrasie verhoog word. Dieselfde studie het gewys dat hoë vlakke van wilde-tipe GR ligand-onafhanklike dimerisering kan toon, 'n voorvereiste vir koöperatiewe ligandbinding. Ten slotte kon die verskuiwings in Hill koëffisiënt, potensie en ligand-onafhanklike dimerisering opgehef word deur die gebruik van 'n GRdim mutant wat nie tot dimerisering in staat is nie. 'n Kern uitkoms van hierdie projek was die formulering van 'n wiskundige model van GR dimerisering en Dex binding. Hierdie model kon die eksperimenteel waargenome skuiwe in potensie en Hill koëffisiënt met verandering in GR konsentrasie naboots, wanneer Dex-GR versadigings-bindingseksperimente gesimuleer word, al was die grootte van hierdie verskuiwings nie dieselfde as in eksperimentele data uit die literatuur nie. Hierdie studie bevestig dus die toename in potensie en Hill koëffisiënt met 'n toename in GR konsentrasie, wat oorspronklik deur Robertson et al. is aangetoon. Verder is hierdie model in staat om GR-GC binding te voorspel in selle met verskillende GR konsentrasies. Daar word berig oor die ontwikkeling van 'n metode om die spesifieke binding in 'n populasie van selle te skaleer tot die relatiewe verspreiding van GR binne daardie populasie. Die onderliggende kinetiese grondslag vir die toename in potensie is in hierdie studie bepaal, nl. 'n GR konsentrasie-afhanklike afname in koff terwyl kon min of meer konstant bly. Hierdie afname in koff word geëlimineer wanneer dimerisering uitgeskakel word, en derhalwe kan die konsentrasie-afhanklike verskuiwing in potensie waarskynlik toegeskryf word aan die dimeriseringsreaksies in beide die klassieke en alternatiewe roetes van GR aktivering. Hierdie projek behels 'n nuwe benadering tot die simulering van GR-GC binding, wat beskou word as 'n noodsaaklike stap in GR aktivering. Die bevindinge toon dat die GC seintransduksie-stelsel meer gevoelig is vir veranderinge in GR konsentrasie as wat voorheen verwag was. Dit het implikasies vir navorsing oor GC seintransduksie, steroïede in die algemeen, asook vir steroïed-terapieë en die ontwikkeling van weerstand teen GCs.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101272
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