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1. Complex transcriptional regulations of a hyperparasitic quadripartite system in giant viruses infecting protists

Author

Alexandra Bessenay, Hugo Bisio, Lucid Belmudes, Yohann Couté, Lionel Bertaux, Jean-Michel Claverie, Chantal Abergel, Sandra Jeudy, and Matthieu Legendre

Subjects
Science
Abstract

Abstract Hyperparasitism is a common pattern in nature that is not limited to cellular organisms. Giant viruses infecting protists can be hyperparasitized by smaller ones named virophages. In addition, both may carry episomal DNA molecules known as transpovirons in their particles. They all share transcriptional regulatory elements that dictate the expression of their genes within viral factories built by giant viruses in the host cytoplasm. This suggests the existence of interactions between their respective transcriptional networks. Here we investigated Acanthamoeba castellanii cells infected by a giant virus (megavirus chilensis), and coinfected with a virophage (zamilon vitis) and/or a transpoviron (megavirus vitis transpoviron). Infectious cycles were monitored through time-course RNA sequencing to decipher the transcriptional program of each partner and its impact on the gene expression of the others. We found highly diverse transcriptional responses. While the giant virus drastically reshaped the host cell transcriptome, the transpoviron had no effect on the gene expression of any of the players. In contrast, the virophage strongly modified the giant virus gene expression, albeit transiently, without altering the protein composition of mature viral particles. The virophage also induced the overexpression of transpoviron genes, likely through the indirect upregulation of giant virus-encoded transcription factors. Together, these analyses document the intricated transcriptionally regulated networks taking place in the infected cell.

Published
2024
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2. Large-scale phosphoproteomics reveals activation of the MAPK/GADD45β/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells

Author

Mohammad Al Tarrass, Lucid Belmudes, Dzenis Koça, Valentin Azemard, Hequn Liu, Tala Al Tabosh, Delphine Ciais, Agnès Desroches-Castan, Christophe Battail, Yohann Couté, Claire Bouvard, and Sabine Bailly

Subjects
BMP9, BMP10, Endothelial cells, Signaling, Phosphoproteomics, MAPK, Medicine, Cytology, QH573-671
Abstract

Abstract Background BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. Methods To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry. Results Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45β. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1. Conclusions Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

Published
2024
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3. Author Correction: Cytoskeleton remodeling induced by SMYD2 methyltransferase drives breast cancer metastasis

Author

Alexandre G. Casanova, Gael S. Roth, Simone Hausmann, Xiaoyin Lu, Ludivine J. M. Bischoff, Emilie M. Froeliger, Lucid Belmudes, Ekaterina Bourova-Flin, Natasha M. Flores, Ana Morales Benitez, Tourkian Chasan, Marcello Caporicci, Jessica Vayr, Sandrine Blanchet, Francesco Ielasi, Sophie Rousseaux, Pierre Hainaut, Or Gozani, Muriel Le Romancer, Yohann Couté, Andres Palencia, Pawel K. Mazur, and Nicolas Reynoird

Subjects
Cytology, QH573-671
Published
2024
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4. Cytoskeleton remodeling induced by SMYD2 methyltransferase drives breast cancer metastasis

Author

Alexandre G. Casanova, Gael S. Roth, Simone Hausmann, Xiaoyin Lu, Ludivine J. M. Bischoff, Emilie M. Froeliger, Lucid Belmudes, Ekaterina Bourova-Flin, Natasha M. Flores, Ana Morales Benitez, Tourkian Chasan, Marcello Caporicci, Jessica Vayr, Sandrine Blanchet, Francesco Ielasi, Sophie Rousseaux, Pierre Hainaut, Or Gozani, Muriel Le Romancer, Yohann Couté, Andres Palencia, Pawel K. Mazur, and Nicolas Reynoird

Subjects
Cytology, QH573-671
Abstract

Abstract Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cell dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cell ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulate lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation lose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

Published
2024
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5. Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes

Author

Florentin Pastor, Emilie Charles, Lucid Belmudes, Hélène Chabrolles, Marion Cescato, Michel Rivoire, Thomas Burger, Guillaume Passot, David Durantel, Julie Lucifora, Yohann Couté, and Anna Salvetti

Subjects
hepatitis B virus, primary human hepatocytes, phosphoproteomics, anti-viral response, DNA damage response, RNA-binding proteins, Microbiology, QR1-502
Abstract

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

Published
2024
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6. Cell-type-specific control of secondary cell wall formation by Musashi-type translational regulators in Arabidopsis

Author

Alicia Kairouani, Dominique Pontier, Claire Picart, Fabien Mounet, Yves Martinez, Lucie Le-Bot, Mathieu Fanuel, Philippe Hammann, Lucid Belmudes, Remy Merret, Jacinthe Azevedo, Marie-Christine Carpentier, Dominique Gagliardi, Yohann Couté, Richard Sibout, Natacha Bies-Etheve, and Thierry Lagrange

Subjects
arabidopsis, musashi, stem, SCW, glucuronoxylan, RNA binding protein, Medicine, Science, Biology (General), QH301-705.5
Abstract

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.

Published
2023
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7. A Proteomics-Based Approach Identifies the NEDD4 Adaptor NDFIP2 as an Important Regulator of Ifitm3 Levels

Author

Federico Marziali, Yuxin Song, Xuan-Nhi Nguyen, Lucid Belmudes, Julien Burlaud-Gaillard, Philippe Roingeard, Yohann Couté, and Andrea Cimarelli

Subjects
IFITM3, virus, NEDD4, NDFIP2, interferon, Microbiology, QR1-502
Abstract

IFITMs are a family of highly related interferon-induced transmembrane proteins that interfere with the processes of fusion between viral and cellular membranes and are thus endowed with broad antiviral properties. A number of studies have shown how the antiviral potency of IFITMs is highly dependent on their steady-state levels, their intracellular distribution and a complex pattern of post-translational modifications, parameters that are overall tributary of a number of cellular partners. In an effort to identify additional protein partners involved in the biology of IFITMs, we devised a proteomics-based approach based on the piggyback incorporation of IFITM3 partners into extracellular vesicles. MS analysis of the proteome of vesicles bearing or not bearing IFITM3 identified the NDFIP2 protein adaptor protein as an important regulator of IFITM3 levels. NDFIP2 is a membrane-anchored adaptor protein of the E3 ubiquitin ligases of the NEDD4 family that have already been found to be involved in IFITM3 regulation. We show here that NDFIP2 acts as a recruitment factor for both IFITM3 and NEDD4 and mediates their distribution in lysosomal vesicles. The genetic inactivation and overexpression of NDFIP2 drive, respectively, lower and higher levels of IFITM3 accumulation in the cell, overall suggesting that NDFIP2 locally competes with IFITM3 for NEDD4 binding. Given that NDFIP2 is itself tightly regulated and highly responsive to external cues, our study sheds light on a novel and likely dynamic layer of regulation of IFITM3.

