A Platform for In Silico Simulation of Protein-peptide Interaction Specificity
Olivier Gagnon
A versatile total synthesis of chrysamide B and dimeric nitrophenyl trans-epoxyamide analogs
Christopher Bérubé
Amyloid nano-assemblies: bioorthogonal conjugation via enzymatic-mediated ligation
Ximena Zottig
An Additive Potential Model for H2O2 Calibrated for Protein Simulations
Esam Orabi
An approach to discover small allosteric modulators of RNases 2, 3, 4 and 5
Marie-Aude Pinoteau
Analyse bio-informatique des gènes impliqués dans la voie métabolique produisant le polyhydroxyalkanoate chez des bactéries prélevées sur des sites industriels.
Samy Ajarrag
ANALYSE DE LA SPÉCIFICITÉ FONCTIONNELLE DES PROTÉINES ADAPTATRICES NCK1 ET NCK2.
Kévin Jacquet
Analyse protéomique des réseaux d'interactions de GRB2 dans le cancer du sein HER2+
Alice Beigbeder
Approche intégrative de recherche d'antibiotique appliquée à Clostridium difficile
Mathieu Larocque
Artificial Crown Ether Ion Channel as Promising Therapeutic Agents
Jean-Daniel Savoie
Automated high-throughput Fucosyltransferase inhibition assay on a chip
Laura Leclerc
Brighter red fluorescent proteins display reduced structural dynamics
Adam M Damry
Caractérisation biochimique du récepteur d’hème Shu1.
Thierry Mourer
Caractérisation de l’activité antipsoriasique de substances naturelles d’origine végétale à l’aide du génie tissulaire
Corinne Bouchard
Catching de novo proteins as they arise in natural populations
Eléonore Durand
CRISPR-PCA: towards high-throughput identification of protein-protein interaction regulators
Philippe Després
Crystal Structure Of Flavoenzyme PieE In Complex With Its Cofactor and Substrate
Mahder Manenda
Crystallogenesis and semi-rational enzyme engineering approach of a new metagenomic lipase
Ngoc Thu Hang PHAM
Designer Biosensors for Engineered Metabolic Pathways and Enzyme Evolution
Mohamed Nasr
Developing a screening platform by Surface Plasmon Resonance (SPR) for the characterization and discovery of enzyme inhibitors
Sarah Melissa Jane Abraham
Development of a high-throughput assay to detect fatty acid decarboxylase activity
Jama Hagi-Yusuf
Développement d’une plateforme de production et de purification de vésicules pseudotypées VSVg
Juliette Champeil
Dibenzofurans and Pseudodepsidones from the Lichen Stereocaulon paschale Collected in Northern Quebec
Claudia Carpentier
DNA-protein conjugates for electrochemical biosensing applications
xiaomeng wang
Effects of Phylogenetic Distance on Protein Dynamics, Antibacterial Activity and Cytotoxicity in Members of the Ribonuclease 3 Subfamily
David N. Bernard
Étude computationnelle de protéines de type Takeout chez la tordeuse des bourgeons de l’épinette
Marie-Ève Picard
Étude RMN du mécanisme de calcium-myristoyl switch de la protéine activatrice de la guanylate cyclase 1
Charlotte Lemay-Lefebvre
Évaluation de l'activité biologique de peptides synthétiques à visée antimicrobienne
Gaëlle Simon
Evolution of the molecular mechanisms of dependency between paralogous genes within the protein interaction network
Axelle Marchant
Fluorinated NMR Probes for the Study of Membrane Topology: Monofluorinated Dimyristoylphosphatidylcholine and Dimyristoylphosphatidylglycerol Lipid Membranes
Marie-Claude Gagnon
Function and engineering of enzymes involved in the glycosylation of natural products
Fathima Mohideen
Harnessing Transamidation and Click Chemistry for Peptide and Protein Labeling
Natalie Rachel
Identification and optimization of inhibitors of type II dihydrofolate reductases, trimethoprim-resistant enzymes
Jacynthe Toulouse
Identification de nouvelles protéines effectrices dans la signalisation des récepteurs Eph.
Sara Banerjee
Identification par protéomique des réseaux de signalisation du récepteur aux androgènes modifiés par l’enzalutamide dans le cancer de la prostate résistant à la castration
Lauriane Vélot
Implementation of a Screening Assay for Hypoxia Induced Factor Inhibitors: Therapeutic Target for Tumor Progression
Billel Djerir
Insights into the contributions of residues Asn-14 and Asn-21 in Islet Amyloid Polypeptide self-assembly
Phuong Trang Nguyen
Interaction membranaire et "calcium-myristoyl switch" de la recoverine
Kim Potvin-Fournier
Interaction of the antimicrobial peptide Esculentin 1b (1-18) with Langmuir monolayers
Isabela Moreira Silva
La lécithine rétinol acyltransférase humaine tronquée : liaison membranaire et de son substrat pour élucider son mécanisme d’activité enzymatique
Sarah Roy
La liaison d’un ligand induit des changements conformationnels chez la xylanase C de Streptomyces lividans.
Louise Roux
La liaison membranaire des protéines S100A10 et annexine A2 intervenant dans la réparation membranaire
Xiaolin YAN
Linear and cyclic peptides as green catalysts for chiral epoxidations
Christopher Bérubé
MD simulations and multivariate statistical analyses uncover cofactor-dependent dynamics in GAPDH
Vinod Parmar
Membrane binding of retinol dehydrogenase 8 and its C-terminal segment
André Hädicke
Modulating activity of membrane active antimicrobial peptides with Zn(II) complex
Pierre-Alexandre Paquet-Côté
Modulation of amyloidogenic kinetics by the interval of fluorescence measurement: a key factor explaining irreproducibility (PROTEO)
Mathew Sebastiao
Monitoring Enzyme Activity at the Nanoscale with DNA Probes
Scott Harroun
NMR-based Fragment Screening to Discover Drug Leads That Target Galectin-7
Yann Ayotte
One-pot, kinetically programmed, reactions for molecular detection directly in whole blood
Guichi Zhu
Optimisation des conditions pour la détermination de la structure tridimensionnelle de la lécithine rétinol acyltransférase tronquée par résonance magnétique nucléaire
Marie-Ève Gauthier
Plateforme analytique du dosage de la cinétique de consommation des acides aminés en culture cellulaire de myoblastes
Marc-Olivier Roseberry
Polyfluorinated hexoses. Preparation and applications.
Vincent Denavit
Potential modulators of human adipocyte metabolism for the prevention of type 2 diabetes
Thierry Chénard
Prédiction de la structure des protéines par apprentissage profond
Félix Pigeon
Préparation d’un vaccin antifongique à base de saccharide à partir d’oligomère de D-glucosamine β(1→4)
Tremblay Thomas
Protein delivery into mammalian cells by an innovative peptide-based shuttle for cell therapy and gene-modified cell therapy
Jean-Pascal Lepetit-Stoffaes
Protein engineering of the CalB lipase to synthesize methyl salicylate
Ying Chew
Protein interaction partners are prime targets for genetic intervention and strong contributors to genetic background effects in a yeast disease model
Véronique Hamel
Recent Developments in the Preparation of Monofluoroalkenes from 3-Fluoro-3-halopropenes
Jean-Denys Hamel
Régulation par phosphorylation de la fonction des domaines SH3 (Src-homology 3) des protéines NCK1/2 via le récepteur tyrosine kinase EphA4
Ugo Dionne
Rôle des récepteurs Eph dans l’établissement et le maintien de la polarité des cellules épithéliales
Noémie Lavoie
Self-assembled cross-β-sheet peptide nanofibers as a novel vaccination platform
Margaryta Babych
Semi-rational evolution of the RhlA enzyme from Pseudomonas aeruginosa for the synthesis of industrially relevant rhamnolipids
Carlos Eduardo DulceyJordan
Shifting the boundaries of experimental studies in engineering P450 enzymatic functions: Combining the benefits of computational and experimental methods
Maximilian Ebert
Str3 is a new protein involved in heme transport in Schizosaccharomyces pombe
Vincent Normant
Structural investigations of supercontracted spider dragline silk
Justine Dionne
Structural study of α-synuclein 71-82, a peptide derived from a protein involved in Parkinson’s disease: interactions with model membranes
Benjamin Martial
Study of the molecular basis of the interspecies communication complex ComRS in Streptococcus sp. through semi-rational mutagenesis
Joaquin Guzman Espinola
Synthesis of all monofluorogalactopyranoses as selective galectin inhibitors
Danny Lainé
Systematic analysis of the expression, solubility and purification of fusion proteins expressed with different tags
Sarah Bernier
The dynamical zinc fingers of Miz-1
Cynthia Tremblay
The Impact of Conformational Entropy on the Accuracy of the Molecular Docking Software FlexAID in Binding Mode Prediction
Louis-Philippe Morency
The rapid evolution of an ohnolog contributes to the ecological specialization of incipient yeast species
Chris Eberlein
THE SYNTHESIS OF KERATAN SULFATE GLYCOSAMINOGLYCANS BY A GLYCOSYNTHASE APPROACH
Xiaohua Zhang
Towards to the understanding of regio-specificity in glycopeptide antibiotic sulfotransferases.
Lei Yang
Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks
Yossef Lopez de los Santos
Understanding the early steps of amyloid assembly by mean of the biarsenical fluorescein dye
Noé Quittot
Utilisation de la spectroscopie Raman pour la détection de l’activité de l’oxyde nitrique synthase in vivo chez les bactéries
Francis Poirier Gravel
Why protein oligomer complexes allow better programmability over dimers and monomers?
Dominic Lauzon

A Platform for In Silico Simulation of Protein-peptide Interaction Specificity


Olivier Gagnon1, Roberto Chica1
1University of Ottawa

Understanding protein-peptide interaction specificity is of primary importance in systems biology research as it can help identify post-translational modification sites and protein binding pairs involved in the regulation of many cellular signaling pathways. Currently, peptide arrays are used to determine binding specificities in vitro. This experimental method has been used extensively throughout the years but remains costly and time-consuming. Here, we developed a platform for the in silico simulation of peptide array experiments that is based on multistate computational protein design. Using a single peptide-bound crystal structure from the PDB as input, our platform generates an ensemble of templates, calculates energies of every single point mutant of the bound peptide within a specific recognition window, and ranks mutations based on their predicted effect on stability of the bound complex. The platform then analyzes the results and outputs a virtual peptide array with spots colored based on their predicted stability as well as a recognition motif for the protein binding domain. We benchmarked the algorithm against various proteins whose specificity was investigated using in vitro peptide arrays such as methyltransferases and Src Homology 3 domains. Receiver operating characteristic curves were calculated to assess the quality of predictions. In all cases, the area under the curve fall between 0.70 and 0.84 with on average 79% of true negatives being correctly rejected and 77% of true positives being correctly accepted. Our method represents a cheap and fast alternative to in vitro peptide arrays, and we are now working on making it available as a web-based platform for the scientific community.

A versatile total synthesis of chrysamide B and dimeric nitrophenyl trans-epoxyamide analogs


Christopher Bérubé1, Claudia Carpentier1, Normand Voyer1
1Université Laval and PROTEO

The two dimeric trans-epoxyamides, Chrysamides A and B have been isolated from a deep-sea-derived Penicillium chrysogenum SCSIO41001. Though nitro compounds are rarely isolated from natural sources, those molecules are of great interest by their wide range of biological activities.

 

A noteworthy feature of our approach is the efficient solid-phase preparation of chiral 2,5-diketopiperazines alanine subunit from N-Boc protected D-Alanine amino acid. Taking advantage of the availability of unnatural chiral amino acids as chiral synthons, the alanine 1,4-piperazine ring was synthesized via chiral diamides reduction from 2,5-diketopiperazine derivatives. The high convergency of the synthetic methodology opens the way to a rapid access of Chrysamides natural products analogs by double coupling reaction as final step. A library englobing stereochemical and structural diversity can be assessed. A diversity of substituted cis and trans 1,4-piperazine ring could be prepared in parallel efficiently and rapidly taking advantage of solid-state synthesis from a rich diversity of natural and non-natural amino acids.

 

Our synthetic strategy gave access to a rapid and efficient preparation of Chrysamide A and B by a convergent approach giving eventually access to a library of analogs of those natural products.

 

 

Chen, S.; Wang, J.; Lin, X.; Zhao, B.; Wei, X.; Li, G.; Kaliaperumal, K.; Liao, S.; Yang, B.; Zhou, X.; Liu, J.; Xu, S.; Liu, Y. Org. Lett. 2016, 18, 3650-3653.

Bérubé, C.; Carpentier, C.; Voyer, N. Tetrahedron Lett., 2017, in press

Amyloid nano-assemblies: bioorthogonal conjugation via enzymatic-mediated ligation


Ximena Zottig1, Marie-Jeanne Archambault1, Isabelle Rouiller2, Steve Bourgault1
1Université du Québec à Montréal 2McGill University

Specific polypeptide sequences that auto-assemble into amyloid fibrils that are characterized by a cross-b-sheet quaternary structure appears attractive for nanomaterials development. These fibrils showed mechanical strength comparable to steel and silk while demonstrating structural plasticity. These properties contribute to the potential of amyloid nanofibrils as promising materials for biomedical and biotechnological applications. In this study, developed a novel biochemical strategy to functionalize amyloid nanofibrils, post-assembly. We investigate an enzymatic approach using a sortase to functionalize amyloid nanofibrils. Staphylococcus aureus sortase (SrtA) is a bacterial transpeptidase that covalently anchors surface proteins to the bacterial cell wall. This is accomplished by cleaving between threonine and glycine at an LPXTG recognition motif to generate an acyl-enzyme intermediate that reacts with a terminal amino group of pentaglycine on the cell wall. The amyloid scaffold was prepared by using peptide sequences derived from known amyloidogenic polypeptides, such as the islet amyloid polypeptide. Peptides were synthesized by solid phase synthesis and the amyloid formation was monitored/screened using thioflavin T (ThT) fluorescence. In order to follow the fibrillization conformational transitions, circular dichroism (CD) was employed whereas transmission electron microscopy (TEM) was used for morphological investigation. The SrtA is produced in-house using a recombinant system and the ligation approach was validated using synthetic peptides, high performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC/MS). To verify our enzymatic-mediated ligation of pre-assembled amyloid structure, green fluorescence protein (GFP) was employed as a model and fused to the surface of fibrils. With this approach, amyloid scaffolds can similarly be decorated using multiple proteins (e.g. promoting cell growing, differentiation factors, and antimicrobial peptides). Overall, the ultimate goal of this project is to develop biocompatible scaffolds for applications in drug delivery and tissue repair/engineering.

 

An Additive Potential Model for H2O2 Calibrated for Protein Simulations


Esam Orabi1, Ann English2
1Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada 2PROTEO

Hydrogen peroxide (H2O2) is the simplest peroxide. Industrially, it is used as an oxidizer, bleaching agent and disinfectant. Biological sources of H2O2 include the spontaneous or catalytic breakdown of the superoxide ion generated during mitochondrial respiration and by NADPH oxidase-dependent mechanisms. H2O2 is cytotoxic at high concentrations, yet recently it has been recognized as an important regulator in eukaryotic signal transduction. Optimization of a force field model for H2O2 is thus important for investigating its biological functions and its interactions with biomolecules. We calibrate a four-site additive model for H2O2 based on the ab initio properties of the monomer and the density and heat of vaporization of liquid H2O2 at the boiling point. The model reproduces the ab initio properties of (H2O2)n, H2O2(H2O)m, and H2O(H2O2)m clusters (n = 1−3 and m = 1,2) calculated at the MP2(full)/6-311++G(3df,3pd) level of theory and yield the structure of the pure liquid plus the densities of the aqueous fluid in good agreement with experimental data. The model also is calibrated to reproduce the ab initio structures and energetics of the H2O2 complexes with the side chains of the 20 naturally occurring amino acids. It is essential for understanding the biological roles of H2O2 and represents an important alternative to experiment for studying the properties of hazardous solutions of pure or concentrated H2O2.

An approach to discover small allosteric modulators of RNases 2, 3, 4 and 5


Marie-Aude Pinoteau1, Donald Gagné2, Steven laplante2, Nicolas Doucet3
1INRS-Université du Québec 2INRS-Institut Armand-Frappier 3INRS-Université du Québec

An approach to discover small allosteric modulators of RNases 2, 3, 4 and 5

 

Marie-Aude Pinoteau1, Donald Gagné1, Steven R. LaPlante1 and Nicolas Doucet1,2,3

 

1INRS – Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, QC, H7V 1B7, Canada

2PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications, 1045 Avenue de la Médecine, Université Laval, Québec, QC, G1V 0A6, Canada.

3GRASP, Groupe de Recherche Axé sur la Structure des Protéines, 3649 Promenade Sir William Osler, McGill University, Montréal, QC, H3G 0B1, Canada.

 

 

Pancreatic-type ribonucleases are involved in RNA degradation and form a superfamily in which all members share similar functional and structural properties. Bovine RNase A is the most investigated enzyme of this family. Previous reports have shown that this enzyme experiences conformational exchange on the millisecond time scale, with the efficiency of product release relying on long-range motions of the distant histidine 48 and loop 1 residues. Such correlated dynamical properties have also been observed in human members of this family, although their involvement in biological function still remains elusive. Among the eight human members of this superfamily, four possess therapeutically relevant activities: RNase 2 (Eosinophil Derived Neurotoxin), RNase 3 (Eosinophil Cationic Protein), RNase 4, and RNase 5 (Angiogenin). These proteins respectively display antibacterial, antiviral, cytotoxic, and angiogenic activities. To potentially modulate their biological activities, allosteric regulation could offer the advantage of fine tuning enzyme activity without direct interaction with the substrate binding site. Fragment based drug discovery (FBDD), a technique that uses high-throughput NMR methodologies to screen small chemical fragments, can be used to identify lead compounds that could act as allosteric modulators. The effect of these chemical entities on protein dynamics and enzymatic activity will be investigated to characterize and fine-tune molecular properties essential for the efficient allosteric modulation of human RNases.

Analyse bio-informatique des gènes impliqués dans la voie métabolique produisant le polyhydroxyalkanoate chez des bactéries prélevées sur des sites industriels.


Samy Ajarrag1,2, Manel Ghribi1,2,3, Fatma Meddeb-Mouelhi1,2,3, Marc Beauregard1,2,3
1Université du Québec à Trois Rivières 2PROTEO 3CRML

Analyse bio-informatique des gènes impliqués dans la voie métabolique produisant le polyhydroxyalkanoate chez des bactéries prélevées sur des sites industriels.

 

Samy Ajarrag1, Manel Ghribi1,2,Fatma Meddeb-Mouelhi1,2 et Marc Beauregard1,2.

 

1- PROTEO, Université Laval

2- CRML, Centre de Recherche sur les Matériaux Lignocellulosiques, Université du Québec à Trois-Rivières,

 

 

 

 

    Les huiles usées de moteur sont des déchets polluants provenant de la vidange des automobiles. Lorsque rejetée dans le réseau des eaux usées, l’huile usagée colmate les filtres dans les stations de traitement de l’eau et perturbe les processus d’épuration biologiques. A cause de ces effets il est primordial de continuer la recherche sur des méthodes de traitement ou de valorisation et pourquoi pas, d’explorer le potentiel d’exploitation qu’offrent ces déchets. La nature étant bien faite, la solution à cette problématique pourrait trouver son origine directement dans ces huiles usagées.

 

Les échantillons environnementaux surtout contaminés par les huiles de moteurs sont très hétérogènes et complexes renferment une communauté microbienne riche et diverse possédant les métabolismes les plus performants et les mieux adaptés aux différentes conditions imposées par leur milieu.

 

Ce projet vise à exploiter la biodiversité des micro-organismes déjà existant dans une usine de traitement des huiles moteurs usagées et à construire une banque de micro-organismes ayant un potentiel pour la dégradation (bio-remédiation) ou dans la valorisation de ce déchet. Pour cela, nous avons prélevé des échantillons à plusieurs sites de l’usine Phoenix (St-Henri, Québec). Dans cette affiche les résultats du criblage des microorganismes qui a été effectué sur des boites de pétri en milieu minimum contenant 10% d’huile de moteur usagée et sur milieu de culture conventionnel à 37°C seront présenté. Plusieurs dizaines de souches bactériennes ont été sélectionnées, purifiés et caractérisées sur la base de leur capacité à croitre et à se développer en présence d’huiles usées de moteur. L’identification génétique par le séquençage de l’ADNr16S ainsi que la caractérisation biochimique de ces microorganismes ont été effectués. Des tests de production de bioplastique, le polyhydroxyalcanoate (PHA), sur milieu solide ont été réalisés pour tenter d’identifier une stratégie possible de valorisation. Enfin, une analyse bio-informatique des séquences des gènes codant pour les enzymes ((comme par exemple la PHA synthase (PhaC)) responsables de la production de PHA des souches identifiées a été effectuée, ce qui permettra d’amplifier les gènes d’intérêt et d’établir un corrélation génotype-phénotype qui permettra d’avancer dans la compréhension de la capacité de ces bactéries à produire du bioplastique.

