Spectrométrie de masse des modifications induites ou post-traductionnelles de protéines : méthodologie et application à des protéines d’intérêt thérapeutique

Protein modifications, whether post-translational (PTMs) or chemically induced, play a crucial role on the activity of proteins. Mass spectrometry (MS) techniques such as HRMS, CID/ETD MS/MS, and biochemistrybased methods for structural and kinetic characterization of protein-ligand complexes and PTMs have been developed. MS combined with several biochemical tools has been used to sequence the proteinase inhibitor gregline and to detect a novel PTM. A similar approach shows that the transposase MOS1, a model for the design of HIV integrase inhibitors, is both phosphorylated and acetylated. For the lyase Abf2, a strategy of trapping, purification, proteolysis, and DNA hydrolysis of the Abf2-DNA covalent complex, coupled to MS analysis, has been developed. Finally, the interaction between the metastasis suppressor hPEBP1 and locostatin was dissected. Upon binding to hPEBP1, locostatin undergoes hydrolysis. To identify the site targeted by locostatin, the conditions of reaction and proteolysis were optimized. The qualitative approach reveals the presence of non-specific reactions, leading to the development of 1) a mathematical model to determine the optimum bound fraction for discriminating the specific site from non-specific sites, and 2) a method for the parallel and exhaustive quantification of the degree of modification of all modified sites of a protein. These tools are widely applicable to covalent protein ligands and/or PTMs.Les modifications de protéines, qu’elles soient post-traductionnelles (PTMs) ou induites chimiquement, ont une influence considérable sur l'activité des protéines. Des méthodes de spectrométrie de masse (MS) HRMS, MS/MS CID et ETD, et de biochimie ont été développées pour la caractérisation structurale et cinétique de complexes protéine-ligand et de PTMs, dans le but de disséquer leur mécanisme et de concevoir des médicaments covalents contre des protéines liant des protéases, des kinases, ou l'ADN. La MS combinée avec des outils biochimiques a permis de séquencer l'inhibiteur de protéases grégline, et de détecter une PTM originale. De même, la transposase MOS1, modèle de l'intégrase du VIH pour la conception d'inhibiteurs, s'avère être à la fois acétylée et phosphorylée. Pour la lyase Abf2, une stratégie de piégeage, purification, protéolyse et hydrolyse ADN du complexe covalent Abf2-ADN, couplée à l’analyse MS, a été développée. Enfin, l’interaction entre le surpresseur de métastase hPEBP1 et la locostatine a été disséquée sur la protéine entière et par approche bottom-up. La locostatine s’hydrolyse en butyrate après fixation. Afin d’identifier le site ciblé par la locostatine, les conditions de réaction et de protéolyse ont été optimisées. La présence de réactions non spécifiques a conduit au développement 1) d'un modèle mathématique permettant de déterminer la fraction de liaison optimale pour discriminer le site spécifique des sites non-spécifiques, et 2) d'une méthode pour la quantification parallèle et exhaustive du degré de modification de tous les sites modifiés d'une protéine. Ces outils sont applicables aux ligands covalents de protéines et/ou à leurs PTMs

Mass spectrometry for induced or post-translational modifications : methodology and application to proteins of therapeutic interest

