Heterologous expression of CTP: Phosphocholine cytidylyltransferase from Plasmodium falciparum rescues Chinese Hamster Ovary cells deficient in the Kennedy phosphatidylcholine biosynthesis pathway

Lívia Marton, Fanni Hajdú, Gergely N. Nagy, Nóra Kucsma, G. Szakács, B. Vértessy

Research output: Contribution to journalArticle

Abstract

The plasmodial CTP:phosphocholine cytidylyltransferase (PfCCT) is a promising antimalarial target, which can be inhibited to exploit the need for increased lipid biosynthesis during the erythrocytic life stage of Plasmodium falciparum. Notable structural and regulatory differences of plasmodial and mammalian CCTs offer the possibility to develop species-specific inhibitors. The aim of this study was to use CHO-MT58 cells expressing a temperature-sensitive mutant CCT for the functional characterization of PfCCT. We show that heterologous expression of wild type PfCCT restores the viability of CHO-MT58 cells at non-permissive (40 °C) temperatures, whereas catalytically perturbed or structurally destabilized PfCCT variants fail to provide rescue. Detailed in vitro characterization indicates that the H630N mutation diminishes the catalytic rate constant of PfCCT. The flow cytometry-based rescue assay provides a quantitative readout of the PfCCT function opening the possibility for the functional analysis of PfCCT and the high throughput screening of antimalarial compounds targeting plasmodial CCT.

Original languageEnglish
Article number8932
JournalScientific Reports
Volume8
Issue number1
DOIs
Publication statusPublished - Dec 1 2018

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Choline-Phosphate Cytidylyltransferase
CHO Cells
Antimalarials
Plasmodium falciparum
Cricetulus
Phosphatidylcholines
Ovary
Temperature
Flow Cytometry
Lipids
Mutation

ASJC Scopus subject areas

  • General

Cite this

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title = "Heterologous expression of CTP: Phosphocholine cytidylyltransferase from Plasmodium falciparum rescues Chinese Hamster Ovary cells deficient in the Kennedy phosphatidylcholine biosynthesis pathway",
abstract = "The plasmodial CTP:phosphocholine cytidylyltransferase (PfCCT) is a promising antimalarial target, which can be inhibited to exploit the need for increased lipid biosynthesis during the erythrocytic life stage of Plasmodium falciparum. Notable structural and regulatory differences of plasmodial and mammalian CCTs offer the possibility to develop species-specific inhibitors. The aim of this study was to use CHO-MT58 cells expressing a temperature-sensitive mutant CCT for the functional characterization of PfCCT. We show that heterologous expression of wild type PfCCT restores the viability of CHO-MT58 cells at non-permissive (40 °C) temperatures, whereas catalytically perturbed or structurally destabilized PfCCT variants fail to provide rescue. Detailed in vitro characterization indicates that the H630N mutation diminishes the catalytic rate constant of PfCCT. The flow cytometry-based rescue assay provides a quantitative readout of the PfCCT function opening the possibility for the functional analysis of PfCCT and the high throughput screening of antimalarial compounds targeting plasmodial CCT.",
author = "L{\'i}via Marton and Fanni Hajd{\'u} and Nagy, {Gergely N.} and N{\'o}ra Kucsma and G. Szak{\'a}cs and B. V{\'e}rtessy",
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AU - Marton, Lívia

AU - Hajdú, Fanni

AU - Nagy, Gergely N.

AU - Kucsma, Nóra

AU - Szakács, G.

AU - Vértessy, B.

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AB - The plasmodial CTP:phosphocholine cytidylyltransferase (PfCCT) is a promising antimalarial target, which can be inhibited to exploit the need for increased lipid biosynthesis during the erythrocytic life stage of Plasmodium falciparum. Notable structural and regulatory differences of plasmodial and mammalian CCTs offer the possibility to develop species-specific inhibitors. The aim of this study was to use CHO-MT58 cells expressing a temperature-sensitive mutant CCT for the functional characterization of PfCCT. We show that heterologous expression of wild type PfCCT restores the viability of CHO-MT58 cells at non-permissive (40 °C) temperatures, whereas catalytically perturbed or structurally destabilized PfCCT variants fail to provide rescue. Detailed in vitro characterization indicates that the H630N mutation diminishes the catalytic rate constant of PfCCT. The flow cytometry-based rescue assay provides a quantitative readout of the PfCCT function opening the possibility for the functional analysis of PfCCT and the high throughput screening of antimalarial compounds targeting plasmodial CCT.

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