Cox, Megan H.
(2019)
ABCG2: the lateral slice hypothesis as a model for multidrug transport.
PhD thesis, University of Nottingham.
Abstract
ABCG2 (also known as BCRP) is an efflux ABC transporter recognised to export a wide range of substances. ABCG2 is widely distributed in humans, predominantly residing within tissues which have protective roles, such as the intestine and blood brain barrier. ABCG2 is known for its role in the clinical phenomenon of multidrug resistance, particularly in limiting the exposure to chemotherapeutic agents. Despite this significant role in tissue exposure to xenobiotics, exactly how ABCG2 binds and transports a wide range of structurally unrelated drug substrates remains unknown.
This project set out to determine the molecular basis of drug polyspecificity and transport in this pharmacologically important membrane protein. Previous research had identified a pair of residues (R482, P485) which influenced substrate selectivity. Based on the existing data, a ‘lateral slice’ hypothesis was proposed,
identifying 10 residues in the transmembrane domain that were predicted through topological mapping, to be in a similar position in the plane of the membrane to R482 and P485 and would subsequently form part of a putative drug binding site. All ‘lateral slice’ residues investigated were mutated to alanine, as it is torsionally neutral and retains the propensity to form α-helices, so is unlikely to cause a structural perturbation. All ‘lateral slice’ ABCG2 mutants were able to express full length ABCG2 protein, that predominantly localised correctly to the plasma membrane. Functional investigations demonstrated the involvement of several of the ‘lateral slice’ residues in drug selectivity and efflux. For example, ABCG2F640A showed just over a 2-fold increase in mitoxantrone efflux compared to the wild type protein and a gain in the ability to efflux a non-native drug substrate; daunorubicin (an approximate increase of 7-fold compared to wild type protein). The interpretation of the ‘lateral slice’ experiment data was facilitated by the publication of structural data regarding the ABCG subfamily of ABC transporters. Structural mapping revealed that several of the functionally significant mutated ‘lateral slice’ residues were located to one patch on the surface of the protein. This observation led to a secondary working hypothesis, that the surface patch/pocket could be a site for drug recognition, so further residues lining this region were investigated in much the same way to the ‘lateral slice’ residues. All bar one of the new ‘binding pocket’ residues had an implication in drug efflux, with one mutation, M548A, interestingly demonstrating an enhanced efflux for mitoxantrone (1.4-fold), an impaired efflux for pheophorbide A (2.4-fold), and a gain in efflux for daunorubicin (4.8-fold) compared to wild type protein, showing drug specificity for the mutated residue. The final ‘binding pocket’ residue, L633, demonstrated disrupted protein biogenesis, suggesting a role in protein stabilisation and/or folding.
All the experimental data from these hypotheses were interpreted in terms of a multisite drug binding model for ABCG2 and supported the proposal of a novel transport hypothesis, in which, residues at a cytoplasmic lipid-exposed surface site act as a recognition/binding site for drugs, and residues within a secondary site form part of a translocation pathway to the extracellular compartment. This model makes a number of testable predictions for future research.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Kerr, Ian D.
Layfield, Robert |
| Keywords: |
Drug polyspecificity; Drug transport; Membrane proteins; Translocation pathway |
| Subjects: |
R Medicine > RM Therapeutics. Pharmacology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
56254 |
| Depositing User: |
Cox, Megan
|
| Date Deposited: |
19 Jul 2019 04:40 |
| Last Modified: |
19 Jul 2021 04:30 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/56254 |
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