Relationship between humoral response against hepatitis C virus and disease overcome

International audienceConclusionHumoral response against hepatitis C virus linear epitopes is partly modified according to the disease state. This study highlights the importance of considering relative quantities of antibodies with different specificities rather than the amount of each antibody.Hepatitis C virus infection leads to liver disease whose severity can range from mild to serious lifelong illness. However the parameters involved in the evolution of the disease are still unknown. Among other factors, the virus-elicited antibody profile is suspected to play a role in the outcome of the disease. Analysis of the relationship between anti-virus antibodies and disease state requires the analysis of a large number of serums from patients (hepatitis C virus+) and of epitopes from the viral proteins. Such a study would benefit from microarray-based screening systems that are appropriate for high-throughput assays.We used a method combining peptide chips and surface plasmon resonance imaging previously shown to be suitable for analyzing complex mediums and detecting peptide-protein interactions. 56 peptides covering the entire viral proteome were grafted on chips and their interaction with antibodies present in the 68 injected serums from infected and non-infected donors was measured. Statistical analyses were conducted to determine a possible relationship between antibodies (specificity and amount) and disease states.A good discrimination between infected and non-infected donors validated our approach, and several correlations between antibodies profiles and clinical parameters have been identified. In particular, we demonstrated that ratios between particular antibodies levels allow for accurate discrimination of patients according to their pathologic states

A Membrane Protein/Signaling Protein Interaction Network for Arabidopsis Version AMPv2

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa

Etude proteomique de la vacuole d'Arabidopsis thaliana en vue de l'identification d'acteurs protéiques impliqués dans la détoxication du cadmium

