3D structure prediction of truncated lecithin retinol acyltransferase using bioinformatics 3D prediction tools combined with experimental secondary structure from NMR. Failed successful predictions or successful failed predictions?
Vincent Boulanger
A comprehensive integration of the Residue Interaction Network and NMR relaxation dispersion approaches to understand the dynamic behavior that modulates the catalytic performance of xylanases
Yossef Lopez de los Santos
A fragment screening approach to discover new allosteric modulators of human RNases 3 and 5
Marie-Aude Pinoteau
Accelerating of the discovery of new monooxygenase variants for industrially relevant oxidation reactions
Olivier Rousseau
Brighter red fluorescent proteins display reduced structural dynamics
Adam M. Damry
Caractérisation structurale de la farnésyle diphosphate synthase de type II chez la tordeuse des bourgeons de l’épinette et évaluation d’inhibiteurs potentiels par arrimage moléculaire
Marie-Ève Picard
ConfBuster Web Server: a free web application for macrocycle conformational search and analysis
Gabriel Bégin
crystallization of non-structural proteins ORF24 and ORF26 of lactococcal phage p2
XIAOJUN ZHU
Dancing Zinc Fingers: How to Reconcile Conformational Exchange Within Zinc Fingers and DNA Binding ?
Cynthia Tremblay
Découverte d’AltLMNA, une protéine provenant d’une deuxième séquence codante fonctionnelle dans le gène Lamine A/C
Hélène Mouilleron
Des peptoïdes perméants comme transporteurs de molécules imperméables à travers la membrane cellulaire.
Andréanne Laniel
Designer Biosensors for Engineered Metabolic Pathways and Enzyme Evolution
Mohamed Nasr
Détermination de la structure tridimensionnelle du mutant S175R de la lécithine rétinol acyltransférase tronquée par résonance magnétique nucléaire
Marie-Ève Gauthier
Determining protein-protein interactions and intracellular organisation of the E.coli enterobactin metabolon through in vivo chemical crosslinking
Sylvie Ouellette
Development of a high-throughput assay to detect fatty acid decarboxylase activity
Jama Hagi-Yusuf
Development of a production and purification process for VSVg pseudotyped gesicles
Juliette Champeil
Development of a purification strategy for recombinant Vesicular stomatitis virus (rVSV) based HIV vaccine candidates
Anahita Bakhshizadeh Gashti
Développement de nouveaux analogues peptidomimétiques de la lactivicine ayant un potentiel antibiotique et inhibiteur de β-lactamases
Pierre-Alexandre Paquet-Côté
Développement d’un vecteur viral à double-cassette pour la visualisation en temps réel de l’adhésion et la prolifération des cellules endothéliales progénitrices
Samuel Daigle
Développement d’une approche à haut débit pour le criblage et la quantification de polyhydroxyalkanoates des bactéries échantillonnées à partir d’huiles usées.
Marianne Héneault
Direct phosphorylation of SH3 domains by tyrosine kinase receptors disassembles ligand-induced signaling networks
Ugo Dionne
Directed Evolution of a Triple-Decker Motif Containing Red Fluorescent Protein
Sandrine Legault
Discovering Drug Seeds by NMR Fragment-Based Lead Discovery
Luciana Coutinho de Oliveira
DNA Probes for Monitoring Enzyme Activity
Scott Harroun
DNA-protein conjugates for electrochemical biosensing applications
xiaomengwang
Duplication of homomeric protein: retention of paralogs and evolution of protein-protein interactions
Axelle Marchant
Dynamics and Stability of Long Telomeric G-Quadruplex DNA Sequences
Simone Carrino
Effect of binding interference on the divergence between paralogous genes that encode homodimers
Angel Fernando Cisneros Caballero
Effet de la vitesse de filage sur la structure moléculaire de fibres de soie d’araignée natives et supercontractées
Jane Gagné
Elucidating the activation mechanism of Tn7 transposition
Yao Shen
Étude de l’expression, de la solubilité, du clivage et de la purification de la rétinol déshydrogénase 8 en fusion avec différentes étiquettes de purification et de solubilisation
Charlotte Lemay-Lefebvre
Etude des transporteurs de Nickel chez Helicolibactrer pylori
Mirana Mirana Rakotoarivony
Évolution des complexes protéiques après hybridation entre espèces
Caroline Berger
Evolution of conformational exchange in a host defense enzyme family
David N. Bernard
Expression and purification of immunologic adjuvant P97c protein from Mycoplasma hyopneumoniae
Laurie Gauthier
Fluorogenic chemical tools based on cysteine labeling to study oligomer formation in amyloid self-assembly
Guillaume Charron
Folding and binding act as determinants of environment specific fitness effects
Rohan Dandage
Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device
Laura Leclerc
Function and engineering of enzymes involved in the glycosylation of natural products
Fathima Mohideen
Functional Characterization of AltB2R, an Alternative Protein Encoded in the Bradykinin B2 Receptor Gene Involved in the B2R Signaling
Maxime Gagnon
Functionalization of amyloid-based nanoparticles
Soultan Al-Halifa
Genetic backgrounds have complex effects on the drug treatment to a human disease mutation
Véronique Hamel
Guiding self-assembly and growth of amyloid-like nanoparticles
Ximena Zottig
Guiding self-assembly and growth of amyloid-like nanoparticles
Ximena Zottig
Homology modeling and semi-rational protein engineering of a new metagenomic lipase
Ngoc Thu Hang PHAM
Hybridization as an adaptive force in response to extreme UV conditions
Carla Bautista Rodríguez
Identification and characterization of a novel mitotic target site for Haspin on Histone H2B
Ibrahim Alharbi
Identification of a molecular hinge controlling the amyloid self-assembly and cytotoxicity of islet amyloid polypeptide
Elizabeth Godin
Identification of structural determinants of biased signaling of the apelin receptor
Laurent Bruneau Cossette
Identification protéomique de nouvelles protéines effectrices dans la signalisation des récepteurs Eph
Sara Banerjee
Impact of the incorporation of a monofluoroalkene moiety on the hydrophobicity of small peptides
José Laxio Arenas
Inhibition and activation mechanism study of the kinase by isothermal titration calorimetry (ITC)
yun wang
Interactions of amyloid peptide AS71-82 with model membranes: structural and morphological study via FTIR and ssNMR
Benjamin Martial
Investigations phytochimiques du Bouleau glanduleux et isolation d’actifs cosméceutiques
Claudia Carpentier
Kinetically Programmed, One-Pot DNA Reactions for Molecular Detection Directly in Whole Blood
Guichi Zhu
La liaison du tout-trans rétinol avec la lécithine rétinol acyltransférase tronquée et ses mutants n’explique pas la faible activité enzymatique des mutants
Sarah Roy
La liaison membranaire de la protéine S100A10 et du peptide d’AHNAK intervenant dans la réparation membranaire
Xiaolin Yan
Le complexe du pore nucléaire de la levure comme système-modèle pour l'étude de la rétention des gènes dupliqués
Simon Aubé
Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η
Christopher J. Wilds
Linear and cyclic peptides as green catalysts for chiral epoxidations
Christopher Bérubé
Measuring covalent inhibitor kinetics using isothermal titration calorimetry
Caroline Dufresne
Measuring Enzyme Kinetics Using Isothermal Titration Calorimetry
Justin Di Trani
Optimisation de l’activité et de la sélectivité d’agonistes des récepteurs neurotensinergiques​
Michael Desgagné
Palladium-Catalyzed Synthesis of Functionalized Monofluoroalkenes
Myriam Drouin
Plasma membrane vesicles derived from mammalian cells to study the perturbative nature of amyloid fibril assembly
Mathew Sebastiao
Préparation de peptides macrocycliques sur résine oxime
Alexandre Borgia
Proteomic analysis of NCK1/2 adaptors reveals a new NCK2-specific role in cell abscission during cytokinesis.
François Chartier
Règles thermodynamiques et cinétiques pour l'assemblage et la régulation de nanomachines polymoléculaires à base d’ADN
Dominic Lauzon
Rôle des protéines S100A16 et Annexine A4 dans le maintien de l’intégrité membranaire
Francis Noël
Rôle des récepteurs Eph dans l’établissement de la polarité des cellules épithéliales
Noémie Lavoie
Self-assembled fibrillar nanostructures for vaccine development
Margaryta Babych
Solid-state NMR study of the microalga Chlamydomonas reinhardtii and its constituents
Alexandre POULHAZAN
Structural and (supra)molecular basis of the cellular toxicity of amyloid fibrils
Phuong Trang Nguyen
STRUCTURAL AND BIOPHYSICAL CHARACTERIZATION OF THE HOMODIMERIC INTERFACE OF HUMAN GALECTIN-7
Myriam Letourneau
Structural and dynamic characterization of UbKEKS, a newly identified ubiquitin encoded in a pseudogene
Patrick Delattre
Structural determinants of conformational exchange in GB1 DANCERs
Mayer Marc
Structure et liaison membranaire de la R9AP, une protéine impliquée dans la phototransduction visuelle
Sarah Bernier
Surexpression et purification de la sous-unité gamma de la transducine, une protéine de la phototransduction visuelle
Alexandre Vaillancourt
Synthèse de glycopeptides comme outils immunogéniques dans la recherche antifongique et antitumorale
Tremblay Thomas
Synthesis of poly-fluorinated glucopyranose derivatives from levoglucosan
Megan Bouchard
Systematic perturbation of yeast essential genes using base editing
Philippe Després
The bacterial protein Curli: expression and characterization for the biomedical application of functional amyloid assemblies
Dominic Arpin
The crystal structure of the Cdc5-Dbf4 complex provides insight into Polo-box domain substrate recognition.
Ahmad Almawi
The first crystal structure of a bacterial acetylcholinesterase
Van Dung Pham
The Impact of Conformational Entropy on the Accuracy of the Molecular Docking Software FlexAID in Binding Mode Prediction
Louis-Philippe Morency
The periplasmic reductase DsbG has a chaperone activity in the elyC mutant of Escherichia coli
Imène Kouidmi
THE SYNTHESIS OF KERATAN SULFATE GLYCOSAMINOGLYCANS BY A GLYCOSYNTHASE APPROACH
Xiaohua Zhang
Unraveling the controversial role of the pseudokinase domain of BUBR1 in mitosis
Luciano Gama Braga
Valorisation des huiles usagées à moteur
manel Ghribi
Vers le développement d’une nouvelle génération d’inhibiteurs de l’oncoprotéine c-Myc
Jean-Michel Moreau

3D structure prediction of truncated lecithin retinol acyltransferase using bioinformatics 3D prediction tools combined with experimental secondary structure from NMR. Failed successful predictions or successful failed predictions?


