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Sci Rep. 2018 Jun 12;8(1):8932. doi: 10.1038/s41598-018-27183-w.

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

Author information

1
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, 1117, Hungary. marton.livia@ttk.mta.hu.
2
Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, 6720, Hungary. marton.livia@ttk.mta.hu.
3
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, 1117, Hungary.
4
Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, 1111, Hungary.
5
Division of Structural Biology, University of Oxford, Roosevelt Drive, Oxford, OX37BN, United Kingdom.
6
Institute of Cancer Research, Medical University Vienna, Vienna, Austria.
7
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, 1117, Hungary. vertessy@mail.bme.hu.
8
Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, 1111, Hungary. vertessy@mail.bme.hu.

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.

PMID:
29895950
PMCID:
PMC5997628
DOI:
10.1038/s41598-018-27183-w
[Indexed for MEDLINE]
Free PMC Article

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