Etude des interactions entre les facteurs cytosoliques du complexe de la NADPH Oxydase

During phagocytose of a micro-organism, the NADPH Oxidase complex of neutrophiles is activated. Reactive oxygen species are then produced and contribute to pathogen destruction. This complex comprises cytosolic factors (p67phox, p40phox, p47phox), a small G protein Rac and a flavocytochrome b558 composed of p22phox and gp91phox. Mutations within the genes encoding some of these proteins lead to a rare but serious disease termed chronic granulomatous disease (CGD). The formation of the ternary complex constituted by the cytosolic factors implies several SH3 domain/polyproline interactions and also an interaction PB1 domain interaction. We structurally characterized by Nuclear Magnetic Resonance the interaction between PB1 domains of p67phox and p40phox. We also studied the consequences of activation on the interactions involving p47phox C-terminal polyproline motif and its SH3 domain partners. Thus, we combined the analysis of the structures of p40phox SH3 domain and p67phox C-terminal SH3 domain, in complex with p47phox C-terminal polyproline, with our affinity measures between these partners at various stages of activation. These data were obtained by intrinsic fluorescence of tryptophan present within SH3 domains.Lors de la phagocytose d'un micro-organisme, le complexe de la NADPH Oxydase des neutrophiles est activé. Il permet alors la production d'espèces réactives de l'oxygène qui contribuent à la destruction du pathogène. Ce complexe est constitué de facteurs cytosoliques (p67phox, p40phox, p47phox), d'une petite protéine G Rac et du flavocytochrome b558 membranaire, lui-même composé de p22phox et gp91phox. Des mutations dans les gènes codant pour certaines de ces protéines conduisent à une maladie génétique rare mais grave!: la granulomatose septique chronique (CGD). Au sein du complexe ternaire formé par les facteurs cytosoliques, il existe des interactions de type domaine SH3/motif polyproline et une interaction entre domaines PB1. Par Résonance Magnétique Nucléaire, nous avons caractérisé d'un point de vue structural l'interaction entre les domaines PB1 de p67phox et p40phox. Nous avons également étudié les conséquences de l'activation sur les interactions entre le motif polyproline C-terminal de p47phox et ses domaines SH3 partenaires. Ainsi, nous avons combiné l'analyse des structures des domaines SH3 de p40phox et SH3 C-terminal de p67phox, en complexe avec le polyproline C-terminal de p47phox, avec nos mesures d'affinité entre ces partenaires à différents stades de l'activation. Ces données ont été obtenues par fluorescence intrinsèque du tryptophane présent au sein des domaines SH3

Etude des interactions entre les facteurs cytosoliques du complexe de la NADPH Oxydase

During phagocytose of a micro-organism, the NADPH Oxidase complex of neutrophiles is activated. Reactive oxygen species are then produced and contribute to pathogen destruction. This complex comprises cytosolic factors (p67phox, p40phox, p47phox), a small G protein Rac and a flavocytochrome b558 composed of p22phox and gp91phox. Mutations within the genes encoding some of these proteins lead to a rare but serious disease termed chronic granulomatous disease (CGD). The formation of the ternary complex constituted by the cytosolic factors implies several SH3 domain/polyproline interactions and also an interaction PB1 domain interaction. We structurally characterized by Nuclear Magnetic Resonance the interaction between PB1 domains of p67phox and p40phox. We also studied the consequences of activation on the interactions involving p47phox C-terminal polyproline motif and its SH3 domain partners. Thus, we combined the analysis of the structures of p40phox SH3 domain and p67phox C-terminal SH3 domain, in complex with p47phox C-terminal polyproline, with our affinity measures between these partners at various stages of activation. These data were obtained by intrinsic fluorescence of tryptophan present within SH3 domains.Lors de la phagocytose d'un micro-organisme, le complexe de la NADPH Oxydase des neutrophiles est activé. Il permet alors la production d'espèces réactives de l'oxygène qui contribuent à la destruction du pathogène. Ce complexe est constitué de facteurs cytosoliques (p67phox, p40phox, p47phox), d'une petite protéine G Rac et du flavocytochrome b558 membranaire, lui-même composé de p22phox et gp91phox. Des mutations dans les gènes codant pour certaines de ces protéines conduisent à une maladie génétique rare mais grave!: la granulomatose septique chronique (CGD). Au sein du complexe ternaire formé par les facteurs cytosoliques, il existe des interactions de type domaine SH3/motif polyproline et une interaction entre domaines PB1. Par Résonance Magnétique Nucléaire, nous avons caractérisé d'un point de vue structural l'interaction entre les domaines PB1 de p67phox et p40phox. Nous avons également étudié les conséquences de l'activation sur les interactions entre le motif polyproline C-terminal de p47phox et ses domaines SH3 partenaires. Ainsi, nous avons combiné l'analyse des structures des domaines SH3 de p40phox et SH3 C-terminal de p67phox, en complexe avec le polyproline C-terminal de p47phox, avec nos mesures d'affinité entre ces partenaires à différents stades de l'activation. Ces données ont été obtenues par fluorescence intrinsèque du tryptophane présent au sein des domaines SH3

