Dataset Information

Mass spectrometry data for Leishmania major proteins obtained by immunoprecipitation with anti-PUF1, anti-PUF3 or pre-immunization antibodies.

ABSTRACT: This project was aimed to the characterization of proteins interacting with the RNA binding proteins PUF1 and PUF3 of Leishmania major (Friedlin strain). For this purpose an immunoprecipitation (IP) strategy was followed. Firstly, protein extracts from L. major promastigotes were incubated with specific antibodies, raised in rabbits, against recombinant proteins PUF1 or PUF3. In parallel, a control IP using a pre-immune serum was performed. After affinity purification of the antibody-protein complexes, proteins were analyzed by Reverse phase- Liquid Chomatography/Mass spectrometry.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Leishmania Major

SUBMITTER: Jose María Requena

LAB HEAD: Jose M. Requena

PROVIDER: PXD022581 | Pride | 2021-05-17

REPOSITORIES: Pride

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Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

Dataset Information

Mass spectrometry data for Leishmania major proteins obtained by immunoprecipitation with anti-PUF1, anti-PUF3 or pre-immunization antibodies.

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