Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.780051
Title: Analysis of ABCG2 pharmacology and oligomerisation by solution based fluorescence correlation spectroscopy
Author: Horsey, Aaron J.
ISNI:       0000 0004 7965 7418
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2019
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Abstract:
ABCG2 is a human ATP-binding cassette (ABC) transporter. It has a broad substrate range that includes a number of small molecule drugs as well as endogenous metabolites. With its expression in barrier tissues (e.g. kidney epithelium) ABCG2 has a strong influence on pharmacokinetics in addition to other physiological roles (e.g. urate excretion). ABCG2 is also expressed in several tumours and confers resistance to various chemotherapy agents. The detailed interactions and mechanisms that support such polyspecificity are not well-established and progress in understanding ABCG2 pharmacology has been limited. The aim of this project was to develop novel, solution-based analysis methods to investigate ABCG2 pharmacology. Styrene-maleic acid copolymer lipid particles (SMALPs) were used to isolate ABCG2 in nanodiscs from a mammalian expression system (HEK293T). SMALP-ABCG2 particles were then characterised by fluorescence correlation spectroscopy (FCS) to investigate the stoichiometry of ABCG2 complexes and the pharmacology of their substrate binding. ABCG2 tagged with GFP or SNAP-tag was expressed in HEK293T cells. The appropriate plasma membrane localisation was demonstrated by confocal microscopy and the activity of the fusion proteins were validated using mitoxantrone efflux assays. ABCG2 fusion proteins were extracted from HEK293T cell membrane preparations using SMALPs. This process yielded solubilised ABCG2 that was analysed by dynamic light scattering and FCS. These analysis confirmed ABCG2 was extracted into single SMALP particles of 10-20nm diameter with < 5% aggregation. Photon counting histogram analysis was performed on FCS data gathered from SMALP-ABCG2 as well as monomeric (CD86) and dimeric (CD28) control proteins also extracted in SMALPs. The brightness of ABCG2 (51000cpms-1) was compared to that of the monomeric control (27000cpms-1) and the dimeric control (60000cpms-1). This revealed that ABCG2 was extracted as a dimer; the minimum functional unit. Subsequent extraction of ABCG2 from larger scale HEK293T membrane preparations facilitated purification of SMALP-ABCG2 by immobilised metal ion affinity chromatography at concentrations of up to 2μM. Binding of a fluorescent drug substrate of ABCG2 (BODIPY-FL-prazosin) to SMALP-ABCG2 was also analysed using FCS. In the presence of SMALP-ABCG2 (initially 20-30nM) both fast-moving free drug (700μm2s-1) and a much slower protein-bound drug (37μm2s-1) species could be quantified. This was extended into assessing substrate and inhibitor interactions where FCS was used in competition binding assays. Inhibition of BODIPY-FL-prazosin binding by unlabelled prazosin was observed, but not by the known transport inhibitor Ko143. Overall, the techniques developed in this project represent an improved workflow for the purification and assessment of protein complex stoichiometry. This extends to a novel platform for investigating substrate and inhibitor binding and recognition mechanisms.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.780051  DOI: Not available
Keywords: QP Physiology
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