Publications by authors named "Marion Decossas"

49 Publications

Investigation of the effects of two major secretory granules components, insulin and zinc, on human-IAPP amyloid aggregation and membrane damage.

Chem Phys Lipids 2021 Jul 19;237:105083. Epub 2021 Apr 19.

Biophysics Program, Department of Chemistry, Biomedical Engineering, and Macromolecular Science and Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address:

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic peptide found in pancreatic islets of type-2 diabetes (T2D) patients. Under certain conditions, hIAPP is able to form amyloid fibrils that play a role in the progression of T2D. hIAPP is synthesized in the β-cell of the pancreas and stored in the secretory granules before being released into the extracellular compartment. It has been suggested that natural stabilizing agents, such as insulin or zinc present in the secretory granules with hIAPP could prevent hIAPP fibril formation. The difference in the amino acid sequences of IAPP among species strongly correlates with amyloidogenicity and toxicity. The residue histidine at position 18 is known to be important in modulating the fibril formation, membrane leakage and toxicity. In this study, we have synthesized four analogues of hIAPP (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and characterized their aggregation with either insulin or zinc in order to determine the effect of the residue-18 on the insulin-IAPP and zinc-IAPP interactions using a variety of biophysical experiments including thioflavin-T fluorescence, transmission electron microscopy imaging, circular dichroism, and NMR spectroscopy. We show that insulin reduced hIAPP fibril formation both in solution and in the presence of membrane and hIAPP-membrane damage and that the interactions are somewhat mediated by the residue-18. In addition, our results reveal that zinc affects the process of hIAPP fibril formation in solution but not in the presence of membrane. Our results indicate that the nature of the residue-18 is important for zinc binding. Based on this observation, we hypothesize that zinc binds to the residues in the N-terminal region of hIAPP, which is not accessible in the presence of membrane due to its strong interaction with lipids.
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http://dx.doi.org/10.1016/j.chemphyslip.2021.105083DOI Listing
July 2021

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

J Biol Chem 2021 Jan-Jun;296:100602. Epub 2021 Mar 27.

Laboratoire de Biogènese Membranaire, UMR 5200, CNRS, Université de Bordeaux, Villenave d'Ornon Cedex, France. Electronic address:

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.
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http://dx.doi.org/10.1016/j.jbc.2021.100602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099651PMC
March 2021

Isolation and CryoTEM of Phages Infecting Bacterial Wine Spoilers.

Bio Protoc 2020 Nov 5;10(21):e3801. Epub 2020 Nov 5.

University of Bordeaux, ISVV, EA4577 Œnologie, USC 1366, INRAE, Villenave d'Ornon, France.

With the objective to isolate phages infecting wine bacterial spoilers, we designed a method for the isolation and purification of phages infecting grape-associated bacteria. The method proved successful to isolate GC1 tectivirus infecting the acetic acid bacterium . The isolated phage represents a new genus within the , named "Gammatectivirus". Using a traditional technique for the concentration of phage particles involving several steps of centrifugation, further insights in the ultrastructure of GC1 could be observed by cryo electron microscopy, saving time and effort. The simple workflow presented may be applied to other viruses infecting bacteria inhabiting other vegetal niches.
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http://dx.doi.org/10.21769/BioProtoc.3801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842372PMC
November 2020

Structural characterization of the EmrAB-TolC efflux complex from E. coli.

Biochim Biophys Acta Biomembr 2021 01 13;1863(1):183488. Epub 2020 Oct 13.

Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany. Electronic address:

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB-TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.
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http://dx.doi.org/10.1016/j.bbamem.2020.183488DOI Listing
January 2021

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.

Nat Commun 2020 10 2;11(1):4948. Epub 2020 Oct 2.

University of Bordeaux, CBMN UMR 5248, Bordeaux INP, F-33600, Pessac, France.

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.
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http://dx.doi.org/10.1038/s41467-020-18770-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532149PMC
October 2020

Optically Active Perovskite CsPbBr Nanocrystals Helically Arranged on Inorganic Silica Nanohelices.

Nano Lett 2020 Dec 5;20(12):8453-8460. Epub 2020 Nov 5.

Chimie et Biologie des Membrance et des Nanoobjets (CBMN), CNRS, University of Bordeaux, Bordeaux INP, UMR 5248, 33607 Pessac, France.

Perovskite nanocrystals (PNCs) exhibit excellent absorption and luminescent properties. Inorganic silica right (or left) handed nanohelices are used as chiral templates to induce optically active properties to CsPbBr PNCs grafted on their surfaces. In suspension, PNCs grafted on the nanohelices do not show any detectable chiroptical properties. In contrast, in a dried film state, they show large circular dichroism (CD) and circularly polarized luminescence (CPL) signals with dissymmetric factor up to 6 × 10. Grazing incidence X-ray scattering, tomography, and cryo-electron microscopy (EM) have shown closely and helically packed PNCs on the dried helices and much more loosely organized PNCs on helices in suspension. Simulations based on the coupled dipole method (CDM) demonstrate that the CD comes from the dipolar interaction between PNC assembled into a chiral structure and the CD decreases with the interparticle distance.
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http://dx.doi.org/10.1021/acs.nanolett.0c02013DOI Listing
December 2020

Regulation of podosome formation in aortic endothelial cells vessels by physiological extracellular cues.

