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GENESTE, Emmanuel; GRELARD, Axelle; DUFOURC, Erick;
Photodegradation of novel oral anticoagulants under sunlight irradiation in aqueous matrices
Chemosphere, 2018, 193, 329-336
Kinetics of photodegradation of novel oral anticoagulants dabigatran, rivaroxaban, and apixaban were studied under simulated solar light irradiation in purified, mineral, and river waters. Dabigatran and rivaroxaban underwent direct photolysis with polychromatic quantum yields of 2.2 x 10(-4) and 4.4 x 10(-2), respectively. The direct photodegradation of apixaban was not observed after 19 h of irradiation. Kinetics of degradation of rivaroxaban was not impacted by the nature of the aqueous matrix while photosensitization from nitrate ions was observed for dabigatran and apixaban dissolved in a mineral water. The photosensitized reactions were limited in the tested river water (Isle River, Perigueux, France) certainly due to the hydroxyl radical scavenging effect of the dissolved organic matter. The study of photoproduct structures allowed to identify two compounds for dabigatran. One of them is the 4-aminobenzamidine while the second one is a cyclization product. In the case of rivaroxaban, as studied by very high field NMR, only one photoproduct was observed i.e. a photoisomer. Finally, seven photoproducts were clearly identified from the degradation of apixaban under simulated solar light. (C) 2017 Elsevier Ltd. All rights reserved.
2 […]
BERBON, Mélanie; HABENSTEIN, Birgit; LOQUET, Antoine;
Detection of side-chain proton resonances of fully protonated biosolids in nano-litre volumes by magic angle spinning solid-state NMR
Journal of biomolecular NMR, 2018, 70, 177-185
We present a new solid-state NMR proton-detected three-dimensional experiment dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C-13C transfer to establish efficient (H)CCH correlations detected on side chain protons. Our approach is demonstrated on the HET-s prion domain in its functional amyloid fibrillar form, fully protonated, with a sample amount of less than 500 g using a MAS frequency of 70kHz. The majority of aliphatic and aromatic side chain protons (70%) are observable, in addition to Halpha resonances, in a single experiment providing a complementary approach to the established proton-detected amide-based multidimensional solid-state NMR experiments for the study and resonance assignment of biosolid samples, in particular for aromatic side chain resonances.
3 […]
HABENSTEIN, Birgit; LOQUET, Antoine;
Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene
Nature Communications, 2018, 9,
Here we present a new approach to diffraction imaging of amyloid fibrils, combining a freestanding graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 A and 2.4 A resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.
4 […]
SAAD, Ahmad; LIET, Benjamin; JOUCLA, Gilles; SANTARELLI, Xavier; TREZEGUET, Véronique;
Role of Glycanation and Convertase Maturation of Soluble Glypican-3 in Inhibiting Proliferation of Hepatocellular Carcinoma Cells
Biochemistry, 2018, 57, 1201-1211
Glypican 3 (GPC3) is a complex heparan sulfate proteoglycan associated with the outer surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. It is also N-glycosylated and processed by a furin-like convertase. GPC3 has numerous biological functions. Although GPC3 is undetectable in normal liver tissue, it is abnormally and highly overexpressed in hepatocellular carcinoma (HCC). Interestingly, proliferation of HCC cells such as HepG2 and HuH7 is inhibited when they express a soluble form of GPC3 after lentiviral transduction. To obtain more insight into the role of some of its post-translational modifications, we designed a mutant GPC3, sGPC3m, without its GPI anchor, convertase cleavage site, and glycosaminoglycan chains. The highly pure sGPC3m protein strongly inhibited HuH7 and HepG2 cell proliferation in vitro and induced a significant increase in their cell doubling time. It changed the morphology of HuH7 cells but not that of HepG2. It induced the enlargement of HuH7 cell nuclear area and the restructuration of adherent cell junctions. Unexpectedly, for both cell types, the levels of apoptosis, cell division, and beta-catenin were not altered by sGPC3m, although growth inhibition was very efficient. Overall, our data show that glycanation and convertase maturation are not required for sGPC3m to inhibit HCC cell proliferation.
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GEAN, Julie; DUFOURC, Erick;
The potent effect of mycolactone on lipid membranes
Plos Pathogens, 2018, 14,
Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 mu M, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.
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MARTINEZ, Denis; HUGUES, Michel; ODAERT, Benoit;
Assignment of H-1, C-13 and N-15 resonances and secondary structure of the Rgd1-RhoGAP domain
Biomolecular Nmr Assignments, 2018, 12, 129-132
The protein Rgd1 is involved in the regulation of cytoskeleton formation and in signalling pathways that control cell polarity and growth in Saccharomyces cerevisiae. Rgd1p is composed of a F-BAR domain required for membrane binding and a RhoGAP domain responsible for activating Rho3p and Rho4p, two GTPases respectively involved in bud growth and cytokinesis. Rgd1p is recruited to the membrane through interactions with phosphoinositide lipids, which bind the two isolated domains and stimulate the RhoGAP activity on Rho4p. As previously shown by crystallography, the membrane-binding F-BAR domain contains a conserved inositol phosphate binding site, which explains the preferential binding of phosphoinositides. In contrast, RhoGAP domains are not expected to bind lipids. In order to unravel this puzzling feature, we solved the three-dimensional structure of the isolated protein and found a cryptic phosphoinositide binding site involving non conserved residues (Martinez et al. 2017). The assignment of the resonances and secondary structure of Rgd1-RhoGAP (aa 450-666) is presented here.
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MARTINEZ, Denis; LEGRAND, Anthony; LAMBERT, Olivier; BERBON, Mélanie; GRELARD, Axelle; LOQUET, Antoine; HABENSTEIN, Birgit;
Coiled-coil oligomerization controls localization of the plasma membrane REMORINs
Journal of structural biology, 2018, 0,
REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo.
LOQUET, Antoine;
Functional Amyloids in Health and Disease
Journal of molecular biology, 2018, 430, 3629-3630
9 […]
LOQUET, Antoine; BERBON, Mélanie; HABENSTEIN, Birgit;
3D structure determination of amyloid fibrils using solid-state NMR spectroscopy
Methods (San Diego, Calif.), 2018, 138, 26-38
The amyloid fold is structurally characterized by a typical cross-beta architecture, which is under debate to represent an energy-favourable folding state that many globular or natively unfolded proteins can adopt. Being initially solely associated with amyloid fibrils observed in the propagation of several neurodegenerative disorders, the discovery of non-pathological (or "functional") amyloids in many native biological processes has recently further intensified the general interest invested in those cross-beta supramolecular assemblies. The insoluble and non-crystalline nature of amyloid fibrils and their usually inhomogeneous appearance on the mesoscopic level pose a challenge to biophysical techniques aiming at an atomic-level structural characterization. Solid-state NMR spectroscopy (SSNMR) has granted breakthroughs in structural investigations on amyloid fibrils ranging from the assessment of the impact of polymorphism in disease development to the 3D atomic structure determination of amyloid fibrils. First landmark studies towards the characterization of atomic structures and interactions involving functional amyloids have provided new impulses in the understanding of the role of the amyloid fold in native biological functions. Over the last decade many strategies have been developed in protein isotope labelling, NMR resonance assignment, distance restraint determination and 3D structure calculation of amyloid fibrils based on SSNMR approaches. We will here discuss the emerging concepts and state-of-the-art methods related to the assessment of amyloid structures and interactions involving amyloid entities by SSNMR.
10 […]
BAUDIN, Antoine; COLLIE, Gavin; CHAIGNEPAIN, Stéphane; HOCQUELLET, Agnès; BERBON, Mélanie; LOQUET, Antoine; GUICHARD, Gilles; ODAERT, Benoit;
1H, 13C, 15N NMR resonance assignments and secondary structure determination of the extra-cellular domain from the human proapoptotic TRAIL-R2 death receptor 5 (DR5-ECD)
Biomolecular NMR assignments, 2018, 12, 309-314
Death receptors (DR) selectively drive cancer cells to apoptosis upon binding to the Tumor necrosis factor-a-Related Apoptosis-Inducing Ligand (TRAIL). Complex formation induces the oligomerization of the death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and transduces the apoptogenic signal to their respective death domains, leading to Death Inducing Signaling Complex (DISC) formation, caspase activation and ultimately cell death. Several crystal structures of the ExtraCellular Domain from Death Receptor 5 (DR5-ECD) have been reported in complex with the TRAIL ligand or anti-DR5 antibodies, but none for the isolated protein. In order to fill this gap and to perform binding experiments with TRAIL peptidomimetics, we have produced isotopically labelled DR5-ECD and started a conformational analysis by using high-field 3D NMR spectroscopy. Herein, we present the first resonance assignment of a TRAIL receptor in solution and the determination of its secondary structure from NMR chemical shifts.
MARTINEZ, Denis; LEGRAND, Anthony; BERBON, Mélanie; GRELARD, Axelle; LAMBERT, Olivier; LOQUET, Antoine; HABENSTEIN, Birgit;
Insights into the structural mechanisms controlling the dynamic nanodomain organization of REMORINs
Febs Open Bio, 2018, 8, 440-440
DUTOUR, Antoine; BERBON, Mélanie; KAUFFMANN, Brice; HABENSTEIN, Birgit; LOQUET, Antoine;
Structural and polymorphic variability of Bacillus subtilis and Bacillus cereus TasA amyloid-like biofilm filaments
Febs Open Bio, 2018, 8, 98-98
Antoine Loquet Group Accueil Colloids for Health & Nutrition Mentions légales Group History Members Publications Highlights Lipids in all their states Membrane Proteins & Killer Peptides NMR of Soft Matter Contact NMR of Molecular Assemblies Lectures
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informations Mentions légales Dernière mise à jour : 06/01/2016

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