The vacuole is an organelle playing an important role in several plant cellular processes including protection against toxins. Among these, cadmium is a major pollutant, affecting plant physiology, and the vacuole is known for its capacity to store it. However, proteins involved in the transport across the tonoplast and in the chelation of cadmium inside the vacuoles are not well known. To better understand the function of this organelle we initiated a proteomics study to characterize vacuoles from Arabidopsis thaliana cells. This has allowed setting up tools and methods useful to study the dynamics of this proteome as a function of a cadmium exposure. In the first part of my work, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis thaliana cell cultures using Ficoll density gradients. Absence of significant contamination by other cellular compartments was validated by Western-blot and enzymatic measurements. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, the protein components present in both membrane and soluble fractions of the Arabidopsis cell vacuoles were analysed, leading to the identification of 689 proteins including 110 transporters. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein-tagged fusion proteins. This first work was completed by a more extensive study of the vacuole proteome, in order to analyse it at a quantitative and organisational levels. IEF / SDS-PAGE were performed with the soluble proteins of vacuoles, resolved spots were quantified using PDQuest software and identified by mass spectrometry (collaboration with LEDyP, CEA Grenoble). Some proteins that were not identified in the previous study were found, leading to a list of 709 vacuolar proteins, among which some cytosolic proteins were identified. A map of a cytosolic protein-enriched fraction was made and compared with that of the soluble vacuolar proteome in order to understand the origin of the presence of the cytosolic proteins in our samples. These data combined with those from partial digestion experiments of intact vacuoles (performed with proteinase K) suggest that the main part of these cytosolic proteins are localized outside the vacuoles and are co-purified because of specific interactions of these proteins with the tonoplast. Some of them are also inside the vacuole, probably in the process of being degraded. Finally, supramolecular organization of the vacuolar proteome was evaluated using native electrophoresis (BN-PAGE), and putative isolated complexes were identified by mass spectrometry. These experiments confirmed the presence of some known complexes (vacuolar ATPase for example) and suggested new complexes containing proteases and/or glycosyl hydrolases. A last aspect of my work was the development of an informatics tool, designed to store and exploit data from high throughput analyses. Such a structure has allowed cross-analysis of data from vacuolar proteomics work together with those from microarray transcriptomics experiments. This led to the identification of several vacuolar proteins transcriptionally regulated during cadmium stress. Among them, DWARF1, which catalyses a key step in the brassinosteroids pathway, was selected to further analyse its possible implication in the mechanisms of cadmium response. We observed that brassinosteroids modulate Arabidopsis thaliana seedlings' response to cadmium, and that this mechanism could not be linked to any known tolerance process, suggesting the existence of a new tolerance / sensitivity factor. This work is a basis for the study of the changes occuring in the vacuolar proteome under cadmium stress, which should lead to a better comprehension of this organelle's involvement in plant response to heavy metals. We set up new methods and tools, and obtained several results that highlight new aspects of plant vacuole physiology.La vacuole est un organite qui joue un rôle important dans de nombreux processus de la cellule végétale, et en particulier dans la protection contre les toxiques cellulaires. Parmi ceux-ci, le cadmium est un polluant courant qui affecte les fonctions physiologiques de la plante. La vacuole est connue pour sa capacité à séquestrer les ions métalliques présents dans le cytosol. Toutefois, les acteurs protéiques de cette compartimentation, et notamment les transporteurs, ne sont pas bien connus. Afin de mieux comprendre le rôle de cet organite dans les mécanismes de protection contre le stress métallique, nous avons mis en place une série d'outils d'analyse du protéome vacuolaire, dans le but de réaliser une caractérisation protéomique de référence de cet organite, utile pour l'étude de sa dynamique en conditions de stress. Mon travail a consisté dans un premier temps à mettre au point une méthode de purification de vacuoles à partir de cellules en culture d'Arabidopsis thaliana. La pureté des échantillons obtenus a été confirmée à l'aide de tests biochimiques (western-blots et mesures d'activités enzymatiques) et nous avons pu initier l'analyse par spectrométrie de masse des constituants protéiques de la membrane et de la fraction soluble de la vacuole. Celle-ci a permis d'identifier 689 protéines non-redondantes, dont 110 transporteurs. La localisation in vivo de 5 d'entre elles a été réalisée via l'expression in planta de ces protéines fusionnées à la GFP. Cette première approche protéomique a été complétée d'une étude plus approfondie du protéome vacuolaire, visant à en acquérir des notions quantitatives et organisationnelles. Pour cela, des gels d'électrophorèses bidimensionnelles (IEF / SDS-PAGE) ont été réalisés à partir de la fraction soluble des vacuoles. Les spots résolus ont été quantifiés via le logiciel d'analyse d'images PDQuest et identifiés par spectrométrie de masse. Cette étude a permis de mettre en évidence un certain nombre de protéines majeures de ce compartiment et a porté le nombre total de protéines vacuolaires identifiées par nos travaux à 709. Quelques protéines connues pour être cytosoliques ont toutefois été retrouvées, et une cartographie du protéome cytosolique a également été réalisée afin de la comparer avec celle du protéome soluble de la vacuole et tenter de mieux comprendre l'origine de ces protéines. Cette analyse a été complétée d'une expérience préliminaire de digestion de vacuoles intactes par la protéinase K. Les résultats obtenus suggèrent la présence de protéines à l'intérieur de la vacuole (probablement en cours de dégradation), et d'autres associées à la face externe du tonoplaste probablement de façon spécifique. Enfin, la présence de complexes protéiques a été évaluée à travers la réalisation d'électrophorèses en conditions non dénaturantes qui ont permis de retrouver des complexes connus (ATPase vacuolaire) mais aussi de mettre en évidence plusieurs complexes putatifs de protéines diverses (protéases, glycosidases ...). Un dernier aspect de mon travail a enfin consisté à développer un outil informatique d'exploitation de données d'analyses à haut débit. La confrontation, grâce à cette structure, des résultats de protéomiques vacuolaires et d'autres d'expression génique lors d'un stress cadmium ont permis d'identifier des protéines présentes dans (ou associées à) la vacuole dont le niveau de transcrit est modulé lors d'un stress. Ces analyses croisées ont notamment mis en évidence la protéine DWARF1, qui catalyse une étape de la biosynthèse des brassinostéroïdes, une classe d'hormones. L'étude de l'implication de cette hormone a alors montré que celle-ci est capable de moduler la tolérance au cadmium de plantules, très probablement via des mécanismes qui n'ont encore jamais été identifiés. L'ensemble de ce travail constitue une base pour l'étude ultérieure de la dynamique du protéome vacuolaire. Il propose des méthodes et des outils d'analyse, ainsi qu'une série de données de référence, pour mieux comprendre les processus vacuolaires de la détoxication métallique, et a d'ores et déjà permis de mettre en évidence des aspects nouveaux du fonctionnement de cet organite

