Quantitation of the effects of cellular glucocorticoid receptor concentration on dimerization

Riekert, Brendon (2021-04)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: The glucocorticoids (GCs) are a group of steroids affecting virtually every cell in the body. The ability of the GCs to modulate the effects of the immune system is of particular interest therapeutically. Due to this characteristic, it is not surprising that the GCs have become the front line treatment in addressing a diverse array of inflammatory diseases ranging from sepsis to asthma. The GCs are known to function by means of their cognate receptor, the glucocorticoid receptor (GR), which resides in the cytoplasm as a monomer, complexed to various chaperone proteins. Once activated by ligand binding, the GR is able to act via two classical pathways. In the first, the activated GR remains in its monomeric form and acts in a trans-repressive manner to attenuate pro-inflammatory signalling and is associated with the positive therapeutic effects of the GCs. Alternatively, the activated GR is able to form a homodimer and activate metabolic pathways through transactivation. This form of activation is associated with the negative side effects of the GCs. The concentration of the GR in the body varies substantially, not only between different tissue types, but also within the same tissue in different disease states. Studies on GR concentration by Robertson et al. [1] has given rise to the ligand-independent dimerization model. This model suggests that higher concentrations of the GR lead to ligand independent dimerization that improves the sensitivity of tissues to GC stimulation through conformational changes that increase affinity of the GR for the ligand. Subsequent research by Barry [2] produced an in silico model of the GR dimerization cycle highlighting the importance of fluctuations in koff as opposed to the kon in affecting the affinity of the receptor for ligands at increasing GR concentrations, stressing the importance of ligand residence time in ligand receptor interactions. The experiments of Robertson et al. [1] were completed using the human GR. However, another pharmacologically sound model is that of the mouse GR, which at present offers the advantage of the recently developed monomeric GR construct, the GRmon and offers superior dimerization resistance when compared to the classical GRdim construct. Furthermore, the new fluorescence correlation spectroscopy technique for the observation of oligomers, the number and brightness assay (N&B), offers a unique opportunity to observe concentration dependent GR oligomerization by a confocal microscope [3]. The current project illustrates a comprehensive means of translating the previously defined physiologically relevant GR concentrations to the confocal microscope using median fluorescence intensity measurement. Furthermore, a method is proposed to use the N&B technique to observe ligand independent dimerization in the mouse model. Finally, a pilot investigation was conducted using the N&B method to capture 30 minutes of the oligomerization behaviour of the mouse GR following stimulation with the GR agonist dexamethasone, and some challenges which could be overcome in future research are highlighted. To our knowledge, this is the first time the N&B technique has been attempted on the African continent.

AFRIKAANSE OPSOMMING: Die glukokortikoïede (GK) is ‘n groep steroïede wat ‘n effek op feitlik elke sel in die liggaam uitoefen. In terme van terapeutiese gebruik is die GK van groot belang weens hul effek op die immuunstelsel. Weens hierdie eienskape is dit geensins verrassend dat die GK in die voorste linie staan as ‘n medikasie vir ‘n wye verskeidenheid siektes van sepsis tot asma nie. Die GK oefen hul effekte deur middel van hul verwante reseptor, die glukokortoïed reseptor (GR) uit, wat as ‘n deel van ‘n metgesel proteïenkompleks in die sitoplasma van die sel voorkom. Sodra geaktiveer deur ligand kan GR volgens een van twee moontlike klasieke roetes optree. Volgens die eerste roete, die trans-onderdrukkende roete, word pro- inflammatoriese seine onderdruk deur middel van monomeriese GR proteïne. Hierdie roete word met die positiewe effekte van GK’s geassosieer. Die tweede moontlike roete, die trans- aktiverende roete, is afhanklik van ‘n dimeriese vorm van die GR, en lei tot transkripsie van metaboliese gene. Die trans roete word met die newe-effekte van die GK geassosieer. Die konsentrasie van die GR in die liggaam wissel nie net afhangende van die weefsel tipe in die liggaam nie, maar ook tussen gesonde en siek weefsel. Onlangse navorsing oor GR konsentrasie deur Robertson et al. [1] het gelei tot die formulering van die ligand-onafhanklike roete van dimerisering. Hierdie roete behels dat hoёr konsentrasies van die GR lei tot ligand- onafhanklike dimerisering wat so die sensitiwiteit vir GK‘s verhoog deur struktuurveranderinge in die GR teweeg te bring wat die affiniteit van die GR vir GK‘s verhoog. Daaropvolgende navorsing deur [2] het gelei tot die daarstelling van ‘n in silico model van die GR dimerisasie siklus. In hierdie model word die onderliggende belangrikheid van veranderings in die koff in pleks van die kon geimpliseer as a ‘n faktor wat die affiniteit van die GR vir die ligand by toenemende konsentrasies GR te beïnvloed. Hierdie studie het dus die belangrikheid van molekulêre verblyftyd beklemtoon. Die eksperimente van Robertson et al. is met die menslikke GR model gedoen. Die muis GR model is egter ook ‘n geldige farmakologiese model wat tans die voordeel bied van die nuwe monomeriese GR konstruk, GRMon, wat dimerisasie beter teenstaan as die klassiek GR dim konstruk. Verder bied die nuwe fluoressensie-korrelasie spektroskopie metode, die aantal en helderheid toets (N&B), ‘n unieke geleentheid waarmee konsentrasie afhanklike GR oligomerisasie m.b.v. die konfokale mikroskoop waargeneem kan word [3]. Hierdie projek sluit ‘n samevatting in wat die navorser in staat stel om vorige gedefinieerde fisiologiese relevante GR konsentrasies oor te dra na die konfokale mikroskoop sisteem d.m.v. mediaan lig intensiteit metings verky vanaf fluoresensie gesorteerde GR sel populasies. Verder word ‘n metode voorgestel om N&B te gebruik om ligand-onafhanklike GR dimerisasie in die muis model te bepaal. Laastens is ‘n loods eksperiment uitgevoer wat die N&B metode gebruik om die oligomerisasie van muis GR oor 30 minute na stimulasie met die GR agonis, deksametasoon, te volg, en uitdagings vir toekomstige navorsing word bespreek. Hierdie is sover ons kon vasstel die eerste poging op die Afrika-vasteland om N&B toe te pas.

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