Etude des interactions entre les facteurs cytosoliques du complexe de la NADPH Oxydase

Lors de la phagocytose d'un micro-organisme, le complexe de la NADPH Oxydase des neutrophiles est activé. Il permet alors la production d'espèces réactives de l'oxygène qui contribuent à la destruction du pathogène. Ce complexe est constitué de facteurs cytosoliques (p67phox, p40phox, p47phox), d'une petite protéine G Rac et du flavocytochrome b558 membranaire, lui-même composé de p22phox et gp91phox. Des mutations dans les gènes codant pour certaines de ces protéines conduisent à une maladie génétique rare mais grave: la granulomatose septique chronique (CGD). Au sein du complexe ternaire formé par les facteurs cytosoliques, il existe des interactions de type domaine SH3/motif polyproline, et une interaction entre domaines PB 1. Par Résonance Magnétique Nucléaire, nous avons caractérisé d'un point de vue structural l'interaction entre les domaines PB1 de p67phox et p40phox. Nous avons également étudié les conséquences de l'activation sur les interactions entre le motif polyproline C-terminal de p47phox et ses domaines SH3 partenaires. Ainsi, nous avons combiné l'analyse des structures des domaines SH3 de p40phox et SH3 C-terminal de p67phox, en complexe avec le polyproline C-terminal de p47phox, avec nos mesures d'affinité entre ces partenaires à différents stades de l'activation. Ces données ont été obtenues par fluorescence intrinsèque du tryptophane présent au sein des domaines SH3.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Influenza virus nucleoprotein: structure, RNA binding, oligomerization and antiviral drug target.

International audienceThe nucleoprotein (NP) of influenza virus covers the viral RNA entirely and it is this NP-RNA complex that is the template for transcription and replication by the viral polymerase. Purified NP forms a dynamic equilibrium between monomers and small oligomers, but only the monomers can oligomerize onto RNA. Therefore, drugs that stabilize the monomers or that induce abnormal oligomerization may have an antiviral effect, as would drugs that interfere with RNA binding. Crystal structures have been produced for monomeric and dimeric mutants, and for trimers and tetramers; high-resolution electron microscopy structures have also been calculated for the viral NP-RNA complex. We explain how these structures and the dynamic oligomerization equilibrium of NP can be and have been used for anti-influenza drug development

Physicochemical characterisation of French royal jelly: Comparison with commercial royal jellies and royal jellies produced through artificial bee-feeding.

International audienceA reliable database is established from the analysis of 500 French royal jellies (RJ). Diversity of geographical origins, harvesting seasons, and forage sources were taken into account. Authenticity intervals for numerous physicochemical parameters have been established from French RJs, obtained without bee-feeding with artificial sugars or proteins. RJs from Italy and non-European countries, derived from feeding experiments, were analysed and compared with the database. Differences in 10-HDA, d13C and sugars (sucrose, erlose, maltose, maltotriose) contents were observed. Sucrose and erlose contents in French RJs were less than 1.8 and 0.4%, respectively, whereas they attained 3.9 and 2.0% in some commercial samples and 7.7 and 1.7% in RJs produced by sugarcane feeding. Maltose and maltotriose contents were less than 1.0 and 0.2% in French RJs, and they can attain levels of 2.6 and 0.4% in commercial samples; they can be as high as 5.5 and 1.7% in RJs obtained by bee-feeding with starch hydrolysate syrups. RJs obtained with and without feeding exhibit d13C values ranging from 26.45 to 23.73%, and from 26.05 to 14.65%, respectively. This study could be used to discriminate different production methods and provide a useful tool for identifying unknown commercial RJs. Highlights A total of 750 royal jellies (RJs) from different origins were studied. Origins included domestic, imported, obtained by bee-feeding. French RJ samples were chemically characterised. Authenticity intervals were calculated for authentic RJs. Sucrose, erlose, maltose, maltotriose and δ13C contents are bee-feeding markers

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains.

International audienceThe neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start

Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects

Structure-Based Discovery of the Novel Antiviral Properties of Naproxen against the Nucleoprotein of Influenza A Virus

International audienceThe nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove

Monomeric Nucleoprotein of Influenza A Virus

International audienceIsolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection