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Elisar Barbar - One of the best experts on this subject based on the ideXlab platform.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating Lyssavirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Francine C. Gérard, Jean-marie Bourhis, Danielle Blondel, Marc Jamin, Elisar BarbarAbstract:The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus Phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.
Nathan E. Jespersen - One of the best experts on this subject based on the ideXlab platform.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating Lyssavirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Francine C. Gérard, Jean-marie Bourhis, Danielle Blondel, Marc Jamin, Elisar BarbarAbstract:The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus Phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.
Marc Jamin - One of the best experts on this subject based on the ideXlab platform.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating Lyssavirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Francine C. Gérard, Jean-marie Bourhis, Danielle Blondel, Marc Jamin, Elisar BarbarAbstract:The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus Phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.
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unphosphorylated rhabdoviridae Phosphoproteins form elongated dimers in solution
Biochemistry, 2007Co-Authors: Francine C A Gerard, Euripedes A Ribeiro, Aurelie A V Albertini, Irina Gutsche, Guiseppe Zaccai, Rob W H Ruigrok, Marc JaminAbstract:The phosphoprotein (P) is an essential component of the replication machinery of rabies virus (RV) and vesicular stomatitis virus (VSV), and the oligomerization of P, potentially controlled by phosphorylation, is required for its function. Up to now the stoichiometry of phosphoprotein oligomers has been controversial. Size exclusion chromatography combined with detection by multiangle laser light scattering shows that the recombinant unphosphorylated Phosphoproteins from VSV and from RV exist as dimers in solution. Hydrodynamic analysis indicates that the dimers are highly asymmetric, with a Stokes radius of 4.8-5.3 nm and a frictional ratio larger than 1.7. Small-angle neutron scattering experiments confirm the dimeric state and the asymmetry of the structure and yield a radius of gyration of about 5.3 nm and a cross-sectional radius of gyration of about 1.6-1.8 nm. Similar hydrodynamic properties and molecular dimensions were obtained with a variant of VSV phosphoprotein in which Ser60 and Thr62 are substituted by Asp residues and which has been reported previously to mimic phosphorylation by inducing oligomerization and activating transcription. Here, we show that this mutant also forms a dimer with hydrodynamic properties and molecular dimensions similar to those of the wild type protein. However, incubation at 30 degrees C for several hours induced self-assembly of both wild type and mutant proteins, leading to the formation of irregular filamentous structures.
Melvin J Glimcher - One of the best experts on this subject based on the ideXlab platform.
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the isolation and characterization of glycosylated Phosphoproteins from herring fish bones
Journal of Biological Chemistry, 2010Co-Authors: Haiyan Zhou, Erdjan Salih, Melvin J GlimcherAbstract:Past studies of bone extracellular matrix Phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish Phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa Phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.
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on the problem of covalent linkages between Phosphoproteins and collagen in bovine dentin and bone
Journal of Bone and Mineral Research, 2009Co-Authors: Melvin J Glimcher, Beatrice Lefteriou, Dora KossivaAbstract:: The majority of Phosphoproteins in bovine bone and dentin are insoluble in EDTA and guanidine hydrochloride (Gu.HCl) at 2 degrees C. After removal of EDTA and Gu.HCl-soluble proteins at 2 degrees C, collagen alpha-chains and alpha-chain polymers were extracted from bovine bone and dentin in Gu.HCl at elevated temperatures and purified by several chromatographic techniques and SDS-PAGE. Small amounts of O-phosphoserine were found in all collagen components. In contrast, O-phosphoserine was not detected in the purified collagen components soluble in EDTA or Gu.HCl at 2 degrees C nor was hydroxyproline detected in the EDTA-soluble Phosphoproteins. In contrast, although the vast majority of EDTA-insoluble collagen and phosphoprotein molecules can be readily dissociated by a variety of molecular sieving and ion-exchange chromatographic procedures, a small number are very strongly associated or covalently cross-linked. These results are consistent with the findings that both hydroxyproline and hydroxylysine are present in purified phosphoprotein components released from the EDTA-insoluble tissue by bacterial collagenase. The hydroxylysine/100 hydroxyproline ratios in the phosphoprotein-collagen complexes are much higher than those in dentin or bone collagens.
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The effect of complexing Phosphoproteins to decalcified collagen on in vitro calcification.
Connective Tissue Research, 2009Co-Authors: A. Endo, Melvin J GlimcherAbstract:Decalcified samples of chicken bone containing Phosphoproteins of varying concentrations were used to assess the effect of Phosphoproteins and of protein-bound Ser(P) and Thr(P) in the in vitro nucleation of a Ca-P solid phase from metastable solutions of Ca and P. Phosphoproteins of bone as well as the Phosphoproteins from egg yolk (phosvitin) were used.Increasing concentrations of phosphoprotein [as measured by the amount of protein bound Ser(P) and Thr(P)] in the decalcified bone particles significantly reduced the time required for nucleation to occur after exposure to metastable solutions of Ca and P (decreased operational lag times). Treatment with wheat germ acid phosphatase markedly reduced the concentration of Ser(P) and Thr(P) in the decalcified bone samples and in the decalcified bone collagen samples complexed with Phosphoproteins (almost to zero). The loss of the organic phosphate groups significantly increased the operational lag time, but did not abolish nucleation of apatite crystals by th...
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Ultrastructural immunolocalization of a major phosphoprotein in embryonic chick bone.
Connective Tissue Research, 2009Co-Authors: Marc D. Mckee, Y. Gotoh, Antonio Nanci, William J. Landis, Louis C. Gerstenfeld, Melvin J GlimcherAbstract:Immunocytochemistry utilizing the protein A-gold technique was used to examine the ultrastructural cellular and extracellular distribution of a major phosphoprotein in chick bone. HCl-extracts of embryonic and neo-natal chick bones contain a major 66kD phosphoprotein (BPP) which was purified and used to raise polyclonal antibodies in rabbits. The mid-diaphyseal regions of 8-, 12- and 18-day embryonic chick tibiae were fixed with 1% glutaraldehyde and embedded in Epon or Lowicryl. Electron microscopy following incubation of tissue sections with the antibody and the protein A-gold complex revealed specific immunolabeling over the rER and Golgi apparatus of osteoblasts and over those areas of bone matrix containing Ca and P as determined by electron probe x-ray microanalysis. These included extracellular areas in the matrix undergoing early mineralization and electron dense patches occurring at the mineralization front and extending throughout the more mature bone regions. Biochemical analyses of bone tissue processed similarly to that used for immunocytochemistry confirmed the retention of phosphoprotein in the tissue. The spatial correlation of phosphoprotein in the extracellular matrix with Ca-P mineral deposits confirms an earlier report using 33Pi and radioautography and may indicate a role for Phosphoproteins in calcification.
Danielle Blondel - One of the best experts on this subject based on the ideXlab platform.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating Lyssavirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Francine C. Gérard, Jean-marie Bourhis, Danielle Blondel, Marc Jamin, Elisar BarbarAbstract:The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus Phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.