Abstract
Severe malaria due to Plasmodium falciparum infection remains a serious threat to health worldwide and new therapeutic targets are highly desirable. Small molecule inhibitors of prenyl transferases, enzymes that catalyze the post-translational isoprenyl modifications of proteins, exhibit potent antimalarial activity. The antimalarial actions of prenyltransferase inhibitors indicate that protein prenylation is required for malaria parasite development. In this study, we used a chemical biology strategy to experimentally characterize the entire complement of prenylated proteins in the human malaria parasite. In contrast to the expansive mammalian and fungal prenylomes, we find that P. falciparum possesses a restricted set of prenylated proteins. The prenylome of P. falciparum is dominated by Rab GTPases, in addition to a small number of prenylated proteins that also appear to function primarily in membrane trafficking. Overall, we found robust experimental evidence for a total of only thirteen prenylated proteins in P. falciparum, with suggestive evidence for an additional two probable prenyltransferase substrates. Our work contributes to an increasingly complete picture of essential, post-translational hydrophobic modifications in blood-stage P. falciparum.
Highlights
Reason, protein farnesyltransferase inhibitors have been extensively explored by the pharmaceutical industry as potential human chemotherapeutics[10,11]
Red blood cells infected with P. falciparum were exposed to the probe in the presence or absence of fosmidomycin (FSM), an established inhibitor of isoprenoid biosynthesis in P. falciparum[23,24], followed by release of the intact parasites via mild detergent treatment
Evidence strongly suggests that protein prenylation is required for asexual development of the P. falciparum malaria parasite; several distinct chemotypes of prenyltransferase inhibitors exhibit potent antimalarial activity[15,16,18,34,35,36]
Summary
Reason, protein farnesyltransferase inhibitors have been extensively explored by the pharmaceutical industry as potential human chemotherapeutics[10,11]. P. falciparum malaria parasites possess protein prenyltransferase activity and have been found to incorporate both farnesyl and geranylgeranyl modifications into protein substrates[12,13]. Since Plasmodium spp. are evolutionarily divergent from organisms used to generate these models, and few prenylated proteins have been experimentally confirmed in malaria parasites, it is not clear how well bioinformatics algorithms perform in predicting prenyltransferase substrates for Plasmodium spp. We use a chemical labeling approach to metabolically tag, potentially, the full complement of prenylated proteins in asexual P. falciparum parasites. Our approach was to metabolically incorporate an alkyne-modified isoprenoid analogue into the pool of prenyltransferase protein substrates. This additional alkyne functional group permits selective binding of prenylated proteins to streptavidin beads, via click chemistry with biotin-azide. The resulting prenylated proteins were identified by subsequent tryptic digestion and LC-MS analysis, coupled with bioinformatics analysis
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