Publications by authors named "Antoine Loquet"

70 Publications

New Insights into Wine Taste: Impact of Dietary Lipids on Sensory Perceptions of Grape Tannins.

J Agric Food Chem 2021 Mar 3. Epub 2021 Mar 3.

Univ. Bordeaux, CNRS, CBMN UMR 5348, IECB, F-33600 Pessac, France.

Wine is very often consumed with a meal. However, although it is well known to tasters that the taste of wine changes in the presence of food, the influence of dietary lipids on wine astringency and bitterness caused by grape tannins is not well established from a molecular point of view. In this context, we investigated wine tannin-lipid interactions by combining biophysical techniques to sensory analysis. Nuclear magnetic resonance and optical and electron microscopy showed an interaction between catechin, a majority component of grape tannins, and lipid droplets from a phospholipid-stabilized oil-in-water emulsion, characterized by (a) an increase in the droplet size in the presence of catechin, (b) slowing of their size growth over time, and (c) an increase in lipid dynamics in the droplet interfacial layer. Those results were strengthened by sensory analysis, which demonstrated that dietary oils decrease the perception of astringency of grape tannin solutions. Our results highlight that dietary lipids are crucial molecular agents impacting our sensory perception during wine consumption.
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http://dx.doi.org/10.1021/acs.jafc.0c06589DOI Listing
March 2021

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Chem Rev 2021 Feb 5;121(4):2545-2647. Epub 2021 Feb 5.

CNRS, UPR9080, Université de Paris, Laboratory of Theoretical Biochemistry, IBPC, Fondation Edmond de Rothschild, PSL Research University, Paris 75005, France.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, , , and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.
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http://dx.doi.org/10.1021/acs.chemrev.0c01122DOI Listing
February 2021

Structural and molecular basis of cross-seeding barriers in amyloids.

Proc Natl Acad Sci U S A 2021 Jan;118(1)

CNRS, Chemistry and Biology of Membranes and Nanoobjects (CBMN) UMR 5348, Institut Europeen de Chimie et Biologie (IECB), University of Bordeaux, F-33600 Pessac, France;

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents.
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http://dx.doi.org/10.1073/pnas.2014085118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817211PMC
January 2021

Identification of NLR-associated Amyloid Signaling Motifs in Bacterial Genomes.

J Mol Biol 2020 11 13;432(23):6005-6027. Epub 2020 Oct 13.

Non-self Recognition in Fungi, Institut de Biochimie et de Génétique Cellulaire, UMR 5095 CNRS, Université de Bordeaux, 1 Rue Camille Saint Saëns, 33077 Bordeaux CEDEX, France. Electronic address:

In filamentous fungi, amyloid signaling sequences allow Nod-like receptors (NLRs) to activate downstream cell-death inducing proteins with HeLo and HeLo-like (HELL) domains and amyloid RHIM and RHIM-related motifs control immune defense pathways in mammals and flies. Herein, we show bioinformatically that analogous amyloid signaling motifs exist in bacteria. These short motifs are found at the N terminus of NLRs and at the C terminus of proteins with a domain we term BELL. The corresponding NLR and BELL proteins are encoded by adjacent genes. We identify 10 families of such bacterial amyloid signaling sequences (BASS), one of which (BASS3) is homologous to RHIM and a fungal amyloid motif termed PP. BASS motifs occur nearly exclusively in bacteria forming multicellular structures (mainly in Actinobacteria and Cyanobacteria). We analyze experimentally a subset of seven of these motifs (from the most common BASS1 family and the RHIM-related BASS3 family) and find that these sequences form fibrils in vitro. Using a fungal in vivo model, we show that all tested BASS-motifs form prions and that the NLR-side motifs seed prion-formation of the corresponding BELL-side motif. We find that BASS3 motifs show partial prion cross-seeding with mammalian RHIM and fungal PP-motifs and that proline mutations on key positions of the BASS3 core motif, conserved in RHIM and PP-motifs, abolish prion formation. This work expands the paradigm of prion amyloid signaling to multicellular prokaryotes and suggests a long-term evolutionary conservation of these motifs from bacteria, to fungi and animals.
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http://dx.doi.org/10.1016/j.jmb.2020.10.004DOI Listing
November 2020

Novel self-replicating α-synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons.

