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Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculo-skeletal disease in humans. The viral RNA genome replication occurs in membrane spherules named replication organelles (ROs). In this work, we investigate the native structural organization of CHIKV ROs in their cellular context usingin situcryogenic-electron microscopy approaches at late replication stage. We observed previously unreported diameter heterogeneity of ROs at the plasma membrane of infected human cells. CHIKV ROs were only marginally detected in cytopathic vacuoles where they are homogeneous in size, suggesting a finely regulated internalization process. Our data show that ROs maintained at the plasma membrane beyond the first viral cycle are dynamically active both in viral RNA replication, in its export to the cell cytosol, but also in the production of viral proteins. We suggest that late CHIKV ROs have an amplifying role or represent an alternative pathway in the production of infectious viral particles. All these observations bring new insight into the CHIKV life cycle in human cells.

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with β-arrestin1. It reveals an atypical position of β-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/β-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R C-terminus are clearly identified and interact extensively with the β-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a striking structural variability among GPCR-arrestin signaling complexes.

International audience; Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5’-monophosphate (UMP) kinase is a promis- ing target for novel antibiotic discovery as it is essential for bacterial sur- vival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regula- tion with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σB factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σB is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.

The septin collar of budding yeast is an ordered array of septin filaments that serves a scaffolding function for the cytokinetic machinery at the bud neck and compartmentalizes the membrane between mother and daughter cell. How septin architecture is aided by septin-binding proteins is largely unknown. Syp1 is an endocytic protein that was implicated in the timely recruitment of septins to the newly forming collar through an unknown mechanism. Using advanced microscopy and in vitro reconstitution assays, we show that Syp1 is able to align laterally and tightly pack septin filaments, thereby forming flat bundles or sheets. This property is shared by the Syp1 mammalian counterpart FCHo2, thus emphasizing conserved protein functions. Interestingly, the septin-bundling activity of Syp1 resides mainly in its intrinsically disordered region. Our data uncover the mechanism through which Syp1 promotes septin collar assembly and offer another example of functional diversity of unstructured protein domains.

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. Here, we address these unusual features by solving the cryo-EM structure of MtbRho at 3.3 Å resolution, providing a new framework for future antibiotic development. The MtbRho hexamer is poised into a pre-catalytic, open-ringed state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency.

Structures of the AVP-V2R-Gs protein complex provide molecular insights into the function of this clinically relevant target., The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein–coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo–electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

Arginine-vasopressin (AVP) is a neurohypophysial peptide known as the antidiuretic hormone. It forms an active signalling complex with the V2 receptor (V2R) and the Gs protein, promoting a cAMP/PKA-dependent aquaporin insertion in apical membranes of principal cells of the renal collecting ducts and ultimately, water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signalling system are still unknown. To fill this gap of knowledge, we report here the structure of the AVP-V2R-Gs complex using cryo-electron microscopy (cryo-EM). Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and NMR spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes and could thus represent distinct complex conformations along the signalling activation pathway. Importantly, as compared to those of other class A GPCR-Gs complexes, the structures revealed an original receptor-Gs interface in which the Gsα subunit penetrates deeper into the active V2R, notably forming an ionic bond between its free C-terminal carboxylic function and the side chain of R137 in the V2R. Interestingly, the structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases with opposite clinical outcomes, cNDI or NSIAD respectively. Our study thus provides important structural insights into the function of this clinically relevant GPCR signalling complex.

We present for the first time the aqueous emulsion radical polymerization of cardanol derived methacrylic monomer (CAMA) in order to pave the way for the replacement of styrene. This monomer was synthesized in two-step routes by epoxidation of cardanol prior to methacrylation. Polymerization of CAMA was studied both in toluene solution and in aqueous emulsion. Radical aqueous emulsion homo- and co-polymerization of CAMA with methyl methacrylate (MMA) were performed with sodium dodecyl sulfate as surfactant. Stable latexes were obtained with particle size between 25 and 75 nm. CAMA and MMA conversions were monitored versus time. Influence of radical initiator was studied on gel content, showing transfer to monomer in the case of persulfate as initiator. Glass transition temperature of homoPoly(CAMA) was determined and is suitable for coating application. Photo-crosslinking was performed by thiol-ene chemistry and allowed yielding cross-linked biobased aromatic coatings.