Vincent Boulanger1, Christian Salesse1, Stephane Gagne2
1Université Laval 2Universite Laval

CONTEXT AND GOAL: The lecithin retinol acyltransferase (LRAT) plays an important function in the visual cycle. It converts all-trans retinol to all-trans retinyl ester by transferring an acyl-group from phosphatidylcholine. This enzyme has a catalytic activity centered on Cys-161. Cys-168, Tyr-154, His-60 and His-72 have also been found important for the function. Since LRAT is a transmembrane protein, the truncated LRAT form (tLRAT) is used in laboratory experiments. No tertiary structure of tLRAT is known at this time but preliminary NMR results have allowed the per-residue experimental determination of its secondary structure. The objective of the project is to combine experimental secondary structure from NMR data with two bioinformatic 3D structure prediction tools (I-TASSER, a template-based method and QUARK, an ab initio method) to predict the 3D structure of tLRAT.

MATERIAL AND METHODS: Standalone package of I-TASSER (version 5.1) and web server version of QUARK were used in this study. Predictions were tested using a number of different parameters. Those included the exclusion of some templates, the use of experimental NMR data, as well as the use of distance restraints between important catalytic reactions residues.

RESULTS: A total number of 18 predictions were performed and analyzed, including 17 I-TASSER predictions. Most of the predictions are primarily based on a homologous, but non-natural, chimeric protein structure and have favorable statistics (high C-score value, estimated TM-score > 0.60 and high number of templates). However, these predictions display an incompatible secondary structure with the experimental NMR data. We also performed 3D predictions that incorporate NMR secondary structure, both with and without different combination of active site distance restraints. Other predictions, such as those with excluded templates with or without distance restraints show less favorable statistics (generally poor C-score ranging from -5 to -2, estimated TM-score < 0.40 and low number of templates), but are compatible with NMR secondary structure data. Most of QUARK results have secondary structure congruent with NMR data.

A comprehensive integration of the Residue Interaction Network and NMR relaxation dispersion approaches to understand the dynamic behavior that modulates the catalytic performance of xylanases


Yossef Lopez de los Santos1, Louise Roux1, Nicolas Doucet2,3
1Institut Armand-Frappier (INRS) 2INRS - University of Quebec 3PROTEO

Proteins are the most ubiquitous and versatile devices in nature. They are involved in all cellular functions, including signaling cascades, cellular communication, metabolism, energy production, etc. These “nano-machines” rely on conformational changes to perform their activities. To understand how the collective motions within the protein structure of xylanases modulate their catalytic performance, we used relaxation dispersion NMR experiments to capture atomic-scale dynamics on a per residue basis and integrated that information trough a holistic approach using a Residue Interaction Network (RIN) analysis. Focusing on endo-xylanases from family 11 (Xln11), we recently uncovered previously uncharacterized conformational exchange occurring on the catalytic time frame (ms) in XlnB2 from S. lividans. A recent report linking conformational dynamics to the functional diversity of the RNase enzyme superfamily leads us to hypothesize that conformational exchange could also be evolutionary conserved among Xln11 homologs. Our proposal merges structural organization and functional features from a “network centric” perspective, aiming to link global network properties and the individual motions of its constituents. Such confluence may help to determine the localization of key residues (or sub-networks) that exert control over protein structure organization and the coordination of motions contributing to catalysis in Xln11 members. Our ultimate objective is to provide the theoretical framework to implement semi-rational design on this relevant group of biotechnological targets. 

A fragment screening approach to discover new allosteric modulators of human RNases 3 and 5


Marie-Aude Pinoteau1, Myriam Letourneau1, Yossef Lopez de los Santos1, Donald Gagné1, Yann Ayotte1, Steven laplante1,2, Nicolas Doucet1,2
1INRS Institut Armand-Frappier 2PROTEO

Pancreatic-type ribonucleases form a broad enzyme superfamily sharing specific structural properties involved in RNA degradation. Among human RNases, some present interesting therapeutic profiles, in particular RNases 3 and 5, also known as eosinophil cationic protein (ECP) and angiogenin, respectively. ECP is produced by eosinophils and exhibits antibacterial, antiviral, and cytotoxic activity. It is associated to a number of human pathologies, including asthma and Crohn’s disease. In contrast, RNase 5 is involved in blood vessel formation (angiogenesis), an important biological phenomenon that requires tight regulation. It is an unusual superfamily member since its ribonucleolytic activity is significantly lower than that of other RNases, albeit mandatory for angiogenic activity. As a result, RNase 5 misregulation has drastically negative effects in tumor growth progression. However, this enzyme also exhibits neuroprotective abilities, giving rise to a possible therapeutic avenue for neurodegenerative diseases such as ALS. Because RNases 3 and 5 are good candidates for drug discovery, the goal of the present work is to uncover allosteric modulators that could exert a controlling influence on specific biological activities of these proteins. Previous studies have demonstrated the dynamic nature of pancreatic-type RNases, which rely on long-range motions for proper enzymatic function. This observation further supports the possibility of modulating their activity through allostery. In the present work, a fragment-based drug discovery (FBDD) strategy is presented to achieve this goal, whereby a small fragment library is screened against a target protein and ligand binding is detected by NMR. In contrast to other methodologies, FBDD offers the opportunity to efficiently explore the available chemical space, while providing a scaffold to optimize modulators with greater affinity. A limited number of RNase 3 and 5 fragments will be presented, in addition to their preliminary characterization.

Accelerating of the discovery of new monooxygenase variants for industrially relevant oxidation reactions


Olivier Rousseau1, Musa Ozboyaci1, Maximilian Ebert1, Daniela Quaglia1, Joelle Pelletier1, Sebastian Pechmann1
1Université de Montréal 2Université de Montréal

Industrial production of chemicals relies largely on organic synthesis despite its inherent environmental impact. Enzymes are an eco-friendly alternative that can simplify complex reaction schemes, however they can be labor intensive to engineer. We have developed two methods to accelerate enzyme engineering based on a cytochrome P450 BM3 (BM3) monooxygenase system.  BM3 belongs to the large family of P450s able to catalyze the challenging regio-, chemo- and stereoselective oxidation of non-activated carbon atoms in a single step and is therefore of great industrial interest.

 

First, we explore the substrate promiscuity of BM3. We screen for increased transformation of indole to indigo and thus find new variants that produce raspberry ketone. We optimized a high-throughput NADPH fluorescence assay to screen a library of 55 variants at five positions in the active site. This primary filter reduced by 42% and 56% the number of samples to analyze on LCMS/GCMS for indigo and raspberry ketone, respectively. We identified 18 variants that efficiently synthesize indigo, including 15 that were not previously reported. Eight among the 18 were also active for raspberry ketone production, demonstrating that high NADPH consumption is an excellent indicator of the oxidation of varied substrates.

 

We then use a multiplexing approach to create, screen and stratify variants at a larger scale. Saturation mutants at 49 positions of BM3 were screened using an easy colorimetric plate assay. Screening revealed 29 new positions where at least one variant produces indigo and more than 11000 colonies were pooled in 3 categories: white, light blue and dark blue. Next-generation sequencing (NGS) provided rapid, cheap and massive identification of more than 200 indigo-producing variants. Active and inactive variants are being further characterized using modeling and Molecular Dynamics simulations.

Brighter red fluorescent proteins display reduced structural dynamics


Adam M. Damry1, Natalie K. Goto1, Roberto A. Chica1
1Université d'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 of the widely-used mCherry monomeric RFP (QY = 0.22), a bright variant mScarlet (QY = 0.70), and a dim variant mRojoA (QY = 0.02). A residue-by-residue comparison using 1H–15N HSQC spectra showed line-width broadening correlating with QY in roughly 10% of backbone amide peaks. As peak line-widths are influenced by microsecond–second timescale motions, T1 and T2 relaxation measurements were performed to probe picosecond–nanosecond timescale dynamics. These measurements showed that apparent correlation time increases with QY in roughly 10% of backbone amide peaks. While many positions selected by these experiments are dispersed throughout the RFP scaffold, the β strand 8-10 region shows a cluster of residues whose dynamics correlate with QY on at least one of these timescales. This indicates a potential link between dynamics in this region and chromophore flexibility and brightness. Our results therefore open the door to the rational design of more rigid, brighter RFPs.

Caractérisation structurale de la farnésyle diphosphate synthase de type II chez la tordeuse des bourgeons de l’épinette et évaluation d’inhibiteurs potentiels par arrimage moléculaire


Marie-Ève Picard1, Audrey Nisole2, Catherine Béliveau2, Stephanie Sen3, Aline Barbar2, Michel Cusson1,2, Rong Shi1
1Département de biochimie, de microbiologie et de bio-informatique, Institut de Biologie Intégrative et des Systèmes, PROTEO, Université Laval 2Ressources Naturelles Canada, Service canadien des forêts, Centre de foresterie des Laurentides 3Department of Chemistry, The College of New Jersey

La farnésyle diphosphate synthase (FPPS) est une enzyme de la classe des prényltransférases qui catalyse la condensation de l'isopentényl diphosphate (IPP, C5) avec le diméthylallyl diphosphate (DMAPP, C5) pour générer le produit FPP (C15). Chez les insectes, la FPPS joue un rôle clé dans la biosynthèse de l'hormone juvénile (JH). Cette hormone assure le maintien des caractères juvéniles lors des mues larvaires et favorise la maturation reproductive des insectes adultes.

 

Le génome des Lépidoptères codent deux paralogues de FPPS très distincts, dont l'un (type II) s'exprime presque exclusivement dans les glandes productrices de JH, les corps allates. Il a été supposé que ce paralogue présentait des caractéristiques structurelles qui permettent la liaison de précurseurs plus volumineux requis pour la biosynthèse des JH à branches éthyliques retrouvées uniquement chez les Lépidoptères.

 

Les FPPSs sont connues pour être inhibées par une classe de médicaments appelés bisphosphonates, utilisés pour traiter une diversité de maladies liées aux os chez les humains. Les structures de complexes de FPPS de type II provenant de la tordeuse des bourgeons de l'épinette, Choristoneura fumiferana (Ordre: Lépidoptères), avec deux bisphosphonates et leur co-substrat naturel IPP ont été déterminées par cristallographie aux-rayons X dans notre laboratoire. La comparaison des structures de C. fumiferana et d'autres FPPS fournit des données clé pour guider la conception d'inhibiteurs présentant une plus grande spécificité par rapport aux FPPSs des Lépidoptères, plus particulièrement celle de la tordeuse des bourgeons de l'épinette, l'une des plus grandes menaces des forêts de conifères en Amérique du Nord.

 

Dans le cadre de ce projet, des essais d’arrimage moléculaire ont été réalisés dans le site actif de la FPPS de type II de la tordeuse des bourgeons de l’épinette à l’aide de l’outil idock. Ainsi, des inhibiteurs connus de FPPS d’autres organismes ont été arrimés dans la FPPS de la tordeuse. À partir de ces résultats, nous avons identifié certaines molécules qui ont le potentiel d’être de meilleurs inhibiteurs de la FPPS de ce ravageur forestier.