Sci Adv 2020 Oct 2;6(40). Epub 2020 Oct 2.

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France.

The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ). The generation of τ fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.
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http://dx.doi.org/10.1126/sciadv.abc4364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852382PMC
October 2020

Tandem NMR and Mass Spectrometry Analysis of Human Nuclear Membrane Lipids.

Anal Chem 2020 05 6;92(10):6858-6868. Epub 2020 May 6.

Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR 5248, CNRS, Université Bordeaux, Institut National Polytechnique Bordeaux, Pessac F-33600, France.

The human nuclear membrane is composed of a double bilayer, the inner membrane being linked to the protein lamina network and the outer nuclear membrane continuous with the endoplasmic reticulum. Nuclear membranes can form large invaginations inside the nucleus; their specific roles still remain unknown. Although much of the protein identification has been determined, their lipid composition remains largely undetermined. In order to understand the mechanical and dynamic properties of nuclear membranes we investigated their lipid composition by two quantitative methods, namely, P and H multidimensional NMR and mass spectrometry, using internal standards. We also developed a nondetergent nuclei extraction protocol allowing to produce milligram quantities of nuclear membrane lipids. We found that the nuclear membrane lipid extract is composed of a complex mixture of phospholipids with different phosphatidylcholine species present in large amounts. Negatively charged lipids, with elevated amounts of phosphatidylinositol (PI), were also present. Mass spectrometry confirmed the phospholipid composition and provided further information on acyl-chain length and unsaturation. Lipid chain lengths ranged between 30 and 38 carbon atoms (two chains summed up) with a high proportion of 34 carbon atom length for most species. PI lipids have high amounts of chain lengths with 36-38 carbons. Independent of the chain length unsaturations were highly elevated with one to two double bonds per lipid species.
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http://dx.doi.org/10.1021/acs.analchem.9b05052DOI Listing
May 2020

The unprecedented membrane deformation of the human nuclear envelope, in a magnetic field, indicates formation of nuclear membrane invaginations.

Sci Rep 2020 03 20;10(1):5147. Epub 2020 Mar 20.

Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR5248, CNRS, Université Bordeaux, INP-Bordeaux, F-33600, Pessac, France.

Human nuclear membrane (hNM) invaginations are thought to be crucial in fusion, fission and remodeling of cells and present in many human diseases. There is however little knowledge, if any, about their lipid composition and dynamics. We therefore isolated nuclear envelope lipids from human kidney cells, analyzed their composition and determined the membrane dynamics after resuspension in buffer. The hNM lipid extract was composed of a complex mixture of phospholipids, with high amounts of phosphatidylcholines, phosphatidylinositols (PI) and cholesterol. hNM dynamics was determined by solid-state NMR and revealed that the lamellar gel-to-fluid phase transition occurs below 0 °C, reflecting the presence of elevated amounts of unsaturated fatty acid chains. Fluidity was higher than the plasma membrane, illustrating the dual action of Cholesterol (ordering) and PI lipids (disordering). The most striking result was the large magnetic field-induced membrane deformation allowing to determine the membrane bending elasticity, a property related to hydrodynamics of cells and organelles. Human Nuclear Lipid Membranes were at least two orders of magnitude more elastic than the classical plasma membrane suggesting a physical explanation for the formation of nuclear membrane invaginations.
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http://dx.doi.org/10.1038/s41598-020-61746-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083927PMC
March 2020

Structures of Type III Secretion System Needle Filaments.

Curr Top Microbiol Immunol 2020 ;427:109-131

University of Bordeaux, CNRS, UMR 5248, European Institute of Chemistry and Biology, 2 rue Robert Escarpit, Pessac, 33607, France.