Published
2023
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8. The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield

Author

Alejandro Villalta, Alain Schmitt, Leandro F Estrozi, Emmanuelle RJ Quemin, Jean-Marie Alempic, Audrey Lartigue, Vojtěch Pražák, Lucid Belmudes, Daven Vasishtan, Agathe MG Colmant, Flora A Honoré, Yohann Couté, Kay Grünewald, and Chantal Abergel

Subjects
mimivirus, giant virus, viral dsDNA organization, cryo-EM, cryo-ET, proteomics, Medicine, Science, Biology (General), QH301-705.5
Abstract

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

Published
2022
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9. A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma

Author

Dayana C Farhat, Matthew W Bowler, Guillaume Communie, Dominique Pontier, Lucid Belmudes, Caroline Mas, Charlotte Corrao, Yohann Couté, Alexandre Bougdour, Thierry Lagrange, Mohamed-Ali Hakimi, and Christopher Swale

Subjects
Toxoplasma gondii, epitranscriptomic, RNA processing, Arabidopsis thaliana, Medicine, Science, Biology (General), QH301-705.5
Abstract

Correct 3’end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

Published
2021
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10. Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Author

Hélène Chabrolles, Héloïse Auclair, Serena Vegna, Thomas Lahlali, Caroline Pons, Maud Michelet, Yohann Couté, Lucid Belmudes, Gilliane Chadeuf, Yujin Kim, Ariel Di Bernardo, Pascal Jalaguier, François-Loïc Cosset, Floriane Fusil, Michel Rivoire, Lee D Arnold, Uri Lopatin, Christophe Combet, Fabien Zoulim, David Grierson, Benoit Chabot, Julie Lucifora, David Durantel, and Anna Salvetti

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Published
2020
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11. No fitness impact of the knockout of the two main components of mimivirus genomic fiber and fibril layer

Author

Jean-Marie Alempic, Hugo Bisio, Alejandro Villalta, Sébastien Santini, Audrey Lartigue, Alain Schmitt, Claire Bugnot, Anna Notaro, Lucid Belmudes, Annie Adrait, Olivier Poirot, Denis Ptchelkine, Cristina De Castro, Yohann Couté, and Chantal Abergel

Abstract

The Mimivirus 1.2Mb genome is organized into a nucleocapsid-like genomic fiber composed of a mixture of two GMC-oxidoreductase paralogs (1). Surprisingly, these proteins also constitute the glycosylated fibril layer that decorates the virion (2). The individual inactivation of each gene by homologous recombination-based knockout (KO) confirmed that they are not essential. The resulting genomic fiber corresponds to a 5- and a 6-start helix, similar to the wild type structures, and is composed of the remaining GMC-oxidoreductase. A model is proposed explaining the transition from a 6- to a 5-start helix and their coexistence. The two single mutants and the double mutant, for which both genes have been KO, showed equivalent fitness in laboratory conditions with no impact on the glycosylation of the layer of fibrils surrounding the capsids. Their major protein components can be easily exchanged, as highlighted by the double KO fibril layer composition. Accordingly, we show that the proteins composing the fibril layer can already be different between different members of the family (moumouviruses and megaviruses), echoing their differences in glycan composition (3, 4). These results obtained on two distinct essential processes (genome packaging and virion infectivity) illustrate the exceptional functional redundancy of theMimiviridae, which may be the major evolutionary force behind their giant genomes.One-Sentence Summary: Functional redundancy warrants mimivirus genomic fiber and fibril layer formation.Subject Area: Biological sciences/Microbiology/Virology/Viral evolution; Biological sciences/Microbiology/Virology/Virus structures

Published
2023

13. Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites

Author

Fabien Sindikubwabo, Shuai Ding, Tahir Hussain, Philippe Ortet, Mohamed Barakat, Sebastian Baumgarten, Dominique Cannella, Andrés Palencia, Alexandre Bougdour, Lucid Belmudes, Yohann Couté, Isabelle Tardieux, Cyrille Y Botté, Artur Scherf, and Mohamed-ali Hakimi

Subjects
histone core modifications, chromatin, gene expression, acetylation, methylation, Toxoplasma gondii, Medicine, Science, Biology (General), QH301-705.5
Abstract

An unusual genome architecture characterizes the two related human parasitic pathogens Plasmodium falciparum and Toxoplasma gondii. A major fraction of the bulk parasite genome is packaged as transcriptionally permissive euchromatin with few loci embedded in silenced heterochromatin. Primary chromatin shapers include histone modifications at the nucleosome lateral surface close to the DNA but their mode of action remains unclear. We now identify versatile modifications at Lys31 within the globular domain of histone H4 that crucially determine genome organization and expression in Apicomplexa parasites. H4K31 acetylation at the promoter correlates with, and perhaps directly regulates, gene expression in both parasites. By contrast, monomethylated H4K31 is enriched in the core body of T. gondii active genes but inversely correlates with transcription, whereas it is unexpectedly enriched at transcriptionally inactive pericentromeric heterochromatin in P. falciparum, a region devoid of the characteristic H3K9me3 histone mark and its downstream effector HP1.

Published
2017
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14. Altiratinib blocks Toxoplasma gondii and Plasmodium falciparum development by selectively targeting a spliceosome kinase

Author

Christopher Swale, Valeria Bellini, Matthew W. Bowler, Nardella Flore, Marie-Pierre Brenier-Pinchart, Dominique Cannella, Lucid Belmudes, Caroline Mas, Yohann Couté, Fabrice Laurent, Artur Scherf, Alexandre Bougdour, Mohamed-Ali Hakimi, 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), European Molecular Biology Laboratory [Grenoble] (EMBL), Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-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), Integrated Structural Biology Grenoble (ISBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Fondation pour la Recherche Medicale (EQU202103012571), Roux-Cantarini Fellowship, ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-18-CE15-0023,HostQuest,Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée(2018), ANR-21-CE35-0010,ApiNewDrug,Identification de nouvelles cibles thérapeutiques parmi des médicaments repositionnables pour traiter des maladies dues à des parasitaire apicomplexes(2021), ANR-20-CE18-0006,EpiKillMal,Cibler la méthylation de l'ADN pour contrer la chimiorésistance du paludisme(2020), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 614880,Hosting Toxo, Laurent, Fabrice, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée - - HostQuest2018 - ANR-18-CE15-0023 - AAPG2018 - VALID, Identification de nouvelles cibles thérapeutiques parmi des médicaments repositionnables pour traiter des maladies dues à des parasitaire apicomplexes - - ApiNewDrug2021 - ANR-21-CE35-0010 - AAPG2021 - VALID, Cibler la méthylation de l'ADN pour contrer la chimiorésistance du paludisme - - EpiKillMal2020 - ANR-20-CE18-0006 - AAPG2020 - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, and ERC Consolidator grant - Hosting Toxo - 614880 - INCOMING