ConfBuster Web Server: a free web application for macrocycle conformational search and analysis


Gabriel Bégin1, Xavier Barbeau1, Antony Vincent1,2, Patrick Lague1
1Université Laval 2INRS-Institut Armand-Frappier

ConfBuster Web Server is a free online resource for searching the lowest energy conformations of macrocycles. This web application is a front-end for ConfBuster, an open-source suite of tools developed by our group. In addition to a user email address, the 3D coordinates of a macromolecule in PDB, SDF, MOL or MOL2 format must be provided as input. The results of the conformational search are sent via an archive file in ZIP format attached to an email. As for the original ConfBuster suite of tools, the results include a set of energy-minimized macrocycle coordinates along with a PyMOL visualization script and graphics for meta-analysis. The present server can be used for free to all users, as well as its source code to efficiently build a similar front-end server. To our knowledge, this is the only web resource for macrocycle conformational search freely accessible to the scientific community.

crystallization of non-structural proteins ORF24 and ORF26 of lactococcal phage p2


XIAOJUN ZHU1, DAOWEI ZHU1, Jérémie Hamel1, Sylvain Moineau1, Rong Shi1
1Universite Laval 2Université Laval 3Université Laval 4Université Laval

Lactococcus lactis is extensively used as the starter cultures for the industry production of an array of milk fermented products. The fermentation can be slowed or even stopped in the presence of the lactococcal phage p2. The infection mechanism, however, is still in mystery. The past decade has seen a remarkable progress in the study of structural proteins of phage p2. These results have provided molecular insights into the structure, assembly and infection mechanism of phage p2. Strikingly, ~45% of 49 proteins encoded by phage p2 genome have unknown function. Almost all of these functionally unknown proteins are non-structural proteins produced intracellularly at the beginning of the phage infection process. Notably, no similar homologs for the majority of these proteins have been structurally characterized.

 

In this study, we utilize X-ray crystallography as the primary tool to characterize the non-structural proteins. To date, the crystals of ORF24 and ORF26(SaV) have been obtained and diffraction data sets have been collected to 2.7Å and 2.3Å, respectively. The determination of the structure of these two proteins is in process.

Dancing Zinc Fingers: How to Reconcile Conformational Exchange Within Zinc Fingers and DNA Binding ?


Cynthia Tremblay1, Martin Montagne1, Danny Letourneau1, Pierre Lavigne1
1IPS - Université de Sherbrooke 2Université de Sherbrooke 3Université de Sherbrooke 4Unibersité de Sherbrooke

The C2H2 Zinc Finger (ZF) motif is the most conserved and most abundant protein fold of our genome, with at least 2% of the annotated human genome encoding for ZFs, e.g. 8080 domains out 1854 genes as noted in 2014. 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. Though, knowing that ZFs containing proteins have an average of 8.5 ZFs per protein, to up to 30 ZFs per protein, it appear highly improbable that each of theses ZFs should recognise DNA in a specific manner and lead to extreme affinity. In order to contribute to 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 use that motion in order to bind less effectively random DNA, without affecting its affinity for its binding sequence.

Découverte d’AltLMNA, une protéine provenant d’une deuxième séquence codante fonctionnelle dans le gène Lamine A/C


Hélène Mouilleron1,3,4, Vivian Delcourt1,2,3,4, Sondos Samandi1,3,4, Jean-François Jacques1,3,4, Xavier Roucou1,3,4
1Faculté de Médecine et des Sciences de la Santé, Département de Biochimie, Pavillon de Recherche Appliquée sur le Cancer, Université de Sherbrooke, Québec, Canada 2Université de Lille, INSERM U1192, Laboratoire Protéomique, Réponse Inflammatoire & Spectrométrie de Masse (PRISM) F-59000 Lille, France 3Regroupement stratégique PROTEO, Université Laval, Québec, Canada 4PROTEOMEUS, Université de Sherbrooke, Québec, Canada

    Les lamines de types A (LMNA), codées par le gène Lamine A/C, sont des composantes de la lamina nucléaire. Cette dernière donne sa forme et sa stabilité à l’enveloppe nucléaire et joue un rôle important dans l’organisation de la chromatine. De plus, de nombreuses mutations dans le gène Lamine A/C ont déjà été identifiées comme étant responsables de laminopathies telles que la Progeria, des cardiomyopathies ou encore des dystrophies musculaires.

 

    Au laboratoire, nous avons ré-analysé le transcriptome de différents organismes et avons détecté des milliers de nouvelles séquences codantes, différentes des séquences codantes de référence. Nous les appelons alternatives. Ces séquences codantes alternatives peuvent se trouver dans des CDSs (Coding DNA Sequences) déjà connues dans les cadres de lecture +2 ou +3 ou dans les UTRs (Untranslated Regions) dans tous les cadres de lectures possibles. Elles codent pour des protéines qui sont donc totalement différentes des protéines de références. Ainsi, nous avons notamment découvert que le gène codant pour les lamines de types A pourrait également coder pour une protéine alternative AltLMNA, à l’intérieur de son CDS. Cette protéine alternative, au même titre que sa protéine de référence, est sujette à des mutations et pourrait donc avoir un rôle à jouer dans diverses pathologies.

 

    Nous avons dans un premier temps montré par immunobuvardage que LMNA et AltLMNA sont coexprimées dans des cellules HeLa. Par la suite, il a été démontré par immunofluorescence que la protéine AltLMNA, tout comme sa protéine de référence, possède une localisation nucléaire. Cependant, bien que la localisation d'AltLMNA soit majoritairement périnucléaire, les deux protéines ne colocalisent pas. Dans le but d’avoir des indices quant à la fonction de cette protéine alternative, les partenaires d’interaction d’AltLMNA ont ensuite été identifiés par co-immunoprécipitation suivie d’une analyse par spectrométrie de masse. Ainsi, nous savons qu'AltLMNA interagit avec des protéines impliquées entre autre dans le cycle cellulaire : PSME3 et CENPV. Au vu de ces résultats, nous avons décidé d'étudier le comportement d’AltLMNA au cours du cycle cellulaire. Pour ce faire, nous utilisons des cellules HeLa synchronisées aux différentes phases du cycle cellulaire à l’aide de plusieurs drogues. Il a ainsi été démontré que la localisation d'AltLMNA est étroitement liée au cycle cellulaire. En phase S, AltLMNA possède une localisation périnucléaire et colocalise avec l'hétérochromatine. De plus, il a été observé qu'AltLMNA transloque du noyau vers les mitochondries durant la mitose et que celle-ci perd ses interactions avec PSME3 et CENPV durant cette phase du cycle.

 

    Enfin, AltLMNA interagit pendant l’interphase avec PSME3, une protéine également impliquée dans la réponse aux cassures double-brin de l’ADN. Nous avons pu constater par immunofluorescence et gel d’électrophorèse en champ pulsé (PFGE) que la surexpression d’AltLMNA conduit en effet à la production de cassures double brin de l’ADN. Bien que ces cassures ne semblent pas faire obstacle de façon significative à la prolifération des cellules, celles-ci pourraient partiellement expliquer les maladies sus-citées telle que la Progeria qui se caractérise par un vieillissement accéléré des enfants atteints dû notamment à la présence de nombreux dommages à l’ADN. Ceci est d’autant plus vrai que nous avons observé qu’AltLMNA s’échappe des noyaux des cellules progériques, qui sont très déformés. En effet, si PSME3 est un acteur important de la réparation des cassures double-brin de l’ADN, le fait qu’AltLMNA l’entraine hors des noyaux en cas de Progeria coïnciderait avec la présence de ces nombreux dommages.    

Des peptoïdes perméants comme transporteurs de molécules imperméables à travers la membrane cellulaire.


Andréanne Laniel1, Étienne Marouseau1, Christine Lavoie1, Éric Marsault1
1Université de Sherbrooke

La membrane cellulaire est la barrière protégeant la cellule. Elle permet de contrôler l’échange entre les milieux extra- et intracellulaire. Il y a donc plusieurs classes de molécules qui ne peuvent la traverser. Ainsi, la découverte de peptides perméants ayant la capacité de traverser les membranes cellulaires a permis d’élargir la gamme de molécules capables de les traverser. Dérivée de ces peptides, la classe des transporteurs riches en guanidines (TRGs), s’est avérée prometteuse pour le transport de divers cargos imperméables. Il a été montré que les TRGs doivent interagir avec les glycosaminoglycanes (GAGs) pour entrer dans la cellule. Dans notre étude, plusieurs constructions synthétiques de TRGs ont donc été préparées pour étudier leur interaction avec les motifs de sulfatation des GAGs, dans le but de comprendre leur structure-perméabilité. Pour ce faire, on a évalué et comparé le niveau de pénétration cellulaire des TRGs, ainsi que leur cytotoxicité chez des cellules HeLa. Par la suite, l’objectif est de caractériser l’internalisation de nos constructions synthétiques dans plusieurs lignées cellulaires humaines.

 

À l’aide de la cytométrie de flux, nous avons testé la pénétration de nos TRGs à 5μM en mesurant la fluorescence moyenne de leur cargo, la fluorescéine. Les résultats ont montré que les composés commençaient à passer significativement la membrane cellulaire à partir de 6 guanidines et avaient une pénétration optimale avec 8 guanidines. Nous observons ainsi une absorption cellulaire maximale pour PGua4 (8 guanidines), avec 80% d’absorption de notre contrôle positif, la nonaarginine (R9).

 

À l’aide de la microscopie confocale, nous avons déterminé la distribution cellulaire de R9 et PGua4 chez des cellules HeLa vivantes. Ces peptoïdes s’accumulent tous deux dans les endosomes précoces et seront majoritairement recyclés à des concentrations de 5μM. Il ne semble pas y avoir d’évasion endosomale à cette concentration, c’est donc pourquoi nous allons établir la courbe de concentration correspondant à l’évasion endosomale de ces deux peptoïdes.

 

Pour l’instant, PGua4 est le TRG pénétrant le mieux dans les cellules HeLa. Plusieurs autres TRGs, pour lesquels on a introduit des modifications structurales vis-à-vis de la structure de PGua4, seront testés et les peptoïdes ayant le taux de pénétration le plus élevé seront caractérisés par microscopie confocale. Cette étude nous permettra de mieux comprendre comment les TRGs entrent dans la cellule, afin d’envisager la conjugaison et la livraison de molécules d’intérêt incapables de traverser les membranes.