Among the Gram-negative bacterial secretion systems, type III secretion systems (T3SS) possess a unique extracellular molecular apparatus called the needle. This macromolecular protein assembly is a nanometre-size filament formed by the helical arrangement of hundreds of copies of a single, small protein, which is highly conserved between T3SSs from animal to plant bacterial pathogens. The needle filament forms a hollow tube with a channel ~20 Å in diameter that serves as a conduit for proteins secreted into the targeted host cell. In the past ten years, technical breakthroughs in biophysical techniques such as cryo-electron microscopy (cryo-EM) and solid-state NMR (SSNMR) spectroscopy have uncovered atomic resolution details about the T3SS needle assembly. Several high-resolution structures of Salmonella typhimurium and Shigella flexneri T3SS needles have been reported demonstrating a common structural fold. These structural models have been used to explain the active role of the needle in transmitting the host-cell contact signal from the tip to the base of the T3SS through conformational changes as well as during the injection of effector proteins. In this chapter, we summarize the current knowledge about the structure and the role of the T3SS needle during T3SS assembly and effector secretion.
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http://dx.doi.org/10.1007/82_2019_192DOI Listing
September 2020

Sensitivity boosts by the CPMAS CryoProbe for challenging biological assemblies.

J Magn Reson 2020 02 23;311:106680. Epub 2019 Dec 23.

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, United States; Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States. Electronic address:

Despite breakthroughs in MAS NMR hardware and experimental methodologies, sensitivity remains a major challenge for large and complex biological systems. Here, we report that 3-4 fold higher sensitivities can be obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe™). Such intensity enhancements permit recording 2D and 3D experiments that are otherwise time-prohibitive, such as 2D N-N proton-driven spin diffusion and N-C double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins - demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of applications, especially for biological systems suffering from low intrinsic sensitivity and at physiological temperatures.
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http://dx.doi.org/10.1016/j.jmr.2019.106680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060763PMC
February 2020

MRI assessment of multiple dipolar relaxation time (T) components in biological tissues interpreted with a generalized inhomogeneous magnetization transfer (ihMT) model.

J Magn Reson 2020 02 14;311:106668. Epub 2019 Dec 14.

Aix Marseille Univ, CNRS, CRMBM UMR 7339, Marseille, France. Electronic address:

T, the relaxation time of dipolar order, is sensitive to slow motional processes. Thus T is a probe for membrane dynamics and organization that could be used to characterize myelin, the lipid-rich membrane of axonal fibers. A mono-component T model associated with a modified ihMT sequence was previously proposed for in vivo evaluation of T with MRI. However, experiments have suggested that myelinated tissues exhibit multiple T components probably due to a heterogeneous molecular mobility. A bi-component T model is proposed and implemented. ihMT images of ex-vivo, fixed rat spinal cord were acquired with multiple frequency alternation rate. Fits to data yielded two Ts of about 500 μs and 10 ms. The proposed model seems to further explore the complexity of myelin organization compared to the previously reported mono-component T model.
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http://dx.doi.org/10.1016/j.jmr.2019.106668DOI Listing
February 2020

Structural dissection of amyloid aggregates of TDP-43 and its C-terminal fragments TDP-35 and TDP-16.

FEBS J 2020 Jun 20;287(12):2449-2467. Epub 2019 Dec 20.

CBMN (UMR5248), Université de Bordeaux - CNRS - IPB, Institut Européen de Chimie et Biologie, Pessac, France.

The TAR DNA-binding protein (TDP-43) self-assembles into prion-like aggregates considered to be the structural hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we use a combination of electron microscopy, X-ray fiber diffraction, Fourier-transform infrared spectroscopy analysis, and solid-state NMR spectroscopy to investigate the molecular organization of different TDP constructs, namely the full-length TDP-43 (1-414), two C-terminal fragments [TDP-35 (90-414) and TDP-16 (267-414)], and a C-terminal truncated fragment (TDP-43 ∆GaroS2), in their fibrillar state. Although the different protein constructs exhibit similar fibril morphology and a typical cross-β signature by X-ray diffraction, solid-state NMR indicates that TDP-43 and TDP-35 share the same polymorphic molecular structure, while TDP-16 encompasses a well-ordered amyloid core. We identified several residues in the so-called C-terminal GaroS2 (368-414) domain that participates in the rigid core of TDP-16 fibrils, underlining its importance during the aggregation process. Our findings demonstrate that C-terminal fragments can adopt a different molecular conformation in isolation or in the context of the full-length assembly, suggesting that the N-terminal domain and RRM domains play an important role in the TDP-43 amyloid transition.
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http://dx.doi.org/10.1111/febs.15159DOI Listing
June 2020

Nanodomain Clustering of the Plant Protein Remorin by Solid-State NMR.

