Abstract
The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.
Highlights
Leishmaniasis is a vector-borne neglected tropical disease (NTD) that affects large populations worldwide and is caused by various species of the protozoan pathogen Leishmania
This analysis revealed that LmjF.21.0725 is similar to the assigned putative leucyl, phenylalanyl-tRNA protein transferase sequences of more distantly related species, including Bodo saltans, a free-living nonparasitic kinetoplastid flagellate, and Phytophthora nicotianae (S2 Table)
Use of the suite “Phyre2 investigator” further confirmed the high quality of the predicted Lmj.21.0725 structure (S2 Fig). These in silico results suggest that LmjF.21.0725 might be a structural homologue of E. coli L/F transferase, even though it possesses low overall sequence homology (Fig 2E)
Summary
Leishmaniasis is a vector-borne neglected tropical disease (NTD) that affects large populations worldwide and is caused by various species of the protozoan pathogen Leishmania. The disease is highly prevalent, with 12 million cases distributed in more than 80 countries and an estimated population of 350 million at risk of infection. A putative aminoacyl-tRNA-protein transferase in Leishmania major manuscript. Jena Bioscience GmbH provided support in the form of salary for R.B. and research materials, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
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