Eur J Cell Biol 2020 May 11;99(4):151084. Epub 2020 May 11.

Université de Bordeaux, F-33000 Bordeaux, France; INSERM U1045, F-33000 Bordeaux, France. Electronic address:

Invadosomes are specialised actin-based dynamic microdomains of the plasma membrane. Their occurrence has been associated with cell adhesion, matrix degrading and mechanosensory functions that make them crucial regulators of cell migration and invasion. Monocytic, cancer cell and Src-transformed cell invadosomes have been extensively described. Less well defined are the structures which form in other cell types, i.e., non-haematopoietic and non-transformed cells, exposed to specific stimuli. We herein describe the specificities of podosomes induced in aortic endothelial cells stimulated with TGFβ in vitro and in conditions that more closely resemble the in vivo situation. These podosomes display the typical architecture of monocytic podosomes. They organise into large rosette-shape superstructures where they exhibit collective dynamic behavior consisting in cycles of formation and regression. At the ultrastructural level, microfilament arrangements in individual podosomes were revealed. Oxygen levels and hemodynamic forces, which are key players in endothelial cell biology, both influence the process. In 3D environment, podosomes appear as globular structures along cellular extensions. A better characterization of endothelial podosomes has far-reaching implications in the understanding and, possibly, in the treatment of some vascular diseases.
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http://dx.doi.org/10.1016/j.ejcb.2020.151084DOI Listing
May 2020

Microfluidic diffusional sizing probes lipid nanodiscs formation.

Biochim Biophys Acta Biomembr 2020 06 20;1862(6):183215. Epub 2020 Feb 20.

Univ Bordeaux, CNRS, CBMN UMR 5248, Bat B14 Allée Geoffroy St Hilaire, F-33600 Pessac, France. Electronic address:

The biophysical characterisation of membrane proteins and their interactions with lipids in native membrane habitat remains a major challenge. Indeed, traditional solubilisation procedures with detergents often causes the loss of native lipids surrounding membrane proteins, which ultimately impacts structural and functional properties. Recently, copolymer-based nanodiscs have emerged as a highly promising tool, thanks to their unique ability of solubilising membrane proteins directly from native membranes, in the shape of discoidal patches of lipid bilayers. While this methodology finally set us free from the use of detergents, some limitations are however associated with the use of such copolymers. Among them, one can cite the tedious control of the nanodiscs size, their instability in basic pH and in the presence of divalent cations. In this respect, many variants of the widely used Styrene Maleic Acid (SMA) copolymer have been developed to specifically address those limitations. With the multiplication of new SMA copolymer variants and the growing interest in copolymer-based nanodiscs for the characterisation of membrane proteins, there is a need to better understand and control their formation. Among the techniques used to characterise the solubilisation of lipid bilayer by amphipathic molecules, cryo-TEM, P NMR, DLS, ITC and fluorescence spectroscopy are the most widely used, with a consensus made in the sense that a combination of these techniques is required. In this work, we propose to evaluate the capacity of Microfluidic Diffusional Sizing (MDS) as a new method to follow copolymer nanodiscs formation. Originally designed to determine protein size through laminar flow diffusion, we present a novel application along with a protocol development to observe nanodiscs formation by MDS. We show that MDS allows to precisely measure the size of nanodiscs, and to determine the copolymer/lipid ratio at the onset of solubilisation. Finally, we use MDS to characterise peptide/nanodisc interaction. The technique shows a promising ability to highlight the pivotal role of lipids in promoting interactions through a case study with an aggregating peptide. This confirmed the relevance of using the MDS and nanodiscs as biomimetic models for such investigations.
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http://dx.doi.org/10.1016/j.bbamem.2020.183215DOI Listing
June 2020

Lipidic Aminoglycoside Derivatives: A New Class of Immunomodulators Inducing a Potent Innate Immune Stimulation.

Adv Sci (Weinh) 2019 Aug 15;6(16):1900288. Epub 2019 Jul 15.

CRCINA, INSERM Université d'Angers, Université de Nantes Boulevard Bénoni Goullin Nantes 44200 France.

