Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei Adenosine Transporter 1 (TbAT1) and the proposal of a structural model for the protein

Munday, J. C. et al. (2015) Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei Adenosine Transporter 1 (TbAT1) and the proposal of a structural model for the protein. Molecular Microbiology, 96(4), pp. 887-900. (doi:10.1111/mmi.12979) (PMID:25708978) (PMCID:PMC4755147)

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Abstract

The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1 -/- T. brucei line. Out of a set of six reported SNPs from a reported ‘resistance allele’, none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [3H]-adenosine and [3H]-diminazene, identical to the tbat1-/- control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [3H]-diminazene, but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Alzahrani, Khalid Jamaan and Munday, Dr Jane and De Koning, Professor Harry and McDonald, Ms Fiona and Settimo, Dr Luca
Authors: Munday, J. C., Tagoe, D. N. A., Eze, A. A., Krezdorn, J. A. M., Rojas López, K. E., Alkhaldi, A. A. M., McDonald, F., Still, J., Alzahrani, K. J., Settimo, L., and De Koning, H. P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Health and Wellbeing > MRC/CSO Unit
Journal Name:Molecular Microbiology
Publisher:John Wiley & Sons Ltd.
ISSN:0950-382X
ISSN (Online):1365-2958
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Molecular Microbiology 96(4):887-900
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
371796The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)085349/Z/08/ZIII - PARASITOLOGY
371798The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)085349/B/08/ZIII - PARASITOLOGY
471291Drug resistance in African trypanosomesHarry De KoningMedical Research Council (MRC)G0701258III - PARASITOLOGY