Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence

BACKGROUND: The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. CONCLUSION/SIGNIFICANCE: BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.

International audienceHepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture-derived HCV (HCVcc). Anti-SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI-specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81-HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti-SR-BI antibodies and SR-BI-specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV-CD81 interaction

Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection

Real-time Monitoring for the Next Core-Collapse Supernova in JUNO

Core-collapse supernova (CCSN) is one of the most energetic astrophysical events in the Universe. The early and prompt detection of neutrinos before (pre-SN) and during the SN burst is a unique opportunity to realize the multi-messenger observation of the CCSN events. In this work, we describe the monitoring concept and present the sensitivity of the system to the pre-SN and SN neutrinos at the Jiangmen Underground Neutrino Observatory (JUNO), which is a 20 kton liquid scintillator detector under construction in South China. The real-time monitoring system is designed with both the prompt monitors on the electronic board and online monitors at the data acquisition stage, in order to ensure both the alert speed and alert coverage of progenitor stars. By assuming a false alert rate of 1 per year, this monitoring system can be sensitive to the pre-SN neutrinos up to the distance of about 1.6 (0.9) kpc and SN neutrinos up to about 370 (360) kpc for a progenitor mass of 30$M_{\odot}$ for the case of normal (inverted) mass ordering. The pointing ability of the CCSN is evaluated by using the accumulated event anisotropy of the inverse beta decay interactions from pre-SN or SN neutrinos, which, along with the early alert, can play important roles for the followup multi-messenger observations of the next Galactic or nearby extragalactic CCSN.Comment: 24 pages, 9 figure

Mise au point d'un bioessai à cellules entières, pour la détection de pollutions, basé sur la technologie du promoteur de stress

L'objectif de ce travail est la mise au point d'un bioessai luminescent, permettant d'apprécier rapidement la toxicité globale d'un milieu complexe, grâce à des systèmes biologiques génétiquement modifiés. Le principe est basé sur l'association d'un promoteur de stress (hsp22 ou hsp23 de drosophile) et d'un gène rapporteur, codant une protéine bioluminescente (luciférase) ou fluorescente (EGFP), insérés dans une matrice cellulaire. Les premiers essais, réalisés grâce à des levures, ont montré que cet organisme ne correspondait pas aux critères de sensibilité de ce système. La génération de promoteurs génétiquement manipulés a permis de produire des clones recombinants de lignées cellulaires humaines (HeLa ou HepG2) et d''améliorer les performances du test. Les résultats en présence de composés toxiques tels que des métaux lourds, des perturbateurs endocriniens et d'échantillons environnementaux, ont permis l'induction de la transcription du gène rapporteur avant la manisfestatin de cytotoxicitéLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Développement de couches lipidiques fonctionnalisées pour la détection spécifique de Brins d'ADN à l'interface air-eau (application à un nouveau concept de biopuce à interface fluide)

Une nouvelle méthodologie analytique basée sur la fonctionnalisation d'une interface fluide et sur la caractérisation de ses propriétés micromécaniques a été développée. Un lipide synthétique porteur d'un groupement spermine cationique, le DOGS, est étalé à l'interface air-eau pour former un film capable de capturer des molécules d'ADN. Différentes analyses, directement menées à l'interface ou après transfert de Langmuir-Blodgett, ont permis de mettre en évidence (i) l'interaction de ces molécules d'ADN ou de polynucléotides courts avec la monocouche de DOGS, (ii) l'impact de cette adsorption d'ADN sur le comportement interfacial du DOGS en monocouche. L'addition séquentielle d'oligonucléotides a permis de mener des études d'hybridation à l'interface, et d'améliorer la spécificité du système. Enfin, un nouveau protocole de caractérisation par rhéologie interfaciale a été appliqué pour la détection des variations visco-élastiques induites par l'immobilisation de polynucléotidesLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Immobilisation de biomolécules par la technique du transfert de macromolécules dans le PDMS pour le développement de biopuces