Front Mol Biosci 2019 15;6:107. Epub 2019 Oct 15.

Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), IECB, CNRS, Université Bordeaux, Institut Polytechnique Bordeaux, Pessac, France.

Nanodomains are dynamic membrane subcompartments, enriched in specific lipid, and protein components that act as functional platforms to manage an abundance of cellular processes. The remorin protein of plants is a well-established nanodomain marker and widely serves as a paradigm to study nanodomain clustering. Located at the inner leaflet of the plasma membrane, remorins perform essential functions during signaling. Using deuterium and phosphorus solid-state NMR, we inquire on the molecular determinants of the lipid-protein and protein-protein interactions driving nanodomain clustering. By monitoring thermotropism properties, lipid acyl chain order and membrane thickness, we report the effects of phosphoinositides and sterols on the interaction of various remorin peptides and protein constructs with the membrane. We probed several critical residues involved in this interaction and the involvement of the coiled-coil homo-oligomerisation domain into the formation of remorin nanodomains. We trace the essential role of the pH in nanodomain clustering based on anionic lipids such as phosphoinositides. Our results reveal a complex interplay between specific remorin residues and domains, the environmental pH and their resulting effects on the lipid dynamics for phosphoinositide-enriched membranes.
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http://dx.doi.org/10.3389/fmolb.2019.00107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803476PMC
October 2019

Mechanisms governing subcompartmentalization of biological membranes.

Curr Opin Plant Biol 2019 12 20;52:114-123. Epub 2019 Sep 20.

Univ. Bordeaux, CNRS, Laboratoire de Biogenèse Membranaire (LBM), UMR 5200, 33140 Villenave d'Ornon, France. Electronic address:

Membranes show a tremendous variety of lipids and proteins operating biochemistry, transport and signalling. The dynamics and the organization of membrane constituents are regulated in space and time to execute precise functions. Our understanding of the molecular mechanisms that shape and govern membrane subcompartmentalization and inter-organelle contact sites still remains limited. Here, we review some reported mechanisms implicated in regulating plant membrane domains including those of plasma membrane, plastids, mitochondria and endoplasmic reticulum. Finally, we discuss several state-of-the-art methods that allow nowadays researchers to decipher the architecture of these structures at the molecular and atomic level.
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http://dx.doi.org/10.1016/j.pbi.2019.08.003DOI Listing
December 2019

Structural insights into the AapA1 toxin of Helicobacter pylori.

Biochim Biophys Acta Gen Subj 2020 01 30;1864(1):129423. Epub 2019 Aug 30.

INSERM, U1212, CNRS UMR 5320, Univ. Bordeaux, Laboratoire ARNA, F-33076 Bordeaux, France. Electronic address:

Background: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear.

Methods: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization. Biophysical properties of the AapA1 interaction with lipid membranes were studied by solution and solid-state NMR spectroscopy, plasmon waveguide resonance (PWR) and molecular modeling.

Results: We show that despite a low hydrophobic index, this toxin has a nanomolar affinity to the prokaryotic membrane. NMR spectroscopy reveals that the AapA1 toxin is structurally organized into three distinct domains: a positively charged disordered N-terminal domain (D), a single α-helix (H), and a basic C-terminal domain (R). The R domain interacts and destabilizes the membrane, while the H domain adopts a transmembrane conformation. These results were confirmed by alanine scanning of the minimal sequence required for toxicity.

Conclusion: Our results have shown that specific amino acid residues along the H domain, as well as the R domain, are essential for the toxicity of the AapA1 toxin.