Development of simple and fully characterized immunomodulatory molecules is an active area of research to enhance current immunotherapies. Monophosphoryl lipid A (MPL), a nontoxic lipidic derivative from bacteria, is the first and currently only adjuvant approved in humans. However, its capacity to induce a potent response against weak immunogenic tumoral-associated antigens remains limited. Herein, a new generation of lipidic immunomodulators to conduct a structure-activity relationship study to determine the minimal structural elements conferring immunomodulatory properties is introduced. Two lead molecules characterized by a short succinyl linker between two oleyl chains and a polar headgroup consisting of either naturally occurring tobramycin (DOST) or kanamycin (DOSK) are identified. These two lipoaminoglycosides self-assemble in very small vesicles. In a wide variety of cells including 3D human cell culture, DOST and DOSK induce the upregulation of proinflammatory cytokines and interferon-inducible proteins in a dose and time-dependent manner via a caveolae-dependent proinflammatory mechanism and phosphatidylinositol phospholipase C activation. Furthermore, after intratumoral administration, these lipoaminoglycosides induce an efficient immune response leading to significant antitumor activity in a mouse breast cancer model. Altogether, these findings indicate that DOST and DOSK are two groundbreaking synthetic lipid immunostimulators that can be used as adjuvants to enhance current immunotherapeutic treatments.
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http://dx.doi.org/10.1002/advs.201900288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702646PMC
August 2019

Involvement of caveolin-1 and CD36 in native LDL endocytosis by endothelial cells.

Biochim Biophys Acta Gen Subj 2019 05 12;1863(5):830-838. Epub 2019 Feb 12.

Institute of Chemistry and Biology of Membranes and Nano-objects (CBMN), UMR 5248, CNRS, University of Bordeaux, INP, F-33600 Pessac, France.

Atherosclerosis is a lipid disease characterized by accumulation of low density lipoprotein (LDL) in the artery wall. The transport of LDL across the endothelium of coronary artery is an initiating event of atherosclerosis, whose mechanism remains poorly understood. In the last decade, it has been shown that in caveolin-1 (Cav-1) deficient mice, LDL infiltration in aorta wall is decreased and CD36 expression in aortas is down-regulated, leading to regression of atherosclerotic lesions. In the present study, we show that native LDL endocytosis is decreased in endothelial cells deficient in Cav-1 or CD36. We demonstrate that Cav-1 and CD36 interact in caveolae-rich domains by different biochemical approaches. In addition, confocal microscopy reveals some colocalization of Cav-1 with CD36. These findings indicate that caveolae and CD36 are involved in native LDL endocytosis and suggest that CD36 might be a good candidate for the transport of native LDL across the endothelium, an early event in atherosclerosis.
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http://dx.doi.org/10.1016/j.bbagen.2019.01.005DOI Listing
May 2019

Minimal nanodisc without exogenous lipids for stabilizing membrane proteins in detergent-free buffer.

Biochim Biophys Acta Biomembr 2019 04 30;1861(4):852-860. Epub 2019 Jan 30.

Univ. Bordeaux, CBMN UMR 5248, Bordeaux INP, F-33600 Pessac, France; CNRS, CBMN UMR5248, F-33600 Pessac, France. Electronic address:

Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio. We present here the use of the scaffold protein MSP without added lipids as a minimal system to stabilize membrane proteins. We show that this method is applicable to α-helical and β-strands transmembrane proteins. This method allowed cryo-electron microscopy structural study of the bacterial transporter MexB. A protein quantification indicates that MexB is stabilized by two MSP proteins. This simplified and efficient method proposes a new advance in harnessing the MSP potential to stabilize membrane proteins.
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http://dx.doi.org/10.1016/j.bbamem.2019.01.013DOI Listing
April 2019

Caveolae-mediated effects of TNF-α on human skeletal muscle cells.

Exp Cell Res 2018 09 18;370(2):623-631. Epub 2018 Jul 18.

University of Bordeaux, Bordeaux Institute of Technology, Chemistry and Biology of Membrane and Nanoobjects (CBMN), CNRS, UMR 5248, F-33600 Pessac, France. Electronic address:

Chronic diseases are characterized by the production of pro-inflammatory cytokines such than TNF-α and are frequently correlated with muscle wasting conditions. Among the pleiotropic effects of TNF-α within the cell, its binding to TNFR1 receptor has been shown to activate sphingomyelinases leading to the production of ceramides. Sphingomyelinases and TNF receptor have been localized within caveolae which are specialized RAFT enriched in cholesterol and sphingolipids. Because of their inverted omega shape, maintained by the oligomerization of specialized proteins, caveolins and cavins, caveolae serve as membrane reservoir therefore providing mechanical protection to plasma membranes. Although sphingolipids metabolites, caveolins and TNF-α/TNFR1 have been shown to independently interfere with muscle physiology, no data have clearly demonstrated their concerted action on muscle cell regeneration. In this context, our study aimed at studying the molecular mechanisms induced by TNF-α at the level of caveolae in LHCN-M2 human muscle satellite cells. Here we showed that TNF-α-induced production of ROS and nSMase activation requires caveolin. More strikingly, we have demonstrated that TNF-α induces the formation of additional caveolae at the plasma membrane of myoblasts. Furthermore, TNF-α prevents myoblast fusion suggesting that inflammation could modulate caveolae organization/function and satellite cell function.
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http://dx.doi.org/10.1016/j.yexcr.2018.07.027DOI Listing
September 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

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action.

