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

Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 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 CNS 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 knockin 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 coaggregated with mutant Notch3ECD and that elimination of 1 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.

CuII -Containing 1-Aminocyclopropane Carboxylic Acid Oxidase Is an Efficient Stereospecific Diels-Alderase.

In the context of developing ecofriendly chemistry, artificial enzymes are now considered as promising tools for synthesis. They are prepared in particular with the aim to catalyze reactions that are rarely, if ever, catalyzed by natural enzymes. We discovered that 1-aminocyclopropane carboxylic acid oxidase reconstituted with CuII served as an efficient artificial Diels-Alderase. The kinetic parameters of the catalysis of the cycloaddition of cyclopentadiene and 2-azachalcone were determined (KM =230 µm, kapp =3 h-1 ), which gave access to reaction conditions that provided quantitative yield and >99 % ee of the (1S,2R,3R,4R) product isomer. This unprecedented performance was rationalized by molecular modeling as only one docking pose of 2-azachalcone was possible in the active site of the enzyme and this was the one that leads to the (1S,2R,3R,4R) product isomer.

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 A2A 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.

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

Type 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.

Lack of interaction between NEMO and SHARPIN impairs linear ubiquitination and NF-κB activation and leads to incontinentia pigmenti.

BACKGROUND: Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO. OBJECTIVES: We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway. METHODS: We sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-ß, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1ß. RESULTS: We identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1ß-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1-/- cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination. CONCLUSION: We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.

Use of fluorescence spectroscopy for quantitative investigations of ubiquitin interactions with the ubiquitin-binding domains of NEMO.

Ubiquitin serves as a signal for a variety of cellular processes and its specific interaction with ubiquitin-binding domain (UBD) regulates key cellular events including protein degradation, cell-cycle control, DNA repair, and kinase activation. Several binding mechanisms for isolated UBDs have been reported in recent years. However, little is known about the mechanism through which proteins containing multiple-UBDs achieve specificity for a particular oligomer of polyUb. The NF-κB essential modulator (NEMO, also known IKKγ), which plays a key role in the NF-κB signaling pathway, belongs to the latter family of proteins since it contains two distal NOA (also known UBAN/CC2-LZ/NUB) and ZF UBDs, separated by an unstructured proline-rich linker of about 40 residues in length. Here, we show a new procedure for fast purification of this bipartite domain. We also describe the use of intrinsic fluorescence spectroscopy for quantitative investigations of ubiquitin interactions between two distal ubiquitin-binding domains of NEMO (NOA and ZF). This spectroscopic method has many advantages over other techniques like GST pulldown and Biacore's SPR for monitoring avid interactions between two UBDs, especially when UBDs are located at significant distance from each other within the protein.

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 M2-1(58-177) 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 M2-1(58-177), 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.

1H, 13C, and 15N resonance assignment of the central domain of hRSV transcription antitermination factor M2-1.

M2-1 is an essential co-factor of the respiratory syncytial virus, an important respiratory pathogen in infants and calves. It acts as a transcription antitermination factor which enhances the processivity of the polymerase. Within the polymerase complex, M2-1 interacts with a second co-factor, the phosphoprotein P. It has been shown previously that P and RNA bind to M2-1 in a competitive manner in vitro and that these properties are related to a central domain located between residues Glu59 and Lys177. Here we report the almost complete (1)H, (13)C and (15)N assignment of a fragment of M2-1 corresponding to this region, for further structure determination and interaction studies.

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 ring-shaped nanoparticles (N(SRS)) 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 N(SRS) either intramuscularly, or both intramuscularly and intranasally using Montanide 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 non-vaccinated control calves. Thus N(SRS) vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction.

The respiratory syncytial virus M2-1 protein forms tetramers and interacts with RNA and P in a competitive manner.

The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and approximately 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.

Small-angle X-ray scattering reveals an extended organization for the autoinhibitory resting state of the p47(phox) modular protein.

In response to microbial infection, neutrophiles promote the assembly of the NADPH oxidase complex in order to produce superoxide anions. This reaction is activated by the association of cytosolic factors, p47(phox), p67(phox), p40(phox), and a small G protein Rac with the membranous heterodimeric flavocytochrome b(558), composed of gp91(phox) and p22(phox). In the activation process, p47(phox) plays a central role as the target of phosphorylations and as a scaffolding protein conducting the translocation and assembly of cytosolic factors onto the membranous components. The PX and tandem SH3s of p47(phox) have been highlighted as being key determinants for the interaction with membrane lipids and the p22(phox) component, respectively. In the resting state, the two corresponding interfaces are thought to be masked allowing its cytoplasmic localization. However, the resting state modular organization of p47(phox) and its autoinhibition mode are still not fully understood despite available structural information on separate modules. More precisely, it raises the question of the mutual arrangement of the PX domain and the tandem SH3 domains in the resting state. To address this question, we have engaged a study of the entire p47(phox) molecule in solution using small-angle X-ray scattering. Despite internal autoinhibitory interactions, p47(phox) adopts an extended conformation. First insights about the domain arrangement in whole p47(phox) can be derived. Our data allow to discard the usual representation of a globular and compact autoinhibited resting state.

Structural studies of the neural-cell-adhesion molecule by X-ray and neutron reflectivity.

The structures of adhesion proteins play an important role in the formation of intercellular junctions and the control of intermembrane spacing. This paper describes the combination of neutron and X-ray specular reflectivity measurements to investigate the structure of the ectodomain of the neural-cell-adhesion molecule (NCAM). The measurements with unmodified NCAM suggest the presence of a bend in the extracellular region. Measurements with the polysialic-acid-modified form of NCAM reveal that, at physiological ionic strength, the carbohydrate chains extend beyond the range of the unmodified protein. The excluded volume of the polymer is also ionic-strength-dependent, as expected for a polyelectrolyte. The structural characteristics obtained from these independent analyses of X-ray and neutron reflectivity data agree with each other, with prior reflectivity studies, and with molecular dimensions obtained from direct-force measurements. These results provide structural insights into the configuration of the NCAM ectodomain and the regulation of NCAM adhesion by post-translational modification.