 

Designer Biosensors for Engineered Metabolic Pathways and Enzyme Evolution


Mohamed Nasr1, Logan Timmins1, David Kwan1, Vincent Martin1
1Concordia University

Synthetic biology techniques aimed at constructing artificial metabolic pathways in genetically modified microorganisms are emerging as important, sustainable methods to produce biofuels, pharmaceuticals and valuable chemicals. To reach industrially relevant scales, challenges related to pathway bottlenecks and system optimization must be addressed. Since these pathways are usually built of multiple enzymes, improving pathway efficiency by processes such as enzyme directed evolution offers a solution to these limitations. However, screening methods for the majority of products of these enzymatic pathways are slow and inefficient. The purpose of this work is to utilize transcriptional repressor-based biosensors, predominantly from the TetR family, to develop fast and high-throughput detection methods of artificial metabolic products. This will be achieved by linking the production of these molecules to a measurable signal, such as fluorescence.

 

Transcriptional repressors in general bind specific effectors or effector families, which limits their usability as biosensors in many engineered pathways. Therefore, this project will aim at expanding the toolbox of repressors available by engineering their effector-binding domains to respond to alternative effector molecules. As a proof of principle, using a semi rational approach, we will engineer repressors 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. Our “designer” biosensors will be utilized to improve yields of an adipic acid-producing yeast strain. Particularly, they will be used as genetic circuits within engineered strains for pathway dynamic control, which is a method for in vivo real-time control of gene expression. As well, these biosensors will be used as screening tools for the directed evolution of pathway enzymes.

Détermination de la structure tridimensionnelle du mutant S175R de la lécithine rétinol acyltransférase tronquée par résonance magnétique nucléaire


Marie-Ève Gauthier1,2,3,4,5, Line Cantin1,2,3, Stephane Gagne3,4,5, 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. Le mécanisme de l’activité enzymatique de la LRAT est toujours inconnu, mais quatre résidus ont été recensés comme étant essentiels à la catalyse (H60, H72, Y154 et C161). Il est donc important de déterminer la structure tridimensionnelle de la LRAT afin de mieux comprendre son activité enzymatique ainsi que l’effet des mutations sur cette enzyme. Dans le cadre de ce projet, la structure tridimensionnelle du mutant S175R, comprenant aussi les mutations C161S et C168S, de la forme tronquée de la LRAT (C161S/C168S/S175R-tLRAT) sera déterminée par résonance magnétique nucléaire (RMN). Antérieurement, il a été déterminé que la mutation S175R menait à une perte complète de l’activité enzymatique de la LRAT, contrairement aux autres mutants, où une activité résiduelle a été observée. L’obtention de la structure tridimensionnelle de ce mutant aidera à comprendre le mécanisme de l’activité enzymatique de la LRAT et l’impact de cette mutation l’activité de la protéine. MATÉRIEL ET MÉTHODES : La C161S/C168S/S175R-tLRAT marquée au 15N et 13C a été purifiée par chromatographie d’affinité. La RMN a été faite avec des spectromètres de 600 MHz et 800 MHz. RÉSULTATS : Les résultats obtenus à ce jour ont été comparés avec ceux de la C161S/C168S-tLRAT. Il a été observé que l’introduction de la mutation S175R menait à des changements au niveau des déplacements chimiques des résidus K95 et G100. Ces premiers résultats apportent des informations sur l’importance de ces résidus au niveau du site catalytique de la LRAT. De plus, les analyses par RMN ont permis de déterminer un contenu important en hélices alpha et pelote statistique dans la structure secondaire de la protéine. CONCLUSION : Même si les calculs pour la détermination de la structure tridimensionnelle sont toujours en cours, il a été possible de prédire l’influence de la mutation S175R sur des résidus qui ne sont connus pour être essentiels à la catalyse. Cela permet de postuler que ces résidus se retrouveraient près du site catalytique de la LRAT. La prédiction de la structure secondaire de la C161S/C168S/S175R-tLRAT a aussi permis d’obtenir des résultats différents des modèles de prédiction.

Determining protein-protein interactions and intracellular organisation of the E.coli enterobactin metabolon through in vivo chemical crosslinking


Sylvie Ouellette1, Peter D. Pawelek1
1Université Concordia

Metals ions such as iron are crucial for bacterial metabolism and growth, and many micro-organisms have evolved mechanisms to scavenge extracellular metals. In low-iron conditions, E. coli synthesizes the catecholate siderophore enterobactin, a high-affinity iron chelator that is secreted in order to acquire extracellular iron. Enterobactin is produced through the sequential activities of six biosynthetic enzymes (EntA-F). We hypothesize that some of these enzymes engage in protein-protein interactions to enhance the efficiency of enterobactin biosynthesis via substrate channeling. Our lab has previously reported in vitro evidence of such interactions (EntB-EntA, EntA-EntE).  The E. coli membrane protein EntS acts as the inner-membrane channel for enterobactin export. We additionally hypothesize that the enterobactin biosynthetic machinery is in direct contact with EntS via protein-protein interactions. To investigate these hypotheses, we are employing in vivo formaldehyde crosslinking to determine the intracellular organisation of the enterobactin biosynthetic enzymes as well as the EntS transporter. Initial in vivo crosslinking results indicated that a recombinant H6-tagged EntB forms higher-order complexes with chromosomally-encoded E. coli proteins expressed during iron restriction. We also developed a detergent screen to find conditions to optimally extract H6-tagged EntS in order to identify crosslinked binding partners.

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


Jama Hagi-Yusuf1, David Kwan1
1Concordia University

 

Biofuels have the potential to move our society’s dependence away from fossil-fuel systems and move towards a cleaner and more renewable energy source. The technology has moved from focussing 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 adapted an existing coupled-enzymatic assay to detect OleTJE activity for further research. In this assay, CO2 is fixed and converted to bicarbonate, which is then consumed along with phosphoenol pyruvate in a phosphoenolpyruvate carboxylase-catalyzed reaction generating oxaloacetate. Coupling this with malate dehydrogenase results in a high-throughput absorbance-based assay to detect OleTJE activity.

Development of a production and purification process for VSVg pseudotyped gesicles


Juliette Champeil1,2,3, Mathias Mangion1,2,3, Rénald Gilbert2,4, Bruno Gaillet1,2,3
1Université Laval 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

One of the bottlenecks in cell therapy is to successfully introduce a DNA inside cells, especially in cells known as difficult to transfect. Nowadays, different technics exist but none is fully satisfying. Therefore, virus-like particles have been developed and have proven to efficiently transfect cells. This study is focusing on the development of a production and purification platform for virus-like particles pseudotyped by the VSVg protein (gesicles). The production process is using a serum-free media, with suspension cells HEK 293 F. In order to efficiently produce gesicles, 3 parameters have been optimized, namely the DNA concentration (1, 2 and 4µg/ml), the cells density (106, 3.106 and 6.106 cells/mL) and the DNA : PEI Ratio (1:1; 1:2; 1:4). Among the 27 protocols obtained, we were able to determine that the best gesicles were produced using a cell density of 3.106 ​ cells/ml, a DNA concentration of 2µg/mL and a DNA: PEI ratio of 1:4. Simultaneously, a two-steps purification process was settled, including an isopycnic ultracentrifugation and a chromatography method. Surprisingly, the protocol based on viruses’ purification, using an anion exchange chromatography with a NaCl elution, has shown to induce a loss in gesicles' efficiency to transfect cells. Therefore, a new purification process, using polybrene, was investigated. This cationic polymer snatches the gesicles from the resin thanks to its positive charges and induces the gesicles’ elution. Gesicles' efficiency to deliver DNA was determined thanks to cells transfection efficiency assays and western blot. Even though this method needs to be optimized, we are now able to purify the gesicles.

Development of a purification strategy for recombinant Vesicular stomatitis virus (rVSV) based HIV vaccine candidates


Anahita Bakhshizadeh Gashti1, Alain Garnier2
1Université Laval 2Laval

Manufacturing of high titer, clinical grade vectors in large scales remains a challenging step in clinical progression and future usage of vaccines in humans. Production of clinical grade vectors suitable for gene therapy, involves engagement of many purification strategies. The multiple purification approaches are applied to eliminate the contaminants of the cell culture supernatant while preserving the functionality of the viral vector. However, unlike other gene therapy vectors such as adenoviruses and retroviruses, purification of Vesicular stomatitis virus for large scale vector production, still remains challenging. Therefore, in this project we aim to develop a purification strategy for recombinant Vesicular stomatitis virus (rVSV) based HIV vaccine candidates for their further use in rodents and non-human primates. For this purpose, virus purification at lab scale (clarification of harvested cell culture supernatant) will be achieved by low speed centrifugation of cell culture supernatant obtained from clinically relevant cell lines, HEK293 and Vero cells, followed by virus concentration through ultracentrifugation on a dense solution. The lab scale protocol will then be compared to ultrafilteration, tangential microfiltration, anion exchange chromatography and anionic membrane adsorption methods that are used for large scale vector production.

Développement de nouveaux analogues peptidomimétiques de la lactivicine ayant un potentiel antibiotique et inhibiteur de β-lactamases


Pierre-Alexandre Paquet-Côté1,2, Camille Lapointe Verreault1,2, Laurie Bédard1,2, Normand Voyer1,2
1Université Laval 2PROTEO

Les maladies causées par des bactéries résistantes sont l’une des principales préoccupations mondiales en santé. Ce problème de résistance grandissant conjointement avec une diminution de nouveaux antibiotiques sur le marché est une menace sérieuse à laquelle il est important de trouver des solutions.

 

Isolée de deux souches bactériennes du sol, la lactivicine est une molécule antibiotique agissant sur les mêmes cibles que les β-lactames (pénicillines), mais possédant un noyau hétérocyclique différent.1 De plus, elle constitue un mime structurel d’un dipeptide. Elle est aussi susceptible aux β-lactamases, les enzymes de dégradation conférant la résistance des bactéries au antibiotiques β-lactames.

 

Pour ce projet, nous avons utilisé la structure de la lactivicine comme modèle pour développer une librairie de molécules analogues. Ces analogues utilisent comme base un cycle sulfahydantoïne qui constitue une plateforme peptidomimétique d’un dipeptide.2 Le cycle sulfahydantoïne permet une fonctionnalisation simple et efficace des substituants ouvrant ainsi la porte à plusieurs analogues structurels de la lactivicine. Ceci est dans le but de pouvoir moduler leur efficacité en tant qu’antibiotique, mais aussi comme potentiel inhibiteur de β-lactamases.

 

La synthèse des analogues sulfahydantoïnes sera abordée suivit des résultats de leur efficacité en tant qu’antibiotiques et inhibiteurs de β-lactamases.

 

 

1- Natsugari, H.; Kawano, Y.; Morimoto, A.; Yoshioka, K.; Ochiai, M. J. Chem. Soc. Chem. Comm. 1987, 62-63.

2- Boudjabi, S.; Dewynter, G.; Voyer, N.; Toupet, L.; Montero, J.-L. Eur J Org Chem 1999, 1999, 2275-2283.