Subjects
[SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, General Medicine
Abstract

The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii , Plasmodium , and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii . We have identified Tg PRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for Tg PRP4K and for its Plasmodium falciparum counterpart, Pf CLK3. Our data identified structural features critical for binding of the other Pf CLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum . Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

Published
2022

15. Altiratinib blocks

Author

Christopher, Swale, Valeria, Bellini, Matthew W, Bowler, Nardella, Flore, Marie-Pierre, Brenier-Pinchart, Dominique, Cannella, Lucid, Belmudes, Caroline, Mas, Yohann, Couté, Fabrice, Laurent, Artur, Scherf, Alexandre, Bougdour, and Mohamed-Ali, Hakimi

Subjects
Plasmodium falciparum, Spliceosomes, Animals, Cryptosporidiosis, Cryptosporidium, Humans, Angiogenesis Inhibitors, Malaria, Falciparum, Protein Kinase Inhibitors, Toxoplasma
Abstract

The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include

Published
2022

16. The giant mimivirus 1.2 Mb genome is elegantly organized into a 30 nm diameter helical protein shield

Author

Leandro F Estrozi, Alain Schmitt, Alejandro Villalta, Emmanuelle RJ Quemin, Jean-Marie Alempic, Audrey Lartigue, Vojtěch Pražák, Lucid Belmudes, Daven Vasishtan, Agathe MG Colmant, Flora A Honoré, Yohann Couté, Kay Grünewald, Chantal Abergel, Information génomique et structurale (IGS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), 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), Universität Hamburg (UHH), Université Grenoble Alpes (UGA), ANR-16-CE11-0033,VIRiON,Etude des protéines Fer-Soufre des virus géants à la recherche de réponses sur l'origine de la vie(2016), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
General Immunology and Microbiology, General Neuroscience, Mimivirus genome organization in the icosahedral virion Virology, viral genome organization, Cryoelectron Microscopy, Genome, Viral, General Medicine, General Biochemistry, Genetics and Molecular Biology, Cro-electron microscopy Mimivirus, genomic fiber, cryo-ET, Capsid, Nucleoproteins, proteomics, Giant Viruses, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, cryo-EM, dsDNA, Mimiviridae, Oxidoreductases, giant virus, helical reconstruction
Abstract

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

Published
2022

18. The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages

Author

Arne L. ten Hoeve, Laurence Braun, Matias E. Rodriguez, Gabriela C. Olivera, Alexandre Bougdour, Lucid Belmudes, Yohann Couté, Jeroen P.J. Saeij, Mohamed-Ali Hakimi, Antonio Barragan, The Wenner-Gren Institute, Stockholm University, University of California [Davis] (UC Davis), University of California (UC), 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), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-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), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), and ANR-18-CE15-0023,HostQuest,Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée(2018)

Subjects
Mice, Cell Movement, [SDV]Life Sciences [q-bio], Virology, Macrophages, Animals, Parasitology, Parasites, Dendritic Cells, Microbiology, Toxoplasma
Abstract

International audience; Upon pathogen detection, macrophages normally stay sessile in tissues while dendritic cells (DCs) migrate to secondary lymphoid tissues. The obligate intracellular protozoan Toxoplasma gondii exploits the trafficking of mononuclear phagocytes for dissemination via unclear mechanisms. We report that, upon T. gondii infection, macrophages initiate the expression of transcription factors normally attributed to DCs, upregulate CCR7 expression with a chemotactic response, and perform systemic migration when adoptively transferred into mice. We show that parasite effector GRA28, released by the MYR1 secretory pathway, cooperates with host chromatin remodelers in the host cell nucleus to drive the chemotacticmigration of parasitized macrophages. During in vivo challenge studies, bone marrow-derived macrophages infected with wild-type T. gondii outcompeted those challenged with MYR1- or GRA28-deficient strains in migrating and reaching secondary organs. This work reveals how an intracellular parasite hijacks chemotaxis in phagocytes and highlights a remarkable migratory plasticity in differentiated cells of the mononuclear phagocyte system.

Published
2022

19. The giant Mimivirus 1.2 Mb genome is elegantly organized into a 30 nm helical protein shield

Author

Alejandro Villalta, Alain Schmitt, Leandro F. Estrozi, Emmanuelle R. J. Quemin, Jean-Marie Alempic, Audrey Lartigue, Vojtěch Pražák, Lucid Belmudes, Daven Vasishtan, Agathe M. G. Colmant, Flora A. Honoré, Yohann Couté, Kay Grünewald, and Chantal Abergel

Subjects
viruses
Abstract

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ∼0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and proteomics revealed that it is encased into a ∼30 nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.One-Sentence SummaryMimivirus genome organization in the icosahedral virion.

Published
2022

20. N-acetylation of secreted proteins in Apicomplexa is widespread and is independent of the ER acetyl-CoA transporter AT1

Author

Mary Akinyi Nyonda, Jean-Baptiste Boyer, Lucid Belmudes, Aarti Krishnan, Paco Pino, Yohann Couté, Mathieu Brochet, Thierry Meinnel, Dominique Soldati-Favre, Carmela Giglione, Meinnel, Thierry, Elucider les fonctions multifacettes des protéines acétyl transferase en réponse au stress et dans la régulation métabolique - - KatNat2017 - ANR-17-CAPS-0001 - ERA-CAPS - VALID, Saclay Plant Sciences - - SPS2010 - ANR-10-LABX-0040 - LABX - VALID, Impact des modifications cotranslationnelles N-terminales plastidiales lors de la réponse au stress - - CANMORE2020 - ANR-20-CE92-0040 - AAPG2020 - VALID, Université de Genève = University of Geneva (UNIGE), 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), Maturation des proteines, destinée cellulaire et thérapeutique (PROMTI), 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), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-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), Fondation ARC (ARCPJA32020060002137), ANR-17-CAPS-0001,KatNat,Elucider les fonctions multifacettes des protéines acétyl transferase en réponse au stress et dans la régulation métabolique(2017), ANR-10-LABX-0040,SPS,Saclay Plant Sciences(2010), and ANR-20-CE92-0040,CANMORE,Impact des modifications cotranslationnelles N-terminales plastidiales lors de la réponse au stress(2020)

Subjects
Male, Mammals, Proteome, Plasmodium berghei, Toxoplasma gondii, Acetylation, Cell Biology, Secretory pathway, Endoplasmic Reticulum, Acetyl Coenzyme A, Animals, Acetyl-CoA transporter, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Female, Parasites, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein Processing, Post-Translational, Toxoplasma, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Abstract

Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. berghei, AT1 was found to be important for growth of asexual blood stages, production of female gametocytes and male gametocytogenesis, implying its requirement for parasite transmission. In the absence of AT1, lysine acetylation and N-terminal acetylation in T. gondii remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.

