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1. Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin

Author

François Bulteau, Michel Thépaut, Maxime Henry, Amandine Hurbin, Laetitia Vanwonterghem, Corinne Vivès, Aline Le Roy, Christine Ebel, Olivier Renaudet, Franck Fieschi, Jean-Luc Coll, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Département de Chimie Moléculaire (DCM), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)

Subjects
MESH: HT29 Cells, Pharmaceutical Science, MESH: Spheroids, Cellular, Mice, Nude, MESH: Flow Cytometry, MESH: Recombinant Proteins, 03 medical and health sciences, Mice, 0302 clinical medicine, Spheroids, Cellular, Drug Discovery, Tn antigen, MESH: Mice, Nude, cancer, MESH: Microscopy, Confocal, Animals, Humans, MESH: Animals, Antigens, Tumor-Associated, Carbohydrate, Lectins, C-Type, MESH: Mice, 030304 developmental biology, 0303 health sciences, MESH: Humans, Microscopy, Confocal, MESH: Antigens, Tumor-Associated, Carbohydrate, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Surface Plasmon Resonance, Flow Cytometry, Recombinant Proteins, 3. Good health, MESH: Surface Plasmon Resonance, C-type lectin, A549 Cells, 030220 oncology & carcinogenesis, Molecular Medicine, Female, MESH: A549 Cells, MESH: Female, HT29 Cells, MESH: Neoplasm Transplantation, MESH: Lectins, C-Type, Neoplasm Transplantation
Abstract

International audience; Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.

Published
2021
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2. A prenylated dsRNA sensor protects against severe COVID-19

Author

Wickenhagen, A, Sugrue, E, Lytras, S, Kuchi, S, Noerenberg, M, Turnbull, ML, Loney, C, Herder, V, Allan, J, Jarmson, I, Cameron-Ruiz, N, Varjak, M, Pinto, RM, Lee, JY, Iselin, L, Palmalux, N, Stewart, DG, Swingler, S, Greenwood, EJD, Crozier, TWM, Gu, Q, Davies, EL, Clohisey, S, Wang, B, Trindade Maranhão Costa, F, Freire Santana, M, de Lima Ferreira, LC, Murphy, L, Fawkes, A, Meynert, A, Grimes, G, Da Silva Filho, JL, Marti, M, Hughes, J, Stanton, RJ, Wang, ECY, Ho, A, Davis, I, Jarrett, RF, Castello, A, Robertson, DL, Semple, MG, Openshaw, PJM, Palmarini, M, Lehner, PJ, Baillie, JK, Rihn, SJ, Wilson, SJ, ISARIC4C Investigators, Carson, G, Alex, B, Bach, B, Barclay, WS, Bogaert, D, Chand, M, Cooke, GS, Docherty, AB, Dunning, J, da Silva Filipe, A, Fletcher, T, Green, CA, Harrison, EM, Ho, AYW, Horby, PW, Ijaz, S, Khoo, S, Klenerman, P, Law, A, Lim, WS, Mentzer, AJ, Merson, L, Meynert, AM, Noursadeghi, M, Moore, SC, Paxton, WA, Pollakis, G, Price, N, Rambaut, A, Russell, CD, Sancho-Shimizu, V, Scott, JT, de Silva, T, Sigfrid, L, Solomon, T, Sriskandan, S, Stuart, D, Summers, C, Tedder, RS, Thomson, EC, Thompson, AAR, Thwaites, RS, Turtle, LCW, Gupta, RK, Palmieri, C, Swann, OV, Zambon, M, Dumas, M-E, Griffin, JL, Takats, Z, Chechi, K, Andrikopoulos, P, Osagie, A, Olanipekun, M, Liggi, S, Lewis, MR, Dos Santos Correia, G, Sands, CJ, Takis, P, Maslen, L, Hardwick, H, Donohue, C, Griffiths, F, Oosthuyzen, W, Donegan, C, Spencer, RG, Norman, L, Pius, R, Drake, TM, Fairfield, CJ, Knight, SR, Mclean, KA, Murphy, D, Shaw, CA, Dalton, J, Girvan, M, Saviciute, E, Roberts, S, Harrison, J, Marsh, L, Connor, M, Halpin, S, Jackson, C, Gamble, C, Plotkin, D, Lee, J, Leeming, G, Wham, M, Hendry, R, Scott-Brown, J, Greenhalf, W, Shaw, V, McDonald, SE, Keating, S, Ahmed, KA, Armstrong, JA, Ashworth, M, Asiimwe, IG, Bakshi, S, Barlow, SL, Booth, L, Brennan, B, Bullock, K, Catterall, BWA, Clark, JJ, Clarke, EA, Cole, S, Cooper, L, Cox, H, Davis, C, Dincarslan, O, Dunn, C, Dyer, P, Elliott, A, Evans, A, Finch, L, Fisher, LWS, Foster, T, Garcia-Dorival, I, Gunning, P, Hartley, C, Jensen, RL, Jones, CB, Jones, TR, Khandaker, S, King, K, Kiy, RT, Koukorava, C, Lake, A, Lant, S, Latawiec, D, Lavelle-Langham, L, Lefteri, D, Lett, L, Livoti, LA, Mancini, M, McDonald, S, McEvoy, L, McLauchlan, J, Metelmann, S, Miah, NS, Middleton, J, Mitchell, J, Murphy, EG, Penrice-Randal, R, Pilgrim, J, Prince, T, Reynolds, W, Ridley, PM, Sales, D, Shaw, VE, Shears, RK, Small, B, Subramaniam, KS, Szemiel, A, Taggart, A, Tanianis-Hughes, J, Thomas, J, Trochu, E, van Tonder, L, Wilcock, E, Zhang, JE, Flaherty, L, Maziere, N, Cass, E, Carracedo, AD, Carlucci, N, Holmes, A, Massey, H, Wrobel, N, McCafferty, S, Morrice, K, MacLean, A, Adeniji, K, Agranoff, D, Agwuh, K, Ail, D, Aldera, EL, Alegria, A, Allen, S, Angus, B, Ashish, A, Atkinson, D, Bari, S, Barlow, G, Barnass, S, Barrett, N, Bassford, C, Basude, S, Baxter, D, Beadsworth, M, Bernatoniene, J, Berridge, J, Berry, C, Best, N, Bothma, P, Chadwick, D, Brittain-Long, R, Bulteel, N, Burden, T, Burtenshaw, A, Caruth, V, Chambler, D, Chee, N, Child, J, Chukkambotla, S, Clark, T, Collini, P, Cosgrove, C, Cupitt, J, Cutino-Moguel, M-T, Dark, P, Dawson, C, Dervisevic, S, Donnison, P, Douthwaite, S, Drummond, A, DuRand, I, Dushianthan, A, Dyer, T, Evans, C, Eziefula, C, Fegan, C, Finn, A, Fullerton, D, Garg, S, Garg, A, Gkrania-Klotsas, E, Godden, J, Goldsmith, A, Graham, C, Hardy, E, Hartshorn, S, Harvey, D, Havalda, P, Hawcutt, DB, Hobrok, M, Hodgson, L, Hormis, A, Jacobs, M, Jain, S, Jennings, P, Kaliappan, A, Kasipandian, V, Kegg, S, Kelsey, M, Kendall, J, Kerrison, C, Kerslake, I, Koch, O, Koduri, G, Koshy, G, Laha, S, Laird, S, Larkin, S, Leiner, T, Lillie, P, Limb, J, Linnett, V, Little, J, Lyttle, M, MacMahon, M, MacNaughton, E, Mankregod, R, Masson, H, Matovu, E, McCullough, K, McEwen, R, Meda, M, Mills, G, Minton, J, Mirfenderesky, M, Mohandas, K, Mok, Q, Moon, J, Moore, E, Morgan, P, Morris, C, Mortimore, K, Moses, S, Mpenge, M, Mulla, R, Murphy, M, Nagel, M, Nagarajan, T, Nelson, M, Norris, L, O'Shea, MK, Otahal, I, Ostermann, M, Pais, M, Panchatsharam, S, Papakonstantinou, D, Paraiso, H, Patel, B, Pattison, N, Pepperell, J, Peters, M, Phull, M, Pintus, S, Singh Pooni, J, Planche, T, Post, F, Price, D, Prout, R, Rae, N, Reschreiter, H, Reynolds, T, Richardson, N, Roberts, M, Roberts, D, Rose, A, Rousseau, G, Ruge, B, Ryan, B, Saluja, T, Schmid, ML, Shah, A, Shanmuga, P, Sharma, A, Shawcross, A, Sizer, J, Shankar-Hari, M, Smith, R, Snelson, C, Spittle, N, Staines, N, Stambach, T, Stewart, R, Subudhi, P, Szakmany, T, Tatham, K, Thompson, C, Thompson, R, Tridente, A, Tupper-Carey, D, Twagira, M, Vallotton, N, Vancheeswaran, R, Vincent-Smith, L, Visuvanathan, S, Vuylsteke, A, Waddy, S, Wake, R, Walden, A, Welters, I, Whitehouse, T, Whittaker, P, Whittington, A, Papineni, P, Wijesinghe, M, Williams, M, Wilson, L, Winchester, S, Wiselka, M, Wolverson, A, Wootton, DG, Workman, A, Yates, B, Young, P, Division of Computational and Systems Medicine, Imperial College London, London, SW7 2AZ, UK, Imperial College London - National Heart and Lung Institute, Centre National de la Recherche Scientifique (CNRS), MRC - University of Glasgow Centre for Virus Research, University of Kent [Canterbury], University of Glasgow, National Institute for Health Research, UK Research and Innovation, UKRI MRC COVID-19 Rapid Response Call, Mentzer, AJ, Investigators, ISARIC4C, Lee, J, and Angus, B