201Tl-labeled Prussian blue and Au core@Prussian blue shell nanoparticles were synthesised and investigated in vivo as nanoprobes by using SPECT-CT scintigraphy. We demonstrate that the size, morphology and nanoparticle coating affect the kinetics of the nanoparticle biodistribution.

BGPI : équipe 2 BGPI : équipe 2; International audience; The Broad bean stain virus (BBSV) is a member of the genus Comovirus infecting Fabaceae. The virus is transmitted through seed and by plant weevils causing severe and widespread disease worldwide. BBSV has a bipartite, positive-sense, single-stranded RNA genome encapsidated in icosahedral particles. We present here the cryo-electron microscopy reconstruction of the BBSV and an atomic model of the capsid proteins re fi ned at 3.22 Å resolution. We identi fi ed residues involved in RNA/capsid interactions revealing a unique RNA genome organization. Inspection of the small coat protein C-terminal domain highlights a maturation cleavage between Leu567 and Leu568 and interactions of the C-terminal stretch with neighbouring small coat proteins within the capsid pentameric turrets. These interactions previously proposed to play a key role in the assembly of the Cowpea mosaic virus suggest a common capsid assembly mechanism throughout all comovirus species.

International audience; In regards to their key role in intercellular communication, extracellular vesicles (EVs) have a strong potential as bio-inspired drug delivery systems (DDS). With the aim of circumventing some of their well-known issues (production yield, drug loading yield, pharmacokinetics), we specifically focused on switching the biological vision of these entities to a more physico-chemical one, and to consider and fine-tune EVs as synthetic vectors. To allow a rational use, we first performed a full physico-chemical (size, concentration, surface charge, cryoTEM), biochemical (western blot, proteomics, lipidomics, transcriptomics) and biological (cell internalisation) characterisation of murine mesenchymal stem cell (mMSC)-derived EVs. A stability study based on evaluating the colloidal behaviour of obtained vesicles was performed in order to identify optimal storage conditions. We evidenced the interest of using EVs instead of liposomes, in regards to target cell internalisation efficiency. EVs were shown to be internalised through a caveolae and cholesterol-dependent pathway, following a different endocytic route than liposomes. Then, we characterised the effect of physical methods scarcely investigated with EVs (extrusion through 50 nm membranes, freeze-drying, sonication) on EV size, concentration, structure and cell internalisation properties. Our extensive characterisation of the effect of these physical processes highlights their promise as loading methods to make EVs efficient delivery vehicles.

The facile one-pot, two-step synthesis of a new bio-sourced monomer derived from cardanol and its radical aqueous miniemulsion polymerization are presented in this work. As an abundant and renewable resource that does not compete with the food supply, cardanol is a compound of great interest for the replacement of petroleum-based materials due to its unique structure and properties. The new cardanol methacrylate (CM) was synthesized through hydroxyethylation of the phenolic hydroxyl group followed by methacrylation. The kinetics of the radical homopolymerization of CM and its copolymerization with methyl methacrylate (MMA) were investigated in toluene at 70 °C. The aqueous miniemulsion homo- and copolymerizations of CM and MMA were carried out at 20 wt% solids content, at 70 °C using 2,2′-azobis(2,4-dimethylvaleronitrile) as a radical initiator, sodium dodecyl sulfate as a surfactant, and hexadecane as a hydrophobe. The latexes were colloidally stable with monomodal particle size distributions and mean particle diameters ranging from 100 to 245 nm. The physical and chemical properties of the resulting polymer films were studied by thermogravimetric analyses and rheology.