An innovative technology based on the direct immobilization of proteins and DNA spots during the PDMS polymerization was developed during this work. The introduced technology was named macromolecules to PDMS transfer and allowed the fabrication of functionalized PDMS surfaces containing active immobilized DNA sequences and proteins for molecular recognition. Then, taking advantage the available PDMS properties, two microfluidics systems were designed and used for the chemiluminescent detection of proteins interactions. Finally, an extension of the possibilities offered by this approach was demonstrated through the replacement of PDMS with a solution of poly(methylmethacrylate) (PMMA) polymerizable to produce PMMA biochips.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Contribution of dynamic and static quenchers for the study of protein conformation in ionic liquids by steady-state fluorescence spectroscopy.

International audienceThe study of protein conformation in ionic liquids (ILs) is crucial to understand enzymatic activity. Steady-state fluorescence is a proven, rapid and easy method to evaluate the protein structure in aqueous solutions, but it is discussed when used in ILs. In this work, the structure of the formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three imidazolium-based ILs (dimethylimidazolium dimethylphosphate [MMIm][Me(2)PO(4)], 1-butyl-3-methylimidazolium acetate [BMIm][CH(3)COO], and dimethylimidazolium methylphosphonate [MMIm][CH(3)HPO(2)(OCH(3))]) is studied by fluorescence spectroscopy. The UV-vis spectroscopic analysis shows that the decrease of the FDH fluorescence is not only due to the high light absorption of these ILs. The Stern-Volmer analysis clearly shows that these ILs are quenchers of the indole fluorescence, while this quenching property is not found when imidazole is used. Fluorescence spectra of the FDH in the presence of the ILs show that a maximal ionic liquid concentration (MILc), which could be used for steady-state fluorescence study, should be defined. Therefore, FDH conformation could not be directly related to the decrease of its fluorescence in ILs. Nevertheless, the structure of the FDH could be evaluated with dynamic and static quenchers like iodide or acrylamide, used below the MILc, demonstrating the relevance of this parameter. The Stern-Volmer constants (K(SV)(Q)), calculated in the presence of the different ILs, demonstrate that these ILs are strong denaturing agents, each one acting with a different mechanism. This report provides a suitable and easy-to-apply method to study any enzyme structures in ILs by steady-state fluorescence

Ionic liquid-inspired cations covalently bound to FDH improve its stability and activity in IL

International audienceIonic liquids (ILs) are important new solvents for electrochemistry and biocatalysis, but dehydrogenases usually do not work in ionic liquids. Adding more than 40 % (v/v) of the water miscible ionic liquid [MMIm][Me2PO4] (MMIm: 1-methyl-3-methyl imidazolium dimethylphosphate) inactivates the formate dehydrogenase (FDH) from Candida boidinii. The grafting of a variety of IL-inspired hydroxylated cations (hydroxyalkyl imidazolium, hydroxylalkyl pyrrolydinium, and cholinium) on the enzyme through lysine coupling was performed to understand the relationship between grafted cation, enzyme activity, and protein structure. As a general trend, the more a cation was kosmotropic (e.g., presenting a high B coefficient), the larger the resulting modifications were. The ability of these enzymes to bind the substrates was studied by fluorescence quenching in the presence of nicotinamide adenine dinucleotide (NAD+) and azide. The dissociation constant for NAD+ was only slightly affected by the grafting of the cations, however, the quenching efficiency was reduced. Azide binding was more affected by the cations. In the presence of 30 % (v/v) [MMIm][Me2PO4], the catalytic efficiency of the wild-type enzyme was reduced by 2.8 fold. In comparison, the catalytic efficiency of the modified FDH was preserved in these conditions and even improved after modification by hydroxypropyl imidazolium. The grafting of the chaotropic cations prevented the unfolding of the FDH due to [MMIm][Me2PO4]