Etude proteomique de la vacuole d'Arabidopsis thaliana en vue de l'identification d'acteurs protéiques impliqués dans la détoxication du cadmium

The vacuole is an organelle playing an important role in several plant cellular processes including protection against toxins. Among these, cadmium is a major pollutant, affecting plant physiology, and the vacuole is known for its capacity to store it. However, proteins involved in the transport across the tonoplast and in the chelation of cadmium inside the vacuoles are not well known. To better understand the function of this organelle we initiated a proteomics study to characterize vacuoles from Arabidopsis thaliana cells. This has allowed setting up tools and methods useful to study the dynamics of this proteome as a function of a cadmium exposure. In the first part of my work, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis thaliana cell cultures using Ficoll density gradients. Absence of significant contamination by other cellular compartments was validated by Western-blot and enzymatic measurements. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, the protein components present in both membrane and soluble fractions of the Arabidopsis cell vacuoles were analysed, leading to the identification of 689 proteins including 110 transporters. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein-tagged fusion proteins. This first work was completed by a more extensive study of the vacuole proteome, in order to analyse it at a quantitative and organisational levels. IEF / SDS-PAGE were performed with the soluble proteins of vacuoles, resolved spots were quantified using PDQuest software and identified by mass spectrometry (collaboration with LEDyP, CEA Grenoble). Some proteins that were not identified in the previous study were found, leading to a list of 709 vacuolar proteins, among which some cytosolic proteins were identified. A map of a cytosolic protein-enriched fraction was made and compared with that of the soluble vacuolar proteome in order to understand the origin of the presence of the cytosolic proteins in our samples. These data combined with those from partial digestion experiments of intact vacuoles (performed with proteinase K) suggest that the main part of these cytosolic proteins are localized outside the vacuoles and are co-purified because of specific interactions of these proteins with the tonoplast. Some of them are also inside the vacuole, probably in the process of being degraded. Finally, supramolecular organization of the vacuolar proteome was evaluated using native electrophoresis (BN-PAGE), and putative isolated complexes were identified by mass spectrometry. These experiments confirmed the presence of some known complexes (vacuolar ATPase for example) and suggested new complexes containing proteases and/or glycosyl hydrolases. A last aspect of my work was the development of an informatics tool, designed to store and exploit data from high throughput analyses. Such a structure has allowed cross-analysis of data from vacuolar proteomics work together with those from microarray transcriptomics experiments. This led to the identification of several vacuolar proteins transcriptionally regulated during cadmium stress. Among them, DWARF1, which catalyses a key step in the brassinosteroids pathway, was selected to further analyse its possible implication in the mechanisms of cadmium response. We observed that brassinosteroids modulate Arabidopsis thaliana seedlings' response to cadmium, and that this mechanism could not be linked to any known tolerance process, suggesting the existence of a new tolerance / sensitivity factor. This work is a basis for the study of the changes occuring in the vacuolar proteome under cadmium stress, which should lead to a better comprehension of this organelle's involvement in plant response to heavy metals. We set up new methods and tools, and obtained several results that highlight new aspects of plant vacuole physiology.La vacuole est un organite qui joue un rôle important dans de nombreux processus de la cellule végétale, et en particulier dans la protection contre les toxiques cellulaires. Parmi ceux-ci, le cadmium est un polluant courant qui affecte les fonctions physiologiques de la plante. La vacuole est connue pour sa capacité à séquestrer les ions métalliques présents dans le cytosol. Toutefois, les acteurs protéiques de cette compartimentation, et notamment les transporteurs, ne sont pas bien connus. Afin de mieux comprendre le rôle de cet organite dans les mécanismes de protection contre le stress métallique, nous avons mis en place une série d'outils d'analyse du protéome vacuolaire, dans le but de réaliser une caractérisation protéomique de référence de cet organite, utile pour l'étude de sa dynamique en conditions de