General Significance: Untangling and understanding the mechanism of action of small membrane-targeting toxins are difficult, but nevertheless contributes to a promising search and development of new antimicrobial drugs.
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http://dx.doi.org/10.1016/j.bbagen.2019.129423DOI Listing
January 2020

Molecular architecture of bacterial amyloids in biofilms.

FASEB J 2019 11 5;33(11):12146-12163. Epub 2019 Aug 5.

L'Institut de Chimie et Biologie des Membranes et des Nano-Objets (CBMN), Unité Mixte de Recherche (UMR) 5248, Centre National de la Recherche (CNRS), University of Bordeaux, Pessac, France.

The formation of biofilms provides structural and adaptive bacterial response to the environment. In species, the biofilm extracellular matrix is composed of exopolysaccharides, hydrophobins, and several functional amyloid proteins. We report, using multiscale approaches such as solid-state NMR (SSNMR), electron microscopy, X-ray diffraction, dynamic light scattering, attenuated total reflection Fourier transform infrared (FTIR), and immune-gold labeling, the molecular architecture of and pathogenic functional amyloids. SSNMR data reveal that the major amyloid component TasA in its fibrillar amyloid form contain β-sheet and α-helical secondary structure, suggesting a nontypical amyloid architecture in . Proteinase K digestion experiments indicate the amyloid moiety is ∼100 aa long, and subsequent SSNMR and FTIR signatures for and TasA filaments highlight a conserved amyloid fold, albeit with substantial differences in structural polymorphism and secondary structure composition. Structural analysis and coassembly data on the accessory protein TapA in and its counterpart camelysin in reveal a catalyzing effect between the functional amyloid proteins and a common structural architecture, suggesting a coassembly in the context of biofilm formation. Our findings highlight nontypical amyloid behavior of these bacterial functional amyloids, underlining structural variations between biofilms even in closely related bacterial species.-El Mammeri, N., Hierrezuelo, J., Tolchard, J., Cámara-Almirón, J., Caro-Astorga, J., Álvarez-Mena, A., Dutour, A., Berbon, M., Shenoy, J., Morvan, E., Grélard, A., Kauffmann, B., Lecomte, S., de Vicente, A., Habenstein, B., Romero, D., Loquet, A. Molecular architecture of bacterial amyloids in biofilms.
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http://dx.doi.org/10.1096/fj.201900831RDOI Listing
November 2019

Tetrameric Aβ40 and Aβ42 β-Barrel Structures by Extensive Atomistic Simulations. II. In Aqueous Solution.

J Phys Chem B 2019 08 24;123(31):6750-6756. Epub 2019 Jul 24.

Laboratory of Theoretical Chemistry , Ton Duc Thang University , Ho Chi Minh City , Vietnam.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular Aβ42 and Aβ40 oligomers and plaques. In a recent computational study, we found that the presence of the residues I41 and A42 increases significantly the propensity of Aβ to form a tetrameric β-barrel structure in a bilayer mimicking a neuronal membrane. In this work, we have determined the propensity of the two Aβ proteins to form tetrameric β-barrel structures in aqueous solution using four atomistic protein fields, that is, Amber99SB-ILDN/TIP3P, OPLS/TIP3P, CHARMM36m/TIP3P-modified, and Amber99SB/DISP. Extensive replica exchange molecular dynamics simulations make it clear that a β-barrel, made of two distinct β-hairpin motifs and an asymmetric arrangement of eight antiparallel β-strands with an inner pore diameter of 0.7 nm, exists transiently for Aβ42 peptide, but this is less the case for Aβ40 peptide, due to the change of the CHC-CHC and the Cter-Cter interfaces. This study has several implications in AD.
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http://dx.doi.org/10.1021/acs.jpcb.9b05288DOI Listing
August 2019

A polymorphic helix of a Salmonella needle protein relays signals defining distinct steps in type III secretion.

PLoS Biol 2019 07 1;17(7):e3000351. Epub 2019 Jul 1.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.
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http://dx.doi.org/10.1371/journal.pbio.3000351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625726PMC
July 2019

The extracellular matrix protects Bacillus subtilis colonies from Pseudomonas invasion and modulates plant co-colonization.