Développement d’un vecteur viral à double-cassette pour la visualisation en temps réel de l’adhésion et la prolifération des cellules endothéliales progénitrices


Samuel Daigle1, Mariève Boulanger1, Mathias Mangion1, Corinne Hoesli1,3, Bruno Gaillet1
1Université Laval 2université Laval 3Université McGill

Les prothèses vasculaires utilisées présentement en médecine sont efficaces pour remplacer des vaisseaux sanguins, mais comportent un certain problème quant à la durée de vie de ces prothèses. En effet, cinq ans après la chirurgie, leur taux de perméabilité atteint en moyenne 50%. [1]. Il serait donc intéressant de corriger ce problème à l’aide de cellules endothéliales progénitrices. (EPC). Ces cellules proviennent de la moelle osseuse et pourrait donc se différencier en cellules endothéliales si l’on réussit, à l’aide de peptides, à les attacher à la surface des prothèses vasculaires. Comme ces cellules EPCs se multiplient rapidement sur plusieurs générations, elles pourraient contribuer à l’endothélialisation des prothèses, et ainsi améliorer leur durée de vie pratique.  Le développement de ces cellules comporte plusieurs étapes, et il serait intéressant de développer un moyen d’identifier ces étapes pour bien suivre le développement voulu en cellules EPCs utiles aux prothèses. Le but de ce projet de recherche est de créer un vecteur viral avec le lentivirus qui permettra d’identifier les EPCs et les cellules endothéliales et de caractériser leurs étapes de développement. Le vecteur comporte deux cassettes. La première cassette comporte la séquence qui codera la GFP (green fluorescent protein), une protéine verte qui est contrôlé par le promoteur eNOS présente dans les cellules endothéliales matures. La deuxième mène à l’expression de la nRFP (red fluorescent protein), une protéine rouge, sous contrôle du promoteur CMV. Cette double cassette permettra de quantifier le nombre de cellules qui exprime le promoteur eNOS, donc qui atteint maturité. La construction de la cassette eNOS-GFP a été entamée. Les étapes suivantes du projet consisteront à y ajouter la cassette CMV-nRFP et à introduire le vecteur complet dans lentivirus.

 

Références

 

[1]       P. Klinkert, PN. Post, PJ. Breaslau, JH. Van Bockel. Eur J Vasc Endovasc Surg. 2004, 27(4), 357-362

Développement d’une approche à haut débit pour le criblage et la quantification de polyhydroxyalkanoates des bactéries échantillonnées à partir d’huiles usées.


Marianne Héneault1,2,3, Manel Ghribi1,2,3, Fatma Meddeb1,2,3, beauregard marc1,2,3
1UQTR 2PROTEO 3CRML, Centre de Recherche sur les Matériaux Lignocellulosiques, Université du Québec à Trois-Rivières

Dans la famille des bioplastiques, les polyhydroxyalcanoates (PHA) sont des polyesters obtenus par fermentation bactérienne ayant l’avantage d’être à la fois biosourcés et biodégradables. Les polyhydroxyalkanoates (PHA) sont des composés organiques polyesters comprenant des hydroxy acides gras synthétisés par la plupart des bactéries soumises à un stress alimentaire pour servir de réserve d’énergie. Ils se déposent sous forme de corps d’inclusion nommés granules de PHA (aussi carbonosomes). Dans le cadre du projet initial, nous avons identifié des souches bactériennes productrices de PHA dans des échantillons d’huile usées de moteur. Selon nos résultats, ces souches sont nombreuses et leurs conditions de croissance semblent déterminantes pour la production de PHA. Dans la présente étude, une mesure quantitative rapide à haut débit de l'accumulation des PHA a été effectuée pour faire le suivi de la production de PHA par ces micro-organismes cribles à partir des huiles usées de moteur.  Des mesures de l’intensité de la fluorescence des cellules ont été réalisées dans une microplaque de 96 puits contenant au début du PHA commercial colorées avec le bleu du Nil. Une corrélation linéaire a été obtenue entre la concentration de PHA intracellulaire et l'intensité de fluorescence, elle représente le potentiel de l'emploi de la méthode du bleu du Nil pour déterminer les concentrations de PHA dans les cellules bactériennes. Par la suite, cette méthode de coloration développée a été utilisée avec succès pour démontrer une corrélation entre la quantité de PHA contenu dans les cellules bactériennes exposées à de l’huile à moteur. Ces résultats préliminaires nous ont permis de conclure que les meilleures souches bactériennes  productrices de PHA parmi les candidats sélectionnés de notre banque de microorganismes appartiennent à la famille des Bacillus sp.

Finalement, il est à noter que cette méthode permet non seulement un criblage a haut débit des bactéries productrices de PHA mais également une quantification du niveau de production de ce biopolymère intracellulaire avant extraction.

Direct phosphorylation of SH3 domains by tyrosine kinase receptors disassembles ligand-induced signaling networks


Ugo Dionne1,2,3, François Chartier1,2,3, Yossef Lopez de los Santos3,4, Noémie Lavoie1,2,3, David N. Bernard3,4, Sara Banerjee1,2,3, François Otis3,8, Kévin Jacquet1,2,3, Michel Tremblay1, Mani Jain3,7, Sylvie Bourassa1, Gerald Gish5, Jean-Philippe Gagné1,2,3, Guy G. Poirier1,2,3,6, Patrick Laprise1,2,6, Normand Voyer3,8, Christian Landry3,7, Nicolas Doucet3,4, Nicolas Bisson1,2,3,6
1Centre de recherche du Centre Hospitalier Universitaire (CHU) de Quebec-Université Laval, Québec, QC, Canada 2Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada 3PROTEO-Quebec Network for Research on Protein Function, Engineering, and Applications 4INRS-Institut Armand-Frappier, Université du Québec, Laval, QC, Canada 5Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex, Toronto, ON, Canada 6Department of Molecular Biology, Medical Biochemistry and Pathology 7Department of Biology, Université Laval, Québec, QC, Canada 8Department of Chemistry, Université Laval, Québec, QC, Canada

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system in metazoans. In particular, receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling events upon their activation by extracellular stimuli. RTK activation creates docking sites required for the assembly of signaling networks on the plasma membrane that drive downstream signaling. However, the mechanisms leading to network disassembly and its consequence remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of specific Tyr residues within Src-Homology (SH) 3 domains. The target of the latter events is an evolutionary-conserved Tyr present in most SH3 domains, including the SH2-SH3 adaptor proteins NCK1/2, which are key hubs for the nucleation of RTK-dependent signaling complexes. We show that the EphA4 RTK directly phosphorylates NCK1/2 SH3 domains on this residue, thus entailing the collapse of NCK-dependent signaling networks and the abrogation of their function, both in vitro and in Drosophila. Analysis of other RTK-SH3 pairings revealed that this process is a common negative regulation mechanism. Our findings uncover a novel, conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining on the plasma membrane in a catalytically active state.

Directed Evolution of a Triple-Decker Motif Containing Red Fluorescent Protein


Sandrine Legault1, Matthew G. Eason1, Erin Nguyen1, Roberto A. Chica1
1Faculty of Science, Department of Chemistry and Biomolecular Sciences, University of Ottawa

Red fluorescent proteins (RFPs) are extensively used in biological research. They are particularly suited to whole-body imaging of research model animals because red light leads to reduced phototoxicity and lower background autofluorescence. However, RFPs generally display lower brightness relative to other members of the GFP superfamily of fluorescent proteins. Therefore, the development of brighter RFP variants is desired. Previously, our lab demonstrated that the quantum yield and overall brightness of RFPs can be increased through the rational design of triple-decker motifs of aromatic rings formed by the chromophore and neighboring residues. Here, we use directed evolution to further improve the characteristics of the mRojo-VHSVF RFP, an mRojoA mutant which contains a triple-decker motif consisting of His and Tyr residues surrounding the chromophore. To do so, we generated libraries of random mutants and screened them for enhanced fluorescence intensity over multiple rounds of fluorescence-activated cell sorting. In the first round, we identified two mutants exhibiting up to 1.6-fold increases in brightness. These variants contain the M150L or E176K/N194Y mutations, which lead to approximately 20% improvements in quantum yield and up to 1.5-fold increases in extinction coefficient. These mutants were used as templates for three subsequent rounds of directed evolution. The last random library yielded one mutant brighter than mRojo-VHSVF with a 20% increase in quantum yield and improved maturation. This variant contains the M150L/F165Y/N194Y/C217S mutations. Three of these have previously been shown to affect the fluorescence and maturation properties in other RFPs. F165Y is a unique mutation that is possibly involved in stabilizing the triple-decker motif through a hydrogen bond network. In addition, it participates in a novel quadruple-decker motif, providing a new template for further RFP engineering.

Discovering Drug Seeds by NMR Fragment-Based Lead Discovery


Luciana Coutinho de Oliveira1, Steven laplante1
1INRS-IAF

Small molecule drugs serve a crucial role for combating diseases. This poster presents how we employ NMR to discover the seeds for new drugs using fragment-based screening. Central to these efforts was the need to create a new curated fragment library, the implementation of a consensus NMR screening strategy, and the design of NMR software for rapid analyses purposes. These tools were also critical for establishing a better understanding of the properties of small molecules when free in solution and for deciphering the various types of binding to macromolecules. Examples are shown where these NMR strategies revealed interesting compound solution behaviors (solubility, aggregation), exposed target protein features (folding and changes), and determined stoichiometric binding attributes that served to prioritize quality ligands for downfield medicinal chemistry purposes.

DNA Probes for Monitoring Enzyme Activity


Scott Harroun1, Xiaomeng Wang1, Arnaud Desrosiers1, Alexis Vallée-Bélisle1
1Université de Montréal

To measure enzymatic activity, one typically needs a different assay for each reaction that can be catalysed by the enzyme. Therefore, a universal approach to monitor catalysis of any substrate of a given enzyme is desirable, especially for applications such as high-throughput screening. In this study, we employ fluorescent DNA-based molecular probes to measure enzymatic activity via their attachment to an enzyme. We demonstrate that our DNA probe only reports signal change due to a local effect during catalysis, possibly from structural destabilization. Our approach correlates well with traditional measurements of enzymatic activity (i.e. KM), thereby confirming its validity to measure kinetics. Various conditions were optimized, including the attachment strategy, DNA composition and length, the effect of steric hindrance, and the ratio of DNA and enzyme added. Our system performs well for at least 100 days, making it convenient once prepared. Furthermore, by employing several fluorophores possessing differing structures and hydrophobic properties, it is possible to probe various regions of the enzyme surface during catalysis.

DNA-protein conjugates for electrochemical biosensing applications


xiaomengwang 1, 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.