Nanomedicine 2018 01 19;14(1):141-151. Epub 2017 Sep 19.

In-Cell-Art, Rezé, France. Electronic address:

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper. Results led to the identification of the aminoglycoside lipid dioleyl phosphoramido ribostamycin (DOPRI) as a potent intracellular delivery system for anti-K8. Electron microscopy revealed that delivered anti-K8 molecules were bound to intermediate filaments in cells. Anti-K8 was bound to the surface of DOPRI vesicles without perturbing lipid organization. Macropinocytosis and caveolin mediated endocytosis contributed to anti-K8 internalization and to filament labeling with a major contribution being made by the caveolin pathway. The results showed that the unique properties of DOPRI were sufficient for efficient intracellular protein delivery without requiring lipid helpers.
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http://dx.doi.org/10.1016/j.nano.2017.09.005DOI Listing
January 2018

Lipid Internal Dynamics Probed in Nanodiscs.

Chemphyschem 2017 Oct 30;18(19):2651-2657. Epub 2017 Jun 30.

CBMN, CNRS., University of Bordeaux, IECB, All. Geoffroy Saint-Hilaire, 34600, Pessac, France.

Nanodiscs offer a very promising tool to incorporate membrane proteins into native-like lipid bilayers and an alternative to liposomes to maintain protein functions and protein-lipid interactions in a soluble nanoscale object. The activity of the incorporated membrane protein appears to be correlated to its dynamics in the lipid bilayer and by protein-lipid interactions. These two parameters depend on the lipid internal dynamics surrounded by the lipid-encircling discoidal scaffold protein that might differ from more unrestricted lipid bilayers observed in vesicles or cellular extracts. A solid-state NMR spectroscopy investigation of lipid internal dynamics and thermotropism in nanodiscs is reported. The gel-to-fluid phase transition is almost abolished for nanodiscs, which maintain lipid fluid properties for a large temperature range. The addition of cholesterol allows fine-tuning of the internal bilayer dynamics by increasing chain ordering. Increased site-specific order parameters along the acyl chain reflect a higher internal ordering in nanodiscs compared with liposomes at room temperature; this is induced by the scaffold protein, which restricts lipid diffusion in the nanodisc area.
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http://dx.doi.org/10.1002/cphc.201700450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697661PMC
October 2017

Hetero-oligomerization between the TNF receptor superfamily members CD40, Fas and TRAILR2 modulate CD40 signalling.

Cell Death Dis 2017 02 9;8(2):e2601. Epub 2017 Feb 9.

Institut de Biologie Moléculaire et Cellulaire UPR 3572 'Immunopathologie et Chimie Thérapeutique' du CNRS, 15 Rue René Descartes, Strasbourg Cedex 67084, France.

TNF receptor superfamily members (TNFRSF) such as CD40, Fas and TRAIL receptor 2 (TRAILR2) participate to the adaptive immune response by eliciting survival, proliferation, differentiation and/or cell death signals. The balance between these signals determines the fate of the immune response. It was previously reported that these receptors are able to self-assemble in the absence of ligand through their extracellular regions. However, the role of this oligomerization is not well understood, and none of the proposed hypotheses take into account potential hetero-association of receptors. Using CD40 as bait in a flow cytometry Förster resonance energy transfer assay, TNFRSF members with known functions in B cells were probed for interactions. Both Fas and TRAILR2 associated with CD40. Immunoprecipitation experiments confirmed the interaction of CD40 with Fas at the endogenous levels in a BJAB B-cell lymphoma cell line deficient for TRAILR2. TRAILR2-expressing BJAB cells displayed a robust CD40-TRAILR2 interaction at the expense of the CD40-Fas interaction. The same results were obtained by proximity ligation assay, using TRAILR2-positive and -negative BJAB cells and primary human B cells. Expression of the extracellular domains of Fas or TRAILR2 with a glycolipid membrane anchor specifically reduced the intrinsic signalling pathway of CD40 in 293T cells. Conversely, BJAB cells lacking endogenous Fas or TRAILR2 showed an increased NF-κB response to CD40L. Finally, upregulation of TRAILR2 in primary human B cells correlated with reduced NF-κB activation and reduced proliferation in response to CD40L. Altogether, these data reveal that selective interactions between different TNFRSF members may modulate ligand-induced responses upstream signalling events.
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http://dx.doi.org/10.1038/cddis.2017.22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386471PMC
February 2017

N-Heterocyclic Carbene-Polyethylenimine Platinum Complexes with Potent in Vitro and in Vivo Antitumor Efficacy.