Subjects
2',5'-Oligoadenylate Synthetase/genetics, L PATHWAY, viruses, SINGLE-NUCLEOTIDE POLYMORPHISM, RNASE-L, RNA, Viral/chemistry, RNA, Double-Stranded/chemistry, Virus Replication, Severity of Illness Index, COVID-19/enzymology, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Chiroptera, Sense (molecular biology), 2',5'-Oligoadenylate Synthetase, MESH: COVID-19, MESH: Animals, MESH: Interferons, ENZYME-ACTIVITY, MESH: 2',5'-Oligoadenylate Synthetase, Endoribonucleases/metabolism, 0303 health sciences, MESH: Protein Prenylation, Multidisciplinary, I INTERFERON, Coronaviridae/enzymology, MESH: Polymorphism, Single Nucleotide, 030302 biochemistry & molecular biology, MESH: Chiroptera, CORONAVIRUS REPLICATION, 3. Good health, Cell biology, Multidisciplinary Sciences, Isoenzymes, RNA silencing, VIRUS NS1 PROTEIN, MESH: RNA, Viral, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Isoenzymes, Science & Technology - Other Topics, RNA, Viral, MESH: 5' Untranslated Regions, [SDV.IMM]Life Sciences [q-bio]/Immunology, SARS-CORONAVIRUS, Gene isoform, MESH: Endoribonucleases, Retroelements, RNase P, Coronaviridae, General Science & Technology, Protein Prenylation, Biology, Polymorphism, Single Nucleotide, Chiroptera/genetics, 03 medical and health sciences, MESH: RNA, Double-Stranded, MESH: Retroelements, SARS-CoV-2/genetics, MESH: Severity of Illness Index, Endoribonucleases, Animals, Humans, MESH: SARS-CoV-2, Interferons/immunology, Gene, ISARIC4C Investigators, 030304 developmental biology, RNA, Double-Stranded, Science & Technology, MESH: Humans, ACCEPTOR SITE, Phosphoric Diester Hydrolases, SARS-CoV-2, fungi, MESH: Virus Replication, RNA, COVID-19, Phosphoric Diester Hydrolases/genetics, Viral replication, A549 Cells, Protein prenylation, SPLICE-SITE POLYMORPHISM, Interferons, MESH: A549 Cells, 5' Untranslated Regions, MESH: Phosphoric Diester Hydrolases, Isoenzymes/genetics, MESH: Coronaviridae
Abstract

