Use of stable CHO pools and CHO TGE to accelerate SARS-CoV-2 vaccine development

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Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca 2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition

Using a genetically encoded sensor to identify inhibitors of Toxoplasma gondii Ca2+ Signalling

This work was supported in part by National Institutes of Health Grants AI-110027 and AI-096836 (to S. N. J. M.) and 1DP5OD017892 (to S. L.).The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition.Publisher PDFPeer reviewe

RPRD1A and RPRD1B Are Human RNA Polymerase II C-Terminal Domain Scaffolds for Ser5 Dephosphorylation

The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2

Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite

ÉTUDE STRUCTURALE DES PROTÉINES DE LA CAPSIDE DE L'ADÉNOVIRUS

Adenoviruses are dsDNA non enveloped icosahedral viruses. Although these viruses are widely studied in the area of gene therapy applications, the lack of fundamental knowledge especially from a structural point of view is probably partly responsible for the limited success of the first trials. As a part of a global structural study of adenoviral proteins, we focused on minor proteins, and particularly on protein IX. 4 trimers of protein IX are found at the center of each face of the capsid. Cryo-electron microscopy 3D reconstructions of human adenovirus in complex with Fabs directed against the protein IX C-terminal region, and a canine adenovirus where the C-terminal domain of protein IX was marked with GFP, enabled us to localise the C-terminal region at the capsid surface. A combination of these results with biophysical analyses of various mutants of protein IX allowed to propose a model of integration of this protein into the capsid surface. In particular we showed that protein IX forms a lattice between the different capsomers.Our work also focused on another structural protein, the short fiber head of avian adenovirus. We solved the crystallographic structure of this protein at 2 Å resolution and compared it with other known fiber head structures. Inspite of a low sequence homology, the tertiary and quaternary structures of fiber heads are conserved within members of the adenovirus family. A phylogenetic tree based on the atomic structures of fiber heads revealed a new link between adenoviruses infecting different species. The structure also allowed us to emit hypotheses on the nature of cellular receptors for avian adenovirus. The third structural protein we took an interest in is human adenovirus type 3 penton base. 12 copies of this pentameric protein further assemble into a highly symmetrical dodecahedron particle. The 80 Å cavity of the dodecahedrons and their ability to enter cells prompted to propose these assemblies as potential gene transfer vehicles for gene therapy. We recently succeeded to improve dodecahedron crystals to obtain X-ray diffraction up to 3.5 Å resolution. The structure of the assembly is being solved.Les adénovirus sont des virus icosaédriques non enveloppés à ADN double brin. Bien que ces virus soient très étudiés dans le but d'une application possible en thérapie génique, le manque de connaissances fondamentales, notamment structurales, est probablement à l'origine du faible succès rencontré lors des premiers essais effectués. Dans le cadre d'un programme général d'étude structurale des protéines de l'adénovirus, nous nous sommes intéressés aux protéines mineures et particulièrement à la protéine IX. Cette dernière est localisée sous forme de 4 trimères au centre de chaque face du virus. Grâce à des reconstructions 3D à partir des images de cryo-microscopie électronique, d'une part, d'un adénovirus humain décoré par des Fabs ciblant le domaine C-terminal de la protéine IX et, d'autre part, d'un adénovirus canin dans lequel le domaine C-terminal de la protéine IX a été fusionné avec la GFP, nous avons pu localiser le domaine C-terminal à la surface de la capside. En combinant ces résultats avec des données biophysiques obtenues pour différents mutants de la protéine IX, nous avons proposé un modèle de son intégration au sein de la capside. Nous avons en particulier montré que la protéine IX formait un maillage localisé entre les différents capsomères. Nous nous sommes également intéressés à une autre protéine structurale de l'adénovirus : la tête de fibre courte de l'adénovirus aviaire. Nous avons résolu la structure cristallographique de cette protéine à 2 Å de résolution. Cette structure a été comparée avec d'autres structures de tête de fibre d'adénovirus déjà connues. Nous avons pu en déduire que, malgré de faibles homologies de séquences, les structures tertiaire et quaternaire des têtes de fibre étaient conservées au sein de la famille des adénovirus. La construction d'un arbre phylogénique basé sur les structures atomiques des têtes de fibre nous a permis de mettre en évidence une nouvelle filiation entre les adénovirus infectant différentes espèces. La structure a également conduit à des hypothèses quand à la nature des récepteurs cellulaires de l'adénovirus aviaire.La troisième protéine structurale à laquelle nous nous sommes intéressés est la base du penton de l'adénovirus humain de sérotype 3. Cette protéine pentamérique peut s'associer par douze pour former un assemblage hautement symétrique : le dodécaèdre. Grâce à leur cavité de 80Å et leur capacité à entrer dans les cellules, ces particules dodécaédriques ont été proposées comme véhicule de transfert de gène en thérapie génique potentielle. Nous avons réussi récemment à améliorer des cristaux de cet assemblage pour arriver à une diffraction des rayons X allant jusqu'à une résolution de 3.5 Å. La résolution de la structure est en cours

