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11 results on '"Sylviane Hoos"'

1. Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

Author

Emeline Vernhes, Linda Larbi Chérif, Nicolas Ducrot, Clément Vanbergue, Malika Ouldali, Lena Zig, N’diaye Sidibe, Sylviane Hoos, Luis Ramirez-Chamorro, Madalena Renouard, Ombeline Rossier, Patrick England, Guy Schoehn, Pascale Boulanger, and Karim Benihoud

Subjects
Immunologic diseases. Allergy, RC581-607, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Abstract The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here, we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunization with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+ T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes a unique DNA-free bacteriophage capsid able to display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.

Published
2024
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2. Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, and Alexandre Chenal

Subjects
Calmodulin, CaM, Calmidazolium, CDZ, Calmodulin antagonist, Structure, Biology (General), QH301-705.5
Abstract

Abstract Background Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. Results We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. Conclusion Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Graphical abstract Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Published
2022
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3. The c‐di‐ <scp>AMP</scp> ‐binding protein <scp>CbpB</scp> modulates the level of <scp>ppGpp</scp> alarmone in Streptococcus agalactiae

Author

Giovanni Covaleda‐Cortés, Ariel Mechaly, Terry Brissac, Heike Baehre, Laura Devaux, Patrick England, Bertrand Raynal, Sylviane Hoos, Myriam Gominet, Arnaud Firon, Patrick Trieu‐Cuot, Pierre Alexandre Kaminski, Biologie des Bactéries pathogènes à Gram-positif - Biology of Gram-Positive Pathogens, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hannover Medical School [Hannover] (MHH), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by grants from the French Government ‘Laboratory of Excellence - Integrative Biology of Emerging Infectious Diseases’ (LabEx IBEID, grant number ANR-10-LABX-62-IBEID to PTC), the Fondation pour la Recherche Médicale (FRM grant number DEQ20181039599 to PTC)., We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we thank the staff of beamlines PROXIMA-1 and PROXIMA-2A for assistance. The authors are grateful to the Staff of the Crystallography platform at the Institut Pasteur for robot-driven crystallization screenings. We acknowledge Eduard Baquero Salazar for his help during the early stage of this work. We also acknowledge the help of Mariette Matondo and Thibault Chaze from the Proteomics facility of Institut Pasteur., and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
CbpB protein, Cyclic diadenosine monophosphate (c‐di‐AMP), [SDV]Life Sciences [q-bio], X‐ray crystallography & binding, Streptococcus, Cell Biology, Potassium homeostasis, CBS domain, Molecular Biology, Biochemistry
Abstract

International audience; Cyclic di-AMP is an essential signaling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Published
2023

5. Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

Author

Emeline Vernhes, Linda Larbi Chérif, Nicolas Ducrot, Malika Ouldali, Lena Zig, N’diaye Sidibe, Sylviane Hoos, Luis Ramirez-Chamorro, Madalena Renouard, Ombeline Rossier, Patrick England, Guy Schoehn, Pascale Boulanger, Karim Benihoud, 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), Aspects métaboliques et systémiques de l'oncogénèse pour de nouvelles approches thérapeutiques (METSY), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-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), This work was supported by the Centre National de la Recherche Scientifique and the University Paris-Saclay. L. L.C., E.V. and N.D. received a fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche. L. L.C.received an additional fellowship from the Ligue Nationale contre le cancer. L.R.-C received a fellowship 'Becas Don Carlos Antonio Lopez' from the Paraguayan Ministry of Education. This work benefited from the Cryo-EM platform of I2BC, supported by the French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-05] and member of IBISA. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG, UAR3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INSB-05-02 & project ID 160 to P. B.) and GRAL, financed within the University Grenoble Alpes graduate school (Écoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation pour la Recherche Médicale (FRM), the fonds FEDER and the GIS-Infrastructures en Biologie Santé et Agronomie (IBiSA)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract

The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunisation with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes the first DNA-free bacteriophage capsid able to irreversibly display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.

Published
2022

6. Structural basis for the allosteric inhibition of UMP kinase from Gram‐positive bacteria, a promising antibacterial target

Author

Patrick Walter, Ariel Mechaly, Julien Bous, Ahmed Haouz, Patrick England, Joséphine Lai‐Kee‐Him, Aurélie Ancelin, Sylviane Hoos, Bruno Baron, Stefano Trapani, Patrick Bron, Gilles Labesse, Hélène Munier‐Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Centre de Biologie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Biophysique Moléculaire (Plate-forme), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS) project (to HML). The project was also supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), the Institut Pasteur and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05. PW was the recipient of a PhD fellowship from the EU H2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and European Project: 675555,H2020,H2020-MSCA-ITN-2015,AEGIS(2016)

Subjects
crystal structure, allostery, [SDV]Life Sciences [q-bio], Cryoelectron Microscopy, Uridine Triphosphate, Cell Biology, nucleotide metabolism, Gram-Positive Bacteria, Biochemistry, Anti-Bacterial Agents, Adenosine Triphosphate, effector, Allosteric Regulation, cryo-EM single particle analysis, Humans, Amino Acid Sequence, Guanosine Triphosphate, enzyme regulation, Nucleoside-Phosphate Kinase, Uridine Monophosphate, Molecular Biology, UMP kinase
Abstract

International audience; Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5’-monophosphate (UMP) kinase is a promis- ing target for novel antibiotic discovery as it is essential for bacterial sur- vival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regula- tion with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Published
2022

10. Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, and Alexandre Chenal

Abstract

Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs.Figure and text for the Table of Contents (ToC)Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.

Published
2022

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