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Title: Function and dimerization of the human multidrug resistance pump, ABCG2
Author: Haider, Ameena Jassim
ISNI:       0000 0004 2711 8391
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2011
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ABCG2 is a half-transporter that belongs to the G-subfamily of ABC (ATP- binding cassette) transporters, which are characterised by their unique domain organisation; an N-terminal nucleotide-binding domain (NBD) followed by a transmembrane domain (TMD) to its C-terminal. ABCG2 has been investigated as a multitask transporter widely distributed in normal tissues as well as overexpressed in cancer stem cells and cancer cell lines to confer protection against different xenobiotics and induce multidrug resistance (MDR), respectively. Since the time it was discovered, several studies investigated the effect of certain residues or subdomains within human ABCG2 affecting its substrate specificity, structure, function as well as its dimerization (or oligomerization) status. This study was designed to identify highly co-evolved residues within human cellular localization and/or function. The study also investigated ABCG2 dimerization via a recently developed tool; bimolecular fluorescence complementation (BiFC), through which visualisation of ABCG2 dimerization in live cells was enabled. According to co-evolutionary investigations, eight novel residues were chosen from a wide range of highly correlated coupled residues shared among ABCG- related sequences, and were used to design single mutants that were tested for ABCG2 expression, sub-cellular localization and function, besides control mutants of known effects. All variants were expressed at a comparable level to wild-type ABCG2R482 except for an I573A mutant that showed altered glycosylation level and enhanced intracellular retention. All mutants were functionally capable of extruding Pheophorbide A (PhA), Mitoxantrone and BODIPY-Prazosin, and inhibited by Fumitremorgin C (FTC), as revealed by fluorescent export studies and flow cytometry, except for two mutants, P485A and M549A, which showed altered inhibition profile with FTC. This study revealed that these two residues could participate in the inhibitor binding site or in the communication between the drug and inhibitor binding sites. Bimolecular fluorescence complementation analyses revealed that dimerization of N-terminally YFP-tagged ABCG2 constructs was specifically localized to the plasma membrane of live cells. However, mutating single residues, previously published to participate in ABCG2 dimer formation, did not significantly alter the BiFC signal. BiFC enabled the qualitative investigation of ABCG2 dimerization and function but was insensitive enough to map single residue changes within the dimer formation interface. This study opens the door for future investigations of conserved residues within human ABCG2 that could increase the depth of understanding allosteric interactions between drug and inhibitor binding sites within the transporter via applying a wider range of specific ABCG2 substrates and inhibitors. It also guides further studies to investigate the dimer formation status of ABCG2 and its possible inhibition to overcome multidrug resistance and failure of chemotherapy in cancer cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Not available Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QU Biochemistry