Bioconjug Chem 2016 08 8;27(8):1942-8. Epub 2016 Aug 8.

Institut de Physique et Chimie des Matériaux de Strasbourg Université de Strasbourg-CNRS UMR 7504 , 23 rue du Loess, BP 43, 67034 Strasbourg cedex 2, France.

The current interest for platinum N-heterocyclic carbene complexes in cancer research stems from their impressive toxicity reported against a range of different human cancer cells. To date, the demonstration of their in vivo efficacy relative to that of established platinum-based drugs has not been specifically addressed. Here, we introduce an innovative approach to increase the NHC-Pt complex potency whereby multiple NHC-Pt(II) complexes are coordinated along a polyethylenimine polymer (PEI) chain. We show that such NHC-Pt(II)-PEI conjugates induce human cancer cell death in vitro and in vivo in a xenograft mouse model with no observable side effects in contrast to oxaliplatin. Additional studies indicate nucleus and mitochondria targeting and suggest various mechanisms of action compared to classical platinum-based anticancer drugs.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00320DOI Listing
August 2016

Synthetic ligands of death receptor 5 display a cell-selective agonistic effect at different oligomerization levels.

Oncotarget 2016 10;7(40):64942-64956

Institut de Biologie Moléculaire et Cellulaire, UMR 3572, Laboratoire d'Immunopathologie et Chimie Thérapeutique, Strasbourg 67084, France.

DR4 (Death Receptor 4) and DR5 (Death Receptor 5) are two potential targets for cancer therapy due to their ability to trigger apoptosis of cancer cells, but not normal ones, when activated by their cognate ligand TRAIL (TNF related apoptosis-inducing ligand). Therapies based on soluble recombinant TRAIL or agonist antibodies directed against one of the receptors are currently under clinical trials. However, TRAIL-R positive tumor cells are frequently resistant to TRAIL induced apoptosis. The precise mechanisms of this resistance are still not entirely understood. We have previously reported on synthetic peptides that bind to DR5 (TRAILmim/DR5) and induce tumor cell apoptosis in vitro and in vivo. Here, we showed that while hexameric soluble TRAIL is able to efficiently kill the DR5 positive lymphoma Jurkat or the carcinoma HCT116, these cells are resistant to apoptosis induced by the divalent form of TRAILmim/DR5 and are poorly sensitive to apoptosis induced by an anti-DR5 agonist monoclonal antibody. This resistance can be restored by the cross-linking of anti-DR5 agonist antibody but not by the cross-linking of the divalent form of TRAILmim/DR5. Interestingly, the divalent form of TRAILmim/DR5 that induced apoptosis of DR5 positive BJAB cells, acts as an inhibitor of TRAIL-induced apoptosis on Jurkat and HCT116 cells. The rapid internalization of DR5 observed when treated with divalent form of TRAILmim/DR5 could explain the antagonist activity of the ligand on Jurkat and HCT116 cells but also highlights the independence of the mechanisms responsible for internalization and activation when triggering the DR5 apoptotic cascade.
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http://dx.doi.org/10.18632/oncotarget.10508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323128PMC
October 2016

Shaping quaternary assemblies of water-soluble non-peptide helical foldamers by sequence manipulation.

Nat Chem 2015 Nov 28;7(11):871-8. Epub 2015 Sep 28.

Université de Bordeaux, CBMN, UMR 5248, Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, Pessac 33607, France.

The design and construction of biomimetic self-assembling systems is a challenging yet potentially highly rewarding endeavour that contributes to the development of new biomaterials, catalysts, drug-delivery systems and tools for the manipulation of biological processes. Significant progress has been achieved by engineering self-assembling DNA-, protein- and peptide-based building units. However, the design of entirely new, completely non-natural folded architectures that resemble biopolymers ('foldamers') and have the ability to self-assemble into atomically precise nanostructures in aqueous conditions has proved exceptionally challenging. Here we report the modular design, formation and structural elucidation at the atomic level of a series of diverse quaternary arrangements formed by the self-assembly of short amphiphilic α-helicomimetic foldamers that bear proteinaceous side chains. We show that the final quaternary assembly can be controlled at the sequence level, which permits the programmed formation of either discrete helical bundles that contain isolated cavities or pH-responsive water-filled channels with controllable pore diameters.
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http://dx.doi.org/10.1038/nchem.2353DOI Listing
November 2015

Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions.

J Struct Biol 2015 Dec 21;192(3):470-477. Epub 2015 Oct 21.

University of Bordeaux, Chemistry and Biology of Membranes and Nanoobjects (CBMN), UMR 5248, Pessac, France; Centre National de la Recherche Scientifique (CNRS), CBMN, UMR 5248, Pessac, France. Electronic address:

Cryo-electron microscopy (cryo-EM) allows the visualization of the cell architecture in its native state. We developed a robust solution to adapt cryo-electron microscopy of vitreous sections (CEMOVIS) to a monolayer of adherent cells using a functionalized polyacrylamide hydrogel growing substrate. We applied this method to reconstitute an endothelial cell monolayer to visualize the morphology of adherens junctions (AJs) which regulate permeability and integrity of the vascular barrier. The fine morphology and ultrastructure of AJs from cultured primary human coronary artery endothelial cells (HCAECs) were analyzed in their native state by using CEMOVIS. Doxycycline and sphingosine-1-phosphate (S1P) are known as efficient regulators of endothelial permeability. Doxycycline and S1P treatments both led to a drastic morphological switch from very uneven to standardized 14-17 nm wide AJs over several microns indicative of a better membrane tethering. Repetitive structures were occasionally noticed within the AJ cleft reflecting a local improved structural organization of VE-cadherin molecules. The ultrastructural stabilization of AJs observed upon treatment likely indicates a better adhesion and thus provides structural clues on the mechanism by which these treatments improve the endothelial barrier function. This method was also successfully extended to a thick epithelial barrier model. We expect our strategy to extend the reliable application of CEMOVIS to virtually any adherent cultured cell systems.
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http://dx.doi.org/10.1016/j.jsb.2015.10.009DOI Listing
December 2015

A cell-penetrating foldamer with a bioreducible linkage for intracellular delivery of DNA.

Angew Chem Int Ed Engl 2015 Sep 5;54(38):11133-7. Epub 2015 Aug 5.

Univ. Bordeaux, CBMN, UMR 5248, Institut Européen de Chimie et Biologie (IECB), 2 rue Robert Escarpit, 33607 Pessac (France).

Despite significant advances in foldamer chemistry, tailored delivery systems based on foldamer architectures, which provide a high level of control over secondary structure, are curiously rare among non-viral technologies for transporting nucleic acids into cells. A potent pH-responsive, bioreducible cell-penetrating foldamer (CPF) was developed through covalent dimerization of a short (8-mer) amphipathic oligourea sequence bearing histidine-type units. This CPF exhibits a high capacity to assemble with pDNA and mediates efficient delivery of nucleic acids into the cell. Furthermore, it does not adversely affect cellular viability and was shown to compare favorably with a cognate peptide transfection agent based on His-rich sequences.
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http://dx.doi.org/10.1002/anie.201504884DOI Listing
September 2015

Oxidative stress induces caveolin 1 degradation and impairs caveolae functions in skeletal muscle cells.

PLoS One 2015 23;10(3):e0122654. Epub 2015 Mar 23.

Univ Bordeaux, Chimie et Biologie des Membranes et Nanoobjets, UMR 5248, F-33600 Pessac, France; CNRS, Chimie et Biologie des Membranes et Nanoobjets, UMR 5248, F-33600 Pessac, France; Bordeaux INP, Chimie et Biologie des Membranes et Nanoobjets, UMR 5248, F-33600 Pessac, France.

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H2O2) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H2O2 treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H2O2 treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H2O2 treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H2O2 decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122654PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370508PMC
February 2016

Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion.

Int J Nanomedicine 2015 19;10:1479-92. Epub 2015 Feb 19.

Institute of Chemistry and Biology of Membranes and Nanoobjects, University of Bordeaux, UMR5248, Pessac, France ; Institute of Chemistry and Biology of Membranes and Nanoobjects, Centre National de la Recherche Scientifique, Institute of Chemistry and Biology of Membranes and Nanoobjects, UMR5248, Pessac, France.

The use of silica nanoparticles for their cellular uptake capability opens up new fields in biomedical research. Among the toxicological effects associated with their internalization, silica nanoparticles induce apoptosis that has been recently reported as a biochemical cue required for muscle regeneration. To assess whether silica nanoparticles could affect muscle regeneration, we used the C2C12 muscle cell line to study the uptake of fluorescently labeled NPs and their cellular trafficking over a long period. Using inhibitors of endocytosis, we determined that the NP uptake was an energy-dependent process mainly involving macropinocytosis and clathrin-mediated pathway. NPs were eventually clustered in lysosomal structures. Myoblasts containing NPs were capable of differentiation into myotubes, and after 7 days, electron microscopy revealed that the NPs remained primarily within lysosomes. The presence of NPs stimulated the formation of myotubes in a dose-dependent manner. NP internalization induced an increase of apoptotic myoblasts required for myoblast fusion. At noncytotoxic doses, the NP uptake by skeletal muscle cells did not prevent their differentiation into myotubes but, instead, enhanced the cell fusion.
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http://dx.doi.org/10.2147/IJN.S74158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340375PMC
August 2016

Interaction of Aβ(1-42) amyloids with lipids promotes "off-pathway" oligomerization and membrane damage.