Étude strcuturale des protéines de la capside de l'adénovirus

Les adénovirus sont des virus icosaédriques non enveloppés à ADN double brin. Bien que ces virus soient très étudiés dans le but d'une application possible en thérapie génique, le manque de connaissances fondamentales, notamment structurales, est probablement à l'origine du faible succès rencontré lors des premiers essais effectués. Dans le cadre d'un programme général d'étude structurale des protéines de l'adénovirus, nous nous sommes intéressés aux protéines mineures et particulièrement à la protéine IX. Cette dernière est localisée sous forme de 4 trimères au centre de chaque face du virus. Grâce à des reconstructions 3D à partir des images de cryo-microscopie électronique, d'une part, d'un adénovirus humain décoré par des Fabs ciblant le domaine C-terminal de la protéine IX et, d'autre part, d'un adénovirus canin dans lequel le domaine C-terminal de la protéine IX a été fusionné avec la GFP, nous avons pu localiser le domaine C-terminal à la surface de la capside. En combinant ces résultats avec des données biophysiques obtenues pour différents mutants de la protéine IX, nous avons proposé un modèle de son intégration au sein de la capside. Nous avons en particulier montré que la protéine IX formait un maillage localisé entre les différents capsomères.Nous nous sommes également intéressés à une autre protéine structurale de l'adénovirus : la tête de fibre courte de l'adénovirus aviaire. Nous avons résolu la structure cristallographique de cette protéine à 2 Å de résolution. Cette structure a été comparée avec d'autres structures de tête de fibre d'adénovirus déjà connues. Nous avons pu en déduire que, malgré de faibles homologies de séquences, les structures tertiaire et quaternaire des têtes de fibre étaient conservées au sein de la famille des adénovirus. La construction d'un arbre phylogénique basé sur les structures atomiques des têtes de fibre nous a permis de mettre en évidence une nouvelle filiation entre les adénovirus infectant différentes espèces. La structure a également conduit à des hypothèses quand à la nature des récepteurs cellule. La troisième protéine structurale à laquelle nous nous sommes intéressés est la base du penton de l'adénovirus humain de sérotype 3. Cette protéine pentamérique peut s'associer par douze pour former un assemblage hautement symétrique : le dodécaèdre. Grâce à leur cavité de 80Å et leur capacité à entrer dans les cellules, ces particules dodécaédriques ont été proposées comme véhicule de transfert de gène en thérapie génique potentielle. Nous avons réussi récemment à améliorer des cristaux de cet assemblage pour arriver à une diffraction des rayons X allant jusqu'à une résolution de 3.5 Å. La résolution de la structure est en cours.Adenoviruses are dsDNA non enveloped icosahedral viruses. Although these viruses are widely studied in the area of gene therapy applications, the lack of fundamental knowledge especially from a structural point of view is probably partly responsible for the limited success of the first trials. As a part of a global structural study of adenoviral proteins, we focused on minor proteins, and particularly on protein IX. 4 trimers of protein IX are found at the center of each face of the capsid. Cryo-electron microscopy 3D reconstructions of human adenovirus in complex with Fabs directed against the protein IX C-terminal region, and a canine adenovirus where the C-terminal domain of protein IX was marked with GFP, enabled us to localise the C-terminal region at the capsid surface. A combination of these results with biophysical analyses of various mutants of protein IX allowed to propose a model of integration of this protein into the capsid surface. In particular we showed that protein IX forms a lattice between the different capsomers. Our work also focused on another structural protein, the short fiber head of avian adenovirus. We solved the crystallographic structure of this protein at 2 Å resolution and compared it with other known fiber head structures. Inspite of a low sequence homology, the tertiary and quaternary structures of fiber heads are conserved within members of the adenovirus family. A phylogenetic tree based on the atomic structures of fiber heads revealed a new link between adenoviruses infecting different species. The structure also allowed us to emit hypotheses on the nature of cellular receptors for avian adenovirus. The third structural protein we took an interest in is human adenovirus type 3 penton base. 12 copies of this pentameric protein further assemble into a highly symmetrical dodecahedron particle. The 80 Å cavity of the dodecahedrons and their ability to enter cells prompted to propose these assemblies as potential gene transfer vehicles for gene therapy. We recently succeeded to improve dodecahedron crystals to obtain X-ray diffraction up to 3.5 Å resolution. The structure of the assembly is being solved.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Unraveling the crystal structure of the HpaA adhesin: insights into cell adhesion function and epitope localization of a Helicobacter pylori vaccine candidate

ABSTRACTHelicobacter pylori is a bacterium that exhibits strict host restriction to humans and non-human primates, and the bacterium is widely acknowledged as a significant etiological factor in the development of chronic gastritis, peptic ulcers, and gastric cancers. The pathogenic potential of this organism lies in its adeptness at colonizing the gastric mucosa, which is facilitated by a diverse repertoire of virulence factors, including adhesins that promote the attachment of the bacteria to the gastric epithelium. Among these adhesins, HpaA stands out due to its conserved nature and pivotal role in establishing H. pylori colonization. Moreover, this lipoprotein holds promise as an antigen for the development of effective H. pylori vaccines, thus attracting considerable attention for in-depth investigations into its molecular function and identification of binding determinants. Here, we present the elucidation of the crystallographic structure of HpaA at 2.9 Å resolution. The folding adopts an elongated protein shape, which is distinctive to the Helicobacteraceae family, and features an apical domain extension that plays a critical role in the cell-adhesion activity on gastric epithelial cells. Our study also demonstrates the ability of HpaA to induce TNF-α expression in macrophages, highlighting a novel role as an immunoregulatory effector promoting the pro-inflammatory response in vitro. These findings not only contribute to a deeper comprehension of the multifaceted role of HpaA in H. pylori pathogenesis but also establish a fundamental basis for the design and development of structure-based derivatives, aimed at enhancing the efficacy of H. pylori vaccines.IMPORTANCEHelicobacter pylori is a bacterium that can cause chronic gastritis, peptic ulcers, and gastric cancers. The bacterium adheres to the lining of the stomach using proteins called adhesins. One of these proteins, HpaA, is particularly important for H. pylori colonization and is considered a promising vaccine candidate against H. pylori infections. In this work, we determined the atomic structure of HpaA, identifying a characteristic protein fold to the Helicobacter family and delineating specific amino acids that are crucial to support the attachment to the gastric cells. Additionally, we discovered that HpaA can trigger the production of TNF-α, a proinflammatory molecule, in macrophages. These findings provide valuable insights into how H. pylori causes disease and suggest that HpaA has a dual role in both attachment and immune activation. This knowledge could contribute to the development of improved vaccine strategies for preventing H. pylori infections

Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.

International audienceA 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily