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Title: Interaction between Plasmodium falciparum chloroquine resistance transporter (PfCRT) and atpase: a mechanistic study on the mechanism of chloroquine resistance and its reversal
Authors: Chow, Tsun Sing
Degree: Ph.D.
Issue Date: 2020
Abstract: Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a transmembrane protein localized at the digestive vacuole (DV) membrane in Plasmodium falciparum (P. falciparum) parasite. Mutations in PfCRT correlated with chloroquine resistance (CQR) in malaria; among which K76T point mutation at the first transmembrane helix plays a critical role. The mutant PfCRT effluxes protonated chloroquine (CQ) from the DV of P.falciparum parasite in an ATP-dependent manner. However, PfCRT has no recognizable nucleotide binding domain to bind and hydrolyze ATP. Recently, PfCRT has been shown to physically and functionally interact with α and β subunits of F-ATPase when PfCRT was overexpressed in Pichia pastoris (P. pastoris). Similar interaction occurred between PfCRT and V-ATPase subunit B in P.falciparum (PfV1B). The interacting site between PfCRT and F-/V-ATPase, however, is unknown. By understanding the interacting site, specific inhibitors may be designed to disrupt PfCRT-V-ATPase interaction in P.falciparum to modulate CQR for future therapeutic use. In this project, the N terminus (1-58), internal loop 4 (265-315) and C terminus (399-424) of PfCRT were proposed to be the interacting domains. These 3 parts of PfCRT have the largest soluble domains and therefore have the highest chance of interacting with other proteins. Peptides derived from these parts were tested for their ability to disrupt PfCRT-F-ATPase interaction in P. pastoris microsome. It was found that both N terminus and C terminus can inhibit disuccinimidyl suberate (DSS)-crosslinking of PfCRT-F-ATPase, [3H]-CQ uptake activity and oligomycin A-inhibitable ATPase activity, suggesting that they were involved in such interaction. Truncation and mutation of PfCRT N and C terminus peptides were used to map the interacting regions in detail. The interaction between PfCRT and F-/V-ATPase was studied using the following 4 assays. (1) Physical interaction between C terminus of PfCRT and PfV1B and (2) physical interaction between PfCRT N terminus (1-20) and PfV1B were studied using pulldown assays. The functional consequence of PfCRT was also measured using (3) [3H]-CQ uptake assay and (4) F1-ATPase activity assay. It was found that 3 separate regions of PfCRT can physically and functionally interact with F-/V-ATPase, namely residues 1-20 of N terminus (MKFASKKNNQKNSSKNDERY), residues 21-40 of N terminus (RELDNLVQEGNGSRLGGGSC) and residues 408-424 of C terminus (NEDSEGELTNVDSIITQ). Peptide derived from residues 408-424 of PfCRT C terminus showed the most promising effect on disruption of PfCRT-F-/V-ATPase interaction including inhibition on PfCRT CQ transport activity (51±5%), binding of PfCRT C terminus to PfV1B (74±7%) and binding of PfCRT N terminus (1-20) to PfV1B (52±4%). Such region also has a mild F1-ATPase stimulatory effect (20±3%). This region, therefore, has a potential to be developed as a peptide to modulate CQ resistance. The above mapping results suggested that V1B can interact with 3 sites of PfCRT (2 in N terminus and 1 in C terminus). Based on this, a model for how PfCRT-V-ATPase interaction can regulate CQ transport is proposed which has three stages, namely N1-, N3- and C-stage. In these 3 stages, residues 1-20, 21-40 and 408-424 of PfCRT was interacting with PfV1B, respectively. Interaction between PfCRT and V-ATPase is slightly different in each stage and the transition between stages might be responsible for the coupling of ATP hydrolysis in V-ATPase and CQ transport activity of PfCRT. PfCRT is in "resting", "inward-facing" and "outward-facing" conformation and V-ATPase is in "open", "tight" and "loose" conformation in these 3 stages, respectively. Such model could explain how PfCRT uses the 3 sites to bind to V-ATPase, changes the conformation of the latter, activates the ATPase activity and couples this to the conformational changes of PfCRT to transport CQ.
Subjects: Drug resistance
Hong Kong Polytechnic University -- Dissertations
Pages: xviii, 177 pages : color illustrations
Appears in Collections:Thesis

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