Identification of the Host Substratome of Leishmania -Secreted Casein Kinase 1 Using a SILAC-Based Quantitative Mass Spectrometry Assay.

Autor:	Smirlis D; Institut Pasteur, Université de Paris, Institut National de Santé et Recherche Médicale INSERM U1201, Unité de parasitologie Moléculaire et Signalisation, Paris, France.; Hellenic Pasteur Institute, Athens, Greece., Dingli F; Laboratoire de Spectrométrie de Masse Protéomique (LSMP), Centre de Recherche, Institut Curie, PSL Research University, Paris, France., Sabatet V; Laboratoire de Spectrométrie de Masse Protéomique (LSMP), Centre de Recherche, Institut Curie, PSL Research University, Paris, France., Roth A; Department of General and Visceral Surgery, Centre of Surgery, University Hospital Ulm, Ulm, Germany., Knippschild U; Department of General and Visceral Surgery, Centre of Surgery, University Hospital Ulm, Ulm, Germany., Loew D; Laboratoire de Spectrométrie de Masse Protéomique (LSMP), Centre de Recherche, Institut Curie, PSL Research University, Paris, France., Späth GF; Institut Pasteur, Université de Paris, Institut National de Santé et Recherche Médicale INSERM U1201, Unité de parasitologie Moléculaire et Signalisation, Paris, France., Rachidi N; Institut Pasteur, Université de Paris, Institut National de Santé et Recherche Médicale INSERM U1201, Unité de parasitologie Moléculaire et Signalisation, Paris, France.
Jazyk:	angličtina
Zdroj:	Frontiers in cell and developmental biology [Front Cell Dev Biol] 2022 Jan 03; Vol. 9, pp. 800098. Date of Electronic Publication: 2022 Jan 03 (Print Publication: 2021).
DOI:	10.3389/fcell.2021.800098
Abstrakt:	Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania . This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as "viral and symbiotic interaction," "actin cytoskeleton organisation" and "apoptosis," which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2022 Smirlis, Dingli, Sabatet, Roth, Knippschild, Loew, Späth and Rachidi.)
Databáze:	MEDLINE
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