International audience; INTRODUCTIONInterferons (IFNs) are cytokines that are rapidly deployed in response to invading pathogens. By initiating a signaling cascade that stimulates the expression of hundreds of genes, IFNs create an antiviral state in host cells. Because IFNs heavily influence COVID-19 outcomes, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication can be inhibited by the antiviral state, it is important to understand how the individual antiviral effectors encoded by IFN-stimulated genes (ISGs) inhibit SARS-CoV-2.RATIONALEWe hypothesized that IFN-stimulated antiviral effectors can inhibit SARS-CoV-2, and that variation at the loci encoding these defenses underlies why some people are more susceptible to severe COVID-19.RESULTSWe used arrayed ISG expression screening to reveal that 2′-5′-oligoadenylate synthetase 1 (OAS1) consistently inhibited SARS-CoV-2 in different contexts. Using CRISPR-Cas9, we found that endogenous OAS1 makes a substantial contribution to the antiviral state by recognizing short stretches of double-stranded RNA (dsRNA) and activating RNase L. We globally mapped where OAS1 binds to SARS-CoV-2 viral RNAs and found that OAS1 binding is remarkably specific, with two conserved stem loops in the SARS-CoV-2 5′-untranslated region (UTR) constituting the principal viral target.OAS1 expression was readily detectable at the sites of infection in individuals who died of COVID-19, and specific OAS1 alleles are known to be associated with altered susceptibility to infection and severe disease. It had previously been reported that alleles containing a common splice-acceptor single nucleotide polymorphism in OAS1 (Rs10774671) were associated with less severe COVID-19. We determined that people with at least one allele with a G at this position could express a prenylated form of OAS1 (p46), whereas other individuals could not. Using a series of mutants, we found that C-terminal prenylation was necessary for OAS1 to initiate a block to SARS-CoV-2. Furthermore, confocal microscopy revealed that prenylation targeted OAS1 to perinuclear structures rich in viral dsRNA, whereas non-prenylated OAS1 was diffusely localized and unable to initiate a detectable block to SARS-CoV-2 replication.The realization that prenylation is essential for OAS1-mediated sensing of SARS-CoV-2 allowed us to examine the transcriptome of infected patients and investigate whether there was a link between the expression of prenylated OAS1 and SARS-CoV-2 disease progression. Analysis of the OAS1 transcripts from 499 hospitalized COVID-19 patients revealed that expressing prenylated OAS1 was associated with protection from severe COVID-19.Because prenylated OAS1 was so important in human cases, we wanted to determine whether horseshoe bats, the likely source of SARS-CoV-2, possessed the same defense. When we examined the genomic region where the prenylation signal should reside, retrotransposition of a long terminal repeat sequence had ablated this signal, preventing the expression of prenylated anti-CoV OAS1 in these bats.CONCLUSIONC-terminal prenylation targets OAS1 to intracellular sites rich in viral dsRNA, which are likely the SARS-CoV-2 replicative organelles. Once in the right place, OAS1 binds to dsRNA structures in the SARS-CoV-2 5′-UTR and initiates a potent block to SARS-CoV-2 replication. Thus, the correct targeting of OAS1 and the subsequent inhibition of SARS-CoV-2 likely underpins the genetic association of alleles containing a G at Rs10774671 with reduced susceptibility to infection and severe disease in COVID-19. Moreover, the conspicuous absence of this antiviral defense in horseshoe bats potentially explains why SARS-CoV-2 is so sensitive to this defense in humans.

Published
2021
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3. Inhibition of the replication of SARS-CoV-2 in human cells by the FDA-approved drug chlorpromazine

Author

Michael Blatzer, Raphaël Gaillard, Gérard Friedlander, Fabien Vinckier, David Attali, Etienne Simon-Loriere, Jeanne Chiaravalli, Anne-Cécile Petit, Laurine Levillayer, Matthieu Prot, Marion Plaze, Fabrice Chrétien, Florent Perin-Dureau, Arnaud Cachia, CCSD, Accord Elsevier, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs - - INCEPTION2016 - ANR-16-CONV-0005 - CONV - VALID, GHU Paris Psychiatrie et Neurosciences, Université Paris Cité (UPCité), Physique pour la médecine (PhysMed Paris), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris] (IP), Neuropathologie expérimentale / Experimental neuropathology, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Génétique fonctionnelle des maladies infectieuses - Functional Genetics of Infectious Diseases, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Criblage chémogénomique et biologique (Plateforme) - Chemogenomic and Biological Screening Platform (PF-CCB), Fondation Ophtalmologique Adolphe de Rothschild [Paris], Laboratoire de psychologie du développement et de l'éducation de l'enfant (LaPsyDÉ - UMR 8240), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de psychiatrie et neurosciences de Paris (IPNP - U1266 Inserm), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), This study has received funding from Institut Pasteur (covid-therap), from the French Government's Investissement d'Avenir programme and as a recognition as a Laboratoire d'Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (Grant No. ANR-10-LABX-62-IBEID). ESL acknowledges funding from the INCEPTION programme (Investissements d'Avenir Grant ANR-16-CONV-0005)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), Université de Paris (UP), Physique pour la médecine (UMR 8063, U1273), Institut Pasteur [Paris], Institut Pasteur [Paris]-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)

