The N-myristoylome of Trypanosoma cruzi

Abstract

Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%).

Role of Proteomics in the Study of Trypanosoma cruzi Biology

“…The greater part of these were proteins of unknown function (32), with the remaining (18) involved in a different spectrum of key cellular and metabolic roles, such as intracellular transport, cell signaling, and protein turnover. In conclusion, the results showed that 0.43-0.46% of the T. cruzi proteome corresponded to N-myristoylated proteins, slightly lower than that reported in other eukaryotic organisms (0.5-1.7%) [47].…”

Myristoylation, an Ancient Protein Modification Mirroring Eukaryogenesis and Evolution

“…These approaches were subsequently completed with subcellular fractionation approaches in plants. Combining the 364 sequences from these studies revealed a similar profile ( Figure 1B) to those observed in each of the nine organisms [7,[13][14][15][16][17][18][19].…”

“…& data from individual or large-scale analysis (with exception of T. gondii, see [93]); data in parentheses refer to those obtained with peptide libraries derived from genuine protein sequences, see references [7,[13][14][15][16][17][18][19]. For A. thaliana and H. sapiens, see Supplementary Datasets 1 & 2 in Ref.…”

Quantitative phosphoproteome and proteome analyses emphasize the influence of phosphorylation events during the nutritional stress of Trypanosoma cruzi: the initial moments of in vitro metacyclogenesis

“…NS is the initial stage of in vitro metacyclogenesis and has been proposed as the trigger that leads to the differentiation of the parasite (Figueiredo et al 2000). The SILAC methodology was first employed in T. cruzi in 2016, using a semidefined medium for cultivation to identify the N-myristoylome of the parasite (Roberts and Fairlamb 2016). Under our conditions, continuous growth and sufficient metabolic protein labeling by SILAC of the parasite were only possible using the defined medium LM-14B (De Paula Lima et al 2014).…”