Published
2022

21. A drug repurposing screen identifies altiratinib as a selective inhibitor of a key regulatory splicing kinase and a potential therapeutic for toxoplasmosis and malaria

Author

Caroline Mas, Dominique Cannella, Artur Scherf, Christopher Swale, Lucid Belmudes, Fabrice Laurent, Mohamed-Ali Hakimi, Matthew W. Bowler, Marie-Pierre Brenier-Pinchart, Yohann Couté, Nardella Flore, Alexandre Bougdour, Valeria Bellini, 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), European Molecular Biology Laboratory [Grenoble] (EMBL), Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-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), Integrated Structural Biology Grenoble (ISBG - UMS 3518 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Infectiologie et Santé Publique (UMR ISP), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), (FRM Equipe # EQU202103012571), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-18-CE15-0023,HostQuest,Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée(2018), ANR-21-CE35-0010,ApiNewDrug,Identification de nouvelles cibles thérapeutiques parmi des médicaments repositionnables pour traiter des maladies dues à des parasitaire apicomplexes(2021), ANR-20-CE18-0006,EpiKillMal,Cibler la méthylation de l'ADN pour contrer la chimiorésistance du paludisme(2020), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 614880,Hosting Toxo, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Integrated Structural Biology Grenoble (ISBG), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Chanteloup, Nathalie Katy, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée - - HostQuest2018 - ANR-18-CE15-0023 - AAPG2018 - VALID, Identification de nouvelles cibles thérapeutiques parmi des médicaments repositionnables pour traiter des maladies dues à des parasitaire apicomplexes - - ApiNewDrug2021 - ANR-21-CE35-0010 - AAPG2021 - VALID, Cibler la méthylation de l'ADN pour contrer la chimiorésistance du paludisme - - EpiKillMal2020 - ANR-20-CE18-0006 - AAPG2020 - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, and ERC Consolidator grant - Hosting Toxo - 614880 - INCOMING

Subjects
0303 health sciences, biology, drug repurposing, Kinase, kinase, Plasmodium falciparum, Intron, Toxoplasma gondii, Computational biology, biology.organism_classification, 3. Good health, Apicomplexa, 03 medical and health sciences, Drug repositioning, splicing, 0302 clinical medicine, CLK3, RNA splicing, parasitic diseases, Protozoa, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Altiratinib, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Introductory paragraphThe apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that infect humans and animals and cause severe parasitic diseases. Available therapeutics against these devastating diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. Here, we use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index. We have identified TgPRP4K of T. gondii as the primary target of altiratinib by genetic target deconvolution, highlighting key residues within the kinase catalytic site that, when mutated, confer resistance to the drug. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K as well as for its P. falciparum counterpart PfCLK3. Our data also point to structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the role of this kinase family in splicing in a broad spectrum of eukaryotes, we have shown that altiratinib causes global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a promising pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

Published
2021

23. A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental genes partitioning inToxoplasma

Author

Thierry Lagrange, Guillaume Communie, Alexandre Bougdour, Dominique Pontier, Yohann Couté, Dayana C. Farhat, Christopher Swale, Matthew W. Bowler, Caroline Mas, Lucid Belmudes, Charlotte Corrao, and Mohamed-Ali Hakimi

Subjects
0303 health sciences, Polyadenylation, RNA, Biology, biology.organism_classification, Phenotype, Cell biology, Transcriptome, 03 medical and health sciences, 0302 clinical medicine, Gene expression, Arabidopsis thaliana, Polyadenylation Pathway, Gene, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Correct 3’end processing of mRNAs is regarded as one of the regulatory cornerstones of gene expression. In a parasite that must answer to the high regulatory requirements of its multi-host life style, there is a great need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly-arranged stage specific genes. In this study, we report on our findings inT. gondiiof an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at theseloci. We identify the core polyadenylation complex withinT. gondiiand establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of theT. gondiiCPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant modelArabidopsis thaliana. We bring Nanopore direct RNA sequencing-based evidence of the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in bothT. gondiiandA. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in bothT. gondiiandA. thaliana, as a guardian against aberrant transcriptional read-throughs.Highlightsm6A is recognized in apicomplexan and plants by CPSF4, a member of the cleavage and polyadenylation complex machinery.The structural insight behind the specificity of the binding of m6A by the CPSF4 YTH subunit are solved by high resolution crystal structures.The m6A-driven 3’end polyadenylation pathway protects transcriptome integrity by restricting transcriptional read-throughs and RNA chimera formation in apicomplexan parasites and plants.

Published
2021

24. The Mimivirus 1.2 Mb dsDNA genome is elegantly organized into a nuclear-like weapon

Author

Alejandro Villalta Casares, Flora Honore, Audrey Lartigue, Yohann Couté, Chantal Abergel, Agathe Colmant, Alain Schmitt, Vojta Prazak, Daven Vasishtan, Kay Gruenewald, Jean-Marie Alempic, Lucid Belmudes, and Emmanuelle R. J. Quemin

Subjects
Mimivirus, biology, Computational biology, biology.organism_classification, Genome
Abstract

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses, initially isolated in Acanthamoeba1. Little is known about the organization of the viral genome inside the membrane limited nucleoid2 and whether unpacking or other rearrangements are required prior to transcription and replication. Here we show that opening of its large icosahedral capsid in vitro leads to the release of electron dense, 30 nm diameter rod-shaped objects that appear to be expelled from the particles and unwinding. We developed a purification procedure and characterized the detailed structure at various stages of decompaction using cryo-electron microscopy single particle analyses and its composition by proteomics. This revealed that the viral genome is encased into a helical protein shell surprisingly made of the two GMC-type oxydoreductases that are also the major components of the glycosylated fibrils surrounding the capsid3. The 1.2 Mb genome is folded to follow a 5- or 6-start left-handed helix, depending on the nature of the GMC-oxydoreductase, with each helical strand lining the interior of the protein shell. Proteomic analyses of the purified genomic fibre revealed the presence of several RNA polymerase subunits as well as additional proteins involved in genome compaction that can fit into the central channel of the protein shield. Such an elegant supramolecular organization represents a remarkable evolutionary solution for packaging while protecting the viral genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. We expect that a dedicated energy-driven machinery is required for the assembly of this rod-shaped giant viral chromosome and its further compaction in the membrane limited electron dense nucleoid, characteristic of the mature Mimivirus particles2,4,5.The parsimonious implication of the same protein in two functionally unrelated substructures of the virion is also unexpected for a giant virus with a thousand genes at its disposal.