 

 

ANALYSE DE LA SPÉCIFICITÉ FONCTIONNELLE DES PROTÉINES ADAPTATRICES NCK1 ET NCK2.


Kévin Jacquet1,2,4, François Chartier1,2,4, Sara Banerjee1,2,4, Nicolas Bisson1,2,3,4
1Centre de recherche sur le cancer 2Regroupement stratégique PROTEO 3Département de biologie moléculaire, de biochimie médicale et de pathologie Université Laval 4Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec

CONTEXTE ET OBJECTIFS : NCK1 et NCK2 sont deux protéines sans activité catalytique appartenant au groupe des protéines adaptatrices. Nous cherchons à démontrer que, contrairement à ce qui est admis, NCK1 et NCK2 ne sont pas totalement redondantes dans leurs fonctions et peuvent chacune posséder leur spécificité.

MATÉRIEL ET MÉTHODES : Différentes approches de spectrométrie de masse (AP-MS et BioID) ont été utilisées pour identifier des partenaires spécifiques de NCK1/2 dans différentes lignées cellulaires. Par immunobuvardage et test de liaison in vitro, nous avons i) confirmé certaines interactions identifiées en MS ii) déterminé lesquelles sont directes et iii) quels domaines de NCK2 sont nécessaires pour les médier. Enfin, par immunofluorescences et enregistrements vidéo, nous avons étudié l’implication spécifique de NCK2 dans une fonction cellulaire donnée : la cytokinèse.

RESULTATS : Nos approches de MS nous ont permis d’identifier de nombreux partenaires spécifiques à chaque NCK. Nous avons entre autres confirmé la liaison directe et spécifique de NCK2 avec PKP4 une protéine impliquée dans la régulation de la cytokinèse. Nos essais ont démontré que la spécificité semble être régulée par les différents domaines SH3 et SH2 de NCK2 et que cette dernière pourrait en plus être modulée par les régions interdomaines qui favoriseraient ou inhiberaient la liaison avec certains partenaires spécifiques. Enfin, nos différentes données de microscopie suggèrent qu’une absence de NCK2 engendre des défauts de cytokinèse.

CONCLUSION : Ces études sur NCK1 et NCK2 nous ont permis d’identifier des partenaires spécifiques à chacun tout en suggérant le mécanisme d’action de NCK2 dans la régulation de la cytokinèse.

Analyse protéomique des réseaux d'interactions de GRB2 dans le cancer du sein HER2+


Alice Beigbeder1,2,3, François Chartier2, Nicolas Bisson1,2,3,4
1Centre de Recherche sur le cancer, Université Laval 2Centre de Recherche du CHU de QUébec, Université Laval, Axe Oncologie 3PROTEO, Université Laval 4Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Faculté de médecine, Université Laval

CONTEXTE/OBJECTIFS : GRB2 est une protéine adaptatrice régulant la transduction du signal en aval de récepteurs tyrosine kinases (RTK). En interagissant avec les RTK et différents effecteurs, GRB2 régule la spécificité et diversité des réponses cellulaires. La suractivation du RTK HER2 dans le cancer du sein (HER2+) est associée à un mauvais pronostic. GRB2 constitue un acteur majeur de la signalisation en aval de ce dernier. L’objectif du projet est de caractériser les réseaux GRB2-dépendants et leur implication dans l’oncogenèse du cancer mammaire HER2+. MÉTHODES : Nous avons utilisé des purifications d’affinité couplées à la spectrométrie de masse pour caractériser l’interactome de GRB2. Nous avons caractérisé les complexes par immunobuvardage, immunofluorescence et essais d’adhésion. RÉSULTATS : Nous avons identifié plus de 30 partenaires de GRB2 dans une lignée de cancer mammaire HER2+, incluant des interacteurs connus (PTPN11) et nouveaux comme MPZL1. Cette dernière est une protéine membranaire impliquée dans l’adhésion cellulaire. Nous avons démontré que son association à GRB2 nécessite sa phosphorylation, le recrutement de la phosphatase PTPN11 et le domaine SH2 de GRB2. Nous avons démontré que la fibronectine induit la nucléation du complexe et le recrutement membranaire de GRB2. Enfin, nous avons démontré que PTPN11 est essentielle à l’adhésion cellulaire, et étudions actuellement l’implication de GRB2 dans ce processus. CONCLUSION : Nous avons caractérisé les interacteurs de GRB2 dans une lignée HER2+, et ainsi pour la première fois mis en évidence le partenaire MPZL1. Le complexe décrit par nos travaux pourrait participer au processus oncogénique via l’adhésion cellulaire.

Approche intégrative de recherche d'antibiotique appliquée à Clostridium difficile


Mathieu Larocque1, Louis-Charles Fortier1, Rafael Najmanovich2
1Université de Sherbrooke 2Université de Montréal

Clostridium difficile est une pathogène nosocomiale du tract intestinal ayant la capacité de sporulé. Principal agent causal des diarrhées associé à la prise d’antibiotique, cette bactérie requiert une perturbation de la flore intestinale normale afin d’infecter son hôte. Elle est donc un problème récurrent dans les hôpitaux où l’on retrouve une très forte concentration d’hôtes vulnérables. Les traitements actuellement utilisés pour contrer cette bactérie sont non-spécifiques et présentent de hauts taux de réinfection (24% pour la vancomycine). La découverte de nouveaux antibiotiques plus performants et mieux adaptés au cycle d’infection du pathogène est donc un besoin urgent.

Pour ce faire, des techniques de biologie des systèmes, de mutagenèse et d’arrimage moléculaire furent utilisées au sein d’un protocole intégratif ayant pour but d’identifier et de caractériser des cibles thérapeutiques mieux adaptées ainsi que des ligands de haute affinité pour ces cibles.

Concrètement, un réseau métabolique de la bactérie fut créé afin de simuler de manière in silico (par des calculs réalisés par ordinateur) la croissance bactérienne. L’impact du retrait individuel de chacun des 806 gènes du modèle sur la capacité de celui-ci à produire les éléments essentiels à la division cellulaire sous différentes conditions fut évalué. Cela nous a permis d’identifier 163 gènes ayant un impact sur la croissance dans au moins une des conditions testées. Le potentiel pharmaceutique de ces gènes fut évalué en portant une attention particulière à l’absence d’homologue chez des bactéries de la flore intestinale normale ayant démontré des effets protecteurs contre C. difficile. En se basant sur ce critère et bien d’autres les gènes nadA et ribD pour furent sélectionnée comme cibles d’intérêts supérieurs et ont été choisi pour valider expérimentalement les prédictions du réseau.

Des souches mutantes pour chacun de ces deux gènes essentiels uniquement en condition non riche furent générées en interrompant la séquence codante du gène par un intron de type II, via le protocole ClosTron. Grâce à ces souches, les expériences réalisées de manière in silico ont pu être reproduites et validées en laboratoire. Ces expériences ont démontré que le réseau pouvait prédire correctement le comportement de souches mutantes dans différents milieux. Des expériences de co-infection avec la souche de type sauvage et le mutant nadA de C. difficile ont été réalisé dans un modèle murin. Cette expérience visant à évaluer l’importance du gène in vivo n’identifia aucun impact relié à la délétion du gène. Cela nous en apprenant un peu plus sur l’interaction complexe qui existe entre le pathogène et son hôte.

En parallèle, un protocole itératif d’arrimage moléculaire fut mis au point afin d’identifier des molécules de haute affinité pour certaines de nos cibles les plus prometteuses. Si nadA ne semble pas être un candidat idéal pour la découverte de molécules active, de nombreux autres gènes identifiés par le réseau pourraient servir de cible à ce protocole. Parmi ceux-ci, les gènes prédits comme essentiels en milieu riche pourraient être utilisés favorablement puisque de tels gènes devraient être essentiels en tout temps (incluant lors de l’infection). 51 de ces 76 prédit comme essentiel milieu riche ont déjà été validé expérimentalement en comparant nos prédictions à une expérience de mutagenèse à haut débit réalisé dans des conditions similaires à nos prédictions par Dembek (Dembek et al. 2015).

Dans ce travail, 163 cibles thérapeutiques potentielles de la bactérie furent identifiées en utilisant un réseau métabolique. Le potentiel pharmaceutique de chacune de ces cibles fut évalué alors que le pouvoir prédictif fut démontré en comparant nos prédictions à des résultats expérimentaux. Des ligands sont en cours d’identification pour nos cibles les plus prometteuses et pourraient mener au développement d’antibiotiques plus spécifique et mieux adapté aux infections par C. difficile.

Artificial Crown Ether Ion Channel as Promising Therapeutic Agents


Jean-Daniel Savoie1, François Otis1, Jochen Bürck2, Anne S. Ulrich2, Christophe Moreau3, Michel Vivaudou3, Normand Voyer1
1Département de chimie and PROTEO, Université Laval, Québec, Canada 2Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany 3Institut de Biologie Structurale (IBS), Groupe Channel, Grenoble, France

Ion channels are transmembrane proteins that regulate the flow of ions through cell membranes and are required for the proper functioning of cells. However, some ion channels such as non-gated nanopores may act as toxins by enabling the uncontrolled passage of ions, destroying the usual electrochemical gradients of a cell and leading to its death. Targeting cancer cells with non-gated nanopores would be very promising for the development of new chemotherapeutic agents to treat cancer. In this intent, a new family of synthetic ion channels was developed in our laboratory : a transmembrane helical peptide framework that bears six crown ethers and create a transmembrane ion channel. Even though biophysical studies have shed light on several aspects of these channels, the mechanism of action by which they incorporate membranes remains unclear. Therefore, in an attempt to assess what drives the incorporation of crown ether-modified peptides into bilayer membranes, we have used oriented circular dichroism (OCD) spectroscopy and the two-electrode voltage clamp method (TEVC). Studies in OCD showed a transmembrane orientation at very low peptide/lipid ratios in lipid bilayers and aggregation at higher ratios, while TEVC showed ionic current in genuine Xenopus laevis oocytes cells. Furthermore, oocytes tend to die after a short period of incubation with the peptide, probably from depletion of energy, thus revealing its potential as a chemotherapeutic agent.

Automated high-throughput Fucosyltransferase inhibition assay on a chip


Laura Leclerc1
1Concordia University

The structure and abundance of different cell-surface carbohydrates—or glycans—heavily influence cellular signalling mechanisms that control cell behavior, growth, and death. In many cancers, modification of these glycans by fucosylation—the addition of a fucose sugar residue—results from the upregulation of fucosyltransferase enzymes. Fucosylation of cell-surface glycans can have many downstream effects in cancer development. For example, one such fucosylated glycan, called sialyl-Lewis X, promotes higher metastatic potential and malignancy. In order to screen for potential inhibitors of the fucosyltransferases involved in the assembly of sialyl-LewisX, we have developed a fluorescence-based inhibition assay for the fucosylation of a labeled synthetic oligosaccharide. Upon treatment with specific glycosidase enzymes, hydrolysis of this oligosaccharide releases fluorescent 4-methylumbelliferone. However, fucosylation of the labeled oligosaccharide prior to this treatment results in a structure that is not recognized by the glycosidases, preventing hydrolysis and its associated fluorescent signal. We demonstrate that this assay can be used to detect the inhibition of a fucosyltransferase, since blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. We aim to harness this inhibition assay on a microfluidic platform which combines digital and droplet microfluidics to achieve precise, automated, high-throughput, low-cost drug discovery. 

Brighter red fluorescent proteins display reduced structural dynamics


Adam M Damry1, Natalie K Goto1, Roberto A Chica1
1University of Ottawa

Red fluorescent proteins (RFPs) are genetically-encoded fluorophores that are extensively used in biological research. For all imaging applications, brighter variants are desired. Brightness is directly proportional to quantum yield (QY), and QY improvements can theoretically be achieved by decreasing dynamics of the chromophore responsible for fluorescence by optimizing packing interactions. Although it has been demonstrated that optimization of local packing interactions around the chromophore intended to reduce its conformational freedom can provide brighter FPs, backbone dynamics at sites both proximal and distal to the chromophore can also influence QY but the magnitude and extent of this effect has never been systematically evaluated. Here, we study this relationship using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations of the widely-used mCherry monomeric RFP (QY = 0.22) and its dim variant mRojoA (QY = 0.02). A residue-by-residue comparison of backbone amide peaks in 1H–15N HSQC spectra shows increased line-width for roughly 20% of the dim mRojoA variant’s peaks relative to mCherry. Since peak line-widths are influenced by microsecond–second timescale motions, this suggests that mRojoA exhibits conformational flexibility in regions of its structure that are not dynamic on this time scale in mCherry. To narrow down the timescale of the observed motions, CPMG relaxation dispersion experiments revealed evidence of millisecond-timescale motion in 22 residues in mRojoA, versus only 7 in mCherry. However, the magnitudes of the relaxation dispersions were relatively small, suggesting that dynamics in these RFPs are dominated by shorter-timescale motions. To probe faster timescales, we ran 20-nanosecond MD simulations on both mCherry and mRojoA, as well as an mRojoA mutant that possesses a quantum yield of 0.05. Root-mean-square fluctuations in residues surrounding the chromophore correlate very strongly (R2 = 0.81) to their QY. To validate this result, we are now investigating nanosecond-picosecond timescale dynamics of these three RFPs by NMR. In the long term, we will use this information to rationally design bright RFP variants.

Caractérisation biochimique du récepteur d’hème Shu1.


Thierry Mourer1, Danny Létourneau1, Louis-Philippe Morency1, Rafael Najmanovich2, Pierre Lavigne1,3, Martin Bisaillon1, Simon Labbe1
1Université de Sherbrooke 2Université de Montréal 3Unibersité de Sherbrooke

L’hème est un micronutriment essentiel aux organismes vivants. Ainsi l’acquisition d’hème exogène joue un rôle crucial pour la survie des microorganismes, y compris les levures. À l’aide de l’organisme modèle Schizosaccharomyces pombe nous avons découvert une toute nouvelle protéine, Shu1, permettant l’acquisition d’hème exogène. En carence de fer, Shu1 se localise à la membrane plasmique et lie une molécule d’hème exogène avec une haute affinité. Après liaison, Shu1 pénètre dans la voie d’endocytose et délivre l’hème au compartiment vacuolaire. Subséquemment, l’hème est expulsé de la vacuole pour nourrir les hémoprotéines cellulaires. Cette voie de nutrition hémique, spécifique aux levures, est conservée chez les champignons filamenteux pathogènes de l’homme. De plus, les orthologues de Shu1 chez Candida albicans, Rbt5 et Pga7, sont connus pour jouer un rôle majeur dans les candidémies. Afin d’identifier des inhibiteurs de la voie d’acquisition d’hème exogène, la protéine Shu1 a été exprimé chez Escherichia coli à l’aide du vecteur pET28a. Après purification et renaturation chimique de Shu1, la fonctionnalité du peptide a été validée par sa capacité de liaison à l’hème. Par la suite, la résolution expérimentale de la structure tridimensionnelle de Shu1 par spectroscopie RMN ainsi que son analyse par criblage informatique contre une librairie de composés organiques permettront d’identifier des ligands alternatifs de la molécule d’hème. Les composés identifiés seront testés avec des essais d’atténuation de croissance fongique sur une souche de S. pombe auxotrophe pour l’hème mais exprimant Shu1 ou Rbt5/Pga7. L’ensemble de ces travaux nous permettra de mieux comprendre les mécanismes homéostatiques de la molécule d’hème et de répondre au besoin grandissant en antifongiques pour lutter contre les levures pathogènes de l’homme résistantes aux traitements classiques.

Caractérisation de l’activité antipsoriasique de substances naturelles d’origine végétale à l’aide du génie tissulaire


Corinne Bouchard1,2,4, Sébastien Cardinal3,4, Roxane Pouliot1,2, Normand Voyer3,4
1Faculté de pharmacie, Université Laval 2CMDGT, Centre de recherche du CHU de Québec-Université Laval, Hôpital de l’Enfant-Jésus, CHU de Québec-Université Laval 3Département de chimie, Faculté des sciences et de génie, Université Laval 4Regroupement stratégique PROTEO, Université Laval

CONTEXTE ET OBJECTIFS : Le psoriasis est une maladie cutanée chronique à caractère inflammatoire. Une lésion psoriasique est caractérisée par un épaississement de l’épiderme causé par l’hyperprolifération et la différenciation hâtive des kératinocytes. Les médicaments actuellement disponibles sur le marché ne font que maîtriser les symptômes de la pathologie et l’utilisation de certains traitements occasionne des effets indésirables non négligeables. L’objectif de ce projet est d’identifier, de source végétale québécoise, des composés naturels ayant un potentiel antipsoriasique dans un contexte d’application dermatologique à l’aide d’un modèle cutané lésionnel reconstruit in vitro. Le projet consiste à étudier l’effet de composés polyphénoliques : le québecol, ses analogues et précurseurs.

 

MATÉRIEL ET MÉTHODES : L’inhibition de la croissance cellulaire des molécules a été évaluée afin de sélectionner les plus prometteuses. Les composés ont ensuite été testés sur des substituts cutanés tridimensionnels sains et psoriasiques produits par génie tissulaire. Le caractère antipsoriasique des molécules polyphénoliques a été validé à l’aide d’analyses histologiques et par immunofluorescence indirecte.

 

RÉSULTATS : Les analyses tendent à mettre en évidence le rétablissement de la différenciation anormale des kératinocytes psoriasiques lorsque ceux-ci sont en contact avec les différents polyphénols. En effet, il y a une diminution de l’épaisseur de l’épiderme vivant des substituts psoriasiques traités avec les composés à l’étude comparativement au contrôle. De plus, les différents marquages immunofluorescents indiquent le rétablissement de l’expression de plusieurs protéines impliquées dans la différenciation cellulaire.

 

CONCLUSION : Les résultats préliminaires avec les molécules étudiées démontrent une action pharmacologique efficace au regard d’un traitement du psoriasis.

Catching de novo proteins as they arise in natural populations


Eléonore Durand1, Isabelle Gagnon-Arsenault1, Alexandre Dubé1, Lou Nielly-Thibault1, Guillaume Charron1, Isabelle Hatin2, Olivier Namy2, Christian R. Landry1
1Institut de Biologie Intégrative et des Systèmes, Département de Biologie, PROTEO, Pavillon Charles-Eugène Marchand, 1030 avenue de la Médecine - Université Laval - Québec (QC) G1V 0A6, Canada 2Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud, France

The emergence of new proteins is a driving engine for the acquisition of adaptive innovations. New proteins may arise de novo from previously non-genic regions or from pre-existing gene structures such as gene duplication. The de novo origination is the initial source of genetic novelties and comparative studies between and within species brought to light the potential contribution of intergenic regions in this process. De novo protein origination involves the acquisition of an intergenic ORF by mutations conferring a gain of in frame start and stop codons, and regulatory sites to allow its transcription and translation. Here we explore the dynamics of recently emerging de novo proteins in natural Saccharomyces paradoxus populations. We first characterized intergenic ORF diversity in 24 S. paradoxus strains, sampled in North America, that are structured in 3 lineages. These lineages are used to investigate an on ongoing speciation event, making them a perfect model in the context of recently emerging proteins and their role in adaptation and potentially speciation. We annotated intergenic ORFs and classified them according to the conservation of their positions. In total, 73,657 ORF families were identified: some (2%) are conserved between S. cerevisiae and S. paradoxus while 28 and 8% are fixed and specific to S. cerevisiae or S. paradoxus respectively. Interestingly, the remaining 62% of intergenic ORFs still segregate among or within the S. paradoxus lineages. Then, we characterized the coding potential of these intergenic ORFs using ribosome profiling and RNA sequencing experiments, and showed that ~30% of intergenic ORFs are transcribed and associated with ribosomes. Among them, 232 are significantly differentially expressed between S. paradoxus lineages, 25% of these differences are due to lineage specific expression gain or increase. This diversity illustrates the power of using population data to investigate the emergence of de novo proteins from scratch, which is expected to be rare. We are now characterizing in vivo a subset of recently emerged de novo protein candidates, and their integration into regulatory networks. Our results show that intergenic regions contain a large pool of ORFs readily available for natural selection to draw from during species formation.