Nat Commun 2019 04 23;10(1):1919. Epub 2019 Apr 23.

Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), 29071, Málaga, Spain.

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.
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http://dx.doi.org/10.1038/s41467-019-09944-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478825PMC
April 2019

Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of .

Front Microbiol 2019 29;10:573. Epub 2019 Mar 29.

Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), Grenoble, France.

The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined , , and approaches to characterize the T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in . These results suggest that the T3SS needle of displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.
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http://dx.doi.org/10.3389/fmicb.2019.00573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455054PMC
March 2019

Tetrameric Aβ40 and Aβ42 β-Barrel Structures by Extensive Atomistic Simulations. I. In a Bilayer Mimicking a Neuronal Membrane.

J Phys Chem B 2019 05 23;123(17):3643-3648. Epub 2019 Apr 23.

Laboratory of Theoretical Chemistry , Ton Duc Thang University , Ho Chi Minh City 75837 , Vietnam.

The amyloid-β (Aβ) 42 oligomers are much more toxic than Aβ40 oligomers in Alzheimer's disease. Numerous experiments indicate that toxicity could involve the formation of pores in membranes, but experimental high-resolution structure determination of these pore-forming Aβ oligomers has been impeded by aggregate heterogeneity. Using extensive atomistic simulations, low-resolution data obtained in lipid bilayers, and other theoretical factors, we proposed atomic structures of Aβ40 and Aβ42 β-barrels in a bilayer mimicking a neuronal membrane. The 3D model, which consists of tetramer subunits, two distinct β-hairpin motifs and an asymmetric arrangement of eight antiparallel β-strands, is drastically destabilized for Aβ40 compared to its Aβ42 counterpart. Our computational modeling has several implications in Alzheimer's disease, sheds light on the amyloid pore hypothesis, and explains the higher deleterious property of Aβ42.
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http://dx.doi.org/10.1021/acs.jpcb.9b01206DOI Listing
May 2019

Molecular mechanisms for the destabilization of model membranes by islet amyloid polypeptide.

Biophys Chem 2019 02 14;245:34-40. Epub 2018 Dec 14.

Center for Incubation, Innovation, Research and Consultancy (CIIRC), Jyothy Institute of Technology, Thataguni, Off Kanakapura Road, Bangalore 560082, Karnataka, India. Electronic address:

Misfolding of human islet amyloid polypeptide (hIAPP) into insoluble aggregates is associated with Type 2 diabetes. It has been suggested that hIAPP toxicity may be due to its accumulation in pancreatic islets, causing membrane disruption and cell permeabilization, however the molecular basis underlying its lipid association are still unclear. Here, we combine solid-state NMR, fluorescence and bright field microscopy to investigate hIAPP - lipid membrane interactions. Real-time microscopy highlights a time-dependent penetration of hIAPP oligomers toward the most buried layers of the lipid vesicles until the membrane disrupts. Deuterium NMR was conducted on liposomes at different hIAPP concentration to probe lipid internal order and thermotropism. The gel-to-fluid phase transition of the lipids is decreased by the presence of hIAPP, and site-specific analysis of the order parameter showed a significant increase of lipid order for the first eight positions of the acyl chain, suggesting a partial insertion of the peptide inside the bilayer. These results offer experimental insight into the membrane destabilization of hIAPP on model membrane vesicles.
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http://dx.doi.org/10.1016/j.bpc.2018.12.002DOI Listing
February 2019

Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions.

EMBO J 2019 02 17;38(3). Epub 2018 Dec 17.