International audience; The biological fate of nanoparticles (NPs) for biomedical applications is highly dependent of their size and charge, their aggregation state and their surface chemistry. The chemical composition of the NPs surface influences their stability in biological fluids, their interaction with proteins, and their attraction to the cell membranes. In this work, core-shell magnetic mesoporous silica nanoparticles (Fe 3 O 4 @MSN), that are considered as potential theranostic candidates, are coated with polyethylene glycol (PEG) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer. Their biological fate is studied in comparison to the native NPs. The physicochemical properties of these three types of NPs and their suspension behavior in different media are investigated. The attraction to a membrane model is also evaluated using a supported lipid bilayer. The surface composition of NPs strongly influences their dispersion in biological fluids mimics, protein binding and their interaction with cell membrane. While none of these types of NPs is found to be toxic on mice four days after intravenous injection of a dose of 40 mg kg 1 of NPs, their surface coating nature influences the in vivo biodistribution. Importantly, NP coated with DMPC exhibit a strong accumulation in liver and a very low accumulation in lung in comparison with nude or PEG ones.

Antioxidant activities of Myrtus communis leaf phenolic compounds (McPCs) were investigated on 2,2 � -9- azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS + •) and oxygen radical absorbance capacity (ORAC) tests or on oxidation of biological models, human low-density lipoprotein (LDL) and phospholipid aqueous dispersion (l-α- phosphatidylcholine stabilized by bile salts). Two extraction techniques, microwave-assisted (MAE) and conventional (CE), were used to isolate McPCs, producing similar results of phenolic compound content. ABTS + • assay showed clearly that myrtle extracts exhibited a stronger scavenging effect than butylated hydroxyanisole and α-tocopherol, with a slight advantage for myrtle CE extract. In ORAC assay, the both McPC extracts were similarly less effective than the pure compounds as caffeic acid and myricitrin (myricetin 3-O-rhamnoside) but stronger than butylated hydroxytoluene. Moreover, myrtle CE and MAE extracts, and myricitrin were able to inhibit similarly the production of conjugated dienes and to prolong the lag phase (Tlag) during Cu 2+ -induced LDL oxidation with a dose-response effect. The cryo-electron microscopy observations on studied phospholipid dispersion stabilized by bile salts (BS) revealed the presence of bilayer vesicles and micelles. In 2,2 � -azobis (2-amidinopropane) hydrochloride-induced phospholipid/BS oxidation, myrtle CE and MAE extracts gave similar effects to α-tocopherol and caffeic acid but myricitrin showed a higher protective effect than myrtle extracts. We showed also that no synergic or additive effect between α-tocopherol and myrtle extracts or caffeic acid in α-tocopherol-enriched phospholipid/BS dispersion, but myricitrin showed an additive effect and thus promoted the total antioxidant activity. These data showed that myrtle extract could be used as potential natural antioxidants, food stabilizers, or natural health products.

SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.

This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), theConseil Régional d'Ile-de-France (Chemical Library Project, grant nos. I 06- 222/R and I 09-1739/ R, which included a postdoctoral fellowship for I.S.-A.) and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR- 10-INSB-05-01. T.A. was a recipient of a PhD fellowship from the Conseil Régional d'Ile-de-France and the ''D.I.M. maladies infectieuses, parasitaires et nosocomiales émergentes'' 2011; International audience; Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in nucleotide biosynthesis studied as an important therapeutic target and its complex functioning in vivo is still puzzling and debated. Here, we highlight the structural basis for the regulation of IMPDHs by MgATP. Our results demonstrate the essential role of the CBS tandem, conserved among almost all IMPDHs. We found that Pseudomonas aeruginosa IMPDH is an octameric enzyme allosterically regulated by MgATP and showed that this octameric organization is widely conserved in the crystal structures of other IMPDHs. We also demonstrated that human IMPDH1 adopts two types of complementary octamers that can pile up into isolated fibers in the presence of MgATP. The aggregation of such fibers in the autosomal dominant mutant, D226N, could explain the onset of the retinopathy adRP10. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or macromolecular assembly.

International audience; Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.