Subjects
0301 basic medicine, Saliva, viruses, [SDV]Life Sciences [q-bio], Repurposing of molecules, Pharmacology, Virus Replication, 0302 clinical medicine, MESH: Chlorocebus aethiops, Chlorocebus aethiops, Medicine, MESH: COVID-19, Pharmacology (medical), Tissue Distribution, MESH: Animals, 030212 general & internal medicine, Chlorpromazine, media_common, virus diseases, General Medicine, respiratory system, Human cells, 3. Good health, [SDV] Life Sciences [q-bio], Infectious Diseases, MESH: Drug Repositioning, medicine.drug, Microbiology (medical), Drug, MESH: Antiviral Agents, media_common.quotation_subject, 030106 microbiology, MESH: Vero Cells, Endocytosis, Antiviral Agents, Article, Cell Line, 03 medical and health sciences, Distribution (pharmacology), Animals, Humans, MESH: SARS-CoV-2, Viability assay, MESH: Tissue Distribution, IC50, Vero Cells, MESH: Humans, business.industry, SARS-CoV-2, MESH: Virus Replication, Drug Repositioning, COVID-19, MESH: Chlorpromazine, In vitro, respiratory tract diseases, COVID-19 Drug Treatment, MESH: Cell Line, A549 Cells, MESH: A549 Cells, business
Abstract

Highlights • Chlorpromazine, an FDA-approved drug, inhibits clathrin-mediated endocytosis • CPZ acts as an antiviral against SARS-CoV-1 and MERS-CoV • We evidenced antiviral activity of CPZ against SARS-CoV-2 in monkey and human cells • Because of CPZ high distribution in lungs, CPZ IC50 may translate in clinical routine • CPZ brain distribution could be of high interest for neurological forms of COVID-19, Introduction Urgent action is needed to fight the ongoing COVID-19 pandemic by reducing the number of infected people along with the infection contagiousness and severity. Chlorpromazine (CPZ), the prototype of typical antipsychotics from the phenothiazine group, is known to inhibit clathrin-mediated endocytosis and acts as an antiviral, in particular against SARS-CoV-1 and MERS-CoV. The aim of this in vitro study was to test CPZ against a SARS-CoV-2 isolate in monkey and human cells. Material and methods Monkey VeroE6 cells and human alveolar basal epithelial A549-ACE2 cells were infected with SARS-CoV-2 in presence of different concentrations of CPZ. Supernatants were harvested at day 2 and analysed by RT-qPCR for the presence of SARS-CoV-2 RNA. Cell viability was assessed on non-infected cells. Results We evidenced an antiviral activity of CPZ against SARS-CoV-2 in monkey VeroE6 cells with an IC50 of 8.2 µM, a CC50 of 13.5µM and a SI of 1.65. In human A549-ACE2 cells, CPZ was also associated with an anti-SARS-CoV-2 activity, with an IC50 of 11.3 µM, a CC50 of 23.1 µM and a SI of 2.04. Discussion Even though the measured SI are low, such IC50 measured in vitro may translate to CPZ dosage used in clinical routine because of CPZ high biodistribution in lungs and in saliva. Also, CPZ brain distribution could be of high interest for treating or preventing the neurological and psychiatric forms of COVID-19. Conclusions These preclinical findings support clinical investigation of the repurposing of CPZ, a largely used drug with mild side effects, in COVID-19 treatment.

Published
2021
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4. The Global Phosphorylation Landscape of SARS-CoV-2 Infection

