Abstract
ABSTRACT Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a Plasmodium-encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates. In eukaryotes, P4-ATPases assure the asymmetric phospholipid distribution in membranes by translocating phospholipids from the outer to the inner leaflet. In this work, we have used a recombinantly-produced P. chabaudi ATP2 (PcATP2), to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A. Purified PcATP2/PcCdc50B complex displays ATPase activity in the presence of either phosphatidylserine or phosphatidylethanolamine. In addition, this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work describes the first biochemical characterization of a Plasmodium lipid flippase, a first step towards the understanding of the essential physiological role of this transporter and towards its validation as a potential antimalarial drug target.
Highlights
Malaria is a global health problem affecting over 200 millions of people worldwide, and responsible in 2018 of nearly 4,05,000 deaths around the globe, mostly children[1]
Single expression vectors: The cDNAs encoding the three ATP2 orthologs were cloned in the pYeDP60 expression vector [31] between the EcoRI and NotI restriction sites, yielding the pYeDP60-ATP2-TEVBAD plasmid that contains in downstream position the tobacco etch virus (TEV) protease cleavage-site coding sequence followed by the biotin acceptor domain (BAD) coding sequence
Homologs to ATP2 are present in the genome of other disease-causing intracellular parasites belonging, to the Apicomplexa phylum like Plasmodium (Figure supplement 1) [8], it is still unknown if these transporters are essential
Summary
Malaria is a global health problem affecting over 200 millions of people worldwide, and responsible in 2018 of nearly 4,05,000 deaths around the globe, mostly children[1]. Five different Plasmodium species infect humans, P. falciparum being the most virulent one, accounting for ∼90% of the mortality caused by this disease. The combination of drugs acting on different targets and/or stages is the most efficient strategy to combat malaria as it minimizes the development of new drug-resistance mechanisms [3]. The cDNA encoding the superfolder Green Fluorescent Protein (GFP) was PCR amplified, introducing a NotI site followed by the human Rhinovirus 3C protease sequence (3C-protease) at the 5′, and the XmaI site at the 3′. The morning the tube was centrifuged at 350 × g for 2 min, the supernatant was removed, and the cells were re-suspended in 100 μL of S6A minimal media (SAU without uracil) and plated on S6A-agar plates. The plates were left in the incubator at 28°C for 3–5 days until the transformed colonies appeared
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