Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei

Abstract

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.

Global profiling of myristoylation inToxoplasma gondiireveals key roles for lipidation in CDPK1 and MIC7 function

“…In light of this, we predict that NMT inhibition would exert severe pleiotropic effects on the Toxoplasma lytic cycle. Although potent and selective TgNMT inhibitors are yet to be reported, extensive work in other protozoan parasites (Schlott et al, 2019;Wright et al, 2014;Wright et al, 2016;Wright et al, 2015) demonstrates that selective NMT inhibition could provide an attractive strategy to combat infection.…”

“…To visualize the extent of myristoylation in T. gondii, we adapted a metabolic tagging approach that has previously been applied to mammalian cells (Broncel et al, 2015;Thinon et al, 2014) and protozoan parasites Wright et al, 2016;Wright et al, 2015). In this workflow, a myristic acid (Myr) analogue containing a terminal alkyne group (YnMyr) is added to cell culture upon infection with Toxoplasma ( Figure 1A).…”

“…reagent 1) that provides only indirect proof of target myristoylation ( Figure 2B), cleavable reagents allow for detection of myristoylated peptides in addition to peptides that originate from the enriched proteins ( Figure 2B). This additional layer of confidence in target identification is especially important given the high level of nonmyristoylation dependent background reported for metabolic tagging with YnMyr (Broncel et al, 2015;Wright et al, 2016;Wright et al, 2015). While reagent 2 has been validated as a tool for myristoylated protein and peptide discovery (Broncel et al, 2015), reagent 3 (Speers and Cravatt, 2005), which is expected to produce less background and improve myristoylated peptide discovery, has not previously been applied to study protein myristoylation.…”

“…N-myristoylation is an irreversible, predominantly co-translational covalent addition of myristic acid to an N-terminal glycine (Boutin, 1997;Gordon et al, 1991). It is catalysed by N-myristoyl transferase (NMT), which is conserved in Toxoplasma and has been reported to be a prominent drug target in fungal (Devadas et al, 1995;Nagarajan et al, 1997), Trypanosome (Frearson et al, 2010;Wright et al, 2016) and Leishmania infections (Hutton et al, 2014;Wright et al, 2015). In Plasmodium falciparum (the causative agent of malaria), inhibition of NMT leads to severe pleiotropic consequences affecting parasite development (Schlott et al, 2019;Wright et al, 2014), highlighting the importance of myristoylation for pathogen survival and progression.…”

Dynamic proteinS-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence

“…The palmitoylome was enriched in membrane transporters, proteases, surface proteins, lipid metabolism enzymes, trafficking proteins, and signaling proteins. Recently, the myristoylome and palmitoylome were both characterized and compared in bloodstream forms by metabolic labeling and click chemistry (Wright et al , 2016). The palmitoylomes of T. cruzi and Leishmania parasites have not been reported, but are actively being pursued in our laboratory.…”