Duplication of homomeric protein: retention of paralogs and evolution of protein-protein interactions


Axelle Marchant1, Lou Nielly-Thibault1, Alexandre Dubé1, Isabelle Gagnon-Arsenault1, Yacine Seffar1, 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

Protein-protein interaction (PPI) network contains significantly more homomers than expected by chance and these homomers have two more partners than proteins that do not interact with themselves (Ispalatov et al 2005). Duplication of a homomer results in a pair of paralogs interacting with each other. The appearance of these pairs happens more frequently than explained by chance and the presence of duplicated proteins self-interacting is higher than singlet proteins suggesting a selective force leading to the retention of both protein copies. Thus, if a homomeric protein is duplicated several times, it would lead to the appearance of a fully interconnected complex. With time, some interactions within the complex evolved due to the divergence of the paralog sequences. Thus, the duplication of proteins forming homomers is suspected of being involved in the appearance of complex networks of proteins. However, the evolutionary path imprinted by PPIs following the duplication of a protein interacting with itself is still poorly understood and several opposing scenarios have been proposed (Kaltenegger and Ober 2015). Here, we studied in Saccharomyces cerevisiae, the PPI profile of a large panel of paralogs from small scale (SSD) and whole genome (WGD) duplications to distinguish the different interaction patterns associated with the different evolutionary scenarios. We focus on homomers and interaction between two paralogs of the same pairs using the protein-fragment complementation assay (PCA). To better understand the evolutionary history of PPIs following duplication, we also compare PPI of paralogs observed in S. cerevisiae with orthologs proteins duplicated or no in more or less distance yeast species. We observed divergent tendencies of pattern of interactions depending on duplication mechanism (SSD versus WGD), age of duplication and coexpression of paralogs. Thus, evolution of the duplication of homomers seems to depend of the duplication context and probably impact differently the formation of protein complexes depending of origin of duplication. We also proposed a model explaining the retention of paralogs from the duplication of homomeric protein. We showed that the presence of interaction between the two paralogs involved selective force of retention. Thanks to PCA technology allowing a high-throughput approach and modelisation, our study brings new answers on the evolution of protein interactions following the duplication of homomers.

Dynamics and Stability of Long Telomeric G-Quadruplex DNA Sequences


Simone Carrino1, Anthony Mittermaier2
1McGill University 2McGill University

DNA G-Quadruplexes are ubiquitous in our genome and are thought to be highly involved in regulatory functions, such as DNA replication and transcription. Of particular interest are telomeric G-Quadruplexes; the ~200-nt long telomere overhang theoretically allows folding of multiple tandem G4 units, forming a so called ‘beads-on-a-string’ structure. In this work, we use a combination of Uv melting experiments, global fitting and simulations to describe the folding pathway of (TTAGGG)8TT. Our data are well described by a model that includes misfolded states characterized by folding of one G4 unit with 5’ and 3’ overhangs of variable length. Extension to longer tandem repeats, described both in terms of kinetic and thermodynamic cooperativity, leads to detailed quantitative predictions of the number, spacing, and dynamic interconversion of telomeric G4 repeats.

Effect of binding interference on the divergence between paralogous genes that encode homodimers


Angel Fernando Cisneros Caballero1,2, Christian Landry1,2
1ULAVAL 2PROTEO

Protein complex formation is a key process for cell biology as many proteins need to physically associate to carry out their functions. Interaction among proteins is therefore an important component of their evolution. We are interested in the evolution of these interactions following gene duplication. Of particular interest are paralogs that derive from the duplication of self-interacting proteins because those give rise to heterodimers of paralogs in addition to two self-interacting paralogs. My objective is to evaluate the effect of the competition between heterodimers and homodimers on their sequence divergence. Such competition introduces a molecular conflict that can lead to several different scenarios, depending on which type of complexes is favored by natural selection over time. I am studying these scenarios and their consequences on the divergence of paralogs by simulating the evolution of protein complexes using crystal structures, and the estimation of binding energies, protein stability, and specific fitness functions. Because a large fraction of genes encode self-interacting proteins, these molecular conflicts may be an important factor in the divergence of paralogous proteins and the evolution of protein interaction networks.

Effet de la vitesse de filage sur la structure moléculaire de fibres de soie d’araignée natives et supercontractées


Jane Gagné1, Thierry Lefèvre1, Michèle Auger1
1Université Laval

La soie d’araignée est une fibre solide, extensible et résistante mécaniquement. Elle est donc particulièrement intéressante. La structure de cette dernière est dictée par la structure primaire des spidroïnes qui la constituent et par le processus de production, notamment par la vitesse de filage. Ainsi, les propriétés mécaniques de la soie varient avec la vitesse de filage. Il en est de même pour sa réponse à l’humidité, plus précisément son amplitude de contraction lorsqu’on expose la fibre à une humidité élevée (phénomène dit de « supercontraction »). La vitesse de filage influence la structure même de la soie, notamment au niveau de l’orientation moléculaire des chaines polypeptidiques ainsi qu’au niveau de la cristallinité.

 

La structure moléculaire de la soie de deux espèces d’araignées est analysée, soitNephila clavipes et Araneus diadematus. Les fibres sont obtenues par filage forcé à des vitesses variant entre 0,3 et 20 cm/s, et sont soumises à des humidités relatives supérieures à 90 %. La caractérisation des fibres est effectuée par spectromicroscopie Raman et par résonance magnétique nucléaire à l’état solide.

Elucidating the activation mechanism of Tn7 transposition


Yao Shen1, Jeremy Caron2, Joseph Peters3, Joaquin Ortega1, Alba Guarne1
1McGill University 2McMaster University 3Cornell University

Transposons are ubiquitous mobile DNA elements with broad effects on bacterial evolution, gene expression and the spread of antibiotic resistance. The bacterial transposon Tn7 is distinguished by its tight control of transposition frequency and unique target selection mechanisms. Five transposon-encoded proteins mediate target selection and immunity: a heterodimeric transposase (TnsA/TnsB), two targeting proteins that direct transposition to either a single chromosomal site (TnsD) or a replicating conjugal plasmid (TnsE) and an adaptor that bridges the donor and target DNA interactions (TnsC). Although no transposition occurs until the proper target DNA has been recognized by either TnsD or TnsE, the gain-of-function variant of TnsC (TnsCA225V) can promote Tn7 transposition independent of the target selector protein. Like MuB and IstB regulator proteins of the Mu and IS21 transposable elements, TnsC is an ATP-dependent DNA-binding protein belonging to AAA+ ATPase superfamily. However, the mechanism underlying how TnsC utilizes nucleotide binding and hydrolysis to interface with target selector protein TnsD/E and target DNA to further activate Tn7 transposition remain poorly understood. Using sizing exclusion chromatography and negative staining electron microscopy (EM), we found that TnsC (both WT and A225V) forms dimers in solution, but heptameric rings upon DNA binding in the presence of AMPPnP. The TnsCA225V variant also forms stable rings in the absence of DNA, but they are octameric. We hypothesize that this DNA-dependent 8- to 7-mer transition mimics the conformational change imposed by the target selector protein (TnsE, TnsD) and can be used to dissect the mechanism of TnsC activation. Our ongoing work of obtaining high-resolution cryo-EM structures of TnsC+DNA+AMPPnP ternary complex and TnsCA225V+AMPPnP complex will delineate the activation mechanism of Tn7 transposition.

Étude de l’expression, de la solubilité, du clivage et de la purification de la rétinol déshydrogénase 8 en fusion avec différentes étiquettes de purification et de solubilisation


Charlotte Lemay-Lefebvre1,2,3, 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 Saint-Sacrement, CHU de Québec-Université Laval 3Regroupement stratégique PROTEO, Université Laval

CONTEXTE ET OBJECTIFS : La rétinol déshydrogénase 8 (RDH8) est la première enzyme entrant en jeu dans le cycle visuel. Son rôle est de réduire le tout-trans rétinal en tout-trans rétinol, ce qui prévient l’accumulation d’A2E, une molécule toxique pour les photorécepteurs, car sa présence peut mener, entre autres, à la dégénérescence maculaire liée à l’âge (DMLA). Il y a cependant une accumulation constante d’A2E dans les photorécepteurs même en présence de RDH8, ce qui suscite des interrogations au niveau de l’efficacité de son activité catalytique. Il faut donc caractériser son activité enzymatique en produisant une protéine soluble et de grande pureté. La RDH8 est cependant insoluble dans le lysat bactérien suite à sa surexpression en fusion avec une étiquette de polyhistidines dans un système d’expression E. coli. Différentes étiquettes de purification/solubilisation pourraient permettre de solubiliser la RDH8 dans le lysat bactérien et ainsi rendre possible la caractérisation de son activité enzymatique. Il est donc primordial d’arriver à obtenir la RDH8 sous une forme soluble en fusion avec une étiquette de purification et de solubilisation qui peut être clivée afin de caractériser l’activité enzymatique de la RDH8 purifiée. MATÉRIEL ET MÉTHODES : La RDH8 en fusion avec les étiquettes His10-MBP, MBP-His10 et TagR-TagR a été clonée dans les vecteurs pET-11a ou pMAL-c2x et ensuite surexprimée dans des cellules E. coli (souche BL21 DE3 RIPL). La purification des différentes protéines de fusion est effectuée par chromatographie d’affinité ou hydrophobe. Le clivage des étiquettes est effectué grâce à la présence d’un site de reconnaissance pour la protéase TEV situé entre la RDH8 et l’étiquette dans chaque protéine de fusion. Le niveau de surexpression et de solubilité de chaque protéine de fusion dans le lysat bactérien ainsi qu’après clivage et purification a été déterminé par électrophorèse sur gel SDS-PAGE pour chaque protéine de fusion. RÉSULTATS : On observe un faible niveau de surexpression de la forme soluble des protéines de fusion. Le plus haut niveau de protéine purifiée et de pureté est obtenu avec la RDH8 en fusion avec le His10-MBP. Le plus haut niveau de surexpression est obtenu avec l’étiquette TagR-TagR. Ce sont aussi des protéines de fusion dont il est possible de cliver l’étiquette. CONCLUSIONS : Les conditions de surexpression et de purification de la RDH8 en fusion avec le TagR-TagR et la His10-MBP doivent être optimisées afin d’obtenir une quantité plus élevée de protéine de fusion avec la plus grande pureté possible. De plus, la stabilité de la RDH8 seule en solution doit être améliorée suite au clivage.

Etude des transporteurs de Nickel chez Helicolibactrer pylori


Mirana Mirana Rakotoarivony1, Zakaria Orfi1, Charles Calmettes1
1INRS Institut Armand Frappier

Helicobacter pilori est une bactérie gram-négative de forme spiralée, elle possède des flagelles qui lui donnent une mobilité dans la muqueuse. Helicobacter pilori est responsable des inflammations de la muqueuse gastrique, conduisant à un cancer gastrique. Plus de 70% des cancers gastriques sont dues à une inflammation chronique de la muqueuse gastrique par H pilori. Le traitement de ce cancer est encore limité. Éradiquer H pilori constitue une meilleure approche pour lutter contre le cancer gastrique (Lu & Li, 2014).

