Laboratoire de Spectrométrie de Masse Protéomique (LSMP)

Publications

Année de publication : 2019

Matteo Fossati, Nora Assendorp, Olivier Gemin, Sabrina Colasse, Florent Dingli, Guillaume Arras, Damarys Loew, Cécile Charrier (2019 Nov 10)

Trans-Synaptic Signaling through the Glutamate Receptor Delta-1 Mediates Inhibitory Synapse Formation in Cortical Pyramidal Neurons.

Neuron : 1081-1094.e7 : DOI : S0896-6273(19)30801-3 En savoir plus
Résumé

Fine orchestration of excitatory and inhibitory synaptic development is required for normal brain function, and alterations may cause neurodevelopmental disorders. Using sparse molecular manipulations in intact brain circuits, we show that the glutamate receptor delta-1 (GluD1), a member of ionotropic glutamate receptors (iGluRs), is a postsynaptic organizer of inhibitory synapses in cortical pyramidal neurons. GluD1 is selectively required for the formation of inhibitory synapses and regulates GABAergic synaptic transmission accordingly. At inhibitory synapses, GluD1 interacts with cerebellin-4, an extracellular scaffolding protein secreted by somatostatin-expressing interneurons, which bridges postsynaptic GluD1 and presynaptic neurexins. When binding to its agonist glycine or D-serine, GluD1 elicits non-ionotropic postsynaptic signaling involving the guanine nucleotide exchange factor ARHGEF12 and the regulatory subunit of protein phosphatase 1 PPP1R12A. Thus, GluD1 defines a trans-synaptic interaction regulating postsynaptic signaling pathways for the proper establishment of cortical inhibitory connectivity and challenges the dichotomy between iGluRs and inhibitory synaptic molecules.

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Ana Uzquiano, Carmen Cifuentes-Diaz, Ammar Jabali, Delfina M Romero, Anne Houllier, Florent Dingli, Camille Maillard, Anne Boland, Jean-François Deleuze, Damarys Loew, Grazia M S Mancini, Nadia Bahi-Buisson, Julia Ladewig, Fiona Francis (2019 Aug 8)

Mutations in the Heterotopia Gene Eml1/EML1 Severely Disrupt the Formation of Primary Cilia.

Cell reports : 1596-1611.e10 : DOI : S2211-1247(19)30882-4 En savoir plus
Résumé

Apical radial glia (aRGs) are predominant progenitors during corticogenesis. Perturbing their function leads to cortical malformations, including subcortical heterotopia (SH), characterized by the presence of neurons below the cortex. EML1/Eml1 mutations lead to SH in patients, as well as to heterotopic cortex (HeCo) mutant mice. In HeCo mice, some aRGs are abnormally positioned away from the ventricular zone (VZ). Thus, unraveling EML1/Eml1 function will clarify mechanisms maintaining aRGs in the VZ. We pinpoint an unknown EML1/Eml1 function in primary cilium formation. In HeCo aRGs, cilia are shorter, less numerous, and often found aberrantly oriented within vesicles. Patient fibroblasts and human cortical progenitors show similar defects. EML1 interacts with RPGRIP1L, a ciliary protein, and RPGRIP1L mutations were revealed in a heterotopia patient. We also identify Golgi apparatus abnormalities in EML1/Eml1 mutant cells, potentially upstream of the cilia phenotype. We thus reveal primary cilia mechanisms impacting aRG dynamics in physiological and pathological conditions.

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Vinay Mandati, Laurence Del Maestro, Florent Dingli, Bérangère Lombard, Damarys Loew, Nicolas Molinie, Stephane Romero, Daniel Bouvard, Daniel Louvard, Alexis M Gautreau, Eric Pasmant, Dominique Lallemand (2019 Jul 13)

Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression.

The Journal of biological chemistry : 12992-13005 : DOI : 10.1074/jbc.RA118.006937 En savoir plus
Résumé

Although Merlin’s function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt, and Hippo pathways is well-documented, in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy, and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora protein kinase A during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in HeLa cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with neurofibromatosis type 2 that display an aberrant phosphorylation profile along with defective α-tubulin-binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and ezrin suggest a potential role for Merlin in cell cycle progression.

