title: Enrichment of Leishmania donovani ATP-binding proteins using a staurosporine capture compound.
abstract: Trypanosomatid parasites of the genus Leishmania cause severe human diseases collectively termed leishmaniasis. Parasite ATP-binding proteins have emerged as potent targets for chemotherapeutic intervention. However, many parasite-specific ATP-binding proteins may escape current efforts in drug target identification, validation and deconvolution due to the lack of sequence conservation and functional annotation of these proteins in early branching eukaryotic trypanosomatids. Here, we selectively enriched for ATP-binding proteins from Leishmania donovani axenic promastigote and amastigote total protein extracts utilizing a Capture Compound™ (CC) linked to the ATP-competitive inhibitor staurosporine. As judged by in-gel kinase activity assay and competitive inhibition with free staurosporine, the CC specifically enriched for parasite phosphotransferases. Comparative nanoLC-MS(n) analysis identified 70 captured proteins, including 24 conserved protein kinases, and 32 hypothetical proteins with potential ATP-binding function. We identified conserved signature sequence motifs characteristic for staurosporine-binding protein kinases, and identified the hypothetical proteins LinJ.20.0280 and LinJ.09.1630 as novel ATP-binding proteins. Thus, functional enrichment procedures such as described here, combined with bio-informatics analyses and activity assays, provide powerful tools for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.
  
  
  CONCLUSIONS
  Functional enrichment using a Capture Compound™ linked to the ATP-competitive inhibitor staurosporine provides a powerful new tool for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.
authors.affiliation: Institut Pasteur, and CNRS URA2581, Department of Parasitology and Mycology, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Leclercq
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bartho
authors.forename: Erik
authors.initials: E
authors.lastname: Duelsner
authors.forename: Lisa
authors.initials: L
authors.lastname: von Kleist
authors.forename: Pier Federico
authors.initials: PF
authors.lastname: Gherardini
authors.forename: Antonio
authors.initials: A
authors.lastname: Palmeri
authors.forename: Manuela
authors.initials: M
authors.lastname: Helmer-Citterich
authors.forename: Sabine
authors.initials: S
authors.lastname: Baumgart
authors.forename: Gerald F
authors.initials: GF
authors.lastname: Späth
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000379:methods; D063990:Gene Ontology; D007893:Leishmania donovani / Q000254:growth & development / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D015800:Protozoan Proteins / Q000302:isolation & purification*; D019311:Staurosporine / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002352:Carrier Proteins; D015800:Protozoan Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D019311:Staurosporine