Published
2021

25. Expanding the Occurrence of Polysaccharides to the Viral World: The Case of Mimivirus

Author

Lucid Belmudes, Annalisa Salis, Chantal Abergel, Michela Tonetti, Antonio Molinaro, Anna Notaro, Gianluca Damonte, Yohann Couté, Cristina De Castro, Maria Elena Laugieri, Notaro, Anna, Couté, Yohann, Belmudes, Lucid, Laugeri, Maria Elena, Salis, Annalisa, Damonte, Gianluca, Molinaro, Antonio, Tonetti, Michela G, Abergel, Chantal, De Castro, Cristina, Information génomique et structurale (IGS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-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), Université de Genève = University of Geneva (UNIGE), Université de Naples, ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 832601,Cells and giant viruses: a win-win co-evolution, Université de Genève (UNIGE), Poirot, Olivier, and VIREVOL - Cells and giant viruses: a win-win co-evolution - 832601 - INCOMING

Subjects
Glycosylation, viruses, Computational biology, Biology, Polysaccharide, Genome, Catalysis, Mimiviru, 03 medical and health sciences, chemistry.chemical_compound, NMR spectroscopy, Polysaccharides, Giant Virus, giant viruses, Research Articles, 030304 developmental biology, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, chemistry.chemical_classification, 0303 health sciences, Mimivirus, glycan, Glycobiology, 030302 biochemistry & molecular biology, General Medicine, General Chemistry, biology.organism_classification, glycans, glycocalyx, Mimiviridae, NMR, 3. Good health, giant viruse, chemistry, Capsid, Viruses, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Research Article
Abstract

The general perception of viruses is that they are small in terms of size and genome, and that they hijack the host machinery to glycosylate their capsid. Giant viruses subvert all these concepts: their particles are not small, and their genome is more complex than that of some bacteria. Regarding glycosylation, this concept has been already challenged by the finding that Chloroviruses have an autonomous glycosylation machinery that produces oligosaccharides similar in size to those of small viruses (6–12 units), albeit different in structure compared to the viral counterparts. We report herein that Mimivirus possesses a glycocalyx made of two different polysaccharides, now challenging the concept that all viruses coat their capsids with oligosaccharides of discrete size. This discovery contradicts the paradigm that such macromolecules are absent in viruses, blurring the boundaries between giant viruses and the cellular world and opening new avenues in the field of viral glycobiology., The giant virus Mimivirus has an icosahedral capsid embedded in a thick layer of fibrils, resembling the glycocalyx of many bacteria. This study revealed that these fibrils are composed by a limited set of carrier proteins which bear two distinct polysaccharides. The presence of polysaccharides refutes the paradigm that such macromolecules are absent in viruses and opens new avenues in the field of glycosciences and viral biology.

Published
2021

26. Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

Author

David S. Grierson, Christophe Combet, Thomas Lahlali, Lee D. Arnold, Héloïse Auclair, Caroline Pons, Julie Lucifora, Yohann Couté, Uri Lopatin, David Durantel, Yujin Kim, Benoit Chabot, Anna Salvetti, Serena Vegna, Hélène Chabrolles, Ariel Di Bernardo, Fabien Zoulim, Michel Rivoire, Pascal Jalaguier, Lucid Belmudes, and Maud Michelet

Subjects
Hepatitis B virus, Gene knockdown, Alternative splicing, RNA-binding protein, Viremia, Biology, medicine.disease_cause, medicine.disease, Interactome, Virology, digestive system diseases, Viral replication, RNA splicing, medicine
Abstract

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.Author SummaryChronic infection with Hepatitis B virus (HBV) affects more than 250 millions of people world-wide and is a major global cause of liver cancer. Current treatments lead to a significant reduction of viremia in patients. However, viral clearance is rarely obtained and the persistence of the HBV genome in the hepatocyte’s nucleus generates a stable source of viral RNAs and subsequently proteins which play important roles in immune escape mechanisms and liver disease progression. Therapies aiming at efficiently and durably eliminating viral gene expression are still required. In this study, we identified the nuclear partners of the HBV Core protein (HBc) to understand how this structural protein, responsible for capsid assembly in the cytoplasm, could also regulate viral gene expression. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs). One of these RBPs, SRSF10, was demonstrated to restrict HBV RNA levels and a drug, able to alter its phosphorylation, behaved as an antiviral compound capable of reducing viral gene expression. Altogether, this study sheds new light novel regulatory functions of HBc and provides information relevant for the development of antiviral strategies aiming at preventing viral gene expression.

Published
2020

27. A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment

Author

Yohann Couté, Dominique Cannella, Christopher Swale, Philippe Ortet, Fabien Sindikubwabo, Céline Dard, Lucid Belmudes, Alexandre Bougdour, Mohamed-Ali Hakimi, Mohamed Barakat, Dayana C. Farhat, Pieter-Jan De Bock, 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), Laboratoire de parasitologie-mycologie, CHU Grenoble, Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Laboratoire d'Ecologie Microbienne de la Rhizosphère et d'Environnements Extrêmes (LEMIRE), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Université Grenoble Alpes (UGA), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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), and ANR-18-CE15-0023,HostQuest,Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée(2018)

Subjects
Models, Molecular, Transcription, Genetic, [SDV]Life Sciences [q-bio], Protozoan Proteins, Antibodies, Protozoan, Applied Microbiology and Biotechnology, Protein Structure, Secondary, chromatin remodeling, Histones, microrchidia protein, Histone code, ComputingMilieux_MISCELLANEOUS, Adenosine Triphosphatases, Genetics, 0303 health sciences, Chromatin, Histone Code, Histone, Toxoplasma, epigenetic, Toxoplasmosis, Protein Binding, Microbiology (medical), Apetala 2, Primary Cell Culture, Immunology, Toxoplasma gondii, Biology, Microbiology, Histone Deacetylases, Article, 03 medical and health sciences, Animals, Humans, Epigenetics, Transcription factor, Gene, 030304 developmental biology, Life Cycle Stages, 030306 microbiology, Cell Biology, Fibroblasts, Cats, biology.protein, gene expression, sexual commitment, Histone deacetylase, Protein Processing, Post-Translational, Transcription Factors
Abstract

Toxoplasma gondii has a complex life cycle that is typified by asexual development that takes place in vertebrates, and sexual reproduction, which occurs exclusively in felids and is therefore less studied. The developmental transitions rely on changes in the patterns of gene expression, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific epigenetic programs for each given stage. Here, we identified the T. gondii microrchidia (MORC) protein as an upstream transcriptional repressor of sexual commitment. MORC, in a complex with Apetala 2 (AP2) transcription factors, was shown to recruit the histone deacetylase HDAC3, thereby impeding the accessibility of chromatin at the genes that are exclusively expressed during sexual stages. We found that MORC-depleted cells underwent marked transcriptional changes, resulting in the expression of a specific repertoire of genes, and revealing a shift from asexual proliferation to sexual differentiation. MORC acts as a master regulator that directs the hierarchical expression of secondary AP2 transcription factors, and these transcription factors potentially contribute to the unidirectionality of the life cycle. Thus, MORC plays a cardinal role in the T. gondii life cycle, and its conditional depletion offers a method to study the sexual development of the parasite in vitro, and is proposed as an alternative to the requirement of T. gondii infections in cats.

Published
2020

28. Comprehensive and comparative exploration of the Atp7b

Author

Maud, Lacombe, Michel, Jaquinod, Lucid, Belmudes, Yohann, Couté, Claire, Ramus, Florence, Combes, Thomas, Burger, Elisabeth, Mintz, Justine, Barthelon, Vincent, Leroy, Aurélia, Poujois, Alain, Lachaux, France, Woimant, and Virginie, Brun

Subjects
Adult, Male, Proteome, Ceruloplasmin, Blood Proteins, Middle Aged, Mice, Mutant Strains, Mice, Inbred C57BL, Disease Models, Animal, Mice, Hepatolenticular Degeneration, Liver, Copper-Transporting ATPases, Animals, Humans, Female, Copper
Abstract

Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b

Published
2019

29. Comprehensive and comparative exploration of the Atp7b(-/-) mouse plasma proteome

Author

Lucid Belmudes, Alain Lachaux, Michel Jaquinod, Maud Lacombe, Yohann Couté, Virginie Brun, Florence Combes, Vincent Leroy, Elisabeth Mintz, Justine Barthelon, Claire Ramus, Aurélia Poujois, Thomas Burger, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Université Grenoble Alpes (UGA), Biologie des Métaux (BioMet), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-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)-Institut de Chimie du CNRS (INC)-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), Clinique Universitaire d'Hépato-Gastroentérologie, CHU Grenoble, National reference Centre for Wilson's Disease [Paris] (CRMR Wilson), Service de neurologie [Univ. Paris VII], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Lariboisière-Fernand-Widal [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Lariboisière-Fernand-Widal [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), National Reference Centre for Wilson's Disease, Hôpital Femme Mère Enfant, ANR-19-P3IA-0003,MIAI,MIAI @ Grenoble Alpes(2019), Biologie des Métaux (BioMet ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-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)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de recherche en astrophysique et planétologie (IRAP), Centre National de la Recherche Scientifique (CNRS)-Observatoire Midi-Pyrénées (OMP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut national des sciences de l'Univers (INSU - CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Laboratoire d'Etude du Rayonnement et de la Matière en Astrophysique (LERMA (UMR_8112)), Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de Paris, PSL Research University (PSL)-PSL Research University (PSL)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Matière et Systèmes Complexes (MSC (UMR_7057)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Service de radiologie et imagerie médicale [Rennes], Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Hôpital Pontchaillou-CHU Pontchaillou [Rennes], CarMeN, laboratoire, MIAI @ Grenoble Alpes - - MIAI2019 - ANR-19-P3IA-0003 - P3IA - VALID, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Observatoire Midi-Pyrénées (OMP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Biophysics, Proteomics, Biochemistry, Biomaterials, 03 medical and health sciences, 0302 clinical medicine, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Gene, biology, Metals and Alloys, Phenotype, Blood proteins, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, Chemistry (miscellaneous), 030220 oncology & carcinogenesis, Proteome, biology.protein, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Liver function, Ceruloplasmin, Function (biology)
Abstract

Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b−/− mouse, a genetic and phenotypic model of WD, was developed to provide new insights into the pathogenic mechanisms of WD. Many plasma proteins are secreted by the liver, and impairment of liver function can trigger changes to the plasma proteome. High standard proteomics workflows can identify such changes. Here, we explored the plasma proteome of the Atp7b−/− mouse using a mass spectrometry (MS)-based proteomics workflow combining unbiased discovery analysis followed by targeted quantification. Among the 367 unique plasma proteins identified, 7 proteins were confirmed as differentially abundant between Atp7b−/− mice and wild-type littermates, and were directly linked to WD pathophysiology (regeneration of liver parenchyma, plasma iron depletion, etc.). We then adapted our targeted proteomics assay to quantify human orthologues of these proteins in plasma from copper-chelator-treated WD patients. The plasma proteome changes observed in the Atp7b−/− mouse were not confirmed in these samples, except for alpha-1 antichymotrypsin, levels of which were decreased in WD patients compared to healthy individuals. Plasma ceruloplasmin was investigated in both the Atp7b−/− mouse model and human patients; it was significantly decreased in the human form of WD only. In conclusion, MS-based proteomics is a method of choice to identify proteome changes in murine models of disrupted metal homeostasis, and allows their validation in human cohorts.

Published
2019

30. Commensalism in the Mimiviridae giant virus family

Author

Sandra Jeudy, Sissel Juul, Matthieu Legendre, Daniel J. Turner, Nadège Philippe, Lucid Belmudes, Lionel Bertaux, Laure Beucher, Jean-Marie Alempic, Jean-Michel Claverie, Yohann Couté, Emanuele G. Biondi, Audrey Lartigue, Sébastien Santini, and Chantal Abergel

Subjects
Virophages, Mimivirus, biology, Zamilon virophage, Virophage, Giant Virus, Mimiviridae, Mobilome, biology.organism_classification, Virology, Virus
Abstract

Acanthamoeba-infecting Mimiviridae belong to three clades: Mimiviruses (A), Moumouviruses (B) and Megaviruses (C). The uniquely complex mobilome of these giant viruses includes virophages and linear 7 kb-DNA molecules called “transpovirons”. We recently isolated a virophage (Zamilon vitis) and two transpovirons (maBtv and mvCtv) respectively associated to B-clade and C-clade mimiviruses. We used the capacity of the Zamilon virophage to replicate both on B-clade and C-clade host viruses to investigate the three partite interaction network governing the propagation of transpovirons. We found that the virophage could transfer both types of transpovirons to B-clade and C-clade host viruses provided they were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate both maBtv and mvCtv, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We performed proteomic comparisons of host viruses and virophage particles carrying or cleared of transpovirons in search of proteins involved in this adaptation process. These experiments revealed that transpoviron-encoded proteins are synthetized during the combined mimiviruses/virophage/transpoviron replication process and some of them are specifically incorporated into the virophage and giant virus particles together with the cognate transpoviron DNA. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate from one host virus to another and where the Zamilon virophage and the transpoviron depend on their host giant virus to replicate, this without affecting the giant virus infectious cycle, at least in laboratory conditions.Author SummaryThe Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build huge viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions (transcription and replication) take place. They are themselves the target of infections by 20 kb-dsDNA virophages, replicating in the giant virus factories. They can also be found associated with 7kb-DNA episomes, dubbed transpovirons. We investigated the relationship between these three players by combining competition experiments involving the newly isolated Zamilon vitis virophage as a vehicle for transpovirons of different origins with proteomics analyses of virophage and giant virus particles. Our results suggest that relationship of the virophage, the transpoviron, and their host giant virus, extend the concept of commensalism to the viral world.

