Molecular rules for selectivity in lipase-catalysed acylation of lysine

International audienceThe selectivity of L-lysine acylation by lauric acid catalysed by Candida antarctica lipase B (CALB) was investigated combining experimental and theoretical methodologies. Experiments showed the near-exclusive acylation of lysine ε-amino group; only traces of product resulting from the acylation of lysine α-amino group were observed fleetingly. Molecular modelling simulations were performed aiming to understand the molecular rules for selectivity. Flexible docking simulations combined with structural investigations into lysine/CALB binding modes also suggested the preferential acylation of lysine ε-amino group without, however, excluding the acylation of the lysine α-amino group. Electrostatic interaction energy between lysine and the residues covering the catalytic cavity was calculated in order to understand the discrimination between the two lysine amino groups. The results suggests that the proximity of the carboxylate group hinders the binding of the substrate in configurations enabling the N-acylation. Key interactions with the polar region covering the catalytic triad were identified and a plausible explanation for selectivity was proposed

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Synthèse enzymatique, modélisation moléculaire et caractérisation d'oligomères de flavonoïdes

Ce travail a pour objectif de mettre au point un procédé d oligomérisation de rutine et d esculine par la laccase de Trametes versicolor. Un procédé de synthèse en parallèle et d analyse en ligne par SEC-UV et par MALDI-TOF a été mis au point. L analyse par MALDI-TOF a révélé la formation d un simple pontage, allant jusqu au degré d oligomérisation 6 pour la rutine et 9 pour l esculine. Un pontage par liaison éther a été observé par FTIR dans le cas des oligorutines. L analyse par RMN a démontré la mise en place de liaisons tant C-C que C-O localisées sur la partie phénolique et la partie sucre des monomères. De faibles pH et températures favorisent l allongement de la chaîne, alors que l augmentation de la constante diélectrique du solvant ou de la température augmente la production des oligomères de rutine. La limitation de la masse de ces oligomères serait due à une inhibition de l enzyme, provoquée par les capacités chélatantes des oligomères. Une diminution du pouvoir antioxydant et une augmentation du pouvoir inhibiteur de la xanthine oxydase ont pu être observées lors de l accroissement de la masse des oligomères de rutine. Ces deux activités sont améliorées lors de l accroissement de la masse des oligomères d esculine. Pour ces deux types d oligomères, la solubilité dans l eau est fortement accrue. Dans le cas des oligorutines, cette forte augmentation a été corrélée à la mise en place d un réseau dense de liaisons hydrogène observé par modélisation moléculaire. Globalement, l approche par modélisation moléculaire dans le vide et dans le solvant a permis de dégager des relations structure-activité, reliant notamment le nombre de liaisons hydrogène à la solubilitéThe aim of this work is the elaboration of rutin and esculin oligomerization process by the laccase from Trametes versicolor. A parallel synthesis process and on-line analysis of reaction media by SEC-UV and MALDI-TOF have been elaborated. The MALDI-TOF analysis has revealed the formation of simple bridges between rutin and esculin units, up to degree of oligomerization of 6 and 9 respectively. An ether bond has been observed by FTIR spectrometry for the rutin oligomers. Finally, the NMR analysis has revealed the formation of C-C and C-O bridges both on phenolic and the sugar parts of the flavonoids. At low pH and temperature, the elongation of the chain is favored, whereas increasing the dielectric constant of the solvent or the temperature favors the production of rutin oligomers. The limitation of oligomers mass is explained by the inhibition of the enzyme, probably due to the highest chelation properties of oligomers. In the case of oligorutin, a decrease of antiradical activity and an increase of xanthine oxidase inhibitory activity have been observed when the oligomers molecular mass increases. In the case of esculin oligomers, these two activities increase with the increase of the oligomers mass. For these two types of oligomers, the water solubility is considerably increased. For the oligorutins, this augmentation has been correlated to a dense network of H-bonds, which has been demonstrated by molecular modeling. Globally, the molecular modeling approach in vacuum and in solvent has allowed to establish structure-activity relationshipNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Modélisation moléculaire de l'acétylation de la quercétine par des lipases (étude des interactions enzyme-substrat)

La quercétine (QCT) est un composé polyphénolique d'origine végétale connu pour ses activités antioxydantes et ses effets bénéfiques sur la santé. Sa solubilité, sa stabilité, sa biodisponibilité et ses activités biologiques peuvent être améliorées par une acylation sélective de ses groupements hydroxylés. Ce travail vise à étudier la possibilité d'une acétylation enzymatique de la QCT par la lipase B de Candida antarctica (CALB), la lipase la plus exploitée industriellement pour des estérifications régio- et énantiosélectives. Dans une perspective d'ingénierie rationnelle de l'enzyme, une démarche de modélisation moléculaire est mise en oeuvre pour mieux comprendre les interactions qui régissent le positionnement et l'orientation du substrat dans le site actif de la lipase. Dans une première partie expérimentale, l'absence d'activité d'acétylation de la CALB envers la QCT, en présence d'un excès d'acétate de vinyle, a été confirmé. Dans une seconde partie, cette inactivité de la CALB a été expliquée à l'aide de simulations de docking et de dynamique moléculaire. Elle résulte d'une orientation inappropriée du donneur d'acyle liée à la sérine catalytique et d'une proximité insuffisante des hydroxyles de la QCT vis-à-vis des résidus catalytiques. L'éloignement de la QCT de la triade catalytique est due à la rigidité de la molécule, l'étroitesse du site actif ainsi qu'à des interactions hydrophobes et électrostatiques entre le substrat et les résidus de la cavité. En revanche, cette approche de simulation moléculaire prédit un bon positionnement des deux substrats dans le site actif de la lipase de Pseudomonas cepacia (PCL), laquelle est capable d'acétyler la QCT. Dans une troisième partie, l'influence de mutations de deux résidus impliqués dans les liaisons de stabilisation hydrophobe de la QCT dans la CALB a été investiguée par simulation. La substitution d'isoleucines par des valines et des alanines conduit à une augmentation du volume de la poche catalytique et une mobilité accrue de la QCT. Mais ces mutations sont insuffisantes pour permettre un positionnement adéquat de l'acétate et de la QCT par rapport à la triade catalytique. La dernière partie focalise sur les interactions électrostatiques entre la QCT et le site actif de CALB. Les orientations du substrat dans la cavité suite à une méthylation ou une acétylation des groupements hydroxyles de la QCT sont préciséesQuercetin (QCT) is a plant-produced polyphenolic compound well-known for its antioxidant activities and beneficial health effects. Its solubility, stability, bioavailability and biological activities may be improved by a selective acylation of its hydroxyl groups. This work aims at studying the possibility of QCT enzymatic acetylation by Candida antarctica lipase B (CALB), the most industrially exploited lipase for regio- and enantioselective esterifications. In prospect of the rational enzyme design, a molecular modeling approach was implemented to understand the interactions that govern the substrate positioning and orientation in the lipase's active site. In a first experimental part, the absence of CALB acetylation activity towards quercetin in excess of vinyl acetate was confirmed. In a second part, this inactivity of CALB was explained by means of docking and molecular dynamics simulations. This results from an inappropriate positioning of the acyl donor linked to the catalytic serine and from an insufficient proximity of QCT hydroxyls vis-à-vis catalytic residues. The distance of QCT from the catalytic triad is due to its rigidity and to the narrow active site as well as to hydrophobic and electrostatic interactions between the substrate and the cavity residues. On the contrary, this molecular simulation approach predicts an appropriate positioning of both substrates in the active site of Pseudomonas cepacia lipase (PCL), which can perform QCT acetylation. In a third part, the impact of mutations of two residues implicated in the stabilization of QCT by hydrophobic interactions in CALB was investigated through simulations. The substitution of isoleucines by alanines and valines led to an increase in the catalytic pocket volume which intensified the mobility of QCT. However, these mutations are insufficient to allow an appropriate positioning of acetate and QCT in relation to the catalytic triad. The last part of this work focuses on the electrostatic interactions between QCT and CALB's active site. The substrate orientation in the cavity following methylation or acetylation of QCT's hydroxyl groups was clarifiedMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Extraction, identification, caractérisation des activités biologiques de flavonoïdes de Nitraria retusa et synthèse de dérivés acylés de ces molécules par voie enzymatique

Ce travail a consisté, dans un premier temps, à extraire et à identifier les flavonoïdes majeurs contenus dans les feuilles de Nitraria retusa et à évaluer leurs activités biologiques. Quatre flavonoïdes ont été identifiés dans les extraits et les fractions obtenus : l isorhamnétine, l isorhamnétine-3-O-glucoside et les deux isomères isorhamnétine-3-O-rutinoside et isorhamnétine-3-O-robinobioside. L étude des activités biologiques des extraits et des fractions de N. retusa a permis d établir une relation linéaire entre leur teneur en flavonoïdes et leurs activités antioxydantes et antiprolifératives, les milieux les plus riches présentant les activités les plus importantes. Ces activités dépendent également de la nature des flavonoïdes présents ; ainsi, la très forte activité d inhibition de la xanthine oxydase relevée pour la fraction au chloroforme et sa grande capacité à piéger le radical DPPH ont été attribuées à sa teneur élevée en isorhamnétine, flavonoïde aglycone présentant une grande analogie structurale avec la quercétine, molécule bien connue pour ses activités antioxydantes. Dans un deuxième temps, l acylation enzymatique de l isoquercitrine, flavonoïde modèle, et de l isorhamnétine-3-O-glucoside a été étudiée pour tenter d améliorer leurs propriétés. L acylation enzymatique de l isoquercitrine par des esters éthyliques d acides gras de différentes longueurs de chaîne, catalysée par la lipase B de Candida antarctica, a montré que les performances de la réaction sont inversement proportionnelles à la longueur de la chaîne du donneur d acyle. Des résultats similaires ont été obtenus lors de l acylation de l isorhamnétine-3-O-glucoside. Les activités des esters d isoquercitrine et d isorhamnétine-3-O-glucoside ont été évaluées et comparées à celles des flavonoïdes non acylés. Les esters ont montré des activités antiprolifératives vis-à-vis de cellules Caco2 et d inhibition de la xanthine oxydase plus importantes que celles des molécules d origine. Finalement, ce travail a permis d apporter des éléments de compréhension de la relation structure-activité de flavonoïdes et de leurs dérivés acylésThe present work firstly consisted in studying the extraction and the identification of major flavonoids contained in Nitraria retusa leaves and evaluating their biological activities. Four flavonoids were identified in extracts and fractions: isorhamnetin, isorhamnetin-3-O-glucoside and the two isomers isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-robinobioside. The evaluation of the biological activities of extracts and fractions of N. retusa allowed to establish a linear relationship between their antioxidant and antiproliferative activities and their total flavonoids content, the most enriched exhibiting the highest activities. The nature of the flavonoids present in the extracts and fractions was shown to be important too. Thus, the strong xanthine oxidase inhibition activity and the high DPPH radical scavenging capacity observed for the chloroform fraction can be attributed to its high content in the aglycone flavonoid isorhamnetin, a structural analogue of quercetin which is well known for its antioxidant activities. In a second part, the enzymatic acylation of isoquercitrin as a model compound and isorhamnetin-3-O-glucoside was studied in order to improve their properties. The enzymatic acylation of isoquercitrin by fatty acid ethyl esters of different chain lengths, catalyzed by the lipase B of Candida antarctica, showed that the performance of the reaction is inversely proportional to the acyl donor chain length. Similar results were obtained when acylating the isorhamnetin-3-O-glucoside. The activities of isoquercitrin and isorhamnetin-3-O-glucoside esters were determined and compared to that of initial flavonoids. Esters exhibited higher antiproliferative towards Caco2 cells and xanthine oxidase inhibition activities than original compounds. Finally, this work led to a better understanding of the structure-activity relationship of flavonoids and their acylated derivativesNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Physicochemical characterization of pectin grafted with exogenous phenols

International audiencePectin is a natural polysaccharide, having valuable properties that enable its use in many industrial fields. The aim of this work was to study the impact of pectin modification with phenols, on the properties of this biopolymer. Results suggested that the enzymatic grafting of ferulic acid (FA) oxidation products onto the pectin altered its morphological surface and its thermal properties. Moreover, modified pectin showed a less hygroscopic behavior when water activity is less than 0.50 and a higher ability to bound water above 0.5. Additionally, modified pectin became less viscous than the native pectin and presented different calciumdependent gelation behavior. Finally, a significant improvement of the antioxidant properties of pectin after functionalization was observed. As a conclusion, the modification of pectin with phenolic compounds appeared as a promising way to produce a polysaccharide with new properties that could enlarge the field of its potential applications

Evaluation des représentations négatives concernant les auteurs de violences sexuelles

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Molecular rules for chemo- and regio-selectivity of Candida antarctica lipase B in peptide acylation reactions

International audienceThe chemo- and regio-selectivity of the lipase B of Candida antarctica (CALB) in peptide acylation by oleic acid was investigated combining experimental and theoretical methodologies. Molecular dynamics and docking simulations were performed to study the selectivity of CALB toward the dipeptide Lysine-Serine at the molecular level. To this end, a model that mimics the acyl-enzyme system was built from CALB crystallographic structure and optimized then to be used as docking target. One main orientation of the peptide within the catalytic cavity was obtained. The lysine side chain was observed to enter the cavity, placing the s-amino group as to be acylated near the catalytic residues. This result was consistent with the N-acylation experimentally observed, showing the robustness of the model. Docking simulations were then applied to the peptides Lysine-Tyrosine-Serine, Serine-Tyrosine-Lysine and Leucine-GlutamineLysine-Tryptophan aiming to predict the selectivity of the reaction. Whatever the peptidic sequence and its constitutive amino acids, the models suggested the preferential N-acylation of the lysine side chain. These theoretical results were in perfect accordance with experimental data showing that Ns-oleoyl-Lys derivatives were the major products

Élaboration d’une échelle des représentations sociales négatives concernant les auteurs de violences sexuelles

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Enzymatic synthesis of geranyl acetate in packed bed reactor in supercritical carbon dioxide under various pressure-temperature conditions and reactor configurations

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