Biomacromolecules 2015 Mar 26;16(3):944-50. Epub 2015 Feb 26.

Chimie et Biologie des Membranes et Nanoobjets, CBMN CNRS UMR 5248, Université de Bordeaux , Allée Geoffroy de Saint Hilaire, 33600 Pessac, France.

The toxicity of amyloids, as Aβ(1-42) involved in Alzheimer disease, is a subject under intense scrutiny. Many studies link their toxicity to the existence of various intermediate structures prior to fiber formation and/or their specific interaction with membranes. In this study we focused on the interaction between membrane models and Aβ(1-42) peptides and variants (L34T, mG37C) produced in E. coli and purified in monomeric form. We evaluated the interaction of a toxic stable oligomeric form (oG37C) with membranes as comparison. Using various biophysical techniques as fluorescence and plasmon waveguide resonance, we clearly established that the oG37C interacts strongly with membranes leading to its disruption. All the studied peptides destabilized liposomes and accumulated slowly on the membrane (rate constant 0.02 min(-1)). Only the oG37C exhibited a particular pattern of interaction, comprising two steps: the initial binding followed by membrane reorganization. Cryo-TEM was used to visualize the peptide effect on liposome morphologies. Both oG37C and mG37C lead to PG membrane fragmentation. The PG membrane promotes peptide oligomerization, implicated in membrane disruption. WT (Aβ(1-42)) also perturbs liposome organization with membrane deformation rather than disruption. For all the peptides studied, their interaction with the membranes changes their fibrillization process, with less fibers and more small aggregates being formed. These studies allowed to establish, a correlation between toxicity, fiber formation, and membrane disruption.
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http://dx.doi.org/10.1021/bm501837wDOI Listing
March 2015

Dermal CD14(+) Dendritic Cell and Macrophage Infection by Dengue Virus Is Stimulated by Interleukin-4.

J Invest Dermatol 2015 Jul 18;135(7):1743-1751. Epub 2014 Dec 18.

Laboratory of Immunopathology and Therapeutic Chemistry, CNRS UPR 3572/Laboratory of Excellence MEDALIS, IBMC, University of Strasbourg, Strasbourg, France. Electronic address:

Dengue virus (DENV) is responsible for the most prevalent arthropod-borne viral infection in humans. Events decisive for disease development occur in the skin after virus inoculation by the mosquito. Yet, the role of human dermis-resident immune cells in dengue infection and disease remains elusive. Here we investigated how dermal dendritic cells (dDCs) and macrophages (dMs) react to DENV and impact on immunopathology. We show that both CD1c(+) and CD14(+) dDC subsets were infected, but viral load greatly increased in CD14(+) dDCs upon IL-4 stimulation, which correlated with upregulation of virus-binding lectins Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN/CD209) and mannose receptor (CD206). IL-4 also enhanced T-cell activation by dDCs, which was further increased upon dengue infection. dMs purified from digested dermis were initially poorly infected but actively replicated the virus and produced TNF-α upon lectin upregulation in response to IL-4. DC-SIGN(+) cells are abundant in inflammatory skin with scabies infection or Th2-type dermatitis, suggesting that skin reactions to mosquito bites heighten the risk of infection and subsequent immunopathology. Our data identify dDCs and dMs as primary arbovirus target cells in humans and suggest that dDCs initiate a potent virus-directed T-cell response, whereas dMs fuel the inflammatory cascade characteristic of dengue fever.
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http://dx.doi.org/10.1038/jid.2014.525DOI Listing
July 2015

Cyclic GMP catabolism up-regulation in MRL/lpr lupus-prone mice is associated with organ remodeling.

Biochim Biophys Acta 2014 Jul 12;1842(7):916-26. Epub 2014 Mar 12.

CNRS-Université de Strasbourg, Biophotonique et Pharmacologie, Faculté de Pharmacie, Illkirch, France. Electronic address:

Production of high titer of antibodies against nuclear components is a hallmark of systemic lupus erythematosus, an autoimmune disease characterized by the progressive chronic inflammation of multiple joints and organs. Organ damage and dysfunction such as renal failure are typical clinical features in lupus. Cell hypermetabolism and hypertrophy can accelerate organ dysfunction. In this study we focus on a specific murine model of lupus, the MRL/lpr strain, and investigated the role of cyclic guanosine monophosphate (cGMP) catabolism in organ remodeling of main target tissues (kidney, spleen and liver) in comparison with age-matched control mice. In MRL/lpr-prone mice, the cGMP-phosphodiesterase (PDE) activities were significantly increased in the kidney (3-fold, P<0.001), spleen (2-fold, P<0.001) and liver (1.6-fold, P<0.05). These raised activity levels were paralleled by both an increased activity of PDE1 in the kidney (associated with nephromegaly) and in the liver, and PDE2 in the spleen of lupus-prone mice. The up-regulation of PDE1 and PDE2 activities were associated with a decrease in intracellular cGMP levels. This underlines an alteration of cGMP-PDE signaling in the kidney, spleen and liver targeting different PDEs according to organs. In good agreement with these findings, a single intravenous administration to MRL/lpr mice of nimodipine (PDE1 inhibitor) but not of EHNA (PDE2 inhibitor) was able to significantly lower peripheral hypercellularity (P=0.0401), a characteristic feature of this strain of lupus-prone mice. Collectively, our findings are important for generating personalized strategies to prevent certain forms of the lupus disease as well as for understanding the role of PDEs and cGMP in the pathophysiology of lupus.
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http://dx.doi.org/10.1016/j.bbadis.2014.03.001DOI Listing
July 2014