Les modifications de protéines, qu’elles soient post-traductionnelles (PTMs) ou induites chimiquement, ont une influence considérable sur l'activité des protéines. Des méthodes de spectrométrie de masse (MS) HRMS, MS/MS CID et ETD, et de biochimie ont été développées pour la caractérisation structurale et cinétique de complexes protéine-ligand et de PTMs, dans le but de disséquer leur mécanisme et de concevoir des médicaments covalents contre des protéines liant des protéases, des kinases, ou l'ADN. La MS combinée avec des outils biochimiques a permis de séquencer l'inhibiteur de protéases grégline, et de détecter une PTM originale. De même, la transposase MOS1, modèle de l'intégrase du VIH pour la conception d'inhibiteurs, s'avère être à la fois acétylée et phosphorylée. Pour la lyase Abf2, une stratégie de piégeage, purification, protéolyse et hydrolyse ADN du complexe covalent Abf2-ADN, couplée à l’analyse MS, a été développée. Enfin, l’interaction entre le surpresseur de métastase hPEBP1 et la locostatine a été disséquée sur la protéine entière et par approche bottom-up. La locostatine s’hydrolyse en butyrate après fixation. Afin d’identifier le site ciblé par la locostatine, les conditions de réaction et de protéolyse ont été optimisées. La présence de réactions non spécifiques a conduit au développement 1) d'un modèle mathématique permettant de déterminer la fraction de liaison optimale pour discriminer le site spécifique des sites non-spécifiques, et 2) d'une méthode pour la quantification parallèle et exhaustive du degré de modification de tous les sites modifiés d'une protéine. Ces outils sont applicables aux ligands covalents de protéines et/ou à leurs PTMs.Protein modifications, whether post-translational (PTMs) or chemically induced, play a crucial role on the activity of proteins. Mass spectrometry (MS) techniques such as HRMS, CID/ETD MS/MS, and biochemistrybased methods for structural and kinetic characterization of protein-ligand complexes and PTMs have been developed. MS combined with several biochemical tools has been used to sequence the proteinase inhibitor gregline and to detect a novel PTM. A similar approach shows that the transposase MOS1, a model for the design of HIV integrase inhibitors, is both phosphorylated and acetylated. For the lyase Abf2, a strategy of trapping, purification, proteolysis, and DNA hydrolysis of the Abf2-DNA covalent complex, coupled to MS analysis, has been developed. Finally, the interaction between the metastasis suppressor hPEBP1 and locostatin was dissected. Upon binding to hPEBP1, locostatin undergoes hydrolysis. To identify the site targeted by locostatin, the conditions of reaction and proteolysis were optimized. The qualitative approach reveals the presence of non-specific reactions, leading to the development of 1) a mathematical model to determine the optimum bound fraction for discriminating the specific site from non-specific sites, and 2) a method for the parallel and exhaustive quantification of the degree of modification of all modified sites of a protein. These tools are widely applicable to covalent protein ligands and/or PTMs

Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features

International audienceThe crystallization and structure determination of integral membrane proteins remains a difficult task relying on a good understanding of the behavior of the protein for success. To date, membrane protein structures are still far outnumbered by soluble protein structures. Mass spectrometry is a powerful and versatile tool offering deep insights into the state of the integral membrane protein the structuralist intends to crystallize. With appropriate sample preparation methods, it provides information that can sometimes prove critical at various stages of the structure determination process, from protein expression to model building. Moreover, valuable knowledge is gained when the identified structural features underlie important functional aspects. Electrospray and matrix assisted laser desorption ionization (MALDI) methods, however, face a particular challenge when dealing with integral membrane proteins. A MALDI method specifically optimized for membrane protein analysis is presented here, with detailed information on the sample preparation and deposition, as well as guidelines for domain determination by limited proteolysis. MALDI-time of flight mass spectrometry can be used to do a proper inventory of initiation sites, to tailor a protein to a stable, well-folded form, and to evaluate selenomethionine replacement. These approaches are illustrated with a few examples drawn from the structural biology of ion channels

A sDOE (simple design-of-experiment) approach for parameter optimization in mass spectrometry. Part 1. Parameter selection and interference effects in topdown ETD fragmentation of proteins in a UHR-QTOF instrument.

International audienceDesign-of-experiment (DOE) approaches, originally conceived by J. Fischer, are widely applied in industry, particularly in the context of production for which have been greatly expended. In a research and development context, DOE can be of great use for method development. Specifically, DOE can greatly speed up instrument parameter optimization by first identifying parameters that are critical to a given outcome, showing parameter interdependency where it occurs, and accelerating optimization of said parameters using matrices of experimental conditions. While DOE approaches have been applied in mass spectrometry experiments, they have so far failed to gain widespread adoption. This could be attributed to the fact that DOE can get quite complex and daunting to the everyday user. Here we make the case that a subset of DOE tools, here after called SimpleDOE (sDOE), can make DOE accessible and useful to the Mass Spectrometry community at large. We illustrate the progressive gains from a purely manual approach to sDOE through a stepwise optimization of parameters affecting the efficiency of top-down ETD fragmentation of proteins on a high-resolution Q-TOF mass spectrometer, where the aim is to maximize sequence coverage of fragmentation events

Protein profiling by MS shows evidence of royal protein pheromones in the cuticle extracts of the termite Reticulitermes flavipes

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Somatic embryogenesis and transformation of the diploid Rosa chinensis cv Old Blush

International audienceSomatic embryogenesis was induced from in vitro-derived leaf explants of Rosa chinensis cultivar (cv) Old Blush. Calli producing embryos with expanded cotyledons (RcOBType1 embryos) were obtained. Further refinements of the callus maintenance medium generated a more typical rose embryogenic callus (RcOBType2) displaying high levels of secondary embryogenesis and embryos with limited cotyledon expansion Agrobacterium tumefaciens-mediated transformation assays using β-glucuronidase (GUS) reporter gene showed that both types of embryos were competent for transformation. Under selection conditions, transformed RcOBType1 explants produced non chimaeric transformed embryos, from which shoots could be adventitiously regenerated. In contrast to RcOBType1, transformed RcOBType2 embryos directly yielded transformed shoots when repeatedly cultured in selective regeneration conditions. Transformation efficiency ranged between three to nine percent and shoots suitable for rooting were obtained within 6-8 months. Transgenic plants were transferred into the greenhouse and molecularly confirmed. The availability of transformation methods in a diploid rose, R. chinensis cv. Old Blush, will be useful for gene functional studies

Reproductives signature revealed by protein profiling and behavioral bioassays in termite

Abstract Proteins are known to be social interaction signals in many species in the animal kingdom. Common mediators in mammals and aquatic species, they have seldom been identified as such in insects' behaviors. Yet, they could represent an important component to support social signals in social insects, as the numerous physical contacts between individuals would tend to favor the use of contact compounds in their interactions. However, their role in social interactions is largely unexplored: are they rare or simply underestimated? In this preliminary study, we show that, in the termite Reticulitermes flavipes, polar extracts from reproductives trigger body-shaking of workers (a vibratory behavior involved in reproductives recognition) while extracts from workers do not. Molecular profiling of these cuticular extracts using MALDI-TOF mass spectrometry reveals higher protein diversity in reproductives than in workers and a sex-specific composition exclusive to reproductives. While the effects observed with extracts are not as strong as with live termites, these results open up the intriguing possibility that social signaling may not be limited to cuticular hydrocarbons or other non-polar, volatile chemicals as classically accepted. Our results suggest that polar compounds, in particular some of the Cuticular Protein Compounds (CPCs) shown here by MALDI to be specific to reproductives, could play a significant role in insect societies. While this study is preliminary and further comprehensive molecular characterization is needed to correlate the body-shaking triggering effects with a given set of polar compounds, this exploratory study opens new perspectives for understanding the role of polar compounds such as proteins in caste discrimination, fertility signaling, or interspecific insect communication

Exposition humaine aux fongicides SDHIs par les denrées alimentaires

International audienceDepuis la fin de la seconde guerre mondiale, l’utilisation de produits phytosanitaires chimiques dans l’agriculture conventionnelle s’est développée et intensifiée. Parmi ces produits, les fongicides sont très souvent employés pour la protection des cultures et/ou pendant le stockage des récoltes afin d’éliminer l’apparition de champignons et de moisissures. La famille de fongicides inhibiteur de la succinate déshydrogénase (SDHI) sont utilisées pour certains depuis la fin des années 1960 et d’autres, plus récent, depuis une dizaine d’année. Le mécanisme d’action des SDHIs est d’inhiber la succinate déshydrogénase, un complexe enzymatique, acteur clé dans la chaine respiratoire mitochondriale et donc essentiel à la vie. Hors la SDH est conservée au cours de l’évolution, on la retrouve donc dans quasiment toutesles espèces vivantes de la plante, animaux jusqu’à l’Homme. Une étude récente (Bénit et al., 2019) établis que les SDH de vers de terre, d’abeilles et des êtres humains sont sensibles à 8 SDHI testé.... Chez l’Homme, l’inhibition complète de l’activité SDH entraine des modifications métabolomique, transcriptomique et épigénomique entrainant le développement de tumeurs et de cancers. Différentes études ont pu montrer que la population française est exposée ou potentiellement exposée aux SDHIs par l’air (surveillance du boscalid et fluopyram, Atmo France et Associations agréées de surveillance de la qualité de l’air, 2019), l’alimentation (surveillance du boscalid, flutolanil et carboxin, ANSES, 2011), l’eau (surveillance du boscalid) et les sols. Dans ce contexte, le projet vise à caractériser l’exposition humaine à 12 SDHIs et leurs produits de transformations dans des matrices alimentaires. Une méthode générique sensible et fiable permettant leur quantifications grâce à l’association d’une étape d’extractions (QuEChERS) et d’une étape analytique par chromatographie liquide couplées à la spectrométrie de masse (LC-MS/MS) sera développée et optimisée. Une fois la méthode validée, elle sera appliquée pour mesurer l’exposition humaine par voie alimentaire (boissons, fruits, légumes, céréales, ...) collectés dans diverses sources d’approvisionnement d’un panier moyen français. Certaines expositions en lien avec la santé de l’abeille (miel, pollen), animale (tissus de poissons) et humaine (urine) seront également mesurées dans un second temps

The Interaction between the Drosophila EAG Potassium Channel and the Protein Kinase CaMKII Involves an Extensive Interface at the Active Site of the Kinase

International audienceThe Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. We have characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggests that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel´s kinase recognition sequence modulates the strength of the interaction between the channel and the kinase

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses

International audienceGenomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements