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1. p53 activation during ribosome biogenesis regulates normal erythroid differentiation

Author

François Morlé, Stéphane Giraudier, Eric Soler, Naomi Taylor, Charlotte Andrieu-Soler, Olivier Hermine, Patrick Mayeux, Célia Floquet, Rose Ann Padua, Frédérique Verdier, Sarah Ducamp, Michaela Fontenay, Mohammad Salma, Elisabeth M. Cramer-Borde, Ismael Boussaid, Anna Raimbault, Isabelle Hatin, Diane d'Allard, Amandine Houvert, Narla Mohandas, Boris Guyot, Emilie-Fleur Gautier, Sandrina Kinet, Marjorie Leduc, Pierre-Emmanuel Gleizes, Jean-Jacques Diaz, Salomé Le Goff, François Guillonneau, Nathalie Montel-Lehry, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Hématopoïèse normale et pathologique : émergence, environnement et recherche translationnelle [Paris] ((UMR_S1131 / U1131)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), New York Blood Center, Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Procédés Alimentaires et Microbiologiques (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, ANR-18-IDEX-0001,Université de Paris,Université de Paris(2018), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Immunology, Ribosome biogenesis, Biology, Biochemistry, Ribosome, Mice, 03 medical and health sciences, 0302 clinical medicine, Erythroid Cells, Downregulation and upregulation, Transcription (biology), Ribosomal protein, hemic and lymphatic diseases, Animals, Humans, Erythropoiesis, Gene, ComputingMilieux_MISCELLANEOUS, Organelle Biogenesis, RNA, Cell Differentiation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Hematology, Hematopoietic Stem Cells, Cell biology, 030104 developmental biology, 030220 oncology & carcinogenesis, Organelle biogenesis, Tumor Suppressor Protein p53, Ribosomes
Abstract

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Published
2021
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2. Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient cells

Author

Michaela Fontenay, Pierre-Julien Viailly, Bertrand Cosson, Patrick Mayeux, Anna Raimbault, Dina Al Dulaimi, Emilie-Fleur Gautier, Barbara Burroni, Isabelle Dusanter-Fourt, Ismael Boussaid, Salomé Le Goff, Célia Floquet, Isabelle Hatin, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
Ribosomal Proteins, 5q- syndrome, Proteome, [SDV]Life Sciences [q-bio], Red Cells, Biology, Ribosomopathies, medicine.disease_cause, Ribosome, Article, 03 medical and health sciences, 0302 clinical medicine, Ribosomal protein, hemic and lymphatic diseases, medicine, Coding region, Humans, Diamond-Blackman, Anemia, Macrocytic, Gene, 030304 developmental biology, Anemia, Diamond-Blackfan, 0303 health sciences, Mutation, Translation (biology), GATA1, Hematology, Cell biology, 030220 oncology & carcinogenesis, Codon usage bias, Myelodysplastic Syndromes, Ribosomes
Abstract

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3’UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3’UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.

Published
2020
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3. Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation

Author

Gaël Nicolas, Thierry Peyrard, Mahmoud Mikdar, Slim Azouzi, Patrick Mayeux, Christine Bole-Feysot, Alexandra Willemetz, Olivier Hermine, Cédric Vrignaud, Marc Cloutier, Emilie-Fleur Gautier, Alexandre Raneri, Virginie Salnot, Maryse St-Louis, Caroline Le Van Kim, Jessica Constanzo-Yanez, Jean-Pierre Cartron, Gabriele Jedlitschky, Nancy Robitaille, Carole Éthier, Patricia Hermand, Patrick Nitschke, Yves Colin, colin, yves, Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Paris Diderot - Paris 7 (UPD7)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université des Antilles (UA), Institut National de la Transfusion Sanguine [Paris] (INTS), Département Centre National de Référence pour les Groupes Sanguins [Paris], Plateforme protéomique 3P5 [Institut Cochin] (3P5), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche sur l'Inflammation (CRI (UMR_S_1149 / ERL_8252 / U1149)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cytokines, hématopoïèse et réponse immune (CHRI), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Medicine Greifswald, Héma-Québec [Québec], Héma-Québec [Montréal], Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), and Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Université des Antilles (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Blood Platelets, 0301 basic medicine, Platelet Aggregation, [SDV]Life Sciences [q-bio], Immunology, ATP-binding cassette transporter, ABCC4, Biology, Biochemistry, 03 medical and health sciences, Cyclic nucleotide, chemistry.chemical_compound, 0302 clinical medicine, Erythroid Cells, Antigen, hemic and lymphatic diseases, medicine, Humans, Gene, integumentary system, Impaired platelet aggregation, Cell Biology, Hematology, medicine.disease, Molecular biology, [SDV] Life Sciences [q-bio], Leukemia, Phenotype, 030104 developmental biology, chemistry, 030220 oncology & carcinogenesis, Blood Group Antigens, biology.protein, CRISPR-Cas Systems, Multidrug Resistance-Associated Proteins, BLOOD Commentary, Gene Deletion, K562 cells
Abstract

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3′,5′-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.

Published
2020
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4. Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

Author

Ralfs Buks, Tracy Dagher, Maria Rotordam, David Monedero Alonso, Sylvie Cochet, Emilie-Fleur Gautier, Philippe Chafey, Bruno Cassinat, Jean-Jacques Kiladjian, Nadine Becker, Isabelle Plo, Stéphane Egée, and Wassim El Nemer

Subjects
Erythrocytes, Reticulocytes, Erythroblasts, Proteome, organelle sorting, QH301-705.5, Intracellular Space, Article, 03 medical and health sciences, 0302 clinical medicine, Erythroid Cells, polycythemia vera, hemic and lymphatic diseases, JAK2V617F, red blood cells, Ca2+, enucleation, reticulocytes, Animals, Homeostasis, Humans, Erythropoiesis, Biology (General), 030304 developmental biology, Cell Size, Organelles, Thrombocytosis, 0303 health sciences, General Medicine, Janus Kinase 2, Intermediate-Conductance Calcium-Activated Potassium Channels, 3. Good health, Mice, Inbred C57BL, Mutation, Calcium, Ribosomes, 030215 immunology
Abstract

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Published
2021
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5. Comprehensive proteomic analysis of murine terminal erythroid differentiation

Author

Narla Mohandas, Frédérique Verdier, Patrick G. Gallagher, Michaela Fontenay, Vincent P. Schulz, Christopher D. Hillyer, C. Lacombe, Patrick Mayeux, Emilie-Fleur Gautier, Ismael Boussaid, Marjorie Leduc, Carine Lefevre, François Guillonneau, Meriem Ladli, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Yale University [New Haven], New York Blood Center, Mayeux, Patrick, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)

Subjects
Proteomics, Erythroblasts, Cell, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Transcriptome, 03 medical and health sciences, Mice, 0302 clinical medicine, Red Cells, Iron, and Erythropoiesis, Erythroid Cells, hemic and lymphatic diseases, medicine, Animals, Humans, Erythropoiesis, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, 030304 developmental biology, 0303 health sciences, biology, GATA1, Hematology, Embryonic stem cell, Cell biology, Chromatin, Histone, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Proteome, biology.protein, Leukemia, Erythroblastic, Acute
Abstract

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell–derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow–derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.

Published
2020
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6. The proteome of neutrophils in sickle cell disease reveals an unexpected activation of interferon alpha signaling pathway

Author

Darragh Duffy, Pierre-Louis Tharaux, Patrick Mayeux, Patricia Hermand, Slim Azouzi, Vincent Bondet, Bérengère Koehl, Emilie-Fleur Gautier, François Guillonneau, Caroline Le Van Kim, Sébastien Dechavanne, Institut National de la Transfusion Sanguine [Paris] (INTS), Université de Paris (UP), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Immunobiologie des Cellules dendritiques, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Paris-Centre de Recherche Cardiovasculaire (PARCC (UMR_S 970/ U970)), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Service d'Hématologie Biologique [Hôpital Robert Debré, Paris], AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), This work was supported by a grant from l’Association Recherche et Transfusion, the Institut National de la Transfusion Sanguine, and the Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051, GR-Ex is funded by the program 'Investissements d’avenir' of the French National Research Agency, reference ANR-11-IDEX-0005-02. The Orbitrap Fusion mass spectrometer was acquired with funds from the FEDER through the 'Operational Programme for Competitiveness and Employment 2007-2013' and from the 'Canceropole Ile de France'., ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Vougny, Marie-Christine, and Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Proteome, Neutrophils, Cell, Red Cells, Alpha interferon, Inflammation, Disease, Anemia, Sickle Cell, 03 medical and health sciences, 0302 clinical medicine, Text mining, Medicine, Humans, Letters to the Editor, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, business.industry, Interferon-alpha, Hematology, 3. Good health, Hemoglobinopathies, medicine.anatomical_structure, Cancer research, [SDV.IMM]Life Sciences [q-bio]/Immunology, medicine.symptom, Signal transduction, business, 030215 immunology, Signal Transduction
Abstract

International audience

Published
2020
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7. Comprehensive Proteomic Analysis of Human Erythropoiesis

Author

Patrick Mayeux, Narla Mohandas, Anna Raimbault, Michael Dussiot, Virginie Salnot, John Hale, Yael Zermati, Sarah Ducamp, Marie-Catherine Giarratana, Luc Douay, François Guillonneau, Marjorie Leduc, Frédérique Verdier, Emilie-Fleur Gautier, Catherine Lacombe, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Red Cell Physiology Laboratory [New York, USA], New York Blood Center, Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ligue Nationale Contre le Cancer - Paris, Ligue Nationnale Contre le Cancer, ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), Institut Cochin ( UM3 (UMR 8104 / U1016) ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratory of Excellence GR-Ex, Sorbonne Paris Cité-Université Paris Descartes - Paris 5 ( UPD5 ) -Imagine Institute, Plateforme protéomique 3P5 [Institut Cochin] ( 3P5 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), New York Blood Center - NYBC, Centre de Recherche Saint-Antoine ( CR Saint-Antoine ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), ANR-11-IDEX-0005-02/11-LABX-0051,GR-Ex,Biogenèse et pathologies du globule rouge ( 2011 ), ANR-11-IDEX-0005-02/11-IDEX-0005,USPC,USPC ( 2011 ), Bos, Mireille, Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID, and Ligue Nationale Contre le Cancer (LNCC)

Subjects
Proteomics, 0301 basic medicine, Cell type, Erythroblasts, Proteome, Cellular differentiation, Biology, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Reticulocyte, hemic and lymphatic diseases, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, Erythropoiesis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, lcsh:QH301-705.5, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cells, Cultured, Erythroid Precursor Cells, Messenger RNA, RNA, Cell Differentiation, Molecular biology, Cell biology, 030104 developmental biology, medicine.anatomical_structure, lcsh:Biology (General)
Abstract

SUMMARY Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis., In Brief Gautier et al. use quantitative mass spectrometry to determine the absolute proteome composition of human erythroid progenitors throughout the differentiation process and the quantitative distribution of proteins between reticulocytes and pyrenocytes after enucleation.

Published
2016
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8. Proteomic Landscape of Neutrophils in Sickle Cell Anemia: An Unexpected Autoimmune Profile

Author

Emilie Fleur Gautier, Darragh Duffy, Yves Colin, Patrick Mayeux, Virginie Salnot, Caroline Le Va Kim, Slim Azouzi, Bérengère Koehl, Pierre-Louis Tharaux, Patricia Hermand-Tournamille, Vincent Bondet, Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université des Antilles (UA), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Immunobiologie des Cellules dendritiques, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Robert Debré, Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Toll-like receptor, Innate immune system, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Immunology, Cell Biology, Hematology, Biology, Biochemistry, 03 medical and health sciences, 030104 developmental biology, Cytokine, Interferon, medicine, biology.protein, L-selectin, Signal transduction, Receptor, Interferon type I, medicine.drug
Abstract

Although sickle cell disease (SCD) is a red cell disorder, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), contribute to its pathophysiology. In particular, activated PMNs have been implicated to play an important role in the initiation and propagation of vaso-occlusive events in SCD. Activated PMNs engage in a complex process of abnormal interactions with activated endothelial cells, platelets and circulating erythrocytes contributing to endothelial injury and decreased blood flow. In the present study, global proteomic analysis was performed using label-free mass spectrometry of PMNs from 4 SCD patients (SS) in steady state and from 4 control subjects (AA). We identified a total of 4,534 proteins both in AA and SS PMNs with 3,080 of these proteins identified in at least three samples for each condition. 50 proteins were significantly over-expressed in SS PMNs compared to AA PMNs (ratio > 1.4). STRING employed to monitor potential interaction between the overexpressed proteins showed that the main interactive clusters consist of STAT1 and STAT2, OAS 1, 2 and 3, and many Interferon Signaling Proteins i.e. IFIT1, IFIT2, IFIT 3, ISG15, ISG20, GBP2, IFI35, MX1 and MX2, TLR8 proteins (Fig. 1). This finding implies a strong activation of the type I interferon (IFN) signaling pathway in the SS PMNs (between 10 and 100-fold increase in SS vs AA). In addition, 33 proteins showed significantly lower expression in SS PMNs compared to AA PMNs. Among these were L-selectin (CD62L) and IL-17 receptor A (IL17RA) (p = 0.01). These findings are consistent with previously described phenotypes of aged neutrophils and acute inflammatory responses in SCD. Similar proteomic analysis performed on PMNs from SS patients treated with hydroxycarbamide (HC, n=4) showed that 14 proteins had significantly lower expression compared to untreated-SS patients (ratio Type I IFNs represent the major effector cytokines of the host immune response against viruses and other intracellular pathogens. These cytokines are produced via activation of STAT1 and of pattern recognition receptors, including the Toll-like receptor signaling network. To determine if type I IFN-α could be detected at the protein level in the plasma of SS patients, we used the novel digital-ELISA technology (SIMOA, Quanterix) developed by Wilson et al (J Lab Autom, 2016). Interestingly, we found an increased level of INFα in plasma from SS patients compared to AA (n=32) (p In summary, our novel proteomic analysis documents a high level expression of interferon signaling proteins, STAT1 and TLR8 in the proteome of neutrophils from SS patients and strongly suggests autoimmune or auto-inflammatory phenomena at basal state in SCD. Our results provide strong support for an important role for the innate immune system in the pathophysiology of SCD. Future studies will help determine the relationship between the plasmatic level of IFN-α and clinical complications and will establish if interferon signaling proteins and IFN-α could represent new therapeutic targets in SCD. Disclosures Hermand-Tournamille: Imara: Research Funding. Le Va Kim:Imara: Research Funding. Koehl:Imara: Research Funding.