Le cancer gastrique apparaît plusieurs années après une infection chronique par H pilori. Le nickel est considéré comme un indicateur de facteur de virulence H pilori. L’hydrogénase [NiFe] est importante pour la colonisation in vivo et l’uréase est indispensable pour résister au pH de l’acide gastrique et pour se proliférer dans l’estomac (Fischer et al., 2016). En effet, l’uréase nécessite la présence du nickel pour catalyser la réaction d’hydrolyse de l’urée en dioxyde de carbone et ammonium. De ce fait, H pilori utilise des systèmes très spécifiques pour importer ces ions nickel. Parmi ces systèmes, les protéines NiuBDE, protéines transporteurs appartenant à la famille ABC. Ceci a permis de faire le lien entre le mode de colonisation de l’estomac par H pilori et l’importance du nickel pour la colonisation de la muqueuse gastrique.

Les principaux objectifs de notre projet sont de cristalliser et d’élucider les structures de NiuB1 et NiuB2. Plusieurs approches seront utilisées pour la cristallisation de ces protéines chez H pilori. La résolution des structures cristallographiques de NiuB1 et NiuB2 permettra d’examiner le site de fixation du nickel, de nous aiguiller sur le mécanisme de capture et d’acquisition du nickel et de déterminer leur importance dans le transport du nickel. Cette hypothèse pourrait être considérée comme une cible thérapeutique potentielle dans le traitement des maladies associées à l’Helicobacter pilori et sera un appui dans la conception d’un médicament dans la lutte contre le cancer gastrique.

Évolution des complexes protéiques après hybridation entre espèces


Caroline Berger1, I. Gagnon-Arsenault1, K-M. Moon2, R.G. Stacey2, L.J. Foster2, C.R. Landry1
1Institut de Biologie Intégrative et des Systèmes, Département de Biologie, Regroupement québécois de recherche sur la fonction, l'ingénierie et les applications des protéines, Université Laval. 2Centre for High-Throughput Biology, Michael Smith Laboratories, University of British Columbia.

Les protéines sont les machines moléculaires qui permettent à la cellule de réaliser une action biologique. Cependant, la plupart d’entre elles ne sont fonctionnelles que lorsqu’elles s’assemblent pour former des complexes protéiques, chacun réalisant une fonction précise (les ribosomes, le protéasome, l’ATP synthase, etc.). Dans le cadre de ce projet et en utilisant comme organisme modèle la levure, nous nous intéressons à comprendre comment la transition d’une protéine individuelle en un assemblage organisé que forment les complexes protéiques peut avoir lieu. Parmi les mécanismes moléculaires possibles, l’hybridation entre espèces a un grand potentiel évolutif puisqu’elle conduit à une duplication de l’ensemble des gènes par allopolyploidisation. Le principe de notre méthodologie est donc de simuler en laboratoire le phénomène d’allopolyploidisation par génération d’hybrides de levures, puis d’étudier l’impact de cette hybridation sur l’organisation des complexes protéiques. En collaboration avec l’équipe de Léonard Foster de l’Université de Colombie-Britannique (UBC) et grâce à un stage que j’ai pu effectuer dans leur laboratoire, nous proposons de mettre au point une méthode d’analyse des complexes protéiques basée sur le marquage des protéines de la levure avec différents isotopes d’acides aminés (Kristensen et al. 2012). Une fois les protéines des levures marquées, leurs complexes protéiques sont séparés selon leur taille par chromatographie d’exclusion stérique (SEC). Les fractions obtenues par chromatographie sont ensuite digérées et analysées par spectrométrie de masse. L’application de cette méthode aux hybrides et à leurs espèces parentales permettra à terme de mieux comprendre les changements survenant à l’échelle des interactions protéiques après hybridation entre espèces. 

Evolution of conformational exchange in a host defense enzyme family


David N. Bernard1,2, Myriam Letourneau1, Purva P. Bhojane3, Khushboo Bafna3,4, Marie-Christine Groleau1, Éric Déziel1, Elizabeth E. Howell3, Pratul Argawal3,4, Nicolas Doucet1,2,5
1INRS-Université du Québec 2PROTEO 3University of Tennessee, Knoxville, TN, USA 4Oak Ridge National Laboratory 5GRASP

Hominidae-exclusive ribonucleases (RNases) from subfamily 3 are enzymes that present low ribonucleolytic activity, display antibacterial properties against both gram-positive and gram-negative bacteria, and cytotoxic properties against both parasitic and host cells. Subfamilies 2 and 3 rapidly diverged following a gene duplication about 50 million years ago, resulting in two distinct enzyme families exhibiting distinct biological functions. It has been shown that conformational exchange was responsible for the rate-limiting step of enzyme turnover in the highly homologous bovine RNase A. Yet, despite sharing a common structure, NMR experiments performed in our laboratory showed that these RNases display different conformational exchange behavior on multiple timescales. To better understand how the biological functions of host defense RNase subfamilies 2 and 3 came to diverge, we have characterized the biological activities of various subfamily members, in addition to their conformational exchange behavior. We also performed enzymatic assays, antibacterial assays against E. coli and S. aureus, cytotoxicity assays against HeLa cells, and characterized their conformational exchange on various timescales using NMR relaxation experiments (CPMG, CEST, ­R1, R2 and NOE). Comparing progressively more distant enzymes to their human counterpart should provide information on how these enzymes could evolve new functions and/or alter their behavior on a molecular level.

Expression and purification of immunologic adjuvant P97c protein from Mycoplasma hyopneumoniae


Laurie Gauthier1, Geneviève Bertheau-Mailhot1, Jessica Dion1, Denis Archambault1, Steve Bourgault1
1Université du Québec à Montréal

The use of an appropriate adjuvant remains an important strategy for the development of effective subunit vaccines. The P97 protein of Mycoplasma hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, is involved in microbial adhesion to respiratory tract epithelial cells. We previously showed that the C-terminal portion of P97 (P97c), when coupled to immunogenic proteins and delivered in vivo with adenovectors, increases the antibody response to these immunogens, suggesting an adjuvant effect of P97c. In this study, the P97c protein was produced and purified to further characterize its immunomodulatory effect. To this end, the gene encoding P97c was cloned into the pGex-4-T1 vector and expression was carried out in BL21 DE3 pLySs bacteria. The P97c portion of the expressed GST-P97c fusion protein was then obtained by using a glutathione agarose column and a protein thrombin cleavage procedure. The purified P97c protein was then analyzed by Western Blot to validate the nature of the protein. Circular dichroism spectrum analysis showed a predominantly α-helix conformation of P97c. By conducting in vitro experiments with RAW-blue cells and HEK-BlueTM TLR5 cells, we demonstrated that P97c triggers TLR5 activation. This result correlated with the CXCL8 cytokine production observed in the HEK-BlueTM TLR5 cells. Taken together, these results confirm the modulatory effect of P97c on the innate immune system and constitute an additional testimony of the adjuvant effect of this protein. Experiments are currently under way to develop new generation vaccines using the P97c protein as an adjuvant.

Fluorogenic chemical tools based on cysteine labeling to study oligomer formation in amyloid self-assembly


Guillaume Charron1, Noé Quittot1, Steve Bourgault1
1Université du Québec à Montréal 2UQAM 3Université 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 different diseases, including type II diabetes (T2D) and Alzheimer's disease. 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 the fluorescent dye, thioflavin T, which recognizes amyloids. However, this probe is insensitive to the formation of pre-fibrillar oligomers. Here, we propose novel strategies for the detection of peptide self-assembly using fluorogenic small molecules whose fluorescence emission are amplified following their binding to inter-molecular cysteines incorporated into the peptide sequence. In this study, we are interested in the self-assembly of islet amyloid polypeptide (IAPP), whose amyloid deposits in the pancreatic islets are associated with T2D. Using either mono-cysteine or bi-cysteine IAPP mutants, we were able to detect pre-fibrillar IAPP aggregates and follow the self-assembly, even in complex biological systems. Our results indicate that with positional scanning of cysteine residues, these detection methods offer unique time-dependent conformational insights of the species assembled throughout the amyloidogenic pathway.

Folding and binding act as determinants of environment specific fitness effects


Rohan Dandage1,2, Kausik Chakraborty1,2
1CSIR- Institute of Genomics and Integrative Biology, Mathura Road Campus, New Delhi, India. 2Academy of Scientific and Innovative Research (AcSIR), New Delhi, India.

Environmental conditions are known to shape natural selection. However, their influence on molecular evolution is still largely unclear. Here, we use a high throughput mutational scanning approach to investigate how representative physical and chemical environments alter mutational fates of an antibiotic resistant gene. We obtained empirical fitness of individual single site mutants of the gene in the environments by carrying out co-culture bulk competitions. Mutant fitness was generally found to differ across environments. Remarkably, observed environmental influence on mutational effects was explanable by relative strengths of protein level structural constraints. This analysis revealed a high degree of predictability: overall strengths of environment-specific selection pressures were determined by the degree of mutational perturbation of protein folding and ligand binding. Overall, our results show that such structural constraints act as determinants of environment specific fitness effects.

Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device


Laura Leclerc1, Guy Soffer1, T.W. Tsai2, C.C.Yu 2, Shih S.C.C.1, Kwan D.H.1
1Concordia University 2National Chung-Cheng University

The structure and abundance of different cell-surface carbohydrates—or glycans—heavily influence signalling mechanisms that control cellular 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, sialyl-LewisX is a fucosylated glycan that promotes higher metastatic potential, drug resistance, 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 the subsequent fluorescent signal. We demonstrate that this assay can be used to detect the inhibition of a fucosyltransferase in a 384-well plate and on a digital microfluidics (DMF) device. Python software was developed to facilitate operation and automation on a DMF chip.

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


Fathima Mohideen1, Joel Richard1, Nathalia Kravchenko1, David 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.

Functional Characterization of AltB2R, an Alternative Protein Encoded in the Bradykinin B2 Receptor Gene Involved in the B2R Signaling


Maxime Gagnon1,2,3,4,5, Martin Savard1,3, Jean-François Jacques1,2,4,5, Fernand Gobeil1,3, Xavier Roucou1,2,4,5
1Université de Sherbrooke 2Pavillon de Recherche Appliquée sur le Cancer 3Institut de Pharmacologie de Sherbrooke 4PROTEO 5Proteomeus

Functional and proteomic approaches have concurrently demonstrated the translation of alternative open reading frames (AltORFs) in addition to the annotated coding sequences (CDSs) present on the same mRNA. These AltORFs may be encoded in the UTRs in any reading frame or overlapping the annotated CDS in a different reading frame. Hence, translation potentially produces multiple proteins in addition to the annotated protein. For clarity, proteins translated from the annotated CDS are termed reference protein and proteins translated from AltORFs are termed alternative proteins.