Author

Yiming Cai, Maya Modak, Sebastian Weigang, Emmie de Wit, Jean K. Lim, Alistair Dunham, Benjamin J. Polacco, Qiongyu Li, Svenja Ulferts, Gwendolyn M. Jang, Aurelien Dugourd, David E. Gordon, Jeffrey Z. Guo, Kirsten Obernier, Sophia Bouhaddou, Elizabeth R. Fischer, Anna Gaulton, Jason C.J. Chang, Bjoern Meyer, Diego Quintero, Julian Knerr, Trupti Patil, Emma J. Manners, Michael C. O’Neal, Monita Muralidharan, Joseph Hiatt, Ajda Rojc, James E. Melnyk, Tanja Kortemme, Benjamin R. tenOever, Thomas Vallet, Rémy Robinot, Cassandra Koh, Benjamin E. Nilsson-Payant, Ruth Hüttenhain, Saker Klippsten, Alicia L. Richards, Eloy Felix, Brian K. Shoichet, Beril Tutuncuoglu, Danielle L. Swaney, Veronica V. Rezelj, Jeffery R. Johnson, Margaret Soucheray, Marisa Goff, R. Dyche Mullins, Kris M. White, Erica Stevenson, Jyoti Batra, Christopher J.P. Mathy, Yuan Zhou, Minkyu Kim, Marco Vignuzzi, Claudia Hernandez-Armenta, Kevan M. Shokat, Julio Saez-Rodriguez, Jacqueline M. Fabius, Timothy McBride, Adolfo García-Sastre, Quang Dinh Tran, Alexandra Hardy, Elena Moreno, Alberto Valdeolivas, Mehdi Bouhaddou, Andrew R. Leach, Melanie Ott, Georg Kochs, Pedro Beltrao, Jiewei Xu, Robyn M. Kaake, Merve Cakir, Ying Shi, Nevan J. Krogan, Lisa Miorin, Danish Memon, David J. Broadhurst, Miguel Correa Marrero, Robert Grosse, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Quantitative Biosciences Institute [UC San Francisco, USA] (QBI), University of California [San Francisco] (UC San Francisco), University of California (UC)-University of California (UC), Gladstone Institutes [San Francisco], European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Howard Hughes Medical Institute (HHMI), University of Freiburg [Freiburg], Virus et Immunité - Virus and immunity (CNRS-UMR3569), Universität Heidelberg [Heidelberg] = Heidelberg University, Heidelberg University Hospital [Heidelberg], Zoic Labs [Culver City, CA], Rocky Mountain Laboratories, Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Centre for Integrative Biological Signalling Studies [Freiburg] (CIBSS), This research was funded by grants from the National Institutes of Health ( P50AI150476 , U19AI135990 , U19AI135972 , R01AI143292 , R01AI120694 , P01A1063302 , and R01AI122747 to N.J.K., 1R01CA221969 and 1R01CA244550 to K.M.S., R01GM133981 to D.L.S., 1F32CA236347-01 to J.E.M., U19AI118610 to J.R.J., and F32CA239333 to M.B.), Defense Advance Research Projects Agency HR0011-19-2-0020 (to N.J.K., A.G.S., and K.M.S.), by the Laboratory for Genomics Research (LGR) Excellence in Research Award (ERA) from the Innovative Genomics Institute at UC Berkeley (grant number 133122P ), by CRIP (Center for Research for Influenza Pathogenesis), a NIAID-supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract HHSN272201400008C ) (to A.G.S.), by supplements to NIAID grant U19AI135972 and DoD grant W81XWH-19-PRMRP-FPA (to A.G.S.), and by the generous support of the JPB Foundation , the Open Philanthropy Project (research grant 2020-215611 [5384] ), and other philanthropic donations (to A.G.S.), by the Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' grant ANR-10-LABX-62-IBEID (to M.V.), by the DFG under Germany's Excellence Strategy ( EXC-2189 , project ID 390939984 to R.G.), by a Starting Grant Award from the European Research Council ( ERC-2014-STG 638884 PhosFunc to P.B.), by the Federal Ministry of Education and Research (BMBF, Computational Life Sciences grant 031L0181B to J.S.R.), by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (to E.R.F. and E.D.W.), and by funding from F. Hoffmann-La Roche and Vir Biotechnology and gifts from The Ron Conway Family . K.M.S. is an investigator of the Howard Hughes Medical Institute., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 638884,H2020,ERC-2014-STG,PhosFunc(2015), Universität Heidelberg [Heidelberg], and Vaccine Research Institute (VRI)

Subjects
Proteomics, MAPK/ERK pathway, MESH: Angiotensin-Converting Enzyme 2, MESH: Casein Kinase II, PIKFYVE, 0302 clinical medicine, MESH: Chlorocebus aethiops, MESH: Protein Kinase Inhibitors, MESH: Animals, Casein Kinase II, Lung, 0303 health sciences, Kinase, MESH: Proteomics, Phosphoproteomics, antiviral, Spike Glycoprotein, Cyclin-Dependent Kinases, 3. Good health, MESH: HEK293 Cells, Spike Glycoprotein, Coronavirus, Phosphorylation, Infection, MESH: Pandemics, p38 mitogen-activated protein kinases, Pneumonia, Viral, MESH: Vero Cells, p38, Antiviral Agents, General Biochemistry, Genetics and Molecular Biology, Article, Betacoronavirus, 03 medical and health sciences, Biodefense, Humans, MESH: SARS-CoV-2, MESH: Humans, MESH: Phosphorylation, Prevention, MESH: Host-Pathogen Interactions, fungi, Receptor Protein-Tyrosine Kinases, AXL, Pneumonia, Virology, MAPK, Coronavirus, MESH: Peptidyl-Dipeptidase A, MESH: Pneumonia, Viral, MESH: Phosphatidylinositol 3-Kinases, A549 Cells, Vero cell, Drug Evaluation, 030217 neurology & neurosurgery, Developmental Biology, MESH: Coronavirus Infections, [SDV]Life Sciences [q-bio], viruses, CDK, Drug Evaluation, Preclinical, MESH: Spike Glycoprotein, Coronavirus, Medical and Health Sciences, p38 Mitogen-Activated Protein Kinases, Phosphatidylinositol 3-Kinases, Chlorocebus aethiops, MESH: COVID-19, Viral, Phosphoinositide-3 Kinase Inhibitors, mass spectrometry, biology, phosphoproteomics, Biological Sciences, Preclinical, MESH: Cyclin-Dependent Kinases, Infectious Diseases, Host-Pathogen Interactions, MESH: Betacoronavirus, MESH: Drug Evaluation, Preclinical, MESH: Receptor Protein-Tyrosine Kinases, MESH: Caco-2 Cells, Angiotensin-Converting Enzyme 2, Coronavirus Infections, MESH: Antiviral Agents, casein kinase II, Peptidyl-Dipeptidase A, Vaccine Related, Cyclin-dependent kinase, Proto-Oncogene Proteins, Animals, Pandemics, Protein Kinase Inhibitors, Vero Cells, MESH: Phosphoinositide-3 Kinase Inhibitors, 030304 developmental biology, SARS-CoV-2, COVID-19, Axl Receptor Tyrosine Kinase, MESH: p38 Mitogen-Activated Protein Kinases, MESH: Proto-Oncogene Proteins, Emerging Infectious Diseases, Good Health and Well Being, HEK293 Cells, biology.protein, MESH: A549 Cells, Caco-2 Cells
Abstract

Summary The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies., Graphical Abstract, Highlights • Phosphoproteomics analysis of SARS-CoV-2-infected cells uncovers signaling rewiring • Infection promotes host p38 MAPK cascade activity and shutdown of mitotic kinases • Infection stimulates CK2-containing filopodial protrusions with budding virus • Kinase activity analysis identifies potent antiviral drugs and compounds, Phosphoproteomics analysis of SARS-CoV-2-infected Vero E6 cells reveals host cellular pathways hijacked by viral infection, leading to the identification of small molecules that target dysregulated pathways and elicit potent antiviral efficacy.