Published
2018
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9. Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

Author

Karine Bailly, Patrick Mayeux, Emilie-Fleur Gautier, Wassim El Nemer, Marjorie Leduc, Sylvie Cochet, François Guillonneau, Narla Mohandas, Catherine Lacombe, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Dynamique des Structures et Interactions des Macromolécules Biologiques (DSIMB), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Red Cell Physiology Laboratory [New York, USA], New York Blood Center, Plateforme protéomique 3P5 [Institut Cochin] (3P5), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Transfusion Sanguine [Paris] (INTS), and CHU Pitié-Salpêtrière [APHP]

Subjects
0301 basic medicine, Erythrocytes, Reticulocytes, Proteome, [SDV]Life Sciences [q-bio], Population, 03 medical and health sciences, chemistry.chemical_compound, Red Cells, Iron, and Erythropoiesis, 0302 clinical medicine, Biotin, Reticulocyte, medicine, Humans, education, ComputingMilieux_MISCELLANEOUS, education.field_of_study, Kinase, Hematology, Cell sorting, Adenosine diphosphate, 030104 developmental biology, medicine.anatomical_structure, chemistry, Biochemistry, 030220 oncology & carcinogenesis, Adenosine triphosphate
Abstract

Reticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.

Published
2018
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10. Upregulation of the CDC25A phosphatase downstream of the NPM/ALK oncogene participates in anaplastic large cell lymphoma enhanced proliferation

Author

Anne Mazars, Anne Fernandez-Vidal, Stéphane Manenti, Bernard Payrastre, Emilie-Fleur Gautier, Gregoire Prevost, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Institut Henri Beaufour (IPSEN), IPSEN-BEAUFOUR, and CHU Toulouse [Toulouse]

Subjects
CDC25A, Oncogene Proteins, Fusion, Cdc25, [SDV]Life Sciences [q-bio], Biology, Mice, Phosphatidylinositol 3-Kinases, 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, hemic and lymphatic diseases, Dual-specificity phosphatase, medicine, Animals, Humans, cdc25 Phosphatases, Anaplastic Lymphoma Kinase, Molecular Biology, Anaplastic large-cell lymphoma, ComputingMilieux_MISCELLANEOUS, PI3K/AKT/mTOR pathway, Cell Proliferation, 030304 developmental biology, 0303 health sciences, ABL, Receptor Protein-Tyrosine Kinases, Myeloid leukemia, Oncogenes, Cell Biology, Protein-Tyrosine Kinases, medicine.disease, Up-Regulation, 3. Good health, CDC25A, lymphoma, PI3K, proliferation, oncogene, tyrosine kinase, 030220 oncology & carcinogenesis, biology.protein, Cancer research, Lymphoma, Large-Cell, Anaplastic, Proto-Oncogene Proteins c-akt, Tyrosine kinase, Signal Transduction, Developmental Biology
Abstract

Here, we demonstrate that the expression of the dual specificity phosphatase CDC25A, a key regulator of cell cycle progression, is deregulated in Ba/F3 cells expressing the oncogenic protein NPM/ALK and in human cell lines derived from NPM/ALK-positive anaplastic large cell lymphomas (ALCL). Both transcriptional and post-translational mechanisms account for the constitutive expression of the protein, and the PI3K/Akt pathway is essential for this process. Importantly, pharmacological inhibition of CDC25 dramatically inhibits the proliferation of NPM/ALK-expressing cells, while moderately affecting the proliferation of control Ba/F3 cells. RNA interference-mediated downregulation of CDC25A confirmed that NPM/ALK-expressing cells are highly dependent on this protein for their proliferation. Moreover, similar PI3K/AKt-mediated constitutive expression of CDC25A takes place down-stream of other hematological oncogenes, including BCR/ABL in Chronic Myeloid Leukemia and FLT3-ITD in Acute Myeloid Leukemia. Altogether, our data point to the functional link between hematopoietic oncogenic tyrosine kinases and the G(1) cell cycle regulator CDC25A, and we propose that this protein may be a potential therapeutic target in ALCL and other hematological malignancies.

Published
2009
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