stress. Mon travail a consisté dans un premier temps à mettre au point une méthode de purification de vacuoles à partir de cellules en culture d'Arabidopsis thaliana. La pureté des échantillons obtenus a été confirmée à l'aide de tests biochimiques (western-blots et mesures d'activités enzymatiques) et nous avons pu initier l'analyse par spectrométrie de masse des constituants protéiques de la membrane et de la fraction soluble de la vacuole. Celle-ci a permis d'identifier 689 protéines non-redondantes, dont 110 transporteurs. La localisation in vivo de 5 d'entre elles a été réalisée via l'expression in planta de ces protéines fusionnées à la GFP. Cette première approche protéomique a été complétée d'une étude plus approfondie du protéome vacuolaire, visant à en acquérir des notions quantitatives et organisationnelles. Pour cela, des gels d'électrophorèses bidimensionnelles (IEF / SDS-PAGE) ont été réalisés à partir de la fraction soluble des vacuoles. Les spots résolus ont été quantifiés via le logiciel d'analyse d'images PDQuest et identifiés par spectrométrie de masse. Cette étude a permis de mettre en évidence un certain nombre de protéines majeures de ce compartiment et a porté le nombre total de protéines vacuolaires identifiées par nos travaux à 709. Quelques protéines connues pour être cytosoliques ont toutefois été retrouvées, et une cartographie du protéome cytosolique a également été réalisée afin de la comparer avec celle du protéome soluble de la vacuole et tenter de mieux comprendre l'origine de ces protéines. Cette analyse a été complétée d'une expérience préliminaire de digestion de vacuoles intactes par la protéinase K. Les résultats obtenus suggèrent la présence de protéines à l'intérieur de la vacuole (probablement en cours de dégradation), et d'autres associées à la face externe du tonoplaste probablement de façon spécifique. Enfin, la présence de complexes protéiques a été évaluée à travers la réalisation d'électrophorèses en conditions non dénaturantes qui ont permis de retrouver des complexes connus (ATPase vacuolaire) mais aussi de mettre en évidence plusieurs complexes putatifs de protéines diverses (protéases, glycosidases ...). Un dernier aspect de mon travail a enfin consisté à développer un outil informatique d'exploitation de données d'analyses à haut débit. La confrontation, grâce à cette structure, des résultats de protéomiques vacuolaires et d'autres d'expression génique lors d'un stress cadmium ont permis d'identifier des protéines présentes dans (ou associées à) la vacuole dont le niveau de transcrit est modulé lors d'un stress. Ces analyses croisées ont notamment mis en évidence la protéine DWARF1, qui catalyse une étape de la biosynthèse des brassinostéroïdes, une classe d'hormones. L'étude de l'implication de cette hormone a alors montré que celle-ci est capable de moduler la tolérance au cadmium de plantules, très probablement via des mécanismes qui n'ont encore jamais été identifiés. L'ensemble de ce travail constitue une base pour l'étude ultérieure de la dynamique du protéome vacuolaire. Il propose des méthodes et des outils d'analyse, ainsi qu'une série de données de référence, pour mieux comprendre les processus vacuolaires de la détoxication métallique, et a d'ores et déjà permis de mettre en évidence des aspects nouveaux du fonctionnement de cet organite

R. S. WebTool, a web server for random sampling-based significance evaluation of pairwise distances.

International audiencePairwise comparison of data vectors represents a large part of computational biology, especially with the continuous increase in genome-wide approaches yielding more information from more biological samples simultaneously. Gene clustering for function prediction as well as analyses of signalling pathways and the time-dependent dynamics of a system are common biological approaches that often rely on large dataset comparison. Different metrics can be used to evaluate the similarity between entities to be compared, such as correlation coefficients and distances. While the latter offers a more flexible way of measuring potential biological relationships between datasets, the significance of any given distance is highly dependent on the dataset and cannot be easily determined. Monte Carlo methods are robust approaches for evaluating the significance of distance values by multiple random permutations of the dataset followed by distance calculation. We have developed R. S. WebTool (http://rswebtool.kwaklab.org), a user-friendly online server for random sampling-based evaluation of distance significances that features an array of visualization and analysis tools to help non-bioinformaticist users extract significant relationships from random noise in distance-based dataset analyses

How to define a rejection class based on model learning?

International audienceIn supervised classification, the learning process typically trains a classifier to optimize the accuracy of classifying data into the classes that appear in the learning set, and only them. While this framework fits many use cases, there are situations where the learning process is knowingly performed using a learning set that only represents the data that have been observed so far among a virtually unconstrained variety of possible samples. It is then crucial to define a classifier which has the ability to reject a sample, i.e., to classify it into a rejection class that has not been yet defined. Although obvious solutions can add this ability a posteriori to a classifier that has been learned classically, a better approach seems to directly account for this requirement in the classifier design. In this paper, we propose an innovative learning strategy for supervised classification that is able, by design, to reject a sample as not belonging to any of the known classes. For that, we rely on modeling each class as the combination of a probability density function (PDF) and a threshold that is computed with respect to the other classes. Several alternatives are proposed and compared in this framework. A comparison with straightforward approaches is also provided

Machine-learning assisted phenotyping: From fungal morphology to mode of action hypothesis

International audienceBeyond growth inhibition, fungicides can also trigger specific morphological modifications visualized under transmitted light microscopy. These morphological changes result from the activity of a given compound via the inhibition of a molecular target, commonly named as its mode of action (MoA). We are hence able to classify different molecules into their respective MoA by observing their phenotypic signature, and even to detect new MoA with unknown phenotypic effect for further deconvolution. The aim of the presented work is to develop a robust method for automated recognition and classification of these phenotypic signatures in order to lead to a Mode of Action hypothesis. We compare two machine-learning methods (Random forest and Convolutional Neural Network) for direct processing of images generated on the grey mold Botrytis cinerea subjected to different antifungal molecules. © Bayer | Abteilung | Verfasser | Datu

New insights into the regulation of phytochelatin biosynthesis in A. thaliana cells from metabolite profiling analyses.

In higher plants and some fungi, heavy metals induce the synthesis of chelating peptides known as phytochelatins (PCs). They are characterized by the general structure (gamma-Glu-Cys)(n)-Gly, but in some plant species, the C-terminal glycine can be replaced by serine, glutamine, glutamate or alanine, leading to iso-phytochelatins (iso-PCs). Although the distribution of iso-PCs is considered to differ from one species to another, we previously showed that Arabidopsis thaliana (A. thaliana) cells are able to synthesize most PC-related peptides (PCs and iso-PCs) described in the literature. We also observed an accumulation of the dipeptide gamma-glutamylcysteine (gamma-EC) when cadmium (Cd) (200 muM) was added to the culture medium, suggesting that either glutathione synthetase or glycine availability could be a limiting factor for the biosynthesis of PC-related peptides. In this context, the aim of the present work was to seek new insights into the regulation of PC synthesis by performing metabolic profiling using liquid chromatography-mass spectrometry. The levels of PC-related peptides and their precursors were measured in A. thaliana cells following Cd exposure. A range of doses (0, 50, 200 and 400 muM CdNO(3)) and kinetic studies (from 1 to 48 h) showed a dose threshold (50 muM CdNO(3)) and a lag time between the appearance of PCs and iso-PCs concomitant with the gamma-EC accumulation induced by Cd, occurring at cadmium concentrations above 50 muM. This accumulation was suppressed by supplementation of the culture medium with 25 mM glycine. Glycine supplementation had a limited impact on the concentrations of glutathione and PCs whereas the levels of most iso-PCs were significantly increased. Taken together, these results indicate that GSH is involved in the biosynthesis of the iso-PCs in vivo, and that the biosynthesis of PC-related peptides is limited by the availability of glycine in the presence of high cadmium concentrations