Published
2020
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5. Pore-forming activity of the $Pseudomonas\ aeruginosa$ type III secretion system translocon alters the host epigenome

Author

Charlotte Lombardi, Alain Filloux, Laurent Dortet, François Cretin, Andréa Dessen, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Vaincre la Mucoviscidose RF20140501133, ANR project PRP1.4 T3SS, European Project: 654909,H2020,H2020-MSCA-IF-2014,T6SS-PSEUDO-LIP(2016), Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Imperial College London, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Medical Research Council (MRC)

Subjects
0301 basic medicine, EFFECTOR TARGETS, NF-KAPPA-B, Moths, medicine.disease_cause, Applied Microbiology and Biotechnology, Virulence factor, Epigenesis, Genetic, Type three secretion system, Histones, INFECTION, Type III Secretion Systems, MESH: Animals, MESH: Epigenesis, Genetic, PHOSPHORYLATION, MESH: Bacterial Proteins, Host cell membrane, MESH: Histones, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, MESH: Moths, EPITHELIAL-CELLS, Translocon, Cell biology, Histone Code, Larva, Host-Pathogen Interactions, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Life Sciences & Biomedicine, Microbiology (medical), GENES, Immunology, Microbiology, 03 medical and health sciences, MESH: Type III Secretion Systems, Bacterial Proteins, Genetics, medicine, Animals, Humans, Secretion, HISTONE H3, PROTEIN PHOSPHATASES, Antigens, Bacterial, Science & Technology, MESH: Humans, Cell Membrane, MESH: Host-Pathogen Interactions, Cell Biology, Epigenome, biochemical phenomena, metabolism, and nutrition, MAPK, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, MESH: Histone Code, A549 Cells, MESH: HeLa Cells, bacteria, MESH: A549 Cells, VI SECRETION, MESH: Larva, MESH: Antigens, Bacterial, HeLa Cells, MESH: Cell Membrane
Abstract

Recent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS ‘needle’ delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB–PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion. The pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon serves as a virulence factor to induce host epigenome modification and promote infection.

Published
2018
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6. Peptidomic analysis of skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae): Insight into the origin of host-defense peptides within the Pipidae and characterization of a proline-arginine-rich peptide

Author

Laurent Coquet, Thierry Jouenne, Maria Luisa Mangoni, Jay D. King, J. Michael Conlon, Laure Guilhaudis, Jérôme Leprince, Samir Attoub, School of Biomedical Sciences [Ulster University, UK], University of Ulster, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Différenciation et communication neuronale et neuroendocrine (DC2N), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plate-Forme de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Polymères Biopolymères Surfaces (PBS), Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS), Plate-forme de Protéomique PISSARO, Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Biochemical Sciences 'Rossi Fanelli', Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], United Arab Emirates University (UAEU), Rare Species Conservatory Foundation (RSCF), They also thank Labex Synorg (ANR-11-LABX-0029) for financial support., The authors thank Clint Guadiana and Colette Hairston Adams, Gladys Porter Zoo for help in collecting the frog species and Laurey Steinke and Michele Fontaine, University of Nebraska Medical Center, Omaha, NE for amino acid composition analysis and Per F. Nielsen, Novo Nordisk for sequence analysis of peak 18 (Fig. 1)., ANR-11-LABX-0029,SYNORG,Synthèse Organique : des molécules au vivant(2011), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Proteomics, Pipidae, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Xenopus, Peptide, MESH: Amino Acid Sequence, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, cellular and molecular neuroscience, MESH: Circular Dichroism, PR-39, MESH: Amphibian Proteins, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Host-defense, MESH: Animals, Micelles, Skin, Pipoidea, chemistry.chemical_classification, biology, Ecology, Circular Dichroism, MESH: Proteomics, Protein primary structure, MESH: Arginine, MESH: Micelles, MESH: Antimicrobial Cationic Peptides, Sodium Dodecyl Sulfate, antimicrobial, frog skin, host-defense, pipidae, pr-39, rhinophrynidae, physiology, biochemistry, endocrinology, Biochemistry, Rhinophrynidae, MESH: Sodium Dodecyl Sulfate, Proline, Pipa, Arginine, Amphibian Proteins, Frog skin, 03 medical and health sciences, MESH: Skin, Animals, Humans, Amino Acid Sequence, MESH: Humans, MESH: Pipidae, MESH: Proline, 030102 biochemistry & molecular biology, Bacteria, biology.organism_classification, MESH: Bacteria, 030104 developmental biology, chemistry, A549 Cells, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Antimicrobial, MESH: A549 Cells, Frog Skin, Antimicrobial Cationic Peptides
Abstract

International audience; The Mexican burrowing toad Rhinophrynus dorsalis is the sole extant representative of the Rhinophrynidae. United in the superfamily Pipoidea, the Rhinophrynidae is considered to be the sister-group to the extant Pipidae which comprises Hymenochirus, Pipa, Pseudhymenochirus and Xenopus. Cationic, α-helical host-defense peptides of the type found in Hymenochirus, Pseudhymenochirus, and Xenopus species (hymenochirins, pseudhymenochirins, magainins, and peptides related to PGLa, XPF, and CPF) were not detected in norepinephrine-stimulated skin secretions of R. dorsalis. Skin secretions of representatives of the genus Pipa also do not contain cationic α-helical host-defense peptides which suggest, as the most parsimonious hypothesis, that the ability to produce such peptides by frogs within the Pipidae family arose in the common ancestor of (Hymenochirus+Pseudhymenochirus)+Xenopus after divergence from the line of evolution leading to extant Pipa species. Peptidomic analysis of the R. dorsalis secretions led to the isolation of rhinophrynin-27, a proline-arginine-rich peptide with the primary structure ELRLPEIARPVPEVLPARLPLPALPRN, together with rhinophrynin-33 containing the C-terminal extension KMAKNQ. Rhinophrynin-27 shows limited structural similarity to the porcine multifunctional peptide PR-39 but it lacks antimicrobial and cytotoxic activities. Like PR-39, the peptide adopts a poly-l-proline helix but some changes in the circular dichroism spectrum were observed in the presence of anionic sodium dodecylsulfate micelles consistent with the stabilization of turn structures.