Early differentiated CD138(high) MHCII+ IgG+ plasma cells express CXCR3 and localize into inflamed kidneys of lupus mice.

PLoS One 2013 8;8(3):e58140. Epub 2013 Mar 8.

CNRS, Institut de Biologie Moléculaire et Cellulaire, Immunopathologie et Chimie Thérapeutique/Laboratory of Excellence Medalis, Strasbourg, France.

Humoral responses are central to the development of chronic autoimmune diseases such as systemic lupus erythematosus. Indeed, autoantibody deposition is responsible for tissue damage, the kidneys being one of the main target organs. As the source of pathogenic antibodies, plasma cells are therefore critical players in this harmful scenario, both at systemic and local levels. The aim of the present study was to analyze plasma cells in NZB/W lupus mice and to get a better understanding of the mechanisms underlying their involvement in the renal inflammation process. Using various techniques (i.e. flow cytometry, quantitative PCR, ELISpot), we identified and extensively characterized three plasma cell intermediates, according to their B220/CD138/MHCII expression levels. Each of these cell subsets displays specific proliferation and antibody secretion capacities. Moreover, we evidenced that the inflammation-related CXCR3 chemokine receptor is uniquely expressed by CD138(high)MHCII(+) plasma cells, which encompass both short- and long-lived cells and mostly produce IgG (auto)antibodies. Expression of CXCR3 allows efficient chemotactic responsiveness of these cells to cognate chemokines, which production is up-regulated in the kidneys of diseased NZB/W mice. Finally, using fluorescence and electron microscopy, we demonstrated the presence of CD138(+)CXCR3(+)IgG(+) cells in inflammatory areas in the kidneys, where they are very likely involved in the injury process. Thus, early differentiated CD138(high)MHCII(+) rather than terminally differentiated CD138(high)MHCII(low) plasma cells may be involved in the renal inflammatory injury in lupus, due to CXCR3 expression and IgG secretion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058140PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592892PMC
September 2013

Cysteine-rich domain 1 of CD40 mediates receptor self-assembly.

J Biol Chem 2013 Apr 5;288(15):10914-22. Epub 2013 Mar 5.

Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, CNRS UPR 9021, 15 rue René Descartes, 67084 Strasbourg, France.

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a K(D) of 0.6 μM. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state.
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http://dx.doi.org/10.1074/jbc.M112.427583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624471PMC
April 2013

Carabin deficiency in B cells increases BCR-TLR9 costimulation-induced autoimmunity.

EMBO Mol Med 2012 Dec 29;4(12):1261-75. Epub 2012 Oct 29.

CNRS UPR9021, IBMC, Strasbourg, France.

The mechanisms behind flares of human autoimmune diseases in general, and of systemic lupus in particular, are poorly understood. The present scenario proposes that predisposing gene defects favour clinical flares under the influence of external stimuli. Here, we show that Carabin is low in B cells of (NZB × NZW) F1 mice (murine SLE model) long before the disease onset, and is low in B cells of lupus patients during the inactive phases of the disease. Using knock-out and B-cell-conditional knock-out murine models, we identify Carabin as a new negative regulator of B-cell function, whose deficiency in B cells speeds up early B-cell responses and makes the mice more susceptible to anti-dsDNA production and renal lupus flare after stimulation with a Toll-like Receptor 9 agonist, CpG-DNA. Finally, in vitro analysis of NFκB activation and Erk phosphorylation in TLR9- and B-cell receptor (BCR)-stimulated Carabin-deficient B cells strongly suggests how the internal defect synergizes with the external stimulus and proposes Carabin as a natural inhibitor of the potentially dangerous crosstalk between BCR and TLR9 pathways in self-reactive B cells.
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http://dx.doi.org/10.1002/emmm.201201595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531602PMC
December 2012

Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions.

EMBO J 2012 Aug 12;31(15):3239-51. Epub 2012 Jun 12.

Bordeaux University, Bordeaux, France.

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.
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http://dx.doi.org/10.1038/emboj.2012.161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411072PMC
August 2012
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