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Mariana L Ferrari, Valérie Malardé, Alexandre Grassart, Laura Salavessa, Giulia Nigro, Stéphane Descorps-Declere, John R Rohde, Pamela Schnupf, Vanessa Masson, Guillaume Arras, Damarys Loew, Philippe J Sansonetti, Nathalie Sauvonnet (2019 Jun 19)

promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Proceedings of the National Academy of Sciences of the United States of America : 13582-13591 : DOI : 10.1073/pnas.1902922116 En savoir plus
Résumé

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

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Anne-Sophie Fiorucci, Clara Bourbousse, Lorenzo Concia, Martin Rougée, Anne-Flore Deton-Cabanillas, Gérald Zabulon, Elodie Layat, David Latrasse, Soon Kap Kim, Nicole Chaumont, Bérangère Lombard, David Stroebel, Sophie Lemoine, Ammara Mohammad, Corinne Blugeon, Damarys Loew, Christophe Bailly, Chris Bowler, Moussa Benhamed, Fredy Barneche (2019 May 23)

Arabidopsis S2Lb links AtCOMPASS-like and SDG2 activity in H3K4me3 independently from histone H2B monoubiquitination.

Genome biology : 100 : DOI : 10.1186/s13059-019-1705-4 En savoir plus
Résumé

The functional determinants of H3K4me3, their potential dependency on histone H2B monoubiquitination, and their contribution to defining transcriptional regimes are poorly defined in plant systems. Unlike in Saccharomyces cerevisiae, where a single SET1 protein catalyzes H3K4me3 as part of COMPlex of proteins ASsociated with Set1 (COMPASS), in Arabidopsis thaliana, this activity involves multiple histone methyltransferases. Among these, the plant-specific SET DOMAIN GROUP 2 (SDG2) has a prominent role.

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Antje Hombach-Barrigah, Katharina Bartsch, Despina Smirlis, Heidi Rosenqvist, Andrea MacDonald, Florent Dingli, Damarys Loew, Gerald F Späth, Najma Rachidi, Martin Wiese, Joachim Clos (2019 Mar 27)

Leishmania donovani 90 kD Heat Shock Protein – Impact of Phosphosites on Parasite Fitness, Infectivity and Casein Kinase Affinity.

Scientific reports : 5074 : DOI : 10.1038/s41598-019-41640-0 En savoir plus
Résumé

Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.

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Elie Hatem, Sandy Azzi, Nadine El Banna, Tiantian He, Amélie Heneman-Masurel, Laurence Vernis, Dorothée Baïlle, Vanessa Masson, Florent Dingli, Damarys Loew, Bruno Azzarone, Pierre Eid, Giuseppe Baldacci, Meng-Er Huang (2019 Feb 20)

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

Journal of the National Cancer Institute : 597-608 : DOI : 10.1093/jnci/djy149 En savoir plus
Résumé

Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing.

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Frederik J Verweij, Celine Revenu, Guillaume Arras, Florent Dingli, Damarys Loew, D Michiel Pegtel, Gautier Follain, Guillaume Allio, Jacky G Goetz, Pascale Zimmermann, Philippe Herbomel, Filippo Del Bene, Graça Raposo, Guillaume van Niel (2019 Feb 13)

Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo.

Developmental cell : 573-589.e4 : DOI : S1534-5807(19)30004-8 En savoir plus
Résumé

Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes.

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CAMPAGNE Antoine, LEE Ming-Kang, ZIELINSKI Dina, MICHAUD Audrey, LE CORRE Stéphanie, DINGLI Florent, CHEN Hong, SHAHIDIAN Lara Z, SERVANT Nicolas, LOEW Damarys, PASMANT Eric, PISTEL-VINAY Sophie, WASSEF Michel, MARGUERON Raphaël (2019 Jan 21)

BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation

Nature Communications : 10 : 1-15 : DOI : 10.1038/s41467-018-08255-x En savoir plus
Résumé

In Drosophila, a complex consisting of Calypso and ASX catalyzes H2A deubiquitination and has been reported to act as part of the Polycomb machinery in transcriptional silencing. The mammalian homologs of these proteins (BAP1 and ASXL1/2/3, respectively), are frequently mutated in various cancer types, yet their precise functions remain unclear. Using an integrative approach based on isogenic cell lines generated with CRISPR/Cas9, we uncover an unanticipated role for BAP1 in gene activation. This function requires the assembly of an enzymatically active BAP1-associated core complex (BAP1.com) containing one of the redundant ASXL proteins. We investigate the mechanism underlying BAP1.com-mediated transcriptional regulation and show that it does not participate in Polycomb-mediated silencing. Instead, our results establish that the function of BAP1.com is to safeguard transcriptionally active genes against silencing by the Polycomb Repressive Complex 1.

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Année de publication : 2018

Żylicz Jan Jakub, Bousard Aurélie, Žumer Kristina, Dossin François, Mohammad Eusra, Teixiera da Rocha Simão, Schwalb Björn, Syx Laurène, Dingli Florent, Loew Damarys, Cramer Patrick, Heard Edith (2018 Dec 21)

The Implication of Early Chromatin Changes in X Chromosome Inactivation

Cell : 176 : 1-16 : DOI : 10.1016/j.cell.2018.11.041 En savoir plus
Résumé

During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.

