Dataset Information

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme.

ABSTRACT: The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

SUBMITTER: Izrael R

PROVIDER: S-EPMC7665022 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme.

Izrael Richard R Marton Lívia L Nagy Gergely N GN Pálinkás Hajnalka L HL Kucsma Nóra N Vértessy Beáta G BG

Scientific reports 20201112 1

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the struct ...[more]

PMID: 33184408

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Project description:Phospholipid biosynthesis plays a key role in malarial infection and is regulated by CCT (CTP:phosphocholine cytidylyltransferase). This enzyme belongs to the group of amphitropic proteins which are regulated by reversible membrane interaction. To assess the role of the putative membrane-binding domain of Plasmodium falciparum CCT (PfCCT), we synthesized three peptides, K21, V20 and K54 corresponding to residues 274-294, 308-327 and 274-327 of PfCCT respectively. Conformational behaviour of the peptides, their ability to bind to liposomes and to destabilize lipid bilayers, and their insertion properties were investigated by different biophysical techniques. The intercalation mechanisms of the peptides were refined further by using surface-pressure measurements on various monolayers at the air/water interface. In the present study, we show that the three studied peptides are able to bind to anionic and neutral phospholipids, and that they present an alpha-helical conformation upon lipid binding. Peptides V20 and the full-length K54 intercalate their hydrophobic parts into an anionic bilayer and, to a lesser extent, a neutral one for V20. Peptide K21 interacts only superficially with both types of phospholipid vesicles. Adsorption experiments performed at the air/water interface revealed that peptide K54 is strongly surface-active in the absence of lipid. Peptide V20 presents an atypical behaviour in the presence of phosphatidylserine. Whatever the initial surface pressure of a phosphatidylserine film, peptide V20 and phosphatidylserine entities seem linked together in a special organization involving electrostatic and hydrophobic interactions. We showed that PfCCT presents different lipid-dependence properties from other studied CCTs. Although the lipid-binding domain seems to be located in the C-terminal region of the enzyme, as with the mammalian counterpart, the membrane anchorage, which plays a key role in the enzyme regulation, is driven by two alpha-helices, which behave differently from one another.

Dataset Information

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme.

Publications

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme.

Similar Datasets

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