Abstract
Plasmodium parasites contain several unique membrane compartments in which prenylated proteins may play important roles in pathogenesis. Protein prenylation has also been proposed as an antimalarial drug target because farnesyltransferase inhibitors cause potent growth inhibition of blood-stage Plasmodium However, the specific prenylated proteins that mediate antimalarial activity have yet to be identified. Given the potential for new parasite biology and elucidating drug mechanism-of-action, we performed a large-scale identification of the prenylated proteome in blood-stage P. falciparum parasites using an alkyne-labeled prenyl analog to specifically enrich parasite prenylated proteins. Twenty high-confidence candidates were identified, including several examples of pathogen-specific prenylation activity. One unique parasite prenylated protein was FYVE-containing coiled-coil protein (FCP), which is only conserved in Plasmodium and related Apicomplexan parasites and localizes to the parasite food vacuole. Targeting of FCP to this parasite-specific compartment was dependent on prenylation of its CaaX motif, as mutation of the prenylation site caused cytosolic mislocalization. We also showed that PfRab5b, which lacks C-terminal cysteines that are the only known site of Rab GTPase modification, is prenylated. Finally, we show that the THQ class of farnesyltransferase inhibitors abolishes FCP prenylation and causes its mislocalization, providing the first demonstration of a specific prenylated protein disrupted by antimalarial farnesyl transferase inhibitors. Altogether, these findings identify prenylated proteins that reveal unique parasite biology and are useful for evaluating prenyltransferase inhibitors for antimalarial drug development.
Highlights
From the ‡Department of Biochemistry, §Pathology, ¶Microbiology and Immunology, and ʈChemical and Systems Biology, Stanford Medical School, Stanford University, Stanford, California 94025
Key questions we sought to answer were: What are the protein substrates of prenyltransferases in Plasmodium? How is protein prenylation in the parasite distinct from that in mammalian cells? Which prenylated proteins are important for mediating the antimalarial activity of FT inhibitors? Though the prenylated proteome is significantly reduced in P. falciparum compared with humans, we provide evidence for parasite-specific prenylation activity, including a novel prenylated protein and a novel site of modification
The THQ Class Farnesyltransferase Inhibitor BMS-386914 Disrupts FYVE-containing coiled-coil protein (FCP) Prenylation and Localization— the mechanism-of-action of antimalarial FT inhibitors depends on the cellular functions of CaaX protein substrates whose prenylation is disrupted, no specific CaaX protein substrate has AlkFOH labeled parasites treated with either 1x EC50 or 10x EC50 BMS-386914 and lonafarnib
Summary
From the ‡Department of Biochemistry, §Pathology, ¶Microbiology and Immunology, and ʈChemical and Systems Biology, Stanford Medical School, Stanford University, Stanford, California 94025. Parasite Proteins—To enable large-scale identification of prenylated proteins, we performed metabolic labeling with an alkyne-modified farnesol analog, AlkFOH, in Plasmodium-infected red blood cells.
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