Annexins are soluble proteins that bind to biological membranes in a Ca(2+)-dependent manner. Annexin-A6 (AnxA6) is unique in the annexin family as it consists of the repeat of two annexin core modules, while all other annexins consist of a single module. AnxA6 has been proposed to participate in various membrane-related processes, including endocytosis and exocytosis, yet the molecular mechanism of association of AnxA6 with biological membranes, especially its ability to aggregate membranes, is still unclear. To address this question, we studied the association of AnxA6 with model phospholipid membranes by combining the techniques of quartz crystal microbalance with dissipation monitoring (QCM-D), (cryo-) transmission electron microscopy (TEM) and atomic force microscopy (AFM). The properties of membrane binding and membrane aggregation of AnxA6 were compared to two reference systems, annexin A5 (AnxA5), which is the annexin prototype, and a chimerical AnxA5-dimer molecule, which is able to aggregate two membranes in a symmetrical manner. We show that AnxA6 presents two modes of association with lipid membranes depending on Ca(2+)-concentration. At low Ca(2+)-concentration ( approximately 60-150microM), AnxA6 binds to membranes via its two coplanar annexin modules and is not able to associate two separate membranes. At high Ca(2+)-concentration ( approximately 2mM), AnxA6 molecules are able to bind two adjacent phospholipid membranes and present a conformation similar to the AnxA6 3D crystallographic structure. Possible biological implications of these novel membrane-binding properties of AnxA6 are discussed.

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri=200) and is complete for Ri=50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of similar to 9 rum between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. (C) 2008 Elsevier Ltd. All rights reserved.

Annexin A5 is a protein that binds to membranes containing negatively charged phospholipids in a calcium-dependent manner. We previously found that annexin A5 self-assembles into two-dimensional (2D) crystals on supported lipid bilayers (SLBs) formed on mica while a monolayer of disordered trimers is formed on SLBs on silica. Here, we investigated in detail and correlated the adsorption kinetics of annexin A5 on SLBs, supported on silica and on mica, with the protein’s 2D self-assembly behavior. For this study, quartz crystal microbalance with dissipation monitoring and ellipsometry were combined with atomic force microscopy. We find, in agreement with previous studies, that the adsorption behavior is strongly dependent on the concentration of dioleoylphosphatidylserine (DOPS) in the SLB and the calcium concentration in solution. The adsorption kinetics of annexin A5 are similar on silica-SLBs and on mica-SLBs, when taking into account the difference in accessible DOPS between silica-SLBs and mica-SLBs. In contrast, 2D crystals of annexin A5 form readily on mica-SLBs, even at low protein coverage (≤10%), whereas they are not found on silica-SLBs, except in a narrow range close to maximal coverage. These results enable us to construct the phase diagram for the membrane binding and the states of 2D organization of annexin A5. The protein binds to the membrane in two different fractions, one reversible and the other irreversible, at a given calcium concentration. The adsorption is determined by the interaction of protein monomers with the membrane. We propose that the local membrane environment, as defined by the presence of DOPS, DOPC, and calcium ions, controls the adsorption and reversibility of protein binding.

All prokaryotic nascent chains must be co-translationally deformylated to allow the further essential removal of the first methionine. This early nascent chain processing is ensured by peptide deformylases (PDFs), an enzyme shown to interact with ribosome at the exit tunnel. Recently, studies demonstrating in one hand the acquisition by different marine phages of modified version of bacterial PDF and in the other hand that also viral proteins undergo deformylation led to propose that phage PDFs could be important for viral fitness. Aside from this intriguing hypothesis, not much is known about these phage PDFs. Here, we show that most viral versions of the PDFs do not contain a C-terminal extension previously proposed to be responsible of direct PDF binding to ribosome. Nonetheless, we show that the smallest identified phage PDF, a fully C-terminally-truncated PDF, is still able to bind directly to ribosome via a new dedicated ribosome-binding domain, playing a master regulator function. Crosslinking expe...

