Reappraisal of Vipera aspis Venom Neurotoxicity

BACKGROUND: The variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA(2) neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA(2) composition of the snakes captured in the same areas. [br/] METHODOLOGY/PRINCIPAL FINDINGS: We conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA(2)s. We used SELDI technology to study the diversity of PLA(2) in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA(2)s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA(2) venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains. [br/] CONCLUSIONS/SIGNIFICANCE: The association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA(2) genome and transcriptome data

The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change

Contribution a l'etude des composants de l'adenyl-cyclase cerebrale

SIGLECNRS T 58548 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Efficient monitoring of enzymatic conjugation reaction by surface-enhanced laser desorption/ionization time of flight mass spectrometry for process optimization.

International audienceEfficient analysis of bioconjugation reactions is one the most challenging task for optimizing and eventually achieving the reproducible production of large amount of conjugates. In particular, the complexity of some reaction mixtures precludes the use of most of the existing methods, because of the presence of large amounts of contaminants. As an alternative method, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for monitoring an in vitro enzymatic transglycosylation of N-acetylgalactosamine (GalNAc) residues to a recombinant mucin protein MUC6. For this reaction, catalyzed by the uridine 5'-diphospho-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), we used either a recombinant ppGalNAc-T1 or a mixture of ppGalNAc-Ts contained in MCF7 tumor cell extracts. In the present study, we show that SELDI-TOF MS offers unique advantages over the traditional methodologies. It is a rapid, accurate, sensitive, reproducible, and very convenient analytical method for monitoring the course of a bioconjugation, even in heterogeneous samples such as cell extracts. SELDI-TOF MS proved very useful for optimizing the reaction parameters of the transglycosylation and for achieving the large scale preparation of Tn antigen-glycosylated mucins for antitumor immunotherapy applications

Identification of Conserved Amino Acid Residues of the Salmonella σS Chaperone Crl Involved in Crl-σS Interactions ▿

Proteins that bind σ factors typically attenuate the function of the σ factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor σS (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of σS-dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a Δcrl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-σS interaction and that residues 1 to 71 in σS are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli

New Markers in Anopheles gambiae Salivary Glands After Plasmodium berghei Infection.

Abstract In malaria, mosquito saliva and salivary glands play central roles in the multi-faceted interactions that occur among the parasite, its vector, and its host. Analyzing the processes involved in the survival and maintenance of the Plasmodium parasite in mosquito organs, and in its transmission into vertebrate hosts, may lead to the identification of new molecular targets for parasite control. We used comparative two-dimensional gel polyacrylamide electrophoresis (2D-PAGE), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), and high-performance liquid chromatography (HPLC), followed by Edman sequencing, to study saliva and salivary gland samples from Anopheles gambiae mosquitoes infected or not with Plasmodium berghei. Quantitative 2D-PAGE profile analysis showed that the intensities of seven spots were affected by the presence of the parasite in the salivary glands. Most of the proteins identified possessed a signal peptide. SELDI-TOF-MS revealed 32 proteins/peptides whose peak intensities differed between the Plasmodium-infected and non-infected control groups. Quantitative comparison of HPLC profiles of low-molecular-weight components from salivary gland extracts revealed several peptides and proteins with levels that were modulated by parasite infection. The results of these complementary approaches suggest that the infection of female A. gambiae mosquitoes by P. berghei alters the production levels of several salivary gland proteins and peptides, some of which (e.g., protein cE5, B3VDI9_ANOGA, and AGAP008216-PA) are known or predicted to be secreted in saliva and involved in blood feeding

Antiviral Drug Discovery Strategy Using Combinatorial Libraries of Structurally Constrained Peptides

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton.

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization