Global proteomic analysis of prenylated proteins in Plasmodium falciparum using an alkyne-modified isoprenoid analogue

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.

Metabolic Labeling of Prenylated Proteins Using Alkyne‐Modified Isoprenoid Analogues

“…Labeled proteins appear as bands in the fluorescence scan due to the conjugated fluorophore. Additionally, treatment with inhibitors of the isoprenoid biosynthesis such as lovastatin (Palsuledesai et al, 2016) and fosmidomycin (Suazo et al, 2016) are employed to reduce the pool of endogenous FPP and GGPP, thereby enhancing the incorporation of the isoprenoid probe to the prenylated proteins. This protocol describes metabolic labeling using the isoprenoid analogues C15AlkOH ( 2 ), C15AlkOPP ( 1 ), C15AlkMPP ( 4 ) and C15AlkOP ( 5 ) in COS-7 cells in the presence or absence of lovastatin.…”

“…The synthesis of C15AlkOPP ( 1 ) and its incorporation in vitro by protein farnesyltransferase was reported in 2007 (Hosokawa et al, 2007) followed by reports of its use in metabolic labeling in 2010 (DeGraw et al, 2010). Since that time, it has been employed for a variety of experiments including differential electrophoresis to study prenylation inhibitors (Palsuledesai, Ochocki, Markowski, & Distefano, 2014), labeling of proteins sensitive to human pathogens (Charron, Li, MacDonald, & Hang, 2013) and the delineation of the prenylome of Plasmodium falciparum , the causative agent of malaria (Gisselberg, Zhang, Elias, & Yeh, 2017; Suazo, Schaber, Palsuledesai, Odom John, & Distefano, 2016). Recently, it has been demonstrated that a-factor, a farnesylated pheromone from yeast retains full activity when the farnesyl group is substituted with several chemically modified isoprenoids used for metabolic labeling.…”

a-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity

“… 16 − 20 Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. 21 − 24 However, at present there is no information about the activity of proteins modified with such groups. Additionally, while in vitro enzymological experiments have provided convincing evidence that prenyltransferases can incorporate such analogs, 25 , 26 there are no reports concerning the ability of the two proteases or the methyltransferase to act on proteins containing these moieties to convert them to their fully processed state.…”

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…Similar expansions of potentially farnesylated proteins could be expected in pathogenic organisms that employ either endogenous or host-mediated farnesylation, such as Plasmodium falciparum, Candida albicans, and Legionella pneumophila. (75,(80)(81)(82)(83)(84)(85) Our expansion of the potential substrates recognized by FTase, supported by the reactivity of human-derived C(x)3X peptide sequences, highlights the importance of continuing studies towards identifying additional new and biologically relevant protein substrates and determining their prenylation state within the cell. In particular, identification and isolation of endogenously prenylated C(x)3X proteins will be essential to understanding the biological impact of prenylation of non-canonical FTase substrates.…”