Published
2017
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7. A RAS-CaMKKβ-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPβ through a novel 3'UTR mechanism

Author

Kevin P. Claffey, Peter F. Johnson, Mesfin Gonit, Myriam Gorospe, Atharva Bhat, Jacqueline Salotti, Jiji Chen, Sandip K. Basu, Benoit Viollet, Mouse Cancer Genetics Program [Frederick, MD, USA] (Center for Cancer Research), National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH)-Center for Cancer Research [Frederick, MD, USA], Lentigen Tech, Inc. [Gaithersburg, MD, USA], Cancer and Inflammation Program [Frederick, MD, USA] (Leidos Biomedical Research Inc.), Frederick National Laboratory for Cancer Research (FNLCR)-NCI-Frederick, Advanced Imaging and Microscopy Resource [Bethesda, MD, USA], National Institutes of Health [Bethesda] (NIH), Laboratory of Genetics [Baltimore, MD, USA] (National Institute on Aging-Intramural Research Program), [Institut Cochin] Département Endocrinologie, métabolisme, diabète (EMD) (EMD), 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), Department of Cell Biology [Farmington, CT, USA], University of Connecticut (UCONN), This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government., and Viollet, Benoit

Subjects
0301 basic medicine, Cancer Research, AMP-Activated Protein Kinases, MESH: Mice, Knockout, law.invention, ELAV-Like Protein 1, Mice, law, Neoplasms, CEBPB, MESH: Animals, MESH: Neoplasms, MESH: AMP-Activated Protein Kinases, 10. No inequality, 3' Untranslated Regions, Cellular Senescence, [SDV.MHEP.EM] Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Mice, Knockout, MESH: ELAV-Like Protein 1, MESH: 3' Untranslated Regions, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Cell biology, MESH: MCF-7 Cells, MESH: HEK293 Cells, MCF-7 Cells, MESH: ras Proteins, Senescence, MESH: Enzyme Activation, MESH: Cell Line, Tumor, Biology, MESH: CCAAT-Enhancer-Binding Protein-beta, 03 medical and health sciences, Cell Line, Tumor, Genetics, Animals, Humans, Molecular Biology, MESH: Mice, MESH: Humans, Three prime untranslated region, Binding protein, CCAAT-Enhancer-Binding Protein-beta, AMPK, MESH: Cellular Senescence, Enzyme Activation, 030104 developmental biology, HEK293 Cells, Cytoplasm, A549 Cells, Cancer cell, NIH 3T3 Cells, ras Proteins, Suppressor, MESH: A549 Cells, MESH: NIH 3T3 Cells
Abstract

International audience; Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPβ through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPβ activation is inhibited by G/U-rich elements (GREs) in its 3'UTR. This mechanism, termed "3'UTR regulation of protein activity" (UPA), maintains C/EBPβ in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPβ UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3'UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm-a fundamental feature of UPA-thereby stimulating C/EBPβ activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca++-CaMKKβ-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPβ activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKβ. Thus, CaMKKβ-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPβ.

Published
2017
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8. In vitro and in vivo evidence for an inflammatory role of the calcium channel TRPV4 in lung epithelium: Potential involvement in cystic fibrosis

Author

Rainer Kiefmann, Lhousseine Touqui, Marie Potier-Cartereau, Christelle Coraux, Cristina Plata, Clémence Henry, Eric Morello, Mustapha Si-Tahar, Antoine Guillon, Emilie Dalloneau, Christophe Vandier, Rose-France Aimar, Maria-Teresa Pérez-Berezo, Frédéric Esnard, Nicolas Cenac, Yongzheng Wu, Miguel A. Valverde, Christian Börnchen, Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Francois Rabelais [Tours], Centre de Physiopathologie Toulouse Purpan (CPTP), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées, Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra [Barcelona] (UPF), Défense innée et inflammation, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Nutrition, croissance et cancer (U 1069) (N2C), Network 'Ion channels and Cancer-Canceropole Grand Ouest', Cancéropôle Grand-Ouest, Plasticité de l'épithélium respiratoire dans les conditions normales et pathologiques - UMR-S 903 (PERPMP), Université de Reims Champagne-Ardenne (URCA)-Centre Hospitalier Universitaire de Reims (CHU Reims)-Institut National de la Santé et de la Recherche Médicale (INSERM)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV), Cardiovascular Research Center Hamburg (CVRC), University Hamburg, Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (UKE), This work was financially supported by the 'Johanna und Fritz Buch-Gedächtnisstiftung.' C. O. Henry was funded by 'Fonds de Dotation Recherche en Santé Respiratoire' (Paris, France), and E. Dalloneau was funded by 'Vaincre la Mucoviscidose' and by a Fédération de recherche en infectiologie travel grant. A. Guillon was funded by an Inserm 'poste d'accueil.' Part of the research was funded by fundings from the French 'Conseil Régional Centre-Val de Loire,' the Spanish Ministry of Economy and Competitiveness(SAF2012-38140), Maria de Maeztu Programme for Units of Excellence in R&D (MDM-2014-0370), Fondo de Investigación Sanitaria(RD12/0042/0014), and FEDER Funds., Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Reims Champagne-Ardenne (URCA)-Université de Reims Champagne-Ardenne (URCA), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, SFR CAP Santé (Champagne-Ardenne Picardie Santé), and Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre Hospitalier Universitaire de Reims (CHU Reims)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Reims Champagne-Ardenne (URCA)