 

CRISPR-PCA: towards high-throughput identification of protein-protein interaction regulators


Philippe Després1,2, Alexandre Dubé1, Nozomu Yachie3, Christian Landry1,4
1IBIS, Université Laval 2Département de Biochimie, Microbiologie et Bio-informatique, Université Laval 3RCAST, University of Tokyo 4Département de Biologie, Université Laval 5Département de Biochimie, Microbiologie et 6Université Laval

La biologie des systèmes et un champ d’étude qui vise à comprendre le fonctionnement des cellules et des organismes en étudiant simultanément le plus grand nombre de leurs composantes possibles. L’un des aspects les plus important de cet objectif est l’étude des protéines et de leurs interactions : ces minuscules machines moléculaires sont responsables de la majorité de ce qui se produit dans la cellule et peuvent s’associer ensemble pour former des complexes ayant des fonctions essentielles. Des problèmes dans le fonctionnement des protéines ou des perturbations dans leurs réseaux de communication sont à l’origine d’un grand nombre de maladies humaines. La plupart des techniques actuelles permettent de tracer une carte reliant les différentes protéines présentes dans une cellule, mais offrent peu d’informations sur la nature du lien qui les unissent. L’objectif de mon projet est de développer une technique permettant de caractériser facilement les relations entre les différentes protéines d’un réseau ou d’un complexe rapidement et à moindre coût. Pour ce faire, nous utiliserons la levure come modèle pour l’humain en étudiant des réseaux et des complexes conservés entre les deux espèces : les informations qui ressortiront de ces tests offriront des informations importantes sur les mécanismes derrière certaines maladies neurodégénératives graves, comme le Parkinson et l’Alzheimer. Cette nouvelle technique, qui unit plusieurs des outils les plus avancés de biologie moléculaire et de bio-informatique présentement utilisés, ouvre la porte à l’exploration en détail des relations entre les protéines, tant dans un contexte sain que lorsqu’il y a maladie.

 

Crystal Structure Of Flavoenzyme PieE In Complex With Its Cofactor and Substrate


Mahder Manenda1, Marie-Ève Picard1, Changsheng Zhang†2, Rong Shi1
1*Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS et PROTEO, Université Laval, Québec, QC, G1V 0A6, Canada 2†Center for Marine Microbiology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China

Flavin-binding protein PieE is involved in post-PKS hydroxylation of certain members of a family of natural products known as piericidins. Due to their structural resemblance to ubiquinone, piericidins are potent inhibitors of bacterial NADH–ubiquinone oxidoreductase and eukaryotic mitochondria. Here we report the crystal structure of the enzyme PieE with bound FAD (2.0Å) along with the structure of the ternary complex of the enzyme with FAD and substrate (2.4Å). PieE is a hexameric enzyme and exhibits three known domains of flavoenzyme monooxygenases: N-terminal FAD-binding domain, a central catalytic domain and a C-terminal thioredoxin-like fold involved in oligomerization. Compared to the FAD-bound structure, substrate binding at the active site causes some perturbations on the positioning of the isoalloxazine ring of FAD, as would be required for catalysis. Residues like R52 and W296 are likely involved in modulating this movement while residues such as F258, M378, V243, and others line up the hydrophobic pocket required to bind the substrate. The residue H54 is highly likely to be involved in the direct activation of the aromatic substrate for the ensuing monooxygenation. Mutagenesis studies aimed at validating functional roles of some of the residues are underway.

 

Crystallogenesis and semi-rational enzyme engineering approach of a new metagenomic lipase


Ngoc Thu Hang PHAM1, Yossef Lopez de los Santos1, Guillaume Brault1, Charles Calmettes1, Nicolas Doucet1
1INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, QC H7V 1B7, Canada

Lipase-based catalysis offers advantages over conventional chemical-based routines, such as eco-friendly production and a decrease in the side products of typical racemic mixtures. The new lipase LipIAF5.2, which was extracted from a local metagenomic study, showed broad substrate specificity and was found to hydrolyze 18 different flavor ester compounds. The enzyme is alkali-thermostable and solvent tolerant, while possessing the ability to synthesize a number of commercially relevant biomolecules. As a result, LipIAF5.2 is a promising candidate for the biosynthesis of flavor and fragrance compounds for the biotechnological industry. To expand on the ability of LipIAF5.2 to synthesize complex flavors, we devised a strategy to evolve the enzyme using a semi-rational protein engineering approach. Since an in-depth knowledge of the enzyme structure and active-site environment is required, crystallogenesis of LipIAF5.2 was first undertaken to resolve its molecular structure by X-ray crystallography. The enzyme was purified by affinity and size-exclusion chromatography, while crystallogenesis was performed in various screening conditions. Co-crystallization of LipIAF5.2 was also performed with phosphonates, substrate analog inhibitors. Based on crystal resolution and predictive molecular homology modeling, a semi-rational engineering strategy will be presented to improve the synthetic activity of LipIAF5.2 against industrially relevant flavor esters.

 

Keywords: Lipases, protein engineering, x-ray crystallography, ester synthesis

Designer Biosensors for Engineered Metabolic Pathways and Enzyme Evolution


Mohamed Nasr1, David Kwan1, Vincent Martin1
1Centre for Applied Synthetic Biology, Department of Biology, Concordia University

Synthetic biology techniques aimed at constructing artificial metabolic pathways in genetically modified microorganisms are emerging as important sustainable methods for the production of biofuels, pharmaceuticals and commodity chemicals. To reach industrially relevant scales however, challenges related to bottlenecks and system optimization must be addressed. Directed evolution offers a solution to these limitations, yet the lack of high-throughput detection methods for the products of these reactions remains a disadvantage. The purpose of this work is to utilize transcriptional factor-based biosensors, particularly from the TetR family of repressors, to link the production of these substances to a signal such as fluorescence or antibiotic resistance.

 

Transcriptional repressors are proteins that regulate genes by binding specific effector molecules, and conditionally binding to DNA. This project aims at expanding the toolbox of repressors available by rationally engineering their effector-binding domains to respond to alternative effector molecules. As a proof of principle, using a combined computational and directed evolution approach, we will engineer biosensors from these proteins to respond to intermediates of an engineered metabolic pathway to adipic acid that has been derived from the shikimate pathway. Adipic acid is a precursor of nylon and plastics and is currently produced unsustainably from petrochemicals, with worldwide annual demands of over 2 million tonnes. In optimizing a biorenewable alternative for the production of adipic acid, our “designer” biosensors will be used as parts within genetic circuits for pathway dynamic control and as tools for the directed evolution of pathway enzymes to ultimately improve yields.

Developing a screening platform by Surface Plasmon Resonance (SPR) for the characterization and discovery of enzyme inhibitors


Sarah Melissa Jane Abraham1,3,4, Jacynthe Toulouse2,3,4, Nathalia Bukar1, Dominic Bastien2, Nathalia Kadnikova1, Jean-François Masson1, Joelle Pelletier1,2,3,4
1Department of Chemistry, University of Montreal 2Department of Biochemistry, University of Montreal 3PROTEO 4CGCC

    The objective of the research is to develop a more sensitive screening method based on a portable Surface Plasmon Resonance (SPR) device, an emerging technology. Our target enzyme is the R67 dihydrofolate reductase (R67 DHFR), which confers bacterial resistance to the antibiotic trimethoprim. Here, the target enzyme is linked to a very thin gold surface with specific plasmonic (optical) properties that are proportional to the mass of bound molecules. This can allow monitoring of binding events to the surface-linked R67 DHFR, and thus permit identification of inhibitors. However, the mass of a typical inhibitor (i.e. 500-1000 g/mol) is too low to result in a significant SPR signal. Therefore, a competitive assay will be developed: a gold nanoparticle carrying a substrate analog will bind the surface-immobilized R67 DHFR, giving a strong SPR signal due to its high mass. Then, upon screening for potential inhibitors, the bound nanoparticles will be displaced from the target enzyme if a molecule provides sufficient affinity. Thus, it is possible to indirectly monitor the binding of an inhibitor to the target. This project aims firstly at testing and validating of the SPR screening approach applied to R67 DHFR, and then applying this methodology to the screening of new inhibitors.

Development of a high-throughput assay to detect fatty acid decarboxylase activity


Jama Hagi-Yusuf1, David Kwan1
1Concordia University 2Centre for Structural and Functional Genomics 3Centre for Applied Synthetic Biology

Biofuels have the potential to move our society’s dependence away from fossil-fuel systems towards cleaner and more renewable energy sources. The technology has moved from focusing on an ethanol-dependent fuel source to more complex and energy rich hydrocarbons, similar to those found in petroleum. Recently, a heme-dependent P450 decarboxylase enzyme, OleTJE, was discovered in the bacterium Jeotgalicoccus sp, which was determined via in vitro and in vivo studies to be capable of catalyzing the decarboxylation of long-chain fatty acids, producing terminal alkenes and CO2, thus making OleTJE an ideal candidate for biofuel production. The development of a high-throughput assay is important in engineering and studying the protein. OleTJE activity can be determined by detecting CO2, a byproduct of the OleTJE-catalyzed conversion of fatty acids into terminal alkenes. Here we developed a high-throughput coupled enzyme assay to detect OleTJE activity. In this assay, CO2 is converted to bicarbonate, which is then consumed along with phosphoenol pyruvate in a phosphoenolpyruvate carboxylase-catalyzed reaction generating oxaloacetate and releasing phosphate. Coupling this with an existing phosphate-dependent assays results in a highly sensitive and high-throughput fluorescence-based assay to detect OleTJE activity.


 

Développement d’une plateforme de production et de purification de vésicules pseudotypées VSVg


Juliette Champeil1,2,3, Mathias Mangion1,2,3, Rénald Gilbert2,4, Bruno Gaillet1,2,3
1Chemical Engineering Department, Université Laval, Quebec, QC, Canada 2Thécell : FRQS Cell and Tissue Therapy Network 3PROTÉO : The Quebec Network for Research on Protein Function, Structure, and Engineering 4Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada

Un complexe formé de vésicules pseudotypées par la protéine d’enveloppe G du virus de la stomatite vésiculaire (VSVg) et d’ADN est capable de transfecter efficacement des cellules. Actuellement, les vésicules (V-VSVg) sont produites par transfection transitoire de cellules HEK293 adhérentes à l’aide un plasmide, codant pour la protéine VSVg, en milieu avec sérum, puis concentrées par ultracentrifugation. Notre étude vise à développer et d’optimiser un système de production de V-VSVg en cellules en suspension (293 F), se cultivant dans un milieu sans sérum, et de mettre en place une méthode de purification. Dans un premier temps, nous avons fait varier trois paramètres de production, à savoir : la densité cellulaire, la concentration d’ADN et le ratio ADN : PEI. Les meilleures conditions de production ont ensuite été déterminées par un test cellulaire et un Western Blot. Dans un second temps, un système de purification en deux étapes, composé d’une ultracentrifugation sur coussin de sucrose suivie d’une chromatographie échangeuses d’ions a été développé. La présence des vésicules et leur efficacité après purification ont été déterminées respectivement par Dot Blot et test cellulaire. Les résultats montrent que cette plateforme est efficace pour purifier de vésicules et que celles-ci conservent leur activité.

Dibenzofurans and Pseudodepsidones from the Lichen Stereocaulon paschale Collected in Northern Quebec


Claudia Carpentier1, Emerson Ferreira Queiroz2, Laurence Marcourt2, Jean-Luc Wolfender2, Jabrane Azelmat1, Daniel Grenier1, Stéphane Boudreau1, Normand Voyer1
1Université Laval 2Université de Genève

Lichens are a symbiotic association of a fungi and an algae or a cyanobacteria.1 In response to environmental stress, lichens produce a vast diversity of bioactive natural products (NPs) to protect itself.1,2 Therefore, lichens of Northen Quebec remain an underexplored source of potential bioactive secondary metabolites due to the extreme growing conditions in the Nunavik region.

 

Chemical investigation of the lichen Stereocaulon paschale has led to the isolation and identification of two new dibenzofurans and 11 knowns lichen metabolites.3 Six pseudodepsidone-type metabolites were identified and derived from the cleavage of the depsidone linkage of lobaric acid, the major compound of the crude extract. Lobaric acid and the pseudodepsidones metabolites demonstrated significant in vitro inhibitory activity against major pro-inflammatory targets (NF-κB, TNF-α, IL-6, IL-8, IL-1β). To further investigate their potency as pharmacological agents, we have developed a synthetic methodology to give access to a variety of lichens metabolites. Isolation, identification, synthesis and inhibitory activity against pro-inflammatory targets will be presented.


1: Oettl, S. K.; Gerstmeier, J.; Khan, S. Y.; et al. PloS one, 2013, 8, e76929.

2: Vasconsuelo, A.; Boland, R. Plant Science, 2007, 172, 861.

3: Carpentier, C.; Ferreira Queiroz, E.; Marcourt, L.; Wolfender, J.-L.; Azelmat, J.; Grenier, D.; Boudreau, S.; Voyer, N. J. Nat. Prod. 2017, 80 , 210.

DNA-protein conjugates for electrochemical biosensing applications


xiaomeng wang1, Alexis Vallée-Bélisle1
1Université de Montréal

Protein-oligonucleotide conjugates (POCs) possess unique properties with broad applications ranging from biomedical diagnostic assays to fundamental research on molecular recognition. The future of this class of molecules is bright, but the tools for making them are far from generic. Different approaches of conjugation, including non-covalent and covalent attachment, typically require modification of the protein. Here, we explore a cheap and universal covalent labelling approach to synthesize a wide range of POCs from non-modified proteins. By using a heterobifunctional cross-linker, we have successfully attached a thiol-modified, redox-labeled single-stranded DNA (ssDNA) to lysine residues of a bacterial fimbriae protein. Using careful choice of reaction conditions (e.g. stoichiometry, time, buffer), we show that we can synthesize a range of conjugated proteins containing different numbers of DNAs. Future efforts will investigate site-specific labelling for different research topics in our lab. Possible approaches to achieve this goal include kinetic control, or by using a NTA-labeled ssDNA1 that can non-covalently attach to a poly-hisdine tag on a protein. This ssDNA1 will hybridize with an ester-functionalized ssDNA2 which will then react with the spatially proximate lysine on the protein, followed by displacement of ssDNA1 to leave the covalently-attached ssDNA2.

Effects of Phylogenetic Distance on Protein Dynamics, Antibacterial Activity and Cytotoxicity in Members of the Ribonuclease 3 Subfamily


David N. Bernard1,2, Myriam Létourneau1, Donald Gagné1,2, Marie-Christine Groleau1, Éric Déziel1, Nicolas Doucet1,2,3
1INRS-Université du Québec 2PROTEO 3GRASP

Enzymes are key players of many important biological processes and understanding their mechanism of action is mandatory for proper pharmaceutical or industrial applications of these macromolecules. In fact, 3D structure, function and dynamics appear to be closely related, recent experimental evidence suggesting that conformational exchange may be involved in promoting catalysis in many enzyme systems, although the mechanisms underlying this atomic flexibility remain unclear. It is still unknown whether sequence and/or structure are evolutionarily conserved to promote flexibility events linked to biological function among protein homologs. In order to tackle these interrogations, we have used NMR to characterize the millisecond timescale conformational exchange in various members of the ribonuclease A superfamily. While these enzymes display very similar structure, their evolutionary distance and diversified biological activities complicate flexibility-function analyses. To solve this issue, we have investigated mammalian homologs of human ribonuclease 3 (Eosinophil Cationic Protein, ECP), comparing the human enzyme with its close ECP homologs from monkeys Macaca fascicularis, Pongo pygmaeus, Pongo abelii and Aotus trivirgatus. Our findings show that conformational exchange in the monkey enzymes strongly resembles that of their human counterpart, with subtle changes in exchange rates and/or localization, thus providing insights into the effects of sequence and phylogenetic diversity on protein dynamics. In parallel, antibacterial assays against E. coli and S. aureus have been performed on these proteins, and we have found that the more the protein sequence diverges from the common ancestor, the more potent its antibacterial activity is. Finally, cytotoxicity of these proteins was evaluated on HeLa cells, and a stark difference was found between human ECP and the monkey enzymes, which were still more potent than RNase A. These experiments are the ground to establish the interdependence that could exist between the functions of these proteins and their atomic flexibility.

Étude computationnelle de protéines de type Takeout chez la tordeuse des bourgeons de l’épinette


Marie-Ève Picard1,4, Catherine Béliveau2, Rong Shi1,3,4, Michel Cusson1,2
1Université Laval 2Ressources naturelles Canada 3Institut de Biologie Intégrative et des Systèmes 4PROTEO

L’hormone juvénile (JH) joue un rôle important dans plusieurs processus chez les insectes. Pour être transportée des corps allates (son lieu de synthèse) aux tissus cibles, elle doit cheminer par l’hémolymphe. Comme la JH est particulièrement hydrophobe, contrairement à l’hémolymphe, l’hormone doit être transportée par des protéines dont c’est la fonction spécifique, les Juvenile Hormone Binding Proteins (JHBPs).

 

À partir de données de séquençage du génome de la tordeuse du bourgeon de l’épinette, des analyses génomiques et transcriptomiques ont notamment permis de mettre en évidence la présence de séquences codant pour plusieurs protéines de type Takeout (TO), toutes homologues aux JHBPs (ci-après, ces protéines sont nommées protéines hypothétiques).

 

La caractérisation de ces protéines pourrait permettre l’identification de nouvelles cibles pour le développement rationnel d’insecticides spécifiques aux Lépidoptères. La première étape consiste en une caractérisation bio-informatique de ces séquences. Ainsi, une analyse structurale a été réalisée avec les structures connues de JHBPs et de TO pour mettre en évidence leurs caractéristiques structurales spécifiques. Les séquences de protéines hypothétiques ont ensuite été alignées avec les séquences représentatives des JHBPs et des TOs dont les structures respectives sont connues. Ces analyses ont permis de mettre en évidence que la majorité des protéines hypothétiques appartiennent à la classe des TOs. Toutefois, une de ces protéines hypothétiques semblerait être une JHBP.

 

Des modèles par homologie de chacune des séquences de protéines hypothétiques ont été construits et ont permis de confirmer les observations réalisées par alignement de séquences. L’hormone juvénile III (JHIII) a par la suite été arrimée dans chacun des modèles obtenus. Les protéines présentant le meilleur potentiel de liaison de la JHIII et présentant le meilleur score de docking seront choisies pour être étudiées expérimentalement à l’aide d’analyses transcriptionnelles.

Étude RMN du mécanisme de calcium-myristoyl switch de la protéine activatrice de la guanylate cyclase 1


Charlotte Lemay-Lefebvre1,2,4, Line Cantin1,2,4, Sarah Bernier1,2,4, Stéphane M. Gagné1,3,4, Christian Salesse1,2,4
1Université Laval 2Département d'ophtalmologie et d’ORL-CCF, Faculté de médecine et CUO-recherche, Hôpital St-Sacrement, Centre de Recherche du CHU de Québec 3Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie 4Regroupement stratégique PROTEO

CONTEXTE ET OBJECTIFS: La protéine activatrice de la guanylate cyclase 1 (GCAP1) est exprimée uniquement dans les photorécepteurs de la rétine. Des mutations dans la séquence codante de cette protéine entraînent la dystrophie autosomique dominante des cônes-bâtonnets. La GCAP1 est sensible au calcium et est impliquée dans la phototransduction visuelle. Des études précédemment effectuées au laboratoire suggèrent que la liaison du calcium par la GCAP1 en présence d’une membrane mène à l’extrusion de son groupement myristoyle, ce qui suggère l’existence d’un calcium-myristoyl switch. Les objectifs de ce projet consistent donc à surexprimer et purifier la GCAP1 marquée avec un groupement 13C-myristoyl et à mesurer des spectres de résonance magnétique nucléaire (RMN) afin d’observer le calcium-myristoyl switch de cette protéine. MATÉRIEL ET MÉTHODES: Le protocole de surexpression et de purification de la GCAP1 a été optimisé afin d’obtenir la plus grande quantité possible de protéine en utilisant le moins de myristoyle tout en maintenant un niveau élevé de myristoylation. La GCAP1 myristoylée est habituellement surexprimée en présence d’un excès de myristoyle pour obtenir une myristoylation élevée. La chromatographie liquide à haute performance a permis de déterminer la quantité minimale de myristoyle requise afin d’obtenir une myristoylation élevée de la GCAP1. On utilise le 13C-myristoyl pour observer son couplage avec les acides aminés localisés dans la poche hydrophobe de la GCAP1 en absence de membranes par RMN et le calcium-myristoyl switch en présence des membranes. Des concentrations au-dessus et en-dessous de la concentration micellaire critique du détergent β-octylglucoside miment respectivement la présence et l’absence de membranes. RÉSULTATS : Les protocoles de surexpression et de purification de la GCAP1 myristoylée  ont été optimisés et la mesure des spectres RMN a été obtenue. CONCLUSION : L’optimisation des protocoles de surexpression et de purification a permis d’obtenir une quantité plus élevée de GCAP1 myristoylée.  Nos mesures en RMN suggèrent qu’il pourrait exister un calcium-myristoyl switch chez la GCAP1 en présence de membranes mais des mesures supplémentaires sont nécessaires pour le déterminer hors de tout doute.

Évaluation de l'activité biologique de peptides synthétiques à visée antimicrobienne


Gaëlle Simon1,3, Nicolas Poulin1, Genevieve Lebel1, Pierre-Alexandre Paquet-Côté1,2, François Otis1, Normand Voyer3, Daniel Grenier1
1Université Laval 2PROTEO 3PROTEO - Université Laval

La résistance bactérienne constitue un enjeu d’importance majeure pour la santé publique. Devant l’apparition de peu de nouveaux traitements, il devient impératif de s’attaquer à la problématique avec des solutions alternatives aux antibiotiques traditionnels. Les peptides antimicrobiens constituent une piste intéressante pour le développement de nouveaux agents thérapeutiques. Dans le but d’étudier la relation structure-activité de ces molécules envers les membranes procaryotes,  une librairie de peptides 14-mères cationiques a été élaborée et a démontré de l’activité antimicrobienne lors de tests préliminaires. La présente étude approfondit la caractérisation de l’activité de ces peptides y en mettant à l’épreuve des microorganismes pathogènes. Comme la majorité des infections dépendent de la formation de biofilms bactériens, l’activité contre ces structures biologiques a aussi fait l’objet d’investigation. Il est généralement assumé que les peptides cationiques amphiphiles, agissant comme perturbateurs membranaires, soient plus actifs contre les bactéries Gram négatives. Pourtant, nous avons trouvé des activités plus importantes envers des pathogènes Gram positifs, notamment envers Staphylococcus aureus, bien connue pour ses multiples résistances. Une évaluation plus poussée de la viabilité cellulaire a par contre confirmé l’affaiblissement important d’E. coli pathogène lorsqu’exposée aux 14-mères.

 

Evolution of the molecular mechanisms of dependency between paralogous genes within the protein interaction network


Axelle Marchant1,2,3, Isabelle Gagnon-Arsenault1,2,3, Christian R. Landry1,2,3
1Département de Biologie - Université Laval 2The Quebec Network for Research on Protein Function, Engineering, and Applications 3Institut de Biologie Intégrative et des Systèmes - Université Laval

Mutational robustness is the ability of cells to perform normal functions in spite of genetic perturbations. Paralogous proteins (derived from gene duplication) that have retained redundant functions can compensate for each other when faced with deleterious mutations and thus contribute to mutational robustness. For instance, deleterious mutations in paralogous genes lead to less severe effects than the mutation of single copy genes in yeast as in humans. It was recently shown that this could be achieved directly at the level of protein-protein interactions due to the ability of paralogs to compensate for each other’s losses.  However, some human genetic diseases are, on the contrary, associated with paralogs. The mechanisms underlying these contradictory observations are still poorly understood. It was recently suggested that paralogs often cannot compensate for each other’s loss but rather depend on each other in the protein-protein interaction network. Our working hypothesis is that the duplication of ancestral genes forming homodimers leads to the formation of heterodimers of paralogs that evolve in a way that makes them dependent on each other. We are studying the mechanisms underlying the emergence of this constraint by reconstructing the evolutionary steps leading this phenomenon. Using Protein Complementation Assays (PCA) and the yeast protein interaction network, we examined the evolution of homodimer and heterodimer interactions after gene duplication to understand how the duplication of homodimers leads to physically dependent paralogs. Our results will help understand how protein interaction networks increase in complexity without necessarily gaining in mutational robustness.

Fluorinated NMR Probes for the Study of Membrane Topology: Monofluorinated Dimyristoylphosphatidylcholine and Dimyristoylphosphatidylglycerol Lipid Membranes


Marie-Claude Gagnon1,2, Sébastien Dautrey1,2, Erik Strandberg3, Parvesh Wadhwani3, Jonathan Zerweck3, Anne S. Ulrich3, Michèle Auger2, Jean-François Paquin1
1Département de chimie, PROTEO, CCVC, Université Laval, Québec, QC, Canada 2Département de chimie, PROTEO, CERMA, CQMF, Université Laval, Québec, QC, Canada 3Karlsruhe Institute of Technology (KIT), IBG-2, Karlsruhe, Germany

Understanding the interactions between lipid membranes and drugs, peptides or proteins is of primary importance to determine their mechanism of action. In this context, solid-state nuclear magnetic resonance spectroscopy (NMR) is a method of choice to study their effects on model membranes. Our goal involves marking, with a fluorine atom, phosphoglycerolipids (DMPC and DMPG) to mimic eukaryotic and prokaryotic cell membranes. The incorporation of 19F in membranes offers several advantages for NMR studies, including its high sensitivity and a 100% natural abundance. In addition, the absence of native 19F in membrane components allows the selective detection of 19F-labelled sites. Literature on polyfluorinated lipids suggests these systems are not valid as model membrane because of significant perturbations in comparison to the non-fluorinated model. Nevertheless, as the perturbation is lowered when reducing the number of fluorine atoms, we focused on monofluorinated phospholipids.

 

A variety of monofluorinated dimyristoylphosphatidylcholine derivatives (F-DMPC) with the fluorine atom located on the acyl chain in position 2 of the glycerol (sn-2) were synthesized [1-2] and the synthesis of F-DMPG is ongoing. Biophysical studies on F-DMPC and mixtures between F-DMPC and DMPC allow to evaluate the validity of the new model membrane by studying various properties. Analyses in infrared spectroscopy (FTIR), solid-state NMR spectroscopy, Langmuir and Brewster angle microscopy has been performed. Although F-DMPC membrane properties are significantly different from those of the non-fluorinated model membrane, a 1:3 F-DMPC/DMPC mixture was shown to be the best compromise between a low perturbation induced by the fluorine atom and a high signal intensity in fluorine NMR spectroscopy. Solid-state NMR results regarding the influence of fluorinated DMPC on the behaviour of antimicrobial peptides in DMPC membranes will also be described. These results suggest that the incorporation of 25% of monofluorinated analogues of DMPC in model membranes does not significantly perturb the properties of the lipid bilayers or the orientation of antimicrobial peptides. [3] Hence, those monofluorinated lipids could be potentially used as probes for the study of membrane topology. Using this new model, efforts toward collaboration projects are ongoing to better understand the mechanism of action of peptides and proteins.

 

1. Guimond-Tremblay, J.; Gagnon, M.-C.; Pineault-Maltais, J.-A.; Turcotte, V.; Auger, M.; Paquin, J.-F. Org. Biomol. Chem. 2012, 10, 1145-1148.

2. Gagnon, M.-C.; Turgeon, B.; Savoie, J.-D.; Parent, J.-F.; Auger, M.; Paquin, J.-F. Org. Biomol. Chem. 2014, 12, 5126-5135.

3. Gagnon, M.-C.; Strandberg, E.; Ulrich, A. S.; Paquin, J.-F.; Auger, M. In preparation.

Function and engineering of enzymes involved in the glycosylation of natural products


Fathima Mohideen1, David H. Kwan1
1Concordia University

Many pharmaceutically active small molecule natural products contain sugar moieties that play an important role in their bioactivity. An example of one class of such molecules is the anthracyclines which include the anticancer doxorubicin. These natural product glycosides are biosynthesized by action of glycosyltransferases (GTs). To modify or improve the bioactivity of these molecules by altering glycosylation, in vitro enzymatic methods could circumvent multistep, labor-intensive routes in organic synthesis. This aim is facilitated by screening and engineering GTs to produce modified glycosides. Thus, I have developed a high-throughput screen for assaying GTs enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This will be a valuable tool for discovering and engineering GTs through directed evolution.

Epirubicin, a semisynthetic derivative of doxorubicin, is a high value anticancer drug with fewer side effects than its parent. It is conventionally produced by replacement of the sugar moiety of doxorubicin through several organic synthetic steps. In genetically modified bacteria, engineered biosynthesis has been demonstrated to produce limited amounts of epirubicin. Low yields from these efforts are likely due to poor activity of exogenous enzymes in this artificial biosynthetic pathway. To address this, we will engineer improved GT enzymes by directed evolution. Towards this aim, using a novel in vitro enzymatic synthesis we have produced modified sugar donor substrates for the GT-catalyzed synthesis of anthracyclines. Our resulting library of sugar donors will be used in high-throughput screens to engineer GTs by directed evolution, including those for the production of epirubicin.

Harnessing Transamidation and Click Chemistry for Peptide and Protein Labeling


Natalie Rachel1, Joelle Pelletier1
1Université de Montréal

Site-specific modification of peptides and proteins allows us to control their chemical, structural, and functional properties, with enzymatic conjugation being a promising alternative to traditional synthetic methodologies. The natural capacity for transglutaminases to catalyze protein cross-linking via the formation of isopeptide bonds appealed to us as a starting point to engineer new biotechnology. We, and others, determined that microbial transglutaminase (MTG) can use a wide range of synthetic amine-containing compounds as substrates, broadening its scope for the modification of glutamine-containing peptides and proteins. To further expand the applicability and chemical diversity of MTG-catalyzed protein labeling, we present combinatorial chemoenzymatic strategies utilizing various bioorthogonal reactions. We characterize a one-pot scheme combining MTG and the copper catalyzed azide-alkyne Huisgen cycloaddition (CuAAC), where we determined conditions in which both reactions are compatible, resulting in conjugation yields of over 90%. Building on these results, three metal-free progressions of the CuAAC were simultaneously or sequentially incorporated in a one-pot format with MTG to effectuate protein labeling. The reactions are rapid and circumvent the any potential incompatibility posed by the copper catalyst. We identify the tetrazine ligation as most reactive, with the Staudinger ligation and strain-promoted azide-alkyne cycloaddition also being viable alternatives, as demonstrated by the fluorescent labeling of two proteins. Owing to MTG’s versatility towards its labeling substrate, our results demonstrate the flexibility of these systems, with the researcher being able to fine tune the reactivity specifics.

Identification and optimization of inhibitors of type II dihydrofolate reductases, trimethoprim-resistant enzymes


Jacynthe Toulouse1,2, Dominic Bastien1,2, Delphine Forge3, Brahm Yachnin2,4, Daniel Deon5, Marc Gagnon5, Edward Ruediger5, Kévin Saint-Jacques6, Thaddeus J. Edens7, Genbin Shi8, Xinhua Ji8, Amee R. Manges9, Anne Marinier5, Albert Berghuis2,4, Jean Jacques Vanden Eynde3, Joelle Pelletier1,2
1Université de Montréal 2PROTEO, the Québec Network for Research on Protein Structure, Function and Engineering 3Laboratoire de chimie organique, Université de Mons, Belgium 4Department of Biochemistry, McGill University, Montréal, Qc, Canada 5Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Qc, Canada, 6Département de chimie, Université de Sherbrooke, Qc, Canada, 7Devil’s Staircase Consulting, BC, Canada 8Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, USA, 9School of Population and Public Health, University of British Columbia, BC, Canada

The widespread use of the antibiotic trimethoprim (TMP) in clinical applications against infections provides positive pressure for the development of antibiotic resistance. TMP inhibits the bacterial chromosomal dihydrofolate reductase (Dfr). The selective pressure produced by TMP has resulted in the emergence of an alternative family, the TMP-resistant plasmid-borne DfrB. There is little knowledge of the prevalence of dfrB genes. A library of hundreds of TMP-resistant samples from clinical E. coli infections was screened in silico for the seven known dfrB genes. The relatively new dfrB4 gene was found as a mobile genetic element in a plasmid with multiple resistance genes.

Previously, we reported the first generation of selective inhibitors towards DfrB1(Bastien et al. (2012)). These complex symmetrical bis-benzimidazole inhibitors inhibit in the low micromolar range(Ki = 2−4 μM). We have built upon our previous work by developing a second generation of inhibitors in which we demonstrated, the tolerance to a variety of linker compositions, an increased potency, while optimal length of the inhibitors remained constant. The 1.75-Å-resolution crystal of DfrB1 with one inhibitor has helped uncover the mode of binding.

In order to circumvent risks associated with taking two drugs, we screened a class of folate-like compounds called bisubstrates. Bisubstrates are known inhibitors of a bacterial enzyme found in the folate pathway. We found bisubstrate inhibitors of DfrB1 in low micromolar range(Ki=10−20 μM).

Amino acid sequences of DfrB1 and of DfrB4 are highly similar (82% identity). Based on this similarity, successful DfrB1 inhibitors will be tested as potential DfrB4 inhibitors.

Identification de nouvelles protéines effectrices dans la signalisation des récepteurs Eph.


Sara Banerjee1,2,4, Nicolas Bisson1,2,3,4
1Centre de recherche sur le cancer de l'Université Laval, Hôtel-Dieu de Québec 2PROTEO 3Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Université Laval 4Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec

OBJECTIF: Les récepteurs Eph (EphR), la plus vaste famille de récepteurs tyrosine kinase, régulent une large gamme de processus cellulaires comme l’adhésion et la migration. Cependant, les mécanismes moléculaires permettant d’assurer les fonctions biologiques de ces récepteurs sont loin d’être compris. Dans cette étude, nous cherchons à identifier de nouveaux acteurs de la signalisation dépendante des EphR pour déterminer leurs fonctions dans la signalisation par ces récepteurs.

MÉTHODES: Premièrement, pour identifier les complexes protéiques associés à chaque EphR, nous avons utilisé une approche novatrice de protéomique appelée BioID. Deuxièmement, les données obtenues pour chaque récepteur ont été analysées grâce à différents outils bio-informatiques. Finalement, afin de valider la pertinence des partenaires identifiés par BioID, nous avons commencé à explorer les effets de perte de fonction de ces candidats par ARN interférents au travers deux essais biologiques, basés sur la répulsion ou la ségrégation cellulaire dépendant des EphR.

RÉSULTATS: Nous avons obtenu un réseau protéique pour les 4 EphR étudiés (EphA4,-B2,-B3,-B4) composé de 188 partenaires dont la majorité n’a jamais été décrite comme interagissant avec ces récepteurs. Les analyses informatiques effectuées ont révélé un groupe de 34 protéines interagissant avec les 4 EphR. Nos résultats préliminaires d’essais biologiques ont démontré que la délétion de certains de ces partenaires comme PARD3 est capable de bloquer la ségrégation cellulaire EphB2 dépendante.

CONCLUSION : Globalement, ces travaux conduiront à une meilleure compréhension des voies de signalisation couplées aux EphR fournissant ainsi un précieux aperçu des mécanismes par lesquels les EphR modifient le comportement cellulaire.

Identification par protéomique des réseaux de signalisation du récepteur aux androgènes modifiés par l’enzalutamide dans le cancer de la prostate résistant à la castration


Lauriane Vélot1,2,3, Frédéric Pouliot1,2,4, Nicolas Bisson1,2,3,4,5
1Centre de Recherche sur la Cancer-Université Laval 2Division Oncologie, Centre de recherche du CHU de Québec-Université Laval, Hôtel Dieu de Québec 3Regroupement stratégique PROTEO, Université Laval 4Département de chirurgie, Faculté de médecine, Université Laval 5Département de biologie moléculaire, de biochimie médicale et de pathologie, Faculté de médecine, Université Laval

Le cancer de la prostate (CaP) est le cancer le plus diagnostiqué chez les hommes au Canada. Comme la prolifération des cellules prostatiques est stimulée par les androgènes (e.g. testostérone) via le récepteur des androgènes (AR), les patients métastatiques sont d’abord castrés puis traités par des anti-androgènes. Cependant, la vaste majorité des CaP deviendront résistants à la castration (CaPRC). De nouveaux « super-anti-androgènes » (e.g. Enzalutamide) ont été développés afin de mieux contrôler les CaPRC. Bien que ces traitements augmentent la survie des patients, ces derniers décèderont quand même de leur maladie à court terme. L’objectif de nos travaux est de mieux comprendre les mécanismes d’activation et de blocage du AR afin d’orienter les patients vers les thérapies existantes les plus adéquates et d’identifier de nouvelles cibles thérapeutiques potentielles.

Nous avons utilisé une approche innovante de protéomique, appelé biotinylation de proximité (BioID), pour définir les réseaux de signalisation AR-dépendants modulés dans les lignées LAPC4 hormono-sensibles, après traitement à l’Enzalutamide.

Nous avons identifié 45 partenaires du AR dans des cellules non stimulées, dont 35 sont de nouveaux interacteurs. Après stimulation androgénique, nous étendons le réseau à 320 protéines, dont 278 (incluant 253 nouveaux interacteurs) sont spécifiques de la stimulation aux androgènes. Après traitement à l’Enzalutamide, nous perdons 259 partenaires, ce qui correspond bien à un état inactivé du réseau AR-dépendant. De façon intéressante, nous avons pu identifier 7 protéines qui interagissent spécifiquement avec AR après traitement à l’Enzalutamide.

Nous avons identifié des protéines impliquées spécifiquement dans la régulation de la signalisation de cellules prostatiques après traitement à l’Enzalutamide. Ces candidats potentiels pourraient être des régulateurs clés dans l’acquisition de la résistance à l’Enzalutamide ou la prédiction  de celle-ci. Par conséquent, ces candidats pourraient devenir des cibles thérapeutiques alternatives pour le traitement du CaPRC.

Implementation of a Screening Assay for Hypoxia Induced Factor Inhibitors: Therapeutic Target for Tumor Progression


Billel Djerir1, Marc P. Lussier1, Steve Bourgault1
1Université du Québec à Montréal

Over the last two decades, numerous studies have elucidated how cancer cells and cancer stem cells can survive and grow in a low oxygen tension environment, or hypoxic, in the middle of a poorly vascularized solid tumor. Recently, it has emerged that the hypoxia-inducible factor (HIF) transcription factors are at the center of such an adaptation. Particularly, the HIF-2α transcription factor appears to control the expression of hundreds of downstream target genes and all protein translation in hypoxic cells. Although it is known that its expression and activity are upregulated in most cancers to accelerate tumorigenesis, there are no available inhibitors of HIF-2α. Thus, HIF-2α inhibitor represents a novel class of anticancer agent with outstanding therapeutic potential. In this context, our study aims at implementing biophysical assays to characterize the kinetics and thermodynamics that drive HIF-2α heterodimerization for potentially supporting the development of small molecules that inhibits HIF-2α PAS-B domain. To reach this goal, we expressed the PAS-B domains of HIF-2α and ARNT in Escherichia coli, followed by affinity chromatography purification, GST-tag cleavage and size exclusion chromatography. Afterwards, the kinetics binding of HIF-2α/ARNT heterodimerization was studied by means of surface plasmon resonance (SPR) and the AlphaScreen assays was set up for the identification of HIF-2α/ARNT dimerization’s superior antagonists with five chemical compounds. Overall, these data dissecting this molecular interaction will support the development of a new generation of potent and selective inhibitors of HIF-2α activation and could ultimately lead to novel cancer treatments.

Insights into the contributions of residues Asn-14 and Asn-21 in Islet Amyloid Polypeptide self-assembly


Phuong Trang Nguyen1, Ximena Zottig1, Mathew Sebastiao1, Steve Bourgault1
1UQAM

Insights into the contributions of residues Asn-14 and Asn-21 in Islet Amyloid Polypeptide self-assembly

Phuong Trang Nguyen, Ximena Zottig, Mathew Sebastiao and Steve Bourgault

Département de chimie, Université du Québec à Montréal, C.P. 8888, Succursale Centre-Ville, Montréal (Québec) H3C 3P8

 

Deamidation of asparagine residue is a spontaneous non-enzymatic post-translational modification that results in the conversion of asparagine into a mixture of aspartic acid and iso-aspartic acid. This chemical conversion is known to modulate protein conformation and physicochemical properties, which could lead to protein misfolding and aggregation. In this study, we investigated the effects of site-specific Asn deamidation on the amyloidogenesis of the aggregation-prone peptide islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptidic hormone whose deposition as insoluble amyloid fibrils is closely associated with type-2 diabetes. Asn residues were successively substituted by an Asp or isoAsp and amyloid formation was evaluated by thioflavin T fluorescence assay, circular dichroism spectroscopy, atomic force microscopy and transmission electron microscopy. Whereas deamidation at position 21 inhibited IAPP conformational conversion and amyloid formation, the mutation N14D hastened self-assembly and led to the formation of long and thick amyloid fibrils. In contrast, IAPP was somewhat tolerant to the successive deamidation of Asn residues 22, 31 and 35. Interestingly, a small molar ratio of IAPP deamidated at position 14 promoted the formation of nucleating species and the elongation from unmodified IAPP. Besides, using rat pancreatic beta-cells, we observed that site-specific deamidation did not prevent nor enhance IAPP-induced toxicity. These data indicate that Asn deamidation can modulate IAPP amyloid formation and fibrils morphology and that the site of modification plays a critical role. Above all, this study reinforces the notion that IAPP amyloidogenesis is governed by precise intermolecular interactions involving specific Asn side chains.

Interaction membranaire et "calcium-myristoyl switch" de la recoverine


Kim Potvin-Fournier1,2, Thierry Lefèvre1, Audrey Picard-Lafond1, Catherine Marcotte1, Caroline Dufresne1, Geneviève Valois-Paillard1,2, Pierre Audet1, Line Cantin2, Christian Salesse2, Michèle Auger1
1Département de chimie, PROTEO, CERMA, CQMF, Université Laval, 1045 avenue de la médecine, Québec, Québec G1V 0A6, Canada 2CUO-recherche, CRCHU de Québec, Hôpital du Saint-Sacrement, 1050, chemin Sainte-Foy, Département d’ophtalmologie, PROTEO, Université Laval, Québec, Québec G1S 4L8, Canada

La recoverine est une protéine périphérique présente dans les photorécepteurs et une neuroprotéine sensible au calcium (NCS). La conformation de plusieurs NCS varie en fonction de la concentration en calcium selon le phénomène de "calcium myristoyl switch". L’étude de la liaison membranaire de la recoverine en absence et en présence de calcium est importante en considérant la composition particulière des membranes des photorécepteurs. En effet, les photorécepteurs contiennent plus de 60% de lipides polyinsaturés ainsi que ~20% de lipides chargés négativement, ce qui pourrait être responsable de moduler l’interaction membranaire, la liaison et l’agrégation de la recoverine. La conformation de la recoverine en présence de membranes lipidiques a été étudiée par spectroscopie infrarouge. L’interaction entre la recoverine et les membranes a été étudiée par la combinaison de la spectroscopie infrarouge et résonance magnétique nucléaire à l’état solide grâce au noyau fluor-19, deutérium (H-2) et phosphore-31. La liaison du calcium par la recoverine est importante pour le changement de conformation de cette protéine et sa stabilité thermique. La charge des lipides influence l’interaction membranaire de la recoverine. L’immobilisation du groupement myristoyl de la recoverine est influencée par la présence d’une membrane et sa fluidité. La diffusion latérale des lipides est affectée par la recoverine. La liaison d’un maximum d’ions calcium par la recoverine permet de maintenir sa stabilité thermique et de favoriser son interaction avec les membranes lipidiques chargées négativement. Des membranes lipidiques dans une phase fluide sont nécessaires pour immobiliser de façon optimale le groupement myristoyle de la recoverine.

Interaction of the antimicrobial peptide Esculentin 1b (1-18) with Langmuir monolayers


Isabela Moreira Silva1,2, Katia Regina Perez1, Christian Salesse2
1Departamento de Biofísica, Escola Paulista de Medicina, UNIFESP, São Paulo, São Paulo, Brasil 2CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, Département d’ophtalmologie, Faculté de médecine, PROTEO, Université Laval, Québec

Because of the increase of microorganisms resistance to antibiotics, it becomes necessary the find new and more effective drugs. In this context, antimicrobial peptides are promising candidates. Esculentin 1b is a 46-amino acid peptide found in the skin secretions of the hybrid anuran Pelophylax lessonae/ridibundus. The region composed of the first 18 amino acids (GIFSKLAGKKLKNLLISG-NH2) is responsible for the antimicrobial activity of this peptide with no significant hemolysis. This work aims to study the mode of action of the antimicrobial peptide Esculentin 1b (1-18) on membranes using a model membrane, i.e. Langmuir monolayers composed of neutral and negatively charged phospholipids bearing saturated and unsaturated fatty acyl chains. For this purpose, the maximum initial pressure (MIP) and secondary structure of the peptide was determined using kinetic measurements and polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS), respectively. The kinetic measurements showed that the peptide can reach higher values of MIP in the presence of negatively charged monolayers, independent of the physical state of the membrane. Furthermore, the comparison between liquid-expanded and liquid-condensed physical states using phospholipids bearing the same polar head group indicated that the peptide binds stronger to lipid monolayers in the liquid-expanded physical state. PM-IRRAS measurements with the peptide in the absence of a lipid monolayer do not result in a defined secondary structure. However, in the presence of negatively charged phospholipid monolayers (POPG and POPC:POPG 1:1), we have observed a β-sheet conformation at low initial pressure (10-15mN/m) and a mixture of different structures at high initial pressures (20-35mN/m). On the other hand, in the presence of neutral phospholipid monolayers (POPC and POPE), the peptide does not present a defined structure, such as in absence of monolayers. These results indicate that Esculentin 1b (1-18) binds more efficiently to negatively charged lipid monolayers, likely because it cannot assume a defined secondary structure in the presence of neutral lipid monolayers. Moreover, the peptide binds preferably with monolayers in the liquid-expanded physical state, thus indicating that the organization of the phospholipid monolayers also affects peptide binding. Esculentin 1b (1-18) can mainly bind to negatively charged monolayers, consistently with the lipid content of bacterial membranes. Financial Support: CNPq, FAPESP, INCT-Fx and Global Affairs Canada - ELAP.

La lécithine rétinol acyltransférase humaine tronquée : liaison membranaire et de son substrat pour élucider son mécanisme d’activité enzymatique


Sarah Roy1,2,3,4,5, Ana Coutinho6, Line Cantin1,3,5, Marie-Ève Gauthier1,2,3,4,5, Manuel Prieto6, Stéphane Gagné2,4,5, Christian Salesse1,3,5
1Département d’ophtalmologie, Faculté de médecine, Université Laval 2Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie, Université Laval 3CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec-Université Laval 4Institut de biologie intégrative et des systèmes de l'Université Laval 5Regroupement stratégique PROTEO, Université Laval 6Institut Superior Técnico, Universidade Lisboa, Lisboa, Portugal

Contexte et objectifs : Le pigment visuel des photorécepteurs est composé d’une protéine et d’un chromophore : un dérivé de la vitamine A, le 11-cis rétinal. L’absorption de la lumière par le pigment visuel mène à l’isomérisation de son chromophore en tout-trans rétinal. Ce rétinoïde est par la suite transporté et transformé au cours du cycle visuel pour régénérer le 11-cis rétinal. La lécithine rétinol acyltransférase (LRAT) est une enzyme qui joue un rôle important dans le cycle visuel. Elle possède deux activités enzymatiques. En premier lieu, elle hydrolyse une chaîne acyle des phospholipides (activité PLA1) pour acyler sa cystéine en position 161. Par la suite, elle transfère ce groupement acyle au tout-trans rétinol pour former le tout-trans rétinyl ester (activité acyltransférase). On ne sait toujours pas pourquoi des mutations de la LRAT mènent à la dégénérescence des photorécepteurs et à une perte de la vision. Nous avons montré auparavant que la LRAT tronquée (tLRAT) a une activité enzymatique très élevée, qu’elle lie fortement les membranes et que ses mutations délétères mènent à une perte presque complète de son activité. Par contre, nous avons montré par résonance magnétique nucléaire (RMN) que ces mutations n’ont pas d’effet sur le repliement global de la protéine. Cependant, nos données en électrophorèse suggèrent que les mutants de la tLRAT possèdent une activité PLA1, mais pas d’activité acyltransférase. L’absence de cette activité pourrait être expliquée par une déficience de leur liaison du tout-trans rétinol. Par ailleurs, nos données en RMN sur la structure de la tLRAT suggèrent la présence d’un segment amphiphile en hélice alpha qui permettrait la liaison de cette protéine aux membranes, ce qui faciliterait son activité enzymatique. Les objectifs de ces travaux consistent donc à 1) comparer la liaison du tout-trans rétinol par la tLRAT sauvage et ses mutants afin d’élucider son mécanisme d’activité enzymatique et 2) déterminer la liaison membranaire du segment amphiphile de la tLRAT.

 

Matériel et méthodes : La liaison du tout-trans rétinol par la tLRAT a été mesurée par spectrofluorimétrie. Le segment amphiphile de la tLRAT a été synthétisé commercialement et nous avons débuté des mesures de sa liaison membranaire en utilisant le modèle membranaire des monocouches de Langmuir.

 

Résultats : Les mesures de spectrofluorimétrie ont permis d’observer une augmentation importante de la fluorescence du tout-trans rétinol suite à sa liaison de la tLRAT. Le graphique de l’intensité de fluorescence en fonction de la concentration en rétinol montre une forme hyperbolique dont le lissage avec un modèle mathématique permet de calculer la constante de dissociation du tout-trans rétinol. Une valeur de 59 ± 11 nM a été obtenue avec la tLRAT sauvage, mais une liaison beaucoup plus faible avec le mutant S175R-tLRAT, qui n’a aucune activité enzymatique. De plus, le segment amphiphile de la LRAT a une structure désordonnée sauf en présence de modèles membranaires où il adopte une structure en hélice alpha. Il lie fortement les monocouches de phospholipides.

 

Conclusion : L’information que nous souhaitons obtenir sur la structure tertiaire de la tLRAT, sa liaison membranaire et la liaison du tout-trans rétinol permettra de mieux comprendre son mécanisme d’activité enzymatique et l’effet de ses mutations sur son activité.

 

Financement : IRSC, FFB, PROTEO, RRSV

La liaison d’un ligand induit des changements conformationnels chez la xylanase C de Streptomyces lividans.


Louise Roux1, Nicolas Doucet1
1INRS-Université du Québec

Les polysaccharides cellulosiques des plantes, comme la cellulase et les hétéroxylanes,  constituent la ressource la plus abondante de carbone organique sur la planète, représentant ainsi une source d'énergie inépuisable pour l'hydrolyse enzymatique dans certains domaines industriels, comme la production de carburant, la préparation des aliments, l'industrie du papier et le traitement médical. Cependant, ces procédés qui pourraient avoir un impact positif important sur l'environnement souffrent de coûts de production élevés. La découverte d’enzymes efficaces et adaptées aux domaines biotechnologiques demeure un défi majeur.

Les xylanases sont des enzymes qui catalysent le clivage hydrolytique de la liaison glycosidique de xylanes. Malgré les nombreux efforts dévoués à l’amélioration catalytique des xylanases, en particulier pour leurs utilisations industrielles, la compréhension restreinte de leurs propriétés moléculaires limite les progrès.

Des études cristallographiques et des simulations de dynamique moléculaire sur plusieurs xylanases GH11 ont proposé l'existence d'un mouvement « ouvert-fermé », via une boucle près du site actif (la boucle « thumb loop »), qui peut jouer un rôle majeur dans la liaison au substrat, la libération du produit et/ou la catalyse. Bien que ces études fournissent des informations sur la flexibilité des protéines, elles n’offrent pas de détails sur les mouvements potentiels survenant lors de la réaction catalytique de l'enzyme, c’est-à-dire sur une échelle de temps de l’ordre de la milliseconde. Une récente étude de la xylanase B2 (XlnB2) de Streptomyces lividans par des expériences de dispersion de relaxation 15N-CPMG a permis d’étudier les changements conformationnels survenant à l’échelle de temps de la catalyse. Dans sa forme libre, les résidus dynamiques sont essentiellement regroupés dans les « doigts » et dans les régions composant la fente catalytique, alors qu’en présence d’un analogue de substrat, le « pouce » présente lui aussi de l’échange conformationnel.

Afin d’établir une relation entre la structure, la fonction et la flexibilité des xylanases GH11 et en particulier la conservation évolutive potentielle du rôle des changements conformationnels dans la réaction catalytique, une étude de la xylanase C de S. lividans (XlnC) a été entreprise. Grâce à des expériences RMN de dispersion de relaxation 15N-CPMG, la dynamique à l’échelle de temps de la milliseconde chez cette enzyme a été évaluée. Contrairement à XlnB2 et malgré 75% d’identité de séquence, une structure et une activité catalytique similaire, la XlnC ne montre pas d’échange conformationnel dans sa forme libre. Ce n’est qu’en présence de xylobiose, un produit de la réaction catalytique, qu’un nombre restreint de résidus subissent des mouvements à cette échelle de temps.

L’analyse de ces résultats de dynamique moléculaire en relation avec l’activité enzymatique devrait permettre d’établir les bases d’une meilleure compréhension de la catalyse enzymatique à l’échelle atomique chez les xylanases de cette famille.

 

La liaison membranaire des protéines S100A10 et annexine A2 intervenant dans la réparation membranaire


Xiaolin YAN1,2,3, Élodie Boisselier1,3
1Département de biochimie microbiologie et bio-informatique, Faculté de sciences et génie, Université Laval 2Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval 3CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec

CONTEXTE ET OBJECTIF : Les protéines appartenant à la famille S100 et les annexines interviennent lors de différents mécanismes membranaires vitaux. En effet, le complexe protéique S100A10/annexine A2 permettrait le recrutement de la protéine AHNAK à la membrane en présence de calcium avant de former une plateforme qui initierait la réparation membranaire. Cependant, aucune donnée moléculaire n’est à ce jour disponible sur la liaison membranaire des différentes protéines de ce complexe. L’objectif général de ces travaux est donc d’étudier la liaison membranaire des protéines S100A10 et annexine A2 et de leur complexe afin de mieux comprendre leur rôle lors du processus de réparation membranaire.

MATÉRIEL ET MÉTHODES : Dans un premier temps, les protéines S100A10 et annexine A2 seront surexprimées et purifiées. Le modèle des monocouches de Langmuir sera ensuite utilisé afin de mimer les membranes cellulaires et ainsi caractériser l’interaction des protéines avec différents phospholipides composant les membranes. La structure secondaire, l’orientation et l’organisation membranaire de ces protéines seront étudiées par spectroscopie infrarouge de réflexion-absorption par modulation de la polarisation. Leur localisation membranaire sera déterminée à travers l’étude de l’influence de la liaison de ces protéines sur l’état physique des phospholipides qui sera réalisée par microscopie à fluorescence.

RÉSULTATS : La protéine S100A10-GST a été surexprimée et purifiée par chromatographie d’affinité. Le clivage de l’étiquette GST a été complété. L’optimisation de la deuxième purification pour obtenir la S100A10 pure est actuellement en cours par chromatographie d’échange d’ions. Lorsque la protéine pure sera disponible, le modèle des monocouches des Langmuir sera mis en place pour étudier sa liaison membranaire. CONCLUSION : Ce projet de recherche permettra de développer les connaissances actuelles sur la liaison membranaire des protéines S100A10 et annexine A2. Nous pourrons ainsi identifier les conditions conduisant à une modification de leur liaison membranaire, et éventuellement, à une perte de fonction. Ainsi, ce projet nous aide à mieux déterminer leurs rôles dans la réparation membranaire ainsi que dans les autres mécanismes physiologiques auxquels ces protéines participent.

Linear and cyclic peptides as green catalysts for chiral epoxidations


Christopher Bérubé1, Xavier Barbeau1, Patrick Lague1, Normand Voyer1
1Université Laval et PROTEO

Enzymes are the most efficient class of catalyst in terms of reactivity and selectivity, particularly in living cells where all reactions occur in aqueous media. Inspired by such features, different biomimetic approaches have been developed to mimick enzymatic activity. An elegant example, reported initially by Julià-Colonna, is the asymmetric epoxidation of electron-deficient enones using polypeptides, which leads to epoxyketones with high yields and optical purities.

 

Inspired by those researches, we developed a novel ecofriendly ways for the chiral epoxidation of a,b-unsaturated ketones in water without any co-solvent. Our hypothesis is based on the use of linear oligopeptides and cyclic peptides as chiral catalysts for the substrates. Conformational analysis and molecular modelling studies demonstrated the importance of both the a-helical structure and the hydrophobic nature of homo-oligopeptide catalysts for reactivity and selectivity as described below.

 

 

 

 

 

PLL efficiently catalyzes the epoxidation of a wide variety of electron-deficient enones, leading to high enantioselectivities.1 Both the conformation and hydrophobic nature of oligopeptide catalysts are essential in order to act as efficient biomimetic catalyst. This new process in water opens the way to easily and rapidly produce a variety of chiral epoxyketones through environmentally benign enantioselective transformations.

 

We also synthesised a library of cyclic dipeptides and evaluated their efficacy as catalysts in the asymmetric epoxidation of trans-chalcone. A thorough investigation relying on structure-activity studies and computational studies provided insights into the mechanism of the process.2-3 The cyclic dipeptide acts as a catalyst by templating a supramolecular arrangement at the aqueous-organic interface required for an efficient transformation to occur.

 

 

1. Bérubé, C.; Barbeau, X.; Lagüe, P.; Voyer, N. Chem. Commun., 2017, DOI:10.1039/C7CC01168G

2. Bérubé, C.; Barbeau, X.; Cardinal, S.; P.L, B.; Bouchard, C.; Delcey, N.; Lagüe, P.; Voyer, N. Spramol. Chem., 2017, 29, 330-349

3. Bérubé, C.; Voyer, N. Synth. Commun. 2016, 46, 395-403.

MD simulations and multivariate statistical analyses uncover cofactor-dependent dynamics in GAPDH


Vinod Parmar1,2, Gilles H Peslherbe 2, Ann M English 1,2
1PROTEO 2Centre for Research in Molecular Modeling, Department of Chemistry and Biochemistry, Concordia University

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-known enzyme that exhibits various moonlighting functions and is regulated by cofactor and product binding. In its homotetrameric form, GAPDH is a NAD+-dependent dehydrogenase that binds its cofactor with negative cooperativity. Loss of NAD+ increases the total protein volume, the number of internal waters and the tendency of the tetramer to dissociate and aggregate. Binding of the reduced cofactor, NADH, a product of GAPDH’s dehydrogenase activity, also exhibits negative cooperativity but it binds 10-fold less tightly than NAD+ and destabilizes the homotetramer. Molecular dynamics (MD) simulations reveal that the NAD-binding domains (NBDs) exhibit higher root-mean-square fluctuation when empty or with NADH bound than with NAD+ bound. Principal component analysis (PCA) of the MD trajectories of each subunit reveals a range of displacements of the four NAD+/NADH-bound NBDs but three of the empty NBDs exhibit the same flexibility, with the fourth being less rigid. PCA also uncovers cofactor-dependent differences in the conformational space accessible to the NBD as well as the S-loop, which partly covers the dehydrogenase active site. Residues in these regions access additional minima when the NBDs are empty or NADH-loaded and the separation between the active-site residues, Cys152-His179, increases. The latter may serve to deactivate and protect the catalytic Cys152 in the absence of NAD+ whereas the cofactor-dependent conformational flexibility of the NBD and S-loop likely serve to regulate GAPDH’s dehydrogenase activity.

 

Membrane binding of retinol dehydrogenase 8 and its C-terminal segment


André Hädicke1, Ana I. A. Coutinho2, Mustapha Lhor1, Line Cantin1, Manuel Prieto2, Christian Salesse1
1CUO-Recherche, Centre de recherche du CHU de Québec-Université Laval, Hôpital St-Sacrement, Québec (QC) G1S 4L8, Canada 2Centro Química-Física Molecular, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049 001 Lisboa

Retinol dehydrogenase 8 (RDH8) is a 312-amino acid (aa) protein involved in the visual cycle. Bound to the outer segment disk membranes of photoreceptors, it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2. RDH8 is a member of the short chain dehydrogenase/reductase family. Its C-terminal segment allows its membrane-anchoring through the presence of an assumed amphipathic α-helix and of 1 to 3 acyl groups at positions 299, 302 and 3043. The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.

To evaluate the membrane binding of RDH8, the full-length protein (aa 1-312), a truncated form (aa 1-296), its C-terminal segment (aa 281-312 and 297-312) as well as different additional variants of this segment. The truncated protein binds membranes less efficiently than the full-length form. Thus, the C-terminal segment of RDH8 is essential for the binding and has thus been further examined. The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and both mutants W289F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment. Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.

An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles. These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp = 1.16 to 1.76. These data are independent of the type of lipid and the presence of a mutation. It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer. The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition of α-helical, β-turn and unordered structures.

 

1. Rattner et al. 2000, J Biol Chem, 275, 11034

2. Saari et al. 1998, Vision Res. 38, 1325

3. Lhor and Salesse 2014, Biochem Cell Biol, 92, 510

Modulating activity of membrane active antimicrobial peptides with Zn(II) complex


Pierre-Alexandre Paquet-Côté1,2, Kellie L. Tuck3, Jean-Philippe Paradis1,2, Bim Graham3, Normand Voyer1,2
1Université Laval 2PROTEO 3Monash University

Antimicrobial peptides hold great promises to combat the important increase of bacterial resistance to antibiotics. However, it is difficult to design selective antimicrobial peptides that target only prokaryotic cells. Towards increasing the selectivity of antimicrobial peptides, we incorporated a Zn(II) bis-dipycolyl amine complex on model peptides that we have designed. The addition of the Zn(II) complex increases the antimicrobial activity and decreases the hemolytic activity without substantial change on the secondary structure. The complex also increases the interaction with anionic model membranes and decreases interactions with zwiterionic model membranes. Therefore, incorporating a Zn(II) complex is an interesting strategy to enhance the selectivity of antimicrobial peptides.

Modulation of amyloidogenic kinetics by the interval of fluorescence measurement: a key factor explaining irreproducibility (PROTEO)


Mathew Sebastiao1, Noé Quittot1, Steve Bourgault1,2
1Université du Québec à Montréal 2Quebec Network for Research on Protein Function, Engineering, and Applications, PROTEO

Misfolding of proteins in the form of amyloid fibrils is associated with numerous pathological states such as the Alzheimer’s disease, spongiform encephalopathies and type II diabetes. The development of pharmaceutical inhibitors of amyloid formation is a very active area of research and requires routine and high-throughput assays to evaluate amyloid fibrillization. Still today, the most common experimental approach to monitor amyloid fibril formation in vitro relies on thioflavin T (ThT), a benzothiazole amyloid-sensitive fluorescent dye. However, it is known that assembly is sensitive to a wide array of parameters, making comparison of experimental results between studies and research groups problematic. We observed that even when all parameters, i.e. temperature, buffer, surface, quiescent condition, are carefully maintained constant, discrepancies among kinetics of amyloid assembly monitored in microplate are observed. These differences between assays, performed under quiescent conditions, were associated with the frequency at which the fluorescence is measured. Monitoring the kinetics of self-assembly of the amyloidogenic peptide islet amyloid polypeptide (IAPP) at shorter intervals dramatically accelerates the rate of fibrillization. This observation was confirmed by transmission electron microscopy and circular dichroism spectroscopy. By means of the 8-anilino-1-naphthalenesulfonic acid (ANS) dye, we observed that this effect is independent of ThT. This effect was attributed to the displacements of the microplate between measurements. Overall, this study emphasizes that amyloid assembly is very sensitive to environmental factors and reinforces the importance of a better standardization in kinetic assays.

Monitoring Enzyme Activity at the Nanoscale with DNA Probes


Scott Harroun1, Arnaud Desrosiers1, Alexis Vallée-Bélisle1
1Université de Montréal

Several studies have recently reported enhanced diffusion of enzymes during exothermic catalysis, but explaining this phenomenon remains controversial. How does heat produced at the active site affect the enzyme and surrounding medium? Is the enzyme’s structure destabilized? These questions are a matter of debate. Here we employ programmable DNA switches to measure structural destabilization, and possibly local temperature rise, in the vicinity of an enzyme. ­The unfolding temperature of DNA stem-loops can be readily tuned by varying their nucleobase composition. By attaching a fluorophore/quencher pair at the extremities of these stem-loops, we obtain a library of fluorescent switches that can act as nanothermometers. We selected a DNA switch with optimal signal sensitivity around 37°C, and anchored it onto an enzyme via the strong biotin-streptavidin interaction. We then measure the effects of heat released during enzyme catalysis by monitoring fluorescence variation. Alkaline phosphatase was chosen because its conversion of para-nitrophenylphosphate to para-nitrophenol is highly exothermic, and this enzyme undergoes enhanced diffusion during this reaction.  We find that the DNA switches attached to the enzyme are destabilized during enzyme catalysis, while control DNA switches not attached to the enzyme (i.e. free in solution) do not undergo destabilization. Along with distance-dependent destabilization, these results suggest that enzyme activity may destabilize structures located in their near vicinity.

NMR-based Fragment Screening to Discover Drug Leads That Target Galectin-7


Yann Ayotte1, Jayadeepa R. Murugesan1, Francois Bilodeau2, Sacha Larda2, Patricia Bouchard2, Nathalie Drouin2, Mélissa Morin2, Steven LaPlante1
1INRS - Institut Armand-Frappier 2NMX Research and Solutions

The pharmaceutical industry is currently undergoing dramatic and fundamental changes. One important consequence is that the responsibility for discovering new chemical matter for initiating drug discovery programs is now falling more and more onto the shoulders of smaller institutions (e.g. biotechs, universities, non-profit institutions). However, these institutions lack the impressive infrastructure, large compound collections (e.g. >1 million) and manpower traditionally employed by large pharma. Fortunately, fragment-based screening represents a potential means for these institutions to meet the needs for identifying these seeds for future medications. Fragment-based lead discovery (FBLD) is a validated strategy which has led to compounds in the clinic/market, but serious technology improvements are needed to make it a more practical strategy. We present our new streamlined approaches which includes the introduction of sensitive and simplified NMR-based screening methods, software that assists in automating the identification of fragment binders, and follow-up strategies that help to filter out problematic/promiscuous ligands. Overall, screening time is reduced, deconvolution efforts are automated, and medicinal chemists can focus on more promising drug seeds. These methods are demonstrated within the context of discovering galectin-7 binders which is a promising target for oncology therapeutics. Galectin-7 is a product of p53-induced gene 1 (PIG 1) and is implicated in apoptosis regulation through JNK activation and cytochrome c release. Therefore, inhibition of galectin bears some potential in order to treat several types of cancer.

One-pot, kinetically programmed, reactions for molecular detection directly in whole blood


Guichi Zhu1, Alexis Vallée-Bélisle2
1Université de Montréal 2Université de Montréal

Human developed chemistry typically relies on complex multiple steps reactions necessitating various cycle of purification and reaction conditions. In contrast, Nature has developed a wide variety of one-pot reactions where multiple reactions are sequencially programmed to achieve complex synthesis in the same location and condition. Here we create a versatile DNA-based one-pot, three reactions, which kinetic can be programmed in order to achieve high yield. Using this strategy in an electrochemical format, we demonstrate the multiplexed, quantitative, one-step detection of various molecules in the low nanomolar range, in less than 10 minutes directly in whole blood. We discuss the potential applications of this one-pot assay in the field of point-of-care diagnostic sensors.

Optimisation des conditions pour la détermination de la structure tridimensionnelle de la lécithine rétinol acyltransférase tronquée par résonance magnétique nucléaire


Marie-Ève Gauthier1,2,3,4,5, Sarah Roy1,2,3,4,5, Line Cantin1,2,3, Stéphane Gagné3,4,5, Christian Salesse1,2,3
1Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval 2CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement, CHU de Québec-Université Laval 3Regroupement stratégique PROTEO 4Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie, Université Laval 5Institut de biologie intégrative et des systèmes, Université Laval

CONTEXTE ET OBJECTIFS : La lécithine rétinol acyltransférase (LRAT) joue un rôle important dans le cycle visuel des rétinoïdes. En effet, elle permet l'estérification du tout-trans rétinol en tout-trans rétinyl ester, une étape cruciale dans le processus de régénération du pigment visuel. Des mutations de substitution de la LRAT humaine mènent à la dégénérescence des photorécepteurs et à des maladies telles que la rétinite pigmentaire et l’amaurose congénitale de Leber. Il est donc important de déterminer la structure tridimensionnelle de la LRAT afin de comprendre l’effet de ces mutations sur son activité enzymatique. La structure de la forme tronquée de la LRAT (tLRAT) et de ses mutants sera donc déterminée par résonance magnétique nucléaire (RMN). Afin d’obtenir des spectres comportant le moins de chevauchements des pics, il faut déterminer la température optimale de mesure des spectres, tout en s’assurant de préserver l’intégrité de la tLRAT pendant une période de quatre semaines. Les objectifs de ce projet consistaient donc à déterminer 1) l’activité enzymatique, 2) la structure secondaire, 3) la masse, 4) le patron d’électrophorèse et 4) les spectres 2D-15N-HSQC de la tLRAT incubée à 25, 35, 37, 40 et 45 °C pendant quatre semaines en utilisant un échantillon à une concentration de 1 mM contenant 50 mM de dithiothréitol. MATÉRIEL ET MÉTHODES : La tLRAT a été purifiée par chromatographie d’affinité. Son activité enzymatique a été déterminée par HPLC et sa structure secondaire par dichroïsme circulaire. La masse a été déterminée par spectrométrie de masse. Les gels d’électrophorèse sont préparés avec 15% d’acrylamide. La RMN a été faite avec un spectromètre de 600 MHz. RÉSULTATS : Les données de dichroïsme circulaire, de spectrométrie de masse et d’activité enzymatique ont permis de conclure que l’incubation de la tLRAT pendant quatre semaines à 25, 35 et 37 °C n’a pas d’effet important sur cette protéine. Cependant, l’incubation de la tLRAT à 45 °C a mené à une baisse importante de son activité enzymatique et une dégradation significative de la protéine. Le chevauchement des pics était plus important à 25 °C. CONCLUSION : L’optimisation des conditions pour déterminer la structure tridimensionnelle de la tLRAT par RMN a permis de sélectionner une température de 35 °C à laquelle la structure et l’activité de la tLRAT sont stables et où un minimum de chevauchement des pics est observé dans les spectres.

Plateforme analytique du dosage de la cinétique de consommation des acides aminés en culture cellulaire de myoblastes


Marc-Olivier Roseberry1, Alexandre Côté1, Alain Garnier1
1Université Laval

Plateforme analytique du dosage de la cinétique de consommation des acides aminés en culture cellulaire de myoblastes

 

Roseberry, M.O., Côté, A., Garnier, A.*

Génie chimique, Université Laval

 

La thérapie cellulaire repose notamment sur la propagation d'une grande quantité de cellules issues de cultures primaires. La connaissance des effets des conditions de culture cellulaire optimales est un élément clé en vue d’une éventuelle mise à l’échelle. Outre les molécules-signal telles que les facteurs de croissance, les hormones et les cytokines, les nutriments cellulaires, tels les sucres et les acides aminés sont des composants essentiels au développement et à la fonction cellulaire. Pour ce qui est des acides aminés, particulièrement, l’absence de groupements chromophores ainsi que la grande différence de polarité de ces composés sont un frein majeur à leur séparation et à leur dosage dans un milieu semi-homogène.

Pour doser les acides aminés dans les milieux de culture cellulaire, nous avons adapté une méthode connue (Li, et al., 2016) qui consiste à augmenter leur sensibilité analytique par une réaction de dérivatisation pré-colonne au 2,4-dinitrofluorobenzène (DNFB). Ce dernier est un réactif ajoutant un groupement chromophore par une réaction de substitution nucléophile avec les groupements chimiques fonctionnels suivants : amines, phénols et thiols.

Nous avons appliqué cette méthode d'analyse des acides aminés à la culture de myoblastes humains utilisés en thérapie cellulaire de la dystrophie musculaire de Duchenne en analysant des échantillons prélevés en cours de culture, afin de déceler des acides aminés limitant(s) ou inhibiteur(s). Plus précisément, notre travail se concentre sur l’analyse de la cinétique de consommation des acides aminés en culture cellulaire de myoblastes humains. Pour accomplir cet objectif, le contenu du milieu de culture a été séparé par phase inverse sur un système HPLC où les conditions d’élutions optimales ont été optimisées. Au meilleur de nos connaissances, aucune étude ne rapporte l'analyse des acides aminés solubles dans en culture primaire de cellules à usage thérapeutique.

Références

 

Li, N., Liu, Y., Zhao, Y. et al. (2016). Simultaneous HPLC Determination of Amino Acids in Tea Infusion Coupled to Pre-column Derivatization with 2,4-Dinitrofluorobenzene. Food Analytical Methods (9), 1307-14.

 

* - Auteur de correspondance

Polyfluorinated hexoses. Preparation and applications.


Vincent Denavit1
1Université Laval

Many biologically active molecules contain sugar units in their molecular structure. Therefore, being able to prepare analogs of these carbohydrates can be of great interest in the research for new drugs, vaccines or treatments. In our research group, we're currently working on the preparation of different sugars with hydroxyl groups substituted by fluorine atoms at various positions. We successfully synthesized the first fully fluorinated hexopyranose and we are working to improve our methods to be able to prepare a variety of analogs of biologically interesting sugars. We are now focused on the development of a method to link these heavily fluorinated sugars to a variety of different medically relevant classes of molecules. In the future, this may open the path for the synthesis of complex biomolecules containing sugars units with improved stability, biological activity and improved pharmacokinetic profile.

Potential modulators of human adipocyte metabolism for the prevention of type 2 diabetes


Thierry Chénard1, André Carpentier2, Frédéric-Simon Hould3, André Tchernof3, Rafael Najmanovich4
1Département de Biochimie, FMSS, Université de Sherbrooke, Qc, Canada 2Département de Médecine, FMSS, Université de Sherbrooke, Qc, Canada 3Institut Universitaire de Cardiologie et de Pneumologie du Québec, Université Laval, Qc, Canada 4Département de Pharmacologie et de Physiologie, FM, Université de Montréal, Qc, Canada

Background: Experimental data in both humans and animal models demonstrates that overexposure of lean tissues to fatty acids (FA) plays an important role in the development of insulin resistance and pancreatic dysfunction. Adipose tissues play a central role in the regulation of circulating FA fluxes. FA storage dysfunction in adipose tissues leads to elevated levels of circulating FA fluxes early in the development of T2D. Two general mechanisms govern adipose tissue expansion and storage of excess FA: 1) hyperplasia (increase of preadipocyte recruitment and differentiation, leading to larger numbers of small adipocytes); and 2) hypertrophy (increase in the size of existing adipocytes leading to larger adipocytes). Hyperplasic dysfunction, that is, impaired preadipocyte recruitment and differentiation into lipid-storing cells leads to a pathologic hypertrophic expansion of adipose tissues that contributes to the development of insulin resistance. Studying mechanisms underlying adipocyte hypertrophy could lead to the identification of potential therapeutic targets for the treatment of these conditions.

Methods: We have generated a human adipocyte metabolic network, iTC1390adip, to identify potential genes coding for enzymes affecting adipose tissue expansion mechanisms (adipocyte hypertrophy and/or hyperplasia). We performed in-silico gene deletion analysis in this network using flux balance analysis. We performed a combined optimization of lipid droplet production and biomass as a reflection of adipose hypertrophy and hyperplasia respectively to detect potential targets that may prevent the development of hypertrophy without affecting hyperplasia. We validated the importance of some of these genes by comparing their expression patterns in subcutaneous or visceral adipose tissues between 16 pairs of obese individuals with and without T2D matched for sex, age and body mass index. Total RNA was extracted from whole tissue and we used RT-qPCR to measure the expression levels of the selected genes.

Results: Thirty-one genes in the network were predicted as having a significant effect on lipid droplet production combined with a small effect on biomass production (Figure). Some of these genes, such as DGAT1 and CEPT1, have experimental evidence supporting the effect predicted by the network analysis while others, such as FADS1, ELOVL1, FAR2, HADHA and HSD17B12, could represent new potential therapeutic targets in adipose tissue remodelling. The gene expression pattern analysis showed that the expression of FADS1 is significantly lower in adipose tissues of diabetic patient compared to their non-diabetic counterpart (p<0.05). On the other hand, ELOVL1, FAR2 and HADHA showed significantly increased expression in diabetic patients (p<0.05). Several other genes showed tendencies toward having increased expression in adipose tissues of diabetic patients compared to non-diabetic patients (p<0.10).

Conclusion: To our knowledge it is the first combined use of two different objective functions in an in-silico gene deletion experiment. These results will guide in-cellulo and ex-vivo studies to validate the newly predicted potential therapeutic targets that may lead to the development of modulators of adipocyte metabolism to prevent the onset of T2D.

Prédiction de la structure des protéines par apprentissage profond


Félix Pigeon1, Kyunghyun Cho3, Guillaume Lamoureux1, Yoshua Bengio2
1Concordia University 2Université de Montréal 3New York University

Le rôle des protéines dans les systèmes biologiques est intimement lié à leur structure tridimensionnelle. Toutefois, un très grand nombre des protéines identifiées par la génomique et la protéomique n’ont pas de structure connue. La prédiction de la structure des protéines est donc d’intérêt fondamental en biologie et en médecine, mais demeure extrêmement difficile à faire en général, étant donné la grande diversité des structures retrouvées dans la nature. Un modèle d’apprentissage profond est développé afin de prédire la structure de protéines mono-domaine uniquement en fonction de leur séquence d’acides aminés. Un réseau de neurones artificiel est entrainé avec 300 000 domaines de la base de données Pfam (divisés en 2000 familles), dont les structures ont été extraites de la base de données ModBase. Ces structures sont classées selon leur degré de difficulté et utilisées graduellement lors de l’entrainement pour faciliter l’apprentissage des structures moins intuitives. La performance du modèle est examinée en fonction de la similarité entre les séquences fournies pour l'entraînement et celles fournies pour le test.

 

 

 

 

 

Préparation d’un vaccin antifongique à base de saccharide à partir d’oligomère de D-glucosamine β(1→4)


Tremblay Thomas1, Vincent Denavit1, Denis Giguere1
1Université Laval

Thomas Tremblay, Vincent Denavit et Denis Giguère

 

Département de chimie, Université Laval, Québec (QC), G1V 0A6

 

Les infections fongiques causées par certains types de champignons font de plus en plus de mortalité à travers les populations. Comme la plupart des antibiotiques utilisés actuellement pour traiter ces infections sont inefficaces, il est donc capital de consacrer du temps à la recherche d’un moyen de combattre ces infections. C’est à l’aide de connaissances sur leur surface, qu’il est possible de favoriser la défense du corps humain contre certains champignons pathogènes. Les principaux composants de leur structure sont la chitine et le chitosane, deux polymères de glucosamine. Il serait donc possible d’introduire ces oligosaccharides dans un potentiel vaccin qui agira comme un antigène de champignon pour ainsi sensibiliser le système immunitaire et inciter celui-ci à produire des anticorps contre ces molécules.

 

Cet antigène sera formé de deux parties distinctes, un polysaccharide composé de trois à cinq monomères de D-glucosamine et un polypeptide de trois à six unités de glycine. Ce travail effectué lors du dernier été est centré sur le développement de ce polypeptide ainsi que son addition sur la position anomérique d’un monosaccharide de D-glucosamine orthogonalement protégé. L’obtention du composé désiré permettra de fixer cette molécule sur des nanoparticules de mosaïque de virus venant de la papaye (PapMV) issues des travaux de notre collaborateur Denis Leclerc (CHUL, Centre de recherche en infectiologie) qui, ensemble, formeront le potentiel vaccin antifongique.

 

 

    

  

  

 

 

 

Protein delivery into mammalian cells by an innovative peptide-based shuttle for cell therapy and gene-modified cell therapy


Jean-Pascal Lepetit-Stoffaes1,2, Thomas Del’Guidice2, Louis-Jean Bordeleau2, Joannie Roberge2, Coraline Lauvaux2, Vanessa Théberge2, Bruno Gaillet1, David Guay2, Alain Garnier1
1Université Laval, Chemical Engineering Department, Quebec, QC, Canada 2Feldan Therapeutics, Quebec, QC, Canada

Cell therapy has been on the rise in recent years. It consists in the modification of therapeutic cells to be engrafted to patients, and is applied in multiple pathologies such as muscular dystrophies. Several treatments are currently developed, involving transcription factors or genome editing. These ex vivo technologies are mainly based on gene transfer to express the therapeutic protein in treated cells, by DNA transfection or viral transduction, causing regulatory limitations for clinical studies.

 

A promising strategy to avoid the use of nucleic acids and viruses is the direct delivery of protein therapeutics. This approach offers several advantages, like the transient time of the treatment and the control of the intracellular dose of the protein. Different delivery systems are available, such as cationic lipids and polymers which are efficient but induce cell toxicity. Moreover, they are unsuitable for safety regulation. The use of electroporation is possible, but limited to a few proteins such as Cas9, and to a few cell types. The use of delivery peptides and proteins linked to the therapeutic cargo can avoid these problems. However, the few existing strategies present cell toxicity and lack of efficiency, mainly caused by endosomal entrapment, slowing down the transfer of these methods toward human researches.

 

Here we report an efficient and applicable peptide-based shuttle which enables the intracellular delivery of native proteins without endosomal entrapment, and is able to aim at different cell compartments.  By a simple co-incubation, this peptide delivered functional proteins in multiple cell lines, human embryonic stem cells, human myoblasts, human mesenchymal stromal cells, human hematopoietic stem cells CD34+ and NK cells. It has been applied for the delivery of fluorescent proteins (GFP, mCherry), a transcription factor (HoxB4), antibodies and Cas9 nuclease. Exempt from chemical modifications and presenting low toxicity, the shuttle and the delivered protein are naturally degraded after their active use, decreasing the regulatory burden associated with human cell therapy. This technology is a promising method which can open new avenues for cell therapy applications, bringing transcription factors development and genome editing technologies to clinical studies.

Protein engineering of the CalB lipase to synthesize methyl salicylate


Ying Chew1, Yossef Lopez de los Santos1, Guillaume Brault1, Nicolas Doucet1
1INRS-Université du Québec

A computationally-guided semi-rational protein design approach was used to improve the enzymatic selectivity and catalytic efficiency of Pseudozyma antarctica (CalB) lipase B to synthesize methyl salicylate. This fatty acid ester has significant relevance as a flavoring and fragrance compound in the biotechnological industry. Moreover, CalB is a lipase widely used for enzymatic hydrolysis and synthesis of esters [1,2,3,4,5], offering potential for flavoring agents biological production. However, the relatively confined organization of its active site precludes the recognition of more complex substrates. To overcome this limitation, in silico docking analyses of the best clones from a previously generated mutant library were undertaken to identify amino acid residues most significantly involved in methyl salicylate precursor binding and recognition. These “hot spots” were subjected to combinatorial mutagenesis to generate a second library of CalB variants. Evaluation of this new CalB generation revealed mutations giving rise to an eight-fold increase of synthesis activity in comparison with the wild type enzyme. Finally, a third generation is being constructed and evaluated with the aim to up-scale production of the most efficient variants. Ultimately, the best CalB variant will serve as a template to develop an E. coli whole-cell catalysis system suitable for industrial enzymatic synthesis of methyl salicylate.

Protein interaction partners are prime targets for genetic intervention and strong contributors to genetic background effects in a yeast disease model


Véronique Hamel1,2,3,4,5, Isabelle Gagnon-Arsenault1,2,3,4,5, Alexandre K Dubé1,2,3,4,5, Marie Filteau1,2,3,4,5,6, Christian R Landry1,2,3,4,5
1Centre de recherche en données massives de l'Université Laval (CRDM) 2Département de biologie 3Institut de Biologie Intégrative et des Systèmes (IBIS) 4PROTEO 5Université Laval 6Département des Sciences des Aliments

Mutations causing genetic diseases can have effects that differ depending on the environment of the individual and his genetic background, creating a need for tailored medical treatments. We still know very little about how the genetic background of an individual causes these differences at the molecular level. In order to gain a better understanding of the molecular mode of action of the genetic modifiers responsible for these effects, we use a yeast model for the Wiskott-Aldrich Syndrome. We found that the disease mutation introduced in the yeast LAS17 gene can be corrected by mutations and a drug treatment in a genetic-background specific manner. Many of the compensatory mutations alleviating the phenotype occur in physical interaction partners of Las17p, suggesting that these are prime targets for medical interventions. The compensatory mutations specific to one of the two genetic backgrounds studied provide an opportunity to map the genetic modifiers by QTL analysis. We identified the loci underlying these modifiers and found several genomic regions, mostly centered around genes in the network of Las17p and the protein targeted by the treatment (genetic or physical interactions). Altogether, our results suggest that interaction partners of proteins involved in genetic diseases are prime target for genetic interventions and may also be an important source of genetic background dependent effects.

Recent Developments in the Preparation of Monofluoroalkenes from 3-Fluoro-3-halopropenes


Jean-Denys Hamel1, Mélissa Cloutier1, Myriam Drouin1, Jean-François Paquin1
1Université Laval 2Université Laval

Owing to their strong structural and electronic similarity with amide bonds, monofluoroalkenes are used as non-hydrolyzable isosteres in drug discovery. However, their synthesis still represents a challenge in certain cases.

 

 

 

 

 

 

 

 

 

 

 

In this context, based on previous work by Gouverneur and our group, the Pt-catalyzed allylic amination of 3,3-difluoropropenes was first explored. The reaction uses commercially available Pt(PPh3)4 as a catalyst, proceeds under mild conditions and showcases an interesting case of C-F bond activation. However, thorough evaluation of the scope of the reaction revealed severe limitations in terms of substrates and nucleophiles tolerated.

 

 

 

 

 

 

 

 

 

 

 

 

 

To counter these limitations, we proposed to use 3-chloro-3-fluoropropenes as monofluoroalkene precursors. The superior nucleofuge character of chlorine over fluorine allows for the clean and selective preparation of β-substituted monofluoroalkenes through a SN2’ substitution reaction under metal-free conditions. Most importantly, this transformation behaves nicely with a broad range of N-, S-, O- and C-nucleophiles. To attain more molecular diversity and thus expand further the generality of this synthetic approach, a few subsequent reactions of selected monofluoroalkenes were also successfully accomplished.

 

 

Régulation par phosphorylation de la fonction des domaines SH3 (Src-homology 3) des protéines NCK1/2 via le récepteur tyrosine kinase EphA4


Ugo Dionne1,3,4,7, François Chartier3,4, Noémie Lavoie1,3,4, David Bernard4,5, Kevin Jacquet1,3,4, Michel Tremblay3, Patrick Laprise1,2,3, Nicolas Doucet4,5, Christian Landry4,6,7, Nicolas Bisson1,2,3,4
1Centre de recherche sur le cancer 2Département de biologie moléculaire, biochimie médicale et pathologie 3Centre de Recherche du CHU de Québec, site HDQ, axe Oncologie, Université Laval 4PROTEO 5INRS 6Département de biologie, Université Laval 7IBIS

Les cellules communiquent avec leur environnement en détectant des signaux extracellulaires via des récepteurs membranaires. Les récepteurs tyrosine kinase (RTK) peuvent intégrer ces signaux et initier plusieurs réponses, comme la prolifération et la survie cellulaire. Les protéines adaptatrices, par exemple NCK1/2, couplent des signaux entre des RTK activés et des effecteurs cytoplasmiques via leurs domaines d’interaction SH2 et SH3 (Src-Homology). Les mécanismes contrôlant le désassemblage de ces complexes sont peu connus. Puisque ces protéines ont un rôle essentiel dans la signalisation dérégulée dans les cellules cancéreuses, il est primordial d’élucider leurs modes de régulation. Une tyrosine phosphorylée, conservée dans l’évolution des domaines SH3 de la levure à l’humain, a été détectée par spectrométrie de masse sur NCK1/2. Le RTK EphA4 a été identifié pour phosphoryler directement ce résidu. Des essais biochimiques in vitro de polarisation de fluorescence et de résonance magnétique nucléaire ont montré que cette phosphorylation inhibe la fonction des domaines SH3 de NCK1/2. Ces résultats ont été confirmés dans des cellules en culture grâce à des expériences de spectrométrie de masse quantitative (SWATH). Des essais d’imagerie à haute résolution ont montré que la phosphorylation des domaines SH3 de NCK2 mène au désassemblage des complexes de signalisation normalement recrutés au RTK EphA4 activé. De plus, cette phosphorylation inhibe les fonctions essentielles de NCK1/2 lors du développement du cerveau de la drosophile. Finalement, les expériences réalisées indiquent que ce mécanisme de régulation est partagé avec d’autres RTK et domaines SH3 chez l’humain. Ces travaux mettent en évidence un nouveau mécanisme de régulation négative de la signalisation cellulaire qui ne requiert pas l’inactivation ou le recyclage des RTK. En effet,  les RTK peuvent non seulement recruter des partenaires suite à leur activation, mais aussi les phosphoryler, pour terminer le signal en aval.

Rôle des récepteurs Eph dans l’établissement et le maintien de la polarité des cellules épithéliales


Noémie Lavoie1,2, Sara Banerjee1,2, Patrick Laprise1,3, Nicolas Bisson1,2,3
1Centre de recherche sur le cancer de l'Université Laval et Centre de recherche du CHU de Québec, Axe Oncologie 2Regroupement québécois de recherche sur la fonction, l'ingénierie et les applications des protéines (PROTEO) 3Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de médecine, Université Laval

La communication intercellulaire est essentielle pour le développement et l’homéostasie des organismes multicellulaires. La transmission des signaux extracellulaires est principalement assurée via la formation et l’activation de complexes signalétiques qui agissent en aval de récepteurs membranaires, par exemple les récepteurs tyrosines kinases (RTK). La plus grande famille de RTK sont les récepteurs Eph, qui sont activés suite au contact de leurs ligands membranaires, les éphrines. La signalisation Eph/éphrines est notamment impliquée dans la régulation de la morphologie et de la motilité cellulaires. Nous avons récemment découvert par spectrométrie de masse que les récepteurs Eph s’associent à des protéines requises pour l’établissement et le maintien de la polarité des cellules épithéliales. L’objectif du projet est d’explorer le rôle des récepteurs Eph dans ces processus. Nous avons d’abord montré que six des quatorze récepteurs Eph sont exprimés dans les cellules épithéliales Caco-2 et MDCK-2. Suite à la mise au point d’une méthode de culture 3D pour ces cellules, nous avons aussi observé par immunofluorescence la localisation subcellulaire de ces récepteurs. Finalement, nous concentrons maintenant nos efforts à étudier les effets d’un gain ou d’une perte de fonction de ces récepteurs sur la polarité cellulaire en utilisant nos modèles 3D. Nos études permettront de comprendre l’implication des récepteurs Eph dans l’établissement et le maintien de la polarité des cellules épithéliales.

Self-assembled cross-β-sheet peptide nanofibers as a novel vaccination platform


Margaryta Babych1, Ximena Zottig1, Geneviève Bertheau Mailhot1, Jessica Dion1, Denis Archambault1, Steve Bourgault1
1UQAM

The development of new vaccination strategies is an important challenge for modern research. In most cases, the protection induced by vaccines is only partial. The efficiency of immunotherapies is often complicated by problems related to the limited immunogenicity of certain antigens. The antigen size and geometry are key elements that contribute to the efficiency of a vaccine by favoring their internalization by dendritic cells via the endosomal pathway.1,2 This work aims at developing a novel vaccine platform based on natural self-assembling peptides. The nanofibrils take the form of a cross-β-sheet conformation as observed in amyloid3 and the N-terminal amine group can be used as a fusion site for the functional epitope. This approach brings the following advantages: the assembly process of the amyloids is spontaneous, the nanostructures exhibit a strong mechanical rigidity and the capacity to form highly ordered structures3. The modulation of amino-acid sequences allows to adjust the physico-chemical properties of the amyloid substance. Equally, a self-assembling peptide domain can be easily added to an epitope by using solid phase peptide synthesis and the synthetic petides can be prepared with a precise chemical composition for optimal epitope exposure. Moreover, the use of self-assembling structures can present the epitopes in a multivalent fashion, leading to a increased recognition by immune cells. Finally, the use of chemically synthesized peptides allows us to avoid the difficult purification and characterization processes. These nanostructures can serve as a platform to anchor heterologous antigens and the incorporation of these materials should improve the delivery efficiency to increase immunogenicity.

 

[1] Hervé et al, J Virol. 2014 Jan;88(1):325-38. [2] Liu Yang et al, Biomater. Sci., 2016, 4, 785–802. [3] Akter R et al, J Diabetes Res. 2016;2016:2798269.

Semi-rational evolution of the RhlA enzyme from Pseudomonas aeruginosa for the synthesis of industrially relevant rhamnolipids


Carlos Eduardo DulceyJordan1, Yossef Lopez de los Santos1, Nicolas Doucet1, Eric Déziel1
1INRS-Université du Québec

Rhamnolipids (RLs) are glycolipidic compounds produced by a few of bacterial species, especially Pseudomonas and Burkholderia spp. These compounds display excellent surfactant properties and environmental advantages. Nevertheless, their high production cost hampers their practical use in industry. RhlA acts as a key enzyme in the RL biosynthesis pathway. This enzyme catalyzes the esterification reaction between two units of 3-hydroxylated fatty acids to form a dimer, β-3-(3-hydroxyalkanoyl) alkanoic acid (HAA), the dilipid precursor of RLs. HAA biosynthesis is the rate-limiting step in the carbon flux toward RL biosynthesis; therefore, our research consists in engineering RhlA from Pseudomonas aeruginosa UCBPP-PA14 (PA14) for a more effective HAA production, which should result in increased RL production. In order to identify residues that increase the catalytic efficacy of PA14 RhlA, we took a three-step semi-rational evolution approach. First, (1) we opted to predict residues that could interact with the substrate as ligand binding site residues. These residues were first subjected to alanine-scanning mutagenesis. As expected, when any of these single mutants were expressed in a polar PA14ΔrhlA strain, the in vivo RL production decreased and in some cases disappeared. Given the loss of activity, a secondary round of mutagenesis was used to evolve the enzyme toward increasing activity. These residues were mutated by residues having similar physicochemical properties. The single mutant M37L showed 25% increase in RL production when compared to the wild-type enzyme. Then, (2) we chose potentially relevant residues based on an RhlA homology model. We selected polar residues that seem to be topologically located in close proximity to the predicted RhlA cap-type domain. Selected residues were subjected to site-directed mutagenesis in which they were replaced by amino acids with similar physiochemical properties. We identified eight mutants that increased RL production in vivo in minimal salt medium supplemented with glycerol as sole carbon source after 72 h of culture. (3) The next step in our research consists in combining the various substitutions identified in previous steps. Here we report a semi-rational protein evolution approach that enables an increased RL production in vivo in P. aeruginosa. Nine engineered RhlA variants were able to increase carbon flux toward HAA biosynthesis and, consequently, a higher RL production. As of yet, we have generated RhlA variants which reached 1.5-fold improvement in RLs compared with the wild-type RhlA. This study provides evidence that protein engineering approaches may be successfully used for RL production improvement and similar studies could be extended to modify the RL congener profile and for optimizing proteins involved in the RL metabolic pathway facilitating high-yield RL production in bacteria.

Shifting the boundaries of experimental studies in engineering P450 enzymatic functions: Combining the benefits of computational and experimental methods


Maximilian Ebert1, Simon Dürr1, Armande Ang Houle1,3, Guillaume Lamoureux2, Joelle Pelletier1
1Université de Montréal 2Concordia University 3CHU Sainte-Justine

While biocatalysis is thought to be the sustainable and benign complement to organic synthesis, past efforts to tailor enzymatic function towards desired reactions have faced a significant knowledge gap. Here we present two distinct, combined computational and experimental protocols to predict the conformation of catalytically-relevant enzyme:ligand complexes, and to quickly reveal substrate access channels even in the absence of a ligand-bound structure. The applied Adaptive Biasing Force (ABF) method is broadly applicable for predicting mutational hotspots in a substrate-specific manner and has the potential to drastically reduce the experimental screening effort to tailor an enzyme to new substrates of interest. Starting with a ligand-free crystal structure, we successfully identified all residues known to be involved in palmitic acid binding to Cytochrome P450 CYP102A1 (BM3). The binding trajectory also revealed a new binding residue, Q73, which we confirmed experimentally. Mapping the substrate access channels of proteins represents a significant challenge. Like many other biocatalysts, P450s contain numerous channels thought to be populated by their substrates, products, solvents, and gases. We identified and predicted correctly multiple ligand migration channels for two bacterial P450s (BM3 and CYP102A5), using Implicit Ligand Sampling (ILS) and free molecular dynamics simulations. Furthermore, calculations of the free energy of gas migration through each channel revealed evidence of the evolution toward O2 binding in conjunction with protection against inhibitory CO and exclusion of N2. These results significantly enhance our understanding of gas migration in proteins and provide insights into the evolution of gas-utilizing enzymes.

Str3 is a new protein involved in heme transport in Schizosaccharomyces pombe


Vincent Normant1, Thierry Mourer1, Simon Labbe1
1Université de Sherbrooke

All eukaryotes require iron.  It serves as catalytic cofactor for a variety of enzymes that are required for essential cellular functions.  Iron is also needed to form heme, which is a prosthetic group that is constituted of a protoporphyrin ring with an iron atom at its center.  Hemoproteins such as cytochromes, hemoglobin, and catalases are required in fundamental biochemical processes, such as respiration, oxygen transport, and disproportionation of hydrogen peroxide, respectively.  As a consequence of the importance to fulfill heme requirement for growth, several organisms have evolved with different means to acquire heme.  In Schizosaccharomyces pombe, the iron-regulated cell surface Shu1 protein is required for assimilation of exogenous hemin.  However, mutational inactivation of Shu1 has revealed the existence of an additional heme uptake system with lower affinity.  In the absence of delta-aminolevulinate (ALA) and presence of 0.075 uM hemin, hem1 shu1 null cells were unable to grow.  Under the same conditions but using a medium supplemented with higher hemin concentrations, growth of hem1 shu1 mutant cells was rescued in a Str3-dependent manner.  A functional Str3-GFP protein localizes to the plasma membrane under low iron conditions.  hem1 shu1 mutant cells expressing Str3 were able to take up the heme analog zinc mesoporphyrin IX (ZnMP).  Consistent with a role for Str3 as hemin transporter, results with hemin-agarose pull down assays showed that Str3 binds to hemin.  Further mapping analysis identified amino acid residues within the C-terminal region of Str3 that are involved in its ability to bind hemin.  Collectively, these results have identified a second acquisition system for hemin that requires the cell-surface Str3 protein when hemin is more abundant.

Structural investigations of supercontracted spider dragline silk


Justine Dionne1, Thierry Lefèvre1, Philippe Bilodeau1, Mathieu Lamarre1, Michèle Auger1
1Département de Chimie, PROTEO, CERMA, CQMF, Université Laval, Québec, QC, Canada, G1V 0A6

Dragline spider silk is composed of fibrous proteins organized into crystalline nanodomains embedded in an amorphous matrix. This assembly confers to silk mechanical properties that surpass those of industrial fibers.

 

In the presence of liquid water or high humidity, the amorphous phase is plasticized, which results in the shrinking of the fiber up to 50% of its initial length. This phenomenon, known as supercontraction, can be reversibly induced and thus paves the way for new applications such as artificial muscles. However, before developing this type of biomaterial, it is imperative to have a better understanding of the supercontraction process at the molecular level.

 

Actually, the impact of supercontraction on the conformation of the proteins constituting the dragline is not well defined. Furthermore, although it has already been established that supercontraction induces a disorientation of the molecular units, this effect has not been quantified yet. The main objective of the project is thus to investigate and quantify structural variations upon shrinking for two orb-web spiders. The experiments were carried out by Raman spectromicroscopy, an efficient tool to probe the molecular structure of a unique silk thread.

 

The data revealed a decrease in the orientation level within the fibers and a slight modification of the β-sheet and unordered structure content upon supercontraction. This analysis also constitutes one of the first studies to quantitatively compare the molecular structure of the dragline silk of two spider species. Besides, it would appear that a greater disorientation is associated to a higher percentage of shrinking.

 

 

 

Structural study of α-synuclein 71-82, a peptide derived from a protein involved in Parkinson’s disease: interactions with model membranes


Benjamin Martial1,2,3,4, Thierry Lefèvre1,2,3,4, Émilie Bruneau1,2,3,4, Laurie Bédard1,2,3,4, Thierry Buffeteau5, Michèle Auger1,2,3,4
1Université Laval 2PROTEO 3CERMA 4CQMF 5Université Bordeaux 1

α-synuclein is an amyloid protein involved in Parkinson’s disease. Aggregates of this protein are found in the brain of the patients, more specifically in nerve tissues. This protein can be found in several forms, from monomers to fibrils. The secondary structure of the latter has been described as a parallel β-sheet structure. Along this 140 amino acids protein, a specific portion plays an important role in the aggregation process: the non-amyloid-β component (NAC, sequence 61-95). Within the very core of this NAC, the sequence 71-82 appears to be crucial in the fibril formation process. It has been shown that α-syn71−82 shares several general properties and structural similarities with its parent protein. However, the behavior of this peptide in the proximity of cellular membranes remains unclear. In the present study, we have investigated the peptide structure when interacting with phospholipid model membranes. While no change in the structure (random coil) was observed upon interactions with neutral membranes, the peptide displays a dramatic structural change and adopts a parallel b-sheet structure in the presence of negatively charged membranes. To further investigate this structure, vibrational circular dichroism (VCD) spectroscopy was used to determine its supramolecular chirality. VCD revealed the presence of twisted fibrils, where the fibril twist chirality (left/right) depends on both the peptide concentration and the peptide-to-lipid molar ratio. The structural similarities shared by the peptide with the parent protein suggest that the former may overall influence the behavior and properties of the latter.

Study of the molecular basis of the interspecies communication complex ComRS in Streptococcus sp. through semi-rational mutagenesis


Joaquin Guzman Espinola1,2, Laura Ledesma2, Laetitia Fontaine2, Patrice Soumillion2, Pascal Hols2
1Université de Montréal 2Université Catholique de Louvain

Natural competence is the genetically specified ability of some cells to take up extracellular DNA. Internalized DNA can then be processed as a nutritional source or integrated in the genome to procure an evolutionary advantage to the host. This process is common among bacteria, especially in the Streptococcus genus where it is tightly regulated and linked to the production of bacterial toxins and the formation of biofilms. The ComRS system is a recently described pheromone quorum-sensing system that controls the development of natural competence in pathogenic strains of the salivarius, mutans, pyogenes and bovis groups of streptococci. Remarkably, depending on the species, ComRS systems can display high permissiveness or highly restricted specificity towards non-cognate pheromones, suggesting their implication in interspecies crosstalk. Site-directed mutagenesis linked to in vivo luciferase reporter assays have been used to monitor the development of natural competence. This allowed us to identify key residues and regions of the ComRS system involved. Moreover, functional chimeric proteins with altered pheromones recognition were successfully created to further elucidate the reason for interspecies recognition. Such knowledge could be capital to design natural competence quenchers preventing biofilm formation and inhibiting the major antibiotic resistance acquisition mechanism of those pathogens.

Synthesis of all monofluorogalactopyranoses as selective galectin inhibitors


Danny Lainé1, Vincent Denavit1, Denis Giguere1
1Université Laval

Galectins are a family of galactose-binding proteins with various biological activities. Galectin-3 is implicated in various cancer and galectin-1 increase HIV-1 attachment to target cells. The discovery of selective inhibitors of galectins could lead to new anti-cancer or anti-viral agents. We propose that substituting the hydroxyl groups on the galactopyranose core will procure analogues with superior stability, biological activity and improved pharmacokinetic profile. Polyfluorinated carbohydrates represent a good strategy to improve protein-carbohydrate affinities via desolvation together with attractive dipolar interactions mediated by polar C-F bonds. Incorporation of fluorine atoms in a pyranose sugar unit does not have a deleterious effect on the interactions with its binding pocket, despite the absence of any hydrogen bond donating capacity and the reduced capacity to accept hydrogen bonds. A chiron approach was used to access all of the monofluorogalactoside analogues. NMR spectroscopy was used to confirm the stereochemistry of the fluorine groups. All the analogues were evaluated over galectin-1 and galectin-3.

 

Systematic analysis of the expression, solubility and purification of fusion proteins expressed with different tags


Sarah Bernier1,2,3, Line Cantin1,2,3, Christian Salesse1,2,3
1Département d'ophtalmologie, Faculté de médecine, Université Laval 2CUO-recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement 3Regroupement stratégique PROTEO, Université Laval

GOAL: Studying proteins is a difficult task since getting a pure protein is very challenging. Different expression systems and tags can be assayed but finding the proper combination to obtain a pure protein can be very tedious. The choice of the purification tag is particularly important in the design of the fusion protein. Still, there is no single tag which yet satisfies all requirements. Consequently, passenger proteins are often fused with more than one tag to get the advantages of each individual tag. However, there is a lack of systematic studies on the impact of using a single tag versus combined tags and on the effect of the position of the tags in the construct. In the present work, these challenges are illustrated by expressing and purifying the membrane-associated protein « RGS9-1 anchor protein (R9AP) » with different tags. R9AP plays an important role in visual phototransduction and is associated to membranes via an alpha helical segment located in its C-terminal. The truncated R9AP protein without its membrane-associated alpha helical segment has been used because the full-length protein is both difficult to express and to solubilize.

 

METHODS: The cDNA of bovine R9AP without its C-terminal segment was cloned into pET-11a, pGEX-4T-1 and pMAL-c2X expression vectors. Different fusion proteins were expressed in Escherichia coli using some of the most commonly used purification tags: maltose-binding protein (MBP), glutathione S-transferase (GST) and polyHis tag. To obtain the different fusion proteins, R9AP was expressed with either a single tag (GST, MBP or polyHis) or a combination of tags (polyHis with either GST or MBP). Nine different fusion proteins were cloned, sequenced and expressed in E. coli BL21 (DE3) RIPL. All fusion proteins were purified using a single-step purification. GST-bR9AP was purified using a GSTrap column and MBP-bR9AP was purified with an amylose column, whereas all other fusion proteins were purified using a Ni Sepharose resin.

 

RESULTS: For single-tagged proteins, R9AP was found to be poorly expressed with a single polyHis tag whereas it is highly expressed in fusion with GST and MBP. Moreover, fusion proteins expressed with MBP and GST are fully soluble. When R9AP was expressed with a combination of tags, fusion proteins with a polyHis at the N-terminal (His-MBP- and His-GST-R9AP) were less expressed than single-tagged proteins. However, both proteins were fully soluble. In contrast, when the polyHis was located in between GST or MBP and the R9AP passenger protein (MBP-His- and GST-His-R9AP), high expression and fully soluble fusion proteins was observed. However, when polyHis was located at the C-terminal (GST-R9AP-His), the fusion protein was mostly insoluble. These results suggest that the polyHis tag can have a strong impact on both the expression and the solubility of the fusion protein, depending of its location. All fusion proteins were successfully purified using standard elution conditions except for the GST-R9AP-His fusion protein that was eluted using SDS.

 

CONCLUSIONS: Small changes in the design of fusion proteins can have a huge impact at different levels. Indeed, different positions and combinations of tags can significantly modulate the expression, solubility and purification of fusion proteins. It is thus essential to assay different designs of fusion proteins in order to get the best expression, solubility and purification yields for a specific protein of interest.

 

FUNDING: IRSC, CRSNG, PROTEO, RRSV

The dynamical zinc fingers of Miz-1


Cynthia Tremblay1,2,3,4, Mikaël Bédard1,2,4, Martin Montagne1,4, Danny Letourneau1,4, Pierre Lavigne1,2,3,4
1Université de Sherbrooke 2PROTEO 3Protéoméus 4IPS - Université de Sherbrooke

The C2H2 Zinc Finger (ZF) motif is the most conserved protein fold with a least 2% of the annotated human genome encoding for ZFs. Since the discovery of their ββα fold, ZFs have been assigned DNA binding and recognition roles in transcriptional regulation, with at least 45% of our transcription factor containing one or more ZFs. More precisely, over 700 hundred genes in the human genome possess ZFs, with an average of 8.5 ZFs per protein, where some of them encode for more than 30 ZFs. Despite their abundance, the function of only a few of those poly-ZF proteins is beginning to be understood. In order to contribute our understanding of the functions of those poly-ZF proteins, we study the structure, dynamics and DNA binding of Miz-1 (Myc Interacting Zing finger protein 1), a transcription factor containing 13 ZF, where the first 12 are consecutive. 

Through the structural study of ZF 1 to 10 (plus 13) of Miz-1 and their biophysical proprieties, we noticed that some of its ZFs show clear divergence from the conserved residues in the ββα core and inter-ZF linkers. Those divergences lead to unsuspected conformational exchange within 3 ZFs, to a compact inter-ZF structure and to predicted repulsion with the phosphodiester DNA backbone. We will present the structural and dynamical proprieties of  the remaining ZFs, i.e. 10 to 12, in absence of DNA. Our results show that ZF 10 and 11 undergo structural fluctuations in the ns-µs-ms timescale. Collectively, our results allow us to propose that Miz-1 could possess functions different than the canonical ZF-DNA binding.

The Impact of Conformational Entropy on the Accuracy of the Molecular Docking Software FlexAID in Binding Mode Prediction


Louis-Philippe Morency1,2,3, Rafael Najmanovich2,3
1Université de Sherbrooke 2Université de Montréal 3PROTÉO

A major goal of molecular docking is to predict the experimentally observed binding mode between a biomolecule, i.e. a polymer of amino or nucleic acids, and a ligand— e.g. small molecules, peptides or nucleic acids. This computational method is used to study the structure of the molecular interactions involved in cell’s essential biological functions. Actual molecular docking methods are developed to evaluate the molecular interactions of a single conformation, a single pose at a time, and they are trained to estimate the enthalpic fraction of the binding free energy. Consequently, most current molecular docking methods fail to efficiently model the entropic contributions, especially those of conformational nature, who are fundamental in molecular recognition events. 

 

Our research group develops FlexAID, an accessible and competitive ligand and biomolecule molecular docking software whose focus is on molecular flexibility. Here we introduce FlexAID’s newest feature that allows its scoring function to estimate the conformational entropy by redefining the static binding mode usually predicted in molecular docking into a dynamic collection of similar poses evaluated altogether. We present the impact of FlexAID’s newest feature on its accuracy in binding mode prediction using three increasingly complex scenarios : the Astex Diverse Set, the Astex Non Native Set and the HAP2 dataset. We show that FlexAID outperforms other open-source molecular docking methods when molecular flexibility is crucial. Furthermore, FlexAID now outputs multiple conformations per binding mode, a novelty that allows the user to visualize the dynamics of the complex studied. We believe that its higher accuracy in complex scenarios, the addition of novel features, e.g. the conformational entropy, its accessibility and its easy-to-use graphical user interface place FlexAID in an interesting position to tackle biologically and pharmacologically relevant situations currently ignored by other molecular docking methods.

 

FlexAID is available as a command-line pre-compiled executable (available at http://bcb.med.usherbrooke.ca/flexaid for Windows, macOS & Linux) or through the NRGsuite, a PyMOL integrated user interface allowing the user to use FlexAID in an intuitive manner with real time visualization. Both the NRGsuite and FlexAID are distributed as open-source software.

The rapid evolution of an ohnolog contributes to the ecological specialization of incipient yeast species


Chris Eberlein1, Lou Nielly-Thibault1, Halim Maaroufi1, Alexandre K. Dubé1, Jean-Baptiste Leducq1, Guillaume Charron1, Christian R. Landry1
1Université Laval

Identifying the molecular changes that lead to ecological specialization during speciation is one of the major goals of molecular evolution. One question that remains to be thoroughly investigated is whether ecological specialization derives strictly from adaptive changes and their associated trade-offs, or from conditionally neutral mutations that accumulate under relaxed selection. We used whole-genome sequencing, genome annotation and computational analyses to identify genes that have rapidly diverged between two incipient species of Saccharomyces paradoxus that occupy different climatic regions along a south-west to north-east gradient. We identified genes that show signatures of adaptation and accelerated rates of amino acid substitutions causing asymmetric evolution between lineages as candidate loci for ecological specialization. This set of genes includes a glycyl-tRNA-synthetase, GRS2, which is known to be heat stress-induced in the model and sister species S. cerevisiae. Molecular modelling, expression analysis and fitness assays suggest that the accelerated evolution of this gene in the Northern lineage may be caused by relaxed selection. GRS2 arose during the whole-genome duplication (WGD) that occurred 100 Mya in the yeast lineage. While its ohnolog GRS1 has been preserved in all post-WGD species, GRS2 has frequently been lost and is evolving rapidly, suggesting that the fate of this ohnolog is still to be resolved. Our results suggest that the asymmetric evolution of GRS2 between the two incipient S. paradoxus species contributes to their restricted climatic distributions and thus that ecological specialization derives at least partly from relaxed selection rather than a molecular trade-off resulting from adaptive evolution.

 

THE SYNTHESIS OF KERATAN SULFATE GLYCOSAMINOGLYCANS BY A GLYCOSYNTHASE APPROACH


Xiaohua Zhang1, Gautier Bailleul 1, David Kwan 1
1Concordia University

Glycosaminoglycans serve an important role in cell communication. Keratan sulfate is an example of a glycosaminoglycan that exhibits varied biological functions, such as neuronal development, and maintenance of corneal matrix structure. It is also known to restrict neural regeneration after injury through the formation of glial scars, and to be overexpressed in some cancers. Elucidating the mechanism by which keratan sulfate mediates signalling by determining the interactions of ligands and binding proteins with specific keratan sulfate sub-structures will lead to therapeutics that may be valuable in treating chronic diseases like neural damage and some forms of cancer.

To do this, homogeneous structures of keratin sulfate with discrete length and defined patterns of sulfation are synthesized using glycosynthases, which are engineered from Keratanase II by mutation of the active-site glutamate or aspartate residue that are involved in catalyzing the formation of the oxazoline intermediate. Sequence alignment analysis of several candidates among glutamate and aspartate residues that are conserved among GH111 enzymes reveal six residues that could be replaced by alanine. Preliminary results showed three of these six mutants are inactive to react with the natural substrate keratane sulfate. These mutants could work as glycosynthases that can synthesize homogeneous keratan sulfate oligo- and polysaccharides of defined length and sulfation pattern, thus they can be used to probe the signals and interactions of specific keratan sulfate structures to determine their biological roles, focusing on neurological function and pathologies including cancers.

Towards to the understanding of regio-specificity in glycopeptide antibiotic sulfotransferases.


Lei Yang1, Mahder Manenda2, Gerry Wright3, Rong Shi2
1Université Laval 2Université Laval 3McMaster University

In recent years, the spread of antibiotic resistant bacteria has become a serious problem in public health. This indicates a continuing and cyclical need for new antibiotics. Glycopeptide antibiotics (GPAs) play an important role in the treatment of infections caused by Gram-positive bacteria. GPA consist a heptapeptide skeleton that is modified by a variety of tailoring enzymes, such as glycosyltransferases, acyltransferase, sulfotransferases, methytransferases, and halogenases. Modifications on specific residues of the peptide core by these enzymes generate extensive chemical and functional diversity of natural GPAs. In this study, we focus on three different sulfotransferases, Teg12, Pek25, and Teg14, which could transfer a sulfate group from the cofactor 3’-phosphoadenosine-5’-phosphosulfate (PAPS) to specific residues on the heptapeptide scaffold of GPAs. Here we report a series of structures: Teg12, Pek25, and Teg14 bound with the cofactor product 3’-phosphoadenosine 5’-phosphate (PAP), respectively; and two ternary complexes of Teg12 with PAP and GPA substrate. Study on the structures of these enzyme-substrate-cofactor product complexes could give us a better understanding of the structural determinants governing their regio-specificities and will provide a solid basis to expand the chemical diversity of sulfated GPA derivatives.

Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks


Yossef Lopez de los Santos1, Guillaume Brault2, Nicolas Doucet1,3,4
1INRS – Institut Armand-Frappier, Université du Québec 2Odyssey Biotech 3GRASP, Groupe de Recherche Axé sur la Structure des Protéines 4PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications

Within the evolvability landscape of protein engineering approaches, the vast majority of mutations yield neutral, deleterious, or destabilizing effects. It has been demonstrated that stabilizing mutations are usually achieved by random mutagenesis, making the identification of improved protein variants an exhaustive and inefficient process. Herein we present a semi-rational combinatorial approach supported by docking simulations and Residue Interaction Networks (RINs) to design smaller and smarter libraries of mutants. Lipase B from Pseudozyma antarctica (CalB) was selected as an industrially relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was to develop CalB variants with enhanced synthetic activity towards aromatic substrates like cinnamic and salicylic acid. We used this combined approach to uncover the importance of residues in the CalB active-site cavity and their contribution to the synthetic reaction (Docking), in addition to calculating the energetic contributions upon site-directed mutagenesis (RINs). As a result, we improved the synthetic activity of CalB from 2% to more than 70% of the total substrate-product conversion ratio. This strategy allowed us to obtain more than 5 CalB variants with enhanced activity toward two bulky substrates after only two rounds of directed evolution. The sequential incorporation of favorable mutations increased our chances of selecting improved CalB variants and reduced screening effort. The use of a ‘bottom-up’ strategy such as the RINs allowed us to further understand the effects of mutations throughout the protein structure, a powerful tool for protein engineering purposes.

Understanding the early steps of amyloid assembly by mean of the biarsenical fluorescein dye


Noé Quittot1, Médina Hicheur1, Mathew Sebastiao1, Steve Bourgault1
1Université du Québec à Montréal

A large number of peptides and proteins are recognized for their ability to self-assemble into amyloid fibrils, whose tissue deposition is associated with degenerative diseases such as type II diabetes (T2D) and Alzheimer's disease (AD). The mechanism of self-assembly of amyloidogenic polypeptides has been studied by various techniques and one of the most widely used is an assay based on a fluorescent dye, thioflavin T, which binds to amyloid fibrils. However, this technique is insensitive to the formation of oligomers. In this context, we propose to develop a new detection method. This method relies on a fluorescent dye, Fluorescein Arsenical Hairpin (FlAsH), whose emission of fluorescence is amplified following its binding to a tetra-cysteic motif. Thus, a monomeric unit possessing two cysteines may lead to the reformation of the tetra-cysteic epitope during its oligomerization. In our study, we are interested in the self-assembly of islet amyloid polypeptide (IAPP), whose amyloid deposits are associated with T2D. Using native cysteines in the N-terminal region of IAPP, we were able to follow the self-assembly of IAPP. By this method, we have determined that IAPP does not form stable oligomers during the lag phase, which reinforces the hypothesis of a nucleated polymerization mechanism. Moreover, this assay allows us to evaluate the impact of amyloid co-factors, such as glycosaminoglycans, on the fibrillization process.

Utilisation de la spectroscopie Raman pour la détection de l’activité de l’oxyde nitrique synthase in vivo chez les bactéries


Francis Poirier Gravel1, Manon Couture1
1Université Laval

La spectroscopie Raman est une approche analytique émergente en microbiologie puisqu’elle s’avère efficace pour différencier rapidement différentes bactéries au niveau du genre, de l’espèce et de la souche1. Nous émettons l’hypothèse que cette approche pourrait permettre de détecter les différences phénotypiques associées à des gènes individuels ou des mutations ponctuelles chez les microorganismes, ouvrant ainsi la voie à des  études de type structure/fonction in vivo d’enzymes bactériennes responsables de la résistance aux antibiotiques et d’autres activités biologiques.

L’oxyde nitrique synthase (NOS) est une enzyme qui synthétise de l’oxyde nitrique (NO), un gaz potentiellement toxique, mais qui, à faible dose, est impliqué dans des activités de régulation chez l’humain et de nombreux autres organismes, dont les bactéries. Cette enzyme utilise un acide aminé, le L-arginine, comme substrat, du NADPH et de l’oxygène moléculaire afin de synthétiser le NO et de la citrulline. Il a été démontré que la synthèse du NO chez les bactéries interfère avec la réaction de Fenton et, de ce fait, protège les bactéries du stress oxydatif et leur confère une résistance accrue aux antibiotiques2. Tout comme ceux de Gusarov et al., nos résultats montrent que la bactérie B. subtilis est rendue plus sensible à un antibiotique, la pyocyanine, lorsque le gène codant la NOS est inactivé et que cet effet peut être renversé en complémentant la souche avec un gène de NOS sous le contrôle d’un promoteur inductible à l’arabinose. Le but du projet était d’examiner ces souches par spectroscopie Raman pour déterminer si elles peuvent être différenciées selon qu’elles synthétisent du NO ou non et ensuite, d’évaluer si l’effet de mutations ponctuelles de l’enzyme peut être détecté.

Un microspectromètre Raman et un laser émettant à 532 nm ont été utilisés pour obtenir les spectres Raman de la souche sauvage B. subtilis JH642, de la souche ΔNOS et de la souche complémentée avec le gène NOS de B. anthracis (baNOS). Les souches ont été testées suite à la croissance en  milieu LB et en milieu LB + arabinose 2%. Les données ont été analysées par la méthode de discrimination des composantes principales (DAPC) des données spectrales, ce qui permet le regroupement de spectres similaires et la dispersion des groupes de spectres qui diffèrent. Les résultats obtenus suggèrent qu’il est possible de regrouper les souches ayant une NOS active (JH642 et baNOS) et de les différencier la souche ΔNOS. La prochaine étape sera de déterminer si l’effet de mutations ponctuelles au site actif et ailleurs, ex. au site de liaison d’un cofacteur et à la surface, permettront de mettre en évidence un phénotype associé à ces mutations.

En conclusion, nous avons montré que la simple activation/inactivation du gène NOS peut être détectée par spectroscopie Raman de B. subtilis, ce qui indique que des mutations ponctuelles de la NOS modifant la catalyse pourraient être détectées de cette façon. Cette approche pourrait donc avoir un impact important pour l’étude structure/fonction d’enzymes, et d’autres protéines en général, puisqu’elle ouvre la porte au criblage rapide de l’effet sur la fonction de mutations dans le contexte physiologique.

1-Stockel, S., Kirchhoff, J. et al. (2016). Journal of Raman Spectroscopy 47, 89-109.

2-Gusarov, I., Shatalin, K. et al. (2009). Science 325, 1380-1384.

Why protein oligomer complexes allow better programmability over dimers and monomers?


Dominic Lauzon1,2, Alexis Vallée-Bélisle1,2
1Université de Montréal 2Laboratoire de Biosenseurs & Nanomachines

Proteins have mutated over millions of years and up to 45% of human enzymes listed have evolved into multimeric complexes. We already know that protein complexes can improve biological input by, for example, increasing the activity of enzyme or by helping regulation by combining specificity, allostery, activation and inhibition.[1] On the other hand, less is known about the thermodynamic advantage or cost related to the use of protein complexes and how their assembly may regulate their function. In this study, we employ a synthetic biochemistry approach to compare the performance of monomeric, dimeric and trimeric complexes. We do so by designing a simple DNA structure (three-way junction) that can be form using one, two or three DNA strands.  DNA represents a material of choice because it enables to control every thermodynamic parameters of the structure through simple mutations (e.g. modify the trimer affinity without affecting the dimer affinity). This contrasts with protein systems where the impacts of mutations are often unpredictable. Using mathematical simulation and experimental studies, we show that trimeric complex can exhibit a much larger window of regulation mechanism compared to dimeric complexes or monomers. Our DNA trimers illustrate binding behaviors going from positive to negative cooperativity with Kobs that cover 4 fold of magnitude. We also identify the ratio of dimeric affinity over trimeric affinity as the key parameter for programming the thresholds and the cooperativity of trimer assembly. Results provided by this study shine a new light on possible regulation mechanism of trimeric system and may help understanding why some proteins have evolved into oligomers.

 

[1] Marianayagam, N. J.; Sunde, M.; Matthews, J. M., The power of two: protein dimerization in biology. Trends in Biochemical Sciences 2004, 29 (11), 618-625.