Antioxidant activities of Myrtus communis leaf phenolic compounds (McPCs) were investigated on 2,2'-9-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS(+) •) and oxygen radical absorbance capacity (ORAC) tests or on oxidation of biological models, human low-density lipoprotein (LDL) and phospholipid aqueous dispersion (L-α-phosphatidylcholine stabilized by bile salts). Two extraction techniques, microwave-assisted (MAE) and conventional (CE), were used to isolate McPCs, producing similar results of phenolic compound content. ABTS(+) • assay showed clearly that myrtle extracts exhibited a stronger scavenging effect than butylated hydroxyanisole and α-tocopherol, with a slight advantage for myrtle CE extract. In ORAC assay, the both McPC extracts were similarly less effective than the pure compounds as caffeic acid and myricitrin (myricetin 3-O-rhamnoside) but stronger than butylated hydroxytoluene. Moreover, myrtle CE and MAE extracts, and myricitrin were able to inhibit similarly the production of conjugated dienes and to prolong the lag phase (Tlag) during Cu(2+)-induced LDL oxidation with a dose-response effect. The cryo-electron microscopy observations on studied phospholipid dispersion stabilized by bile salts (BS) revealed the presence of bilayer vesicles and micelles. In 2,2'-azobis (2-amidinopropane) hydrochloride-induced phospholipid/BS oxidation, myrtle CE and MAE extracts gave similar effects to α-tocopherol and caffeic acid but myricitrin showed a higher protective effect than myrtle extracts. We showed also that no synergic or additive effect between α-tocopherol and myrtle extracts or caffeic acid in α-tocopherol-enriched phospholipid/BS dispersion, but myricitrin showed an additive effect and thus promoted the total antioxidant activity. These data showed that myrtle extract could be used as potential natural antioxidants, food stabilizers, or natural health products.We show that microwave-assisted extraction could be an alternative method for plant phenolic compound recovery allowing important gain in time extraction.We report inhibition of low-density lipoprotein oxidation in vitro initiated by Cu(2+) ions. We report that myrtle extract may be a source of natural antioxidants to counteract phospholipid peroxidation as well as α-tocopherol.

International audience; A series of poly(ethylene glycol)-polylactide-poly(ethylene glycol) (PEG-PLA-PEG) triblock copolymers with symmetric or asymmetric chain structures were synthesized by combination of ring-opening polymerization and copper-catalyzed click chemistry. The resulting copolymers were used to prepare self-assembled aggregates by dialysis. Various architectures such as nanotubes, polymersomes and spherical micelles were observed from transmission electron microscopy (TEM), cryo-TEM and atomic force microscopy (AFM) measurements. The formation of diverse aggregates is explained by modeling from the angle of both geometry and thermodynamics. From the angle of geometry, a "blob" model based on the Daoud-Cotton model for star polymers is proposed to describe the aggregate structures and structural changes with copolymer composition and molar mass. In fact, the copolymer chains extend in aqueous medium to form single layer polymersomes to minimize the system's free energy if one of the two PEG blocks is short enough. The curvature of polymersomes is dependent on the chain structure of copolymers, especially on the length of PLA blocks. A constant branch number of aggregates (f) is thus required to preserve the morphology of polymersomes. Meanwhile, the aggregation number (Nagg) determined from the thermodynamics of self-assembly is roughly proportional to the total length of polymer chains. Comparing f to Nagg, the aggregates take the form of polymersomes if Nagg ≈ f, and change to nanotubes if Nagg > f to conform to the limits from both curvature and aggregation number. The length of nanotubes is mainly determined by the difference between Nagg and f. However, the hollow structure becomes unstable when both PEG segments are too long, and the aggregates eventually collapse to yield spherical micelles. Therefore, this work gives new insights into the self-assembly behavior of PEG-PLA-PEG triblock copolymers in aqueous solution which present great interest for biomedical and pharmaceutical applications.

The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21(1-552) (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21(1-552) trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21(1-552) trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21(1-552) are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.

International audience; In this study, we report a rapid sonochemical synthesis of monodisperse nonaggregated Fe3O4@SiO2 magnetic nanoparticles (NPs). We found that coprecipitation of Fe(II) and Fe(III) in aqueous solutions under the effect of power ultrasound yields smaller Fe3O4 NPs with a narrow size distribution (4−8 nm) compared to the silent reaction. Moreover, the coating of Fe3O4 NPs with silica using an alkaline hydrolysis of tetraethyl orthosilicate in ethanol−water mixture is accelerated many-fold in the presence of a 20 kHz ultrasonic field. The thickness of the silica shell can be easily controlled in the range of several nanometers during sonication. Mössbauer spectra revealed that nonsuperparamagnetic behavior of obtained core−shell NPs is mostly related to the dipole−dipole interactions of magnetic cores and not to the particle size effect. Core−shell Fe3O4@SiO2 NPs prepared with sonochemistry exhibit a higher magnetization value than that for NPs obtained under silent conditions owing to better control of the deposited silica quantities as well as to the high speed of sonochemical coating, which prevents the magnetite from oxidizing.

The Annexin-A5 (Anx5) protein is a specific marker of the exposure of phosphatidylserine molecules at the surface of cells, which occurs in processes such as apoptosis and platelet activation. Decoration of self-assembled block copolymer nanostructures by Anx5 is of particular interest in micelle-mediated target drug delivery or in vivo magnetic resonance imaging, the Anx5 imparting (bio)functionality to the system. In this work, the reversible binding of the Anx5 onto polystyrene-b-poly(2-phosphatethyl methacrylate-co-2-hydroxyethyl methacrylate) (PS-b-P(PEMA-co-HEMA)) block copolymer micelles in the presence of Ca2+ ions is described using Quartz crystal microbalance with dissipation monitoring (QCM-D) and polyacrylamide gel electrophoresis (PAGE) analysis. QCM-D experiments confirmed the binding process as well as its reversibility and dependence on the characteristics of macromolecular assemblies, such as the number of phosphonic diacid groups (Pmic) and hydrodynamic diameter (2RH). A linear relationship between the amount of micelles and the amount of protein bound onto the micelle surface until a saturation point was established by QCM-D. The amount of Anx5 bound to PS-b-P(PEMA-co-HEMA) micelles was successfully quantified by PAGE experiments in nondenaturing conditions, which also corroborated that the binding process is mediated by Ca2+ ions. The ability of such surface (bio)-functionalized nanoparticle systems to stabilize and transport hydrophobic loads was highlighted by transmission electron microscopy (TEM) of assemblies with entrapped iron oxide particles.

The formation of protein-decorated nanosized polymeric particles is described. Annexin-A5 protein binding to micellar aggregates having poly((1-ethoxycarbonyl)vinylphosphonic diacid) (PECVPD) and poly(n-butyl acrylate) (PBuA) as corona- and core-forming segments, respectively, was evidenced by Static and Dynamic Light Scattering (SDLS), cryo-Transmission Electron Microscopy (cryo-TEM), and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) experiments. These new objects find potential applications, for example, in micelle-mediated target drug delivery and controlled fabrication of biochips.

International audience; The micellization behavior of a diblock copolymer comprising a highly hydrophilic and biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC corona-forming block and a pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) core-forming block (PMPC-b-PDPA) has been studied by static and dynamic light scattering (SDLS), transmission electron microscopy (TEM), and potentiometry. Self-assembly of PMPC-b-PDPA copolymers with two different DPA volume fractions (Phi DPA) leads to narrowly distributed and structurally distinct spherical micelles, as evidenced by their molecular weight (M-w,M-mic), aggregation number (N,(agg),,), hydrodynamic radius (RH), corona width (M, and core radius (R-c). The excellent potential of these pH-responsive micelles as nanosized drug delivery vehicles was illustrated by the encapsulation of dipyridamole (DIP), a model hydrophobic drug that dissolves in acid solutions and becomes insoluble above pH 5.8, which is comparable to the pK(a),, of the PDPA block. The influence of micelle structure (namely M-w,(mic), N (agg), RH, W, and R,) on drug loading content, drug loading efficiency, partition coefficient, and release kinetics was investigated and confirmed by fluorescence spectroscopy studies. The maximum dipyridamole loadings within PMPC30-b-PDPA(30) (RH = 14.0 nm; W = 4.8 nm; R-c = 9.2 nm) and PMPC30-b-PDPA(60) (RH = 27.1 nm; W = 11.0 nm; R, = 16.1 nm) tr:ticelles were 7 and 12% w/w(p), respectively. This preferential solubilization of DIP into micelles formed by copolymer chains having longer core-forming blocks (i.e., possessing larger core volumes) reflects the larger partition coefficient (K-v) of DIP between the aqueous phase and PMPC(30)b-PDPA(60) inicelles (Kv = 5.7 x 101) compared to PMPC30-b-PDPA(30) micelles (Kv = 1.1 x 10(4)). This enhanced ability Of PMPC30-b-PDPA(60) aggregates to entrap/stabilize small hydrophobic molecules also produces slower release kinetics. Rapid release can be triggered by lowering the pH to induce micellar dissociation.

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg(2+), were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV(I), a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.