A new subunit vaccine based on nucleoprotein nanoparticles confers partial clinical and virological protection in calves against bovine respiratory syncytial virus

Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ringshaped nanoparticles (NSRS) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with NSRS either intramuscularly, or both intramuscularly and intranasally using MontanideTM ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the nonvaccinated control calves. Thus NSRS vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1

Le complexe de la NADPH oxydase : études structurales des facteurs cytosoliques.

The NADPH oxidase complex of neutrophils catalyses the formation of superoxydeions involved in the elimination of pathogenes. It contains two membrane proteins (gp91phox,p22phox), three cytosolic factors (p47phox, p67phox, p40phox), and a small G protein, Rac. In orderto understand interactions and structural modifications, structural studies of full-lengthcytosolic factors alone and complexed have been undertaken. Two approaches have beenselected : electron microscopy and small angle X-ray scattering. The shape of p47phox andp67phox have been calculated using X-ray scattering.Le complexe de la NADPH oydase des neutrophiles catalyse la formation des ionssuperoxydes nécessaires à la destruction des pathogènes. Il est constitué de deux protéinesmembranaires (gp91phox, p22phox), de trois facteurs cytosoliques (p47phox, p67phox, p40phox) etd'une petite protéine G, Rac. Afin de comprendre les interactions et les réarrangementsstructuraux mis en jeu lors de l'activation du complexe, des études structurales des facteurscytosoliques entiers seuls et en complexe ont été menées. Deux approches ont été choisies : lamicroscopie électronique et la diffusion des rayons X aux petits angles. Cette dernièreapproche a permis de calculer une enveloppe pour p47phox et p67phox

Le complexe de la NADPH oxydase : études structurales des facteurs cytosoliques.

The NADPH oxidase complex of neutrophils catalyses the formation of superoxydeions involved in the elimination of pathogenes. It contains two membrane proteins (gp91phox,p22phox), three cytosolic factors (p47phox, p67phox, p40phox), and a small G protein, Rac. In orderto understand interactions and structural modifications, structural studies of full-lengthcytosolic factors alone and complexed have been undertaken. Two approaches have beenselected : electron microscopy and small angle X-ray scattering. The shape of p47phox andp67phox have been calculated using X-ray scattering.Le complexe de la NADPH oydase des neutrophiles catalyse la formation des ionssuperoxydes nécessaires à la destruction des pathogènes. Il est constitué de deux protéinesmembranaires (gp91phox, p22phox), de trois facteurs cytosoliques (p47phox, p67phox, p40phox) etd'une petite protéine G, Rac. Afin de comprendre les interactions et les réarrangementsstructuraux mis en jeu lors de l'activation du complexe, des études structurales des facteurscytosoliques entiers seuls et en complexe ont été menées. Deux approches ont été choisies : lamicroscopie électronique et la diffusion des rayons X aux petits angles. Cette dernièreapproche a permis de calculer une enveloppe pour p47phox et p67phox

Le complexe de la NADPH oxydase (études structurales des facteurs cytosoliques)

Le complexe de la NADPH oydase des neutrophiles catalyse la formation des ions superoxydes nécessaires à la destruction des pathogènes. Il est constitué de deux protéines membranaires (gp91phox, p22phox), de trois facteurs cytosoliques (p47phox. p67phox, p40phox) et d'une petite protéine G. Rac. Afin de comprendre les interactions et les réarrangements structuraux mis en jeu lors de l'activation du complexe, des études structurales des facteurs cytosoliques entiers seuls et en complexe ont été menées. Deux approches ont été choisies: la microscopie électronique et la diffusion des rayons X aux petits angles. Cette dernière approche a permis de calculer une enveloppe pour p47phox et p67phox.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-d-arabinonhydrazide

International audienceType I phosphomannose isomerases (PMIs) are zinc-dependent monofunctional metalloenzymes catalysing the reversible isomerization of d-mannose 6-phosphate to d-fructose 6-phosphate. 5-Phospho-d-arabinonhydrazide (5PAHz), designed as an analogue of the enediolate high-energy intermediate, strongly inhibits PMI from Candida albicans (CaPMI). In this study, we report the 3D crystal structure of CaPMI complexed with 5PAHz at 1.85 Å resolution. The high-resolution structure suggests that Glu294 is the catalytic base that transfers a proton between the C1 and C2 carbon atoms of the substrate. Bidentate coordination of the inhibitor explains the stereochemistry of the isomerase activity, as well as the absence of both anomerase and C2-epimerase activities for Type I PMIs. A detailed mechanism of the reversible isomerization is proposed

CuII ‐Containing 1‐Aminocyclopropane Carboxylic Acid Oxidase Is an Efficient Stereospecific Diels–Alderase

International audienc

Receptor-Based Artificial Metalloenzymes on Living Human Cells

Artificial metalloenzymes are known to be promising tools for biocatalysis, but their recent compartmentalization has led to compatibly with cell components thus shedding light on possible therapeutic applications. We prepared and characterized artificial metalloenzymes based on the A_2A adenosine receptor embedded in the cytoplasmic membranes of living human cells. The wild type receptor was chemically engineered into metalloenzymes by its association with strong antagonists that were covalently bound to copper­(II) catalysts. The resulting cells enantioselectively catalyzed the abiotic Diels–Alder cycloaddition reaction of cyclopentadiene and azachalcone. The prospects of this strategy lie in the organ-confined in vivo preparation of receptor-based artificial metalloenzymes for the catalysis of reactions exogenous to the human metabolism. These could be used for the targeted synthesis of either drugs or deficient metabolites and for the activation of prodrugs, leading to therapeutic tools with unforeseen applications

Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL Authors

International audienceLoss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the two core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet, the relationship between NOTCH3 receptor activity, Notch3ECD accumulation and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the central nervous system of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knock-in Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD co-aggregated with mutant Notch3ECD and that elimination of one copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies

Receptor-Based Artificial Metalloenzymes on Living Human Cells

Artificial metalloenzymes are known to be promising tools for biocatalysis, but their recent compartmentalization has led to compatibly with cell components thus shedding light on possible therapeutic applications. We prepared and characterized artificial metalloenzymes based on the A_2A adenosine receptor embedded in the cytoplasmic membranes of living human cells. The wild type receptor was chemically engineered into metalloenzymes by its association with strong antagonists that were covalently bound to copper­(II) catalysts. The resulting cells enantioselectively catalyzed the abiotic Diels–Alder cycloaddition reaction of cyclopentadiene and azachalcone. The prospects of this strategy lie in the organ-confined in vivo preparation of receptor-based artificial metalloenzymes for the catalysis of reactions exogenous to the human metabolism. These could be used for the targeted synthesis of either drugs or deficient metabolites and for the activation of prodrugs, leading to therapeutic tools with unforeseen applications