Enrichment of Leishmania donovani ATP-binding proteins using a staurosporine capture compound

Autor: Antonio Palmeri, Erik Duelsner, Olivier Leclercq, Lisa von Kleist, Manuela Helmer-Citterich, Gerald F. Späth, Sabine Baumgart, Pier Federico Gherardini, Kathrin Bartho
Přispěvatelé: Parasitologie moléculaire et Signalisation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Caprotec Bioanalytics GmbH, Centre for Molecular Bioinformatics, Università degli Studi di Roma Tor Vergata [Roma], We thank Sophie Veillault for the editorial help. This work was supported by the 7th Framework Programme of the European Commission through a grant to the LEISHDRUG (223414) consortium, by the Agence Nationale de Recherche through a grant to the ANR-11-RPIB-0016 TRANSLEISH consortium, and the French Government's Investissements d'Avenir program: Laboratoire d'Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR-10-LABX-62-IBEID)., ANR-11-RPIB-0016,TRANSLEISH,Découverte de nouvelles protéines kinases chez Leishmania donovani à partir des inhibiteurs tête de série issus d'un criblage phénotypique(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 223414,EC:FP7:HEALTH,FP7-HEALTH-2007-B,LEISHDRUG(2008), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2013
Předmět:
Proteomics
MESH: Gene Ontology
Biophysics
Leishmania donovani
Protozoan Proteins
MESH: Carrier Proteins
Biochemistry
DNA-binding protein
Mass Spectrometry
Protein kinase
03 medical and health sciences
Adenosine Triphosphate
Tandem Mass Spectrometry
MESH: Adenosine Triphosphate
medicine
Staurosporine
Kinase activity
Protein kinase A
Amastigote
MESH: Protein Kinases
MESH: Protozoan Proteins
030304 developmental biology
0303 health sciences
MESH: Leishmania donovani
biology
Kinase
Settore BIO/11
MESH: Proteomics
030302 biochemistry & molecular biology
MESH: Tandem Mass Spectrometry
biology.organism_classification
Capture Compound
Gene Ontology
[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology
MESH: Staurosporine
Sequence motif
Carrier Proteins
Protein Kinases
MESH: Chromatography
Liquid
medicine.drug
Chromatography
Liquid
Zdroj: Journal of Proteomics; Vol 86
Journal of Proteomics
Journal of Proteomics, Elsevier, 2013, 86, pp.97-104. ⟨10.1016/j.jprot.2013.05.002⟩
Journal of Proteomics, 2013, 86, pp.97-104. ⟨10.1016/j.jprot.2013.05.002⟩
ISSN: 1874-3919
1876-7737
DOI: 10.1016/j.jprot.2013.05.002
Popis: International audience; 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.BIOLOGICAL SIGNIFICANCE: 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.
Databáze: OpenAIRE
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