CNRS, UMR 5095, European Institute of Chemistry and Biology, University of Bordeaux, Pessac, France

While Rho GTPases are indispensible regulators of cellular polarity, the mechanisms underlying their anisotropic activation at membranes have been elusive. Using the budding yeast Cdc42 GTPase module, which includes a guanine nucleotide exchange factor (GEF) Cdc24 and the scaffold Bem1, we find that avidity generated via multivalent anionic lipid interactions is a critical mechanistic constituent of polarity establishment. We identify basic cluster (BC) motifs in Bem1 that drive the interaction of the scaffold-GEF complex with anionic lipids at the cell pole. This interaction appears to influence lipid acyl chain ordering, thus regulating membrane rigidity and feedback between Cdc42 and the membrane environment. Sequential mutation of the Bem1 BC motifs, PX domain, and the PH domain of Cdc24 lead to a progressive loss of cellular polarity stemming from defective Cdc42 nanoclustering on the plasma membrane and perturbed signaling. Our work demonstrates the importance of avidity via multivalent anionic lipid interactions in the spatial control of GTPase activation.
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http://dx.doi.org/10.15252/embj.201899652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356062PMC
February 2019

Functional Amyloids in Health and Disease.

J Mol Biol 2018 10 25;430(20):3629-3630. Epub 2018 Jul 25.

Grupo de Microbiología y Patología Vegetal-Unidad Asociada al CSIC, Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.

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http://dx.doi.org/10.1016/j.jmb.2018.07.024DOI Listing
October 2018

H, C, N NMR resonance assignments and secondary structure determination of the extra-cellular domain from the human proapoptotic TRAIL-R2 death receptor 5 (DR5-ECD).

Biomol NMR Assign 2018 10 5;12(2):309-314. Epub 2018 Jun 5.

Chimie et Biologie des Membranes et des Nano-objets (CBMN), Université de Bordeaux - CNRS - Bordeaux INP, UMR 5248, Bâtiment B14, Allée Geoffroy Saint Hilaire, 33600, Pessac Cedex, France.

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.
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http://dx.doi.org/10.1007/s12104-018-9828-1DOI Listing
October 2018

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene.

Nat Commun 2018 05 9;9(1):1836. Epub 2018 May 9.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron, 22607, Hamburg, Germany.

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing 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 Å and 2.4 Å 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.
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http://dx.doi.org/10.1038/s41467-018-04116-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943278PMC
May 2018

3D structure determination of amyloid fibrils using solid-state NMR spectroscopy.

Methods 2018 04 6;138-139:26-38. Epub 2018 Apr 6.

Institute of Chemistry and Biology of Membranes and Nanoobjects, Institut Européen de Chimie et Biologie (CNRS UMR 5248, Université de Bordeaux), 33600 Pessac, France. Electronic address:

The amyloid fold is structurally characterized by a typical cross-β 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-β 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.
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http://dx.doi.org/10.1016/j.ymeth.2018.03.014DOI Listing
April 2018

Detection of side-chain proton resonances of fully protonated biosolids in nano-litre volumes by magic angle spinning solid-state NMR.

J Biomol NMR 2018 03 3;70(3):177-185. Epub 2018 Mar 3.

Institute of Chemistry & Biology of Membranes & Nanoobjects, (UMR5248 CBMN), CNRS, Université Bordeaux, Institut Européen de Chimie et Biologie, 33600, Pessac, France.

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 µg using a MAS frequency of 70 kHz. The majority of aliphatic and aromatic side chain protons (70%) are observable, in addition to Hα 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.
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http://dx.doi.org/10.1007/s10858-018-0168-3DOI Listing
March 2018

Coiled-coil oligomerization controls localization of the plasma membrane REMORINs.

J Struct Biol 2019 04 23;206(1):12-19. Epub 2018 Feb 23.

Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), IECB, CNRS, Universite Bordeaux, Institut Polytechnique Bordeaux, All. Geoffroy Saint-Hilaire, 33600 Pessac, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jsb.2018.02.003DOI Listing
April 2019

Erratum to: Bacterial Filamentous Appendages Investigated by Solid-State NMR Spectroscopy.

Methods Mol Biol 2017 ;1615:E1

Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), CNRS, University of Bordeaux, Institut Européen de Chimie et Biologie, All. Geoffroy Saint-Hilaire, 33600, Pessac, France.

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http://dx.doi.org/10.1007/978-1-4939-7033-9_37DOI Listing
January 2017