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Elie Hatem, Sandy Azzi, Nadine El Banna, Tiantian He, Amélie Heneman-Masurel, Laurence Vernis, Dorothée Baïlle, Vanessa Masson, Florent Dingli, Damarys Loew, Bruno Azzarone, Pierre Eid, Giuseppe Baldacci, Meng-Er Huang (2018 Nov 20)

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

Journal of the National Cancer Institute : DOI : 10.1093/ije/djy149 En savoir plus
Résumé

Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing.

Replier
Antoine Forget, Loredana Martignetti, Stéphanie Puget, Laurence Calzone, Sebastian Brabetz, Daniel Picard, Arnau Montagud, Stéphane Liva, Alexandre Sta, Florent Dingli, Guillaume Arras, Jaime Rivera, Damarys Loew, Aurore Besnard, Joëlle Lacombe, Mélanie Pagès, Pascale Varlet, Christelle Dufour, Hua Yu, Audrey L Mercier, Emilie Indersie, Anaïs Chivet, Sophie Leboucher, Laura Sieber, Kevin Beccaria, Michael Gombert, Frauke D Meyer, Nan Qin, Jasmin Bartl, Lukas Chavez, Konstantin Okonechnikov, Tanvi Sharma, Venu Thatikonda, Franck Bourdeaut, Celio Pouponnot, Vijay Ramaswamy, Andrey Korshunov, Arndt Borkhardt, Guido Reifenberger, Patrick Poullet, Michael D Taylor, Marcel Kool, Stefan M Pfister, Daisuke Kawauchi, Emmanuel Barillot, Marc Remke, Olivier Ayrault (2018 Sep 12)

Aberrant ERBB4-SRC Signaling as a Hallmark of Group 4 Medulloblastoma Revealed by Integrative Phosphoproteomic Profiling.

Cancer cell : 379-395.e7 : DOI : S1535-6108(18)30356-8 En savoir plus
Résumé

The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.

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Nassrallah Amr, Rougée Martin, Bourbousse Clara, Drevensek Stephanie, Fonseca Sandra, Iniesto Elisa, Ait-Mohamed Ouardia, Deton-Cabanillas Anne-Flore, Zabulon Gerald, Ahmed Ikhlak, Stroebel David, Masson Vanessa, Lombard Berangere, Eeckhout Dominique, Gevaert Kris, Loew Damarys, Genovesio Auguste, Breyton Cecile, de Jaeger Geert, Bowler Chris, Rubio Vicente, Barneche Fredy (2018 Sep 7)

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis

eLIFE : DOI : 10.7554/eLife.37892 En savoir plus
Résumé

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status

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Verweij Frederik J, Revenu Celine, Arras Guillaume, Dingli Florent, Loew Damarys, Follain Gautier, Allio Guillaume, Goetz Jacky G., Herbomel Philippe, Del Bene Filippo, Raposo Graça, van Niel Guillaume (2018 Jul 30)

Live tracking of inter-organ communication by endogenous exosomes in vivo

BioRxiv : DOI : 10.1101/380311 En savoir plus
Résumé

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Laencina Laura, Dubois Violaine, Le Moigne Vincent , Viljoen Albertus, Majlessi Laleh, Pritchard Justin, Bernut Audrey, Piel Laura, Roux Anne-Laure, Gaillard Jean-Louis, Lombard Bérengère, Loew Damarys, Rubin Eric J., Brosch Roland, Kremer Laurent, Herrmann Jean-Louis and Girard-Misguich Fabienne (2018 Jan 17)

Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus

Proceedings of the National Academy of Sciences of the United States of America : 115 : E1002-E1011 : DOI : 10.1073/pnas.1713195115 En savoir plus
Résumé

The coevolution of mycobacteria and amoebae seems to have contributed to shaping the virulence of nontuberculous mycobacteria in macrophages. We identified a pool of genes essential for the intracellular survival of Mycobacterium abscessus inside amoebae and macrophages and discovered a hot spot of transposon insertions within the orthologous ESX-4 T7SS locus. We generated a mutant with the deletion of a structural key ESX component, EccB4. We demonstrate rupture of the phagosomal membrane only in the presence of an intact eccB4 gene. These results suggest an unanticipated role of ESX-4 T7SS in governing the intracellular behavior of a mycobacterium. Because M. abscessus lacks ESX-1, it is tempting to speculate that ESX-4 operates as a surrogate for ESX-1 in M. tuberculosis.

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