Subjects
0301 basic medicine, Male, Pathology, Chemokine, Cystic Fibrosis, Physiology, [SDV]Life Sciences [q-bio], Cystic Fibrosis Transmembrane Conductance Regulator, MESH: Cystic Fibrosis/pathology, chemokines, MESH: Rats, Sprague-Dawley, Cystic fibrosis, MESH: Mice, Knockout, Rats, Sprague-Dawley, 0302 clinical medicine, MESH: TRPV Cation Channels/physiology, MESH: Cystic Fibrosis/metabolism, MESH: Cystic Fibrosis Transmembrane Conductance Regulator/metabolism, MESH: Animals, ComputingMilieux_MISCELLANEOUS, Mice, Knockout, biology, 3. Good health, medicine.anatomical_structure, 030220 oncology & carcinogenesis, MESH: Alveolar Epithelial Cells/metabolism, Female, medicine.symptom, Signal transduction, signal transduction, Pulmonary and Respiratory Medicine, TRPV4, medicine.medical_specialty, Mice, 129 Strain, MESH: Cystic Fibrosis/immunology, TRPV Cation Channels, Inflammation, MESH: Calcium Signaling, eicosanoids, 03 medical and health sciences, MESH: Mice, 129 Strain, In vivo, MESH: Mice, Inbred C57BL, Physiology (medical), medicine, Animals, Humans, Calcium Signaling, Lung, MESH: Humans, Calcium channel, lung inflammation, Cell Biology, medicine.disease, MESH: Male, Mice, Inbred C57BL, 030104 developmental biology, A549 Cells, Alveolar Epithelial Cells, Immunology, biology.protein, calcium channel, MESH: A549 Cells, MESH: Female
Abstract

Cystic fibrosis (CF) is an inherited disease associated with chronic severe lung inflammation, leading to premature death. To develop innovative anti-inflammatory treatments, we need to characterize new cellular and molecular components contributing to the mechanisms of lung inflammation. Here, we focused on the potential role of “transient receptor potential vanilloid-4” (TRPV4), a nonselective calcium channel. We used both in vitro and in vivo approaches to demonstrate that TRPV4 expressed in airway epithelial cells triggers the secretion of major proinflammatory mediators such as chemokines and biologically active lipids, as well as a neutrophil recruitment in lung tissues. We characterized the contribution of cytosolic phospholipase A2, MAPKs, and NF-κB in TRPV4-dependent signaling. We also showed that 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, i.e., four natural lipid-based TRPV4 agonists, are present in expectorations of CF patients. Also, TRPV4-induced calcium mobilization and inflammatory responses were enhanced in cystic fibrosis transmembrane conductance regulator-deficient cellular and animal models, suggesting that TRPV4 is a promising target for the development of new anti-inflammatory treatments for diseases such as CF.

Published
2016
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9. Troponin-I enhances and is required for oncogenic overgrowth

Author

Sergio Casas-Tintó, Manuel Serrano, Antonio Maraver, Alberto Ferrús, Herrada, Anthony, Instituto Cajal, Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), and Spanish National Cancer Research Center (CNIO)

Subjects
0301 basic medicine, Male, MESH: Tumor Burden, Lung Neoplasms, Drosophila Cancer Cell competition Cell proliferation, Cell, Gene Expression, Animals, Genetically Modified, Drosophila Proteins, MESH: Animals, cell competition, musculoskeletal system, MESH: RNAi Therapeutics, Tumor Burden, medicine.anatomical_structure, Drosophila melanogaster, Oncology, cardiovascular system, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Rap1, Female, RNA Interference, Drosophila, MESH: Troponin I, MESH: Xenograft Model Antitumor Assays, MESH: Gene Expression, MESH: Drosophila Proteins, MESH: RNA Interference, Mice, Nude, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, MESH: Drosophila melanogaster, MESH: Animals, Genetically Modified, 03 medical and health sciences, Downregulation and upregulation, [SDV.CAN] Life Sciences [q-bio]/Cancer, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Carcinoma, medicine, MESH: Mice, Nude, Animals, Humans, cancer, Gene, MESH: Humans, Cell growth, Troponin I, Cancer, medicine.disease, Xenograft Model Antitumor Assays, MESH: Male, MESH: Lung Neoplasms, 030104 developmental biology, RNAi Therapeutics, cell proliferation, Cell culture, A549 Cells, Immunology, Cancer research, MESH: A549 Cells, MESH: Female, Priority Research Paper
Abstract

Human tumors of various tissue origins show an intriguing over-expression of genes not considered oncogenes, such as that encoding Troponin-I (TnI), a wellknown muscle protein. Out of the three TnI genes known in humans, the slow form, TNNI1, is affected the most. Drosophila has only one TnI gene, wupA. Here, we studied excess- and loss-of function of wupA in Drosophila, and assayed TNNI1 down regulation in human tumors growing in mice. Drosophila TnI excess-of-function increases proliferation and potentiates oncogenic mutations in Ras, Notch and Lgl genes. By contrast, TnI loss-of-function reduces proliferation and antagonizes the overgrowth due to these oncogenic mutations. Troponin-I defective cells undergo Flower- and Sparc-dependent cell competition. TnI can localize to the nucleus and its excess elicits transcriptional up-regulation of InR, Rap1 and Dilp8, which is consistent with the increased cell proliferation. Human tumor cell lines treated with a human Troponin-I peptide arrest in G/G. In addition, proliferation of non-smallcell lung carcinoma xenografts in mice is restrained by TNNI1 down-regulation. Thus, Troponin-I reveals a novel function in cell proliferation that may be of therapeutic interest in certain types of cancer.

Published
2016
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