Published
2019
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31. Exploration of the propagation of transpovirons within Mimiviridae reveals a unique example of commensalism in the viral world

Author

Daniel J. Turner, Emanuele G. Biondi, Sandra Jeudy, Laure Beucher, Jean-Marie Alempic, Nadège Philippe, Matthieu Legendre, Audrey Lartigue, Chantal Abergel, Lionel Bertaux, Sissel Juul, Sébastien Santini, Lucid Belmudes, Jean-Michel Claverie, Yohann Couté, Information génomique et structurale (IGS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), 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), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de chimie bactérienne (LCB), Duke University [Durham], Oxford Nanopore Technologies, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Université Grenoble Alpes (UGA), The Fondation Bettencourt Schueller (OTP51251) and the Provence-Alpes-Côte-d’Azur région (2010 12125), ANR-11-INBS-0013,IFB (ex Renabi-IFB),Institut français de bioinformatique(2011), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Etude de la dynamique des protéomes (EDyP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-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 (UGA)-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), ANR: 11-INBS-0013,ANR-11-INSB-0013,Institut français de bioinformatique (ReNaBi-IFB), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Équipe Services et Architectures pour Réseaux Avancés (LAAS-SARA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and 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 (UGA)

Subjects
Virophages, viruses, Acanthamoeba, Microbiology, Virus, Article, 03 medical and health sciences, Zamilon virophage, Virology, Mimiviridae, Giant Virus, Symbiosis, Cellular microbiology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, Mimivirus, biology, 030306 microbiology, Virophage, biology.organism_classification, Commensalism, Evolutionary biology, Giant Viruses, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Abstract

Acanthamoeba-infecting Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions take place. They are themselves the target of infections by 20-kb-dsDNA virophages, replicating in the giant virus factories and can also be found associated with 7-kb-DNA episomes, dubbed transpovirons. Here we isolated a virophage (Zamilon vitis) and two transpovirons respectively associated to B- and C-clade mimiviruses. We found that the virophage could transfer each transpoviron provided the host viruses were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron originally isolated from the corresponding virus was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate each transpoviron, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We analysed the proteomes of host viruses and virophage particles in search of proteins involved in this adaptation process. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate and where the Zamilon virophage and the transpoviron depend on the giant virus to replicate, without affecting its infectious cycle.

Published
2019

32. Bone Morphogenetic Protein 9 Is a Paracrine Factor Controlling Liver Sinusoidal Endothelial Cell Fenestration and Protecting Against Hepatic Fibrosis

Author

Lucid Belmudes, Olivier Boillot, Agnès Desroches-Castan, Jean-Jacques Feige, Jean-Yves Scoazec, Emmanuelle Tillet, Yohann Couté, Marie Ouarné, Nicolas Ricard, Sabine Bailly, and Christine Mallet

Subjects
0301 basic medicine, Liver Cirrhosis, Proteomics, Activin Receptors, Type II, Mice, Transgenic, Biology, Statistics, Nonparametric, Tissue Culture Techniques, 03 medical and health sciences, Paracrine signalling, Mice, Random Allocation, 0302 clinical medicine, Fibrosis, medicine, Growth Differentiation Factor 2, Hepatic Stellate Cells, Animals, Humans, RNA, Messenger, Cells, Cultured, Regulation of gene expression, Hepatology, GATA4, Endothelial Cells, Activin receptor, medicine.disease, Cell biology, Endothelial stem cell, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, Gene Expression Regulation, Hepatic stellate cell, 030211 gastroenterology & hepatology, Hepatic fibrosis
Abstract

Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15 weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.

Published
2018

33. A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma

Author

Lucid Belmudes, Guillaume Communie, Dayana C. Farhat, Thierry Lagrange, Alexandre Bougdour, Dominique Pontier, Yohann Couté, Matthew W. Bowler, Charlotte Corrao, Christopher Swale, Mohamed-Ali Hakimi, Caroline Mas, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Universitaire [Grenoble] (CHU)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Laboratoire européen de biologie moléculaire - European Molecular Biology Laboratory (EMBL Grenoble), European Molecular Biology Laboratory [Grenoble] (EMBL), Institut Laue-Langevin (ILL), ILL, Laboratoire Génome et développement des plantes (LGDP), Université de Perpignan Via Domitia (UPVD)-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Université Grenoble Alpes (UGA), Integrated Structural Biology Grenoble (ISBG - UMS 3518 ), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-10-INSB-0801 ProFI ANR-10-INSB-0801, ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-18-CE15-0023,HostQuest,Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée(2018), ANR-10-LABX-0041,TULIP,Towards a Unified theory of biotic Interactions: the roLe of environmental(2010), ANR-18-EURE-0019,TULIP-GSR,The Toulouse-Perpignan(2018), 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), Integrated Structural Biology Grenoble (ISBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Rosalie, Martin, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée - - HostQuest2018 - ANR-18-CE15-0023 - AAPG2018 - VALID, Towards a Unified theory of biotic Interactions: the roLe of environmental - - TULIP2010 - ANR-10-LABX-0041 - LABX - VALID, and The Toulouse-Perpignan - - TULIP-GSR2018 - ANR-18-EURE-0019 - EURE - VALID

Subjects
Models, Molecular, Polyadenylation, Arabidopsis thaliana, Arabidopsis, Transcriptome, 0302 clinical medicine, Gene expression, Biology (General), 0303 health sciences, Microbiology and Infectious Disease, Membrane Glycoproteins, biology, General Neuroscience, Cleavage And Polyadenylation Specificity Factor, Zinc Fingers, General Medicine, Phenotype, epitranscriptomic, Cell biology, EPITRANSCRIPTOMIC MODIFICATIONS, Medicine, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], RNA Splicing Factors, Toxoplasma, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Research Article, QH301-705.5, Science, Toxoplasma gondii, Nerve Tissue Proteins, M6A, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Genes, Developmental, RNA, Messenger, YTH DOMAIN, Gene, 030304 developmental biology, Binding Sites, General Immunology and Microbiology, Sequence Analysis, RNA, RNA, Methyltransferases, biology.organism_classification, RNA processing, Gene Expression Regulation, Reading, Other, 030217 neurology & neurosurgery
Abstract

Correct 3’end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

Published
2021

34. Synthesis of Artemisinin-Derived Dimers, Trimers and Dendrimers: Investigation of Their Antimalarial and Antiviral Activities Including Putative Mechanisms of Action

Author

Maria Leidenberger, Lucid Belmudes, Friedrich Hahn, Yohann Couté, Oliver Friedrich, Tony Fröhlich, Svetlana B. Tsogoeva, Barbara Kappes, Manfred Marschall, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), 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)-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 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), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
0301 basic medicine, Dendrimers, medicine.medical_treatment, Dimer, Dihydroartemisinin, Cytomegalovirus, Trimer, 01 natural sciences, Antiviral Agents, Catalysis, 03 medical and health sciences, chemistry.chemical_compound, Antimalarials, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Humans, Antimalarial Agent, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, biology, 010405 organic chemistry, Drug discovery, Chemistry, Organic Chemistry, Plasmodium falciparum, Succinates, General Chemistry, biology.organism_classification, In vitro, Artemisinins, 3. Good health, 0104 chemical sciences, 030104 developmental biology, Biochemistry, Glycoprotein
Abstract

Generation of dimers, trimers and dendrimers of bioactive compounds is an approach that has recently been developed for the discovery of new potent drug candidates. Herein, we present the synthesis of new artemisinin-derived dimers and dendrimers and investigate their action against malaria parasite Plasmodium falciparum 3D7 strain and human cytomegalovirus (HCMV). Dimer 7 was the most active compound (EC50 1.4 nm) in terms of antimalarial efficacy and was even more effective than the standard drugs dihydroartemisinin (EC50 2.4 nm), artesunic acid (EC50 8.9 nm) and chloroquine (EC50 9.8 nm). Trimer 4 stood out as the most active agent against HCMV in vitro replication and exerted an EC50 value of 0.026 μm, representing an even higher activity than the two reference drugs ganciclovir (EC50 2.60 μm) and artesunic acid (EC50 5.41 μm). In addition, artemisinin-derived dimer 13 and trimer 15 were for the first time both immobilized on TOYOPEARL AF-Amino-650M beads and used for mass spectrometry-based target identification experiments using total lysates of HCMV-infected primary human fibroblasts. Two major groups of novel target candidates, namely cytoskeletal and mitochondrial proteins were obtained. Two putatively compound-binding viral proteins, namely major capsid protein (MCP) and envelope glycoprotein pUL132, which are both essential for HCMV replication, were identified.

Published
2018

36. Author response: Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites

Author

Lucid Belmudes, Dominique Cannella, Cyrille Y. Botté, Artur Scherf, Tahir Hussain, Fabien Sindikubwabo, Sebastian Baumgarten, Andrés Palencia, Mohamed Barakat, Yohann Couté, Alexandre Bougdour, Isabelle Tardieux, Shuai Ding, Philippe Ortet, and Mohamed-Ali Hakimi

Subjects
0301 basic medicine, Histone H4, 03 medical and health sciences, 030104 developmental biology, Lateral surface, Expression (architecture), Biology, Genome structure, Cell biology
Published
2017

37. The Long Hunt for pssR—Looking for a Phospholipid Synthesis Transcriptional Regulator, Finding the Ribosome

Author

Lucid Belmudes, Julia Bartoli, Yohann Couté, Laetitia My, Emmanuelle Bouveret, Julie P. M. Viala, Aix Marseille Université (AMU), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), 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), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and 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])

Subjects
0301 basic medicine, Operon, 030106 microbiology, Mutant, Biology, medicine.disease_cause, Microbiology, 03 medical and health sciences, Gene expression, Transcriptional regulation, medicine, Escherichia coli, Molecular Biology, Gene, ComputingMilieux_MISCELLANEOUS, Phospholipids, Diacylglycerol kinase, Mutation, Escherichia coli Proteins, Translation (biology), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Gene Expression Regulation, Bacterial, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Biochemistry, 13. Climate action, Ribosomes, Research Article, Transcription Factors
Abstract

The phospholipid (PL) composition of bacterial membranes varies as a function of growth rate and in response to changes in the environment. While growth adaptation can be explained by biochemical feedback in the PL synthesis pathway, recent transcriptome studies have revealed that the expression of PL synthesis genes can also be tuned in response to various stresses. We previously showed that the BasRS two-component pathway controls the expression of the diacylglycerol kinase gene, dgkA , in Escherichia coli (A. Wahl, L. My, R. Dumoulin, J. N. Sturgis, and E. Bouveret, Mol Microbiol, 80:1260–1275, 2011, https://doi.org/10.1111/j.1365-2958.2011.07641.x ). In this study, we set up a strategy to identify the mutation responsible for the upregulation of pssA observed in the historical pssR1 mutant and supposedly corresponding to a transcriptional repressor (C. P. Sparrow and J. Raetz, J Biol Chem, 258:9963–9967, 1983). pssA encodes phosphatidylserine synthase, the first step of phosphatidylethanolamine synthesis. We showed that this mutation corresponded to a single nucleotide change in the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by the rrnC operon. We further demonstrated that this mutation enhanced the translation of pssA . Though this effect appeared to be restricted to PssA among phospholipid synthesis enzymes, it was not specific, as evidenced by a global effect on the production of unrelated proteins. IMPORTANCE Bacteria adjust the phospholipid composition of their membranes to the changing environment. In addition to enzymatic regulation, stress response regulators control specific steps of the phospholipid synthesis pathway. We wanted to identify a potential regulator controlling the expression of the phosphatidylserine synthase gene. We showed that it was not the previously suggested hdfR gene and instead that a mutation in the anti-Shine-Dalgarno sequence of 16S RNA was responsible for an increase in pssA translation. This example underlines the fact that gene expression can be modulated by means other than specific regulatory processes.

Published
2017

38. Identification of hepatitis B virus core protein nuclear interacting factors points to RNA binding proteins as major regulators of HBV replication

Author

David S. Grierson, Lee D. Arnold, Anna Salvetti, Benoit Chabot, Yohann Couté, Hélène Chabrolles, Lucid Belmudes, Y. Kim, Christophe Combet, David Durantel, Serena Vegna, Julie Lucifora, A. Héloïse, and Fabien Zoulim

Subjects
Hepatology, Hepatitis B virus core, RNA-binding protein, Identification (biology), Biology, Hbv replication, Virology
Published
2018

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