Our studies focus on the multicoding potential of G protein-coupled receptor (GPCR) genes. This superfamily of protein, containing over 830 members, is widely studied for its therapeutics potential. We predict that over 4 600 AltORFs are potentially encoded on GPCR genes’ transcript, of which nearly 36% overlap the reference CDS. This means the study of these ~600 GPCRs using a heterologous expression system potentially includes the expression of over 1 600 AltORFs. Amongst those is the gene coding for the bradykinin B2 receptor (B2R) which codes for an AltORF termed AltB2R. As a proof of concept, our study focuses on the functional characterization of AltB2R.

Our results demonstrate that AltB2R is co-expressed with B2R from the same coding sequence. AltB2R, a cytoplasmic protein, interacts with its reference protein as shown by bi-molecular fluorescence complementation. Functional assays investigating B2R signalling cascade were performed on stable cell lines; one over-expressing AltB2R and one negative control. Endogenous B2R stimulation led to a significantly stronger MAPKs activation in cells over-expressing AltB2R. Moreover, intracellular calcium signalling in response to agonist stimulation was increased in the presence of AltB2R. Next, we designed a mutated B2R (B2R*) where the amino acid sequence of B2R was unchanged, but we mutated a CAA codon of AltB2R to the TAA stop codon. This construction allows for the expression of B2R while effectively stopping the expression of AltB2R.  Functional assays comparing B2R and B2R* constructs showed a decreased MAPKs activation in B2R* transfected cells following agonist stimulation. Furthermore, these cells showed a significantly decreased IP3 generation in response to B2R stimulation. However, there was no difference in ligand binding nor B2R internalization efficiency. Finally, the AltB2R protein has been identified in different brain and breast cancers by mass spectrometry and immunohistochemistry using a custom antibody specifically targeting AltB2R.

These results indicate that AltB2R is a novel regulator of B2R signalling. Since B2R is known for being involved in certain types of cancer (notably brain cancer), our future studies will aim at determining whether AltB2R plays an integral role in these pathologies.

Functionalization of amyloid-based nanoparticles


Soultan Al-Halifa1,2, Ximena Zottig1,2, Laurie Gauthier1,2,3, Margaryta Babych1,2, Denis Archambault3, Steve Bourgault1,2
1Department of Chemistry, Université du Québec à Montréal, Montreal, QC, Canada, H3C 3P8 2Quebec Network for Research on Protein Function, Engineering and Applications, PROTEO 3Department of Biological Sciences, Université du Québec à Montréal, Montreal, QC, Canada, H3C 3P8

Self-assembled nanostructures hold great potential in biomedical applications. This is due to the recent advances in nanoengineering, which allows the design of assemblies with different sizes, shapes, hydrophobicity and surface properties. In particular, self-assembling peptides are attractive due to their straightforward and affordable synthesis, their biocompatibility, their biodegradability, and their versatility, offering a control over the physicochemical properties of the monomeric unit at the molecular level. Moreover, self-assembling peptides can form a variety of structures including micelles, fibers, rods, ribbons, tapes, and vesicles which makes them highly attractive in nanomedicine. In particular, amyloidogenic peptides are interesting do to their ability to form strong and stable nanostructures (mostly fibrils) with enhanced resistance against enzymatic degradation. In our lab, we recently showed that amyloid fibrils based on a peptide sequence derived from a 37-residue hormone, islet amyloid polypeptide, can be controlled via electrostatic interactions. This strategy led to the development of stable and cytocompatible nanorods (NRs) with a height of 2 ± 0.6 nm and a length of 15 ± 5 nm. Herein, we show that these nanorods can be functionalized with biomolecules for specific purposes. In general, functionalization can be achieved by synthesizing a chimeric peptide containing the self-assembling motif and the bioactive molecules, or by postgrafting strategies using chemoselective and orthogonal reactions. In the present work, we combined both functionalization strategies to engineer amyloid-based nanorods presenting multiple copies of different molecules including an epitope, lactose for hepatocyte targeting, and a dye for imaging (scheme below). The resulting functionalized nanorods showed a height of 6 ± 1 nm and a length of 56 ± 25 nm, were cytocompatible and could be taken up by J774 cells, thus highlighting their potential in vaccinology.

Genetic backgrounds have complex effects on the drug treatment to a human disease mutation


Véronique Hamel1,2,3,4, Marie Filteau5, Isabelle Gagnon-Arsenault1,2,3,4, Alexandre K Dubé1,2,3,4, Christian R Landry1,2,3,4
1Institut de Biologie Intégrative et des Systèmes 2Département de Biologie, Université Laval 3PROTEO 4CRDM 5Département des Sciences des Aliments, Université Laval

Mutations causing genetic diseases can have different effects that depend on the individuals’ environments and their genetic backgrounds, creating a need for personalized treatments. In order to develop these treatments, it is necessary to better understand at the molecular level how genetic backgrounds modify the responses to these treatments. To better understand the molecular mode of action of modifier genes, we use a model of Wiskott-Aldrich Syndrome in the yeast Saccharomyces cerevisiaeinvolving the LAS17gene. Following experimental evolution, we found that the disease phenotype can be corrected by mutations and a drug treatment in a genetic-background specific manner. Most genes affected by these compensatory mutations encode Las17p physical interaction partners, excluding the gene that codes for the drug-targeted protein, suggesting potential indirect relationships.The drug response specific to one of the two genetic backgrounds studied provides an opportunity to map the genetic modifiers by QTL analysis. We identified several loci underlying these modifiers, mostly including genes in the protein or genetic interaction network of Las17p and of the protein phosphatase targeted by the treatment. We found that the abundance of End3p could have effects on the function of the LAS17 mutant gene. This result suggests that protein balance of Las17p interaction partners could play a role in modifying the Wiskott-Aldrich Syndrome phenotypes at the cellular level. Altogether, our results suggest that interaction partners are prime target for medical interventions and that they are probably an important source of genetic background-dependent effects.

Guiding self-assembly and growth of amyloid-like nanoparticles


Ximena Zottig1,2, Soultan Al-Halifa1,2, Margaryta Babych1,2, Noé Quittot1,2, Steve Bourgault1,2
1Université du Québec à Montréal 2Proteo

The design of self-assembled supramolecular architecture is of great interest for a variety of applications such as drug and gene delivery, vaccine design, tissue engineering, enzyme catalysis and biosensors. Polypeptides that can self-assemble into amyloid-like assemblies offer many advantages to generate tailored nanoparticles including, functionalization, high mechanical resistance, biocompatibility and enzymatic stability. Nonetheless, the control over the self-assembly and the difficulty of predicting the final supramolecular organization from the peptide sequence constitute major issues. In this study, we develop a novel strategy to control the size, shape and heterogeneity of amyloid-like nanoparticles. This approach is based on electrostatic interactions, which govern nanoparticles growth and morphology. Self-assembling peptides were engineered by end-capping an amyloidogenic peptide with a charged residue. Transmission electron microscopy and atomic force microscopy showed different self-assembled nanostructures, including well-defined rod-like (100 ±1 nm and 144 ± 4 nm), rope-like (700 - 800 nm) and ribbon-like (3500 - 6500 nm) fibrils. Circular dichroism and Fourier-transform infrared spectroscopy revealed an overall parallel b-sheet fibril structure. Unexpectedly, we found that the rod-like nanofibrils exhibited distinctive properties compared to prototypical amyloids. For instance lower thermostability and a poor response to the amyloid dye thioflavin T was observed. In addition, cytotoxicity assays demonstrated that these rod-like fibrils are biocompatible. These results highlight the potential of using electrostatic interactions to precisely control the size, shape and surface properties of amyloid-based fibrils, which are important parameters for the design of functional amyloid fibrils in the biomedical field.  

Guiding self-assembly and growth of amyloid-like nanoparticles


Ximena Zottig1,2, Soultan Al Halifa1,2, Margaryta Babych1,2, Noé Quittot1,2, Steve Bourgault1,2
1Université du Québec à Montréal 2PROTEO

The design of self-assembled supramolecular architecture is of great interest for a variety of applications such as drug and gene delivery, vaccine design, tissue engineering, enzyme catalysis and biosensors.Polypeptides that can self-assemble into amyloid-like assemblies offer many advantages to generate tailored nanoparticles including, functionalization, high mechanical resistance, biocompatibility and enzymatic stability. Nonetheless, the control over the self-assembly and the difficulty of predicting the final supramolecular organization from the peptide sequence constitute major issues. In this study, we develop a novel strategy to control the size, shape and heterogeneity of amyloid-like nanoparticles. This approach is based on electrostatic interactions, which govern nanoparticles growth and morphology. Self-assembling peptides were engineered by end-capping an amyloidogenic peptide with a charged residue. Transmission electron microscopy and atomic force microscopy showed different self-assembled nanostructures, including well-defined rod-like (l00 ±1 nm and 144 ±4 nm), rope-like (700 - 800 nm) and ribbon-like (3500 - 6500 nm) fibrils. Circular dichroism and Fourier-transform infrared spectroscopy revealed an overall parallel b-sheet fibril structure. Unexpectedly, we found that the rod-like nanofibrils exhibited distinctive properties compared to prototypical amyloids. For instance lower thermostability and a poor response to the amyloid dye thioflavin T was observed. In addition, cytotoxicity assays demonstrated that these rod-like fibrils are biocompatible. These results highlight the potential of using electrostatic interactions to precisely control the size, shape and surface properties of amyloid-based fibrils, which are important parameters for the design of functional amyloid fibrils in the biomedical field. 

Homology modeling and semi-rational protein engineering of a new metagenomic lipase


Ngoc Thu Hang PHAM1, Yossef Lopez de los Santos1, Guillaume Brault1, Charles Calmettes1, Nicolas Doucet1
1Université du Québec, INRS - Institut Armand-Frappier

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 flavor ester specificity. This 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 biotechnological applications. 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 strategy. This method relies on good structural knowledge of the protein, which can be solved by X-ray crystallography. However, protein crystals grow in a very small range of challenging and time consuming experimental conditions. Hence, the design of a homology model has been undertaken as a complementary approach for the molecular-based investigation of LipIAF5.2. Various homology models were generated using i-Tasser, Robetta, and Swiss-Model platforms. These prediction models were then evaluated using a variety of structure assessment tools to select the best LipIAF52 homology model. Based on predictive molecular homology modeling, a semi-rational engineering strategy is presented to improve the synthetic activity of LipIAF5.2 for industrially relevant flavor esters.

 

Keywords: Lipases, protein engineering, ester synthesis, homology modeling, structure assessment

Hybridization as an adaptive force in response to extreme UV conditions


Carla Bautista Rodríguez1,2,3,4, Souhir Marsit1,2,3,4, Christian Landry1,2,3,4,5
1Université Laval, IBIS, Landry Lab 2ULAVAL 3Département de biologie 4PROTEO 5Département de biochimie, microbiologie et bio-informatique

The heterogeneous environment can end up presenting extreme conditions as high ultraviolet (UV) radiation. The presence of high UV radiation represents a particular challenge in terms of adaptability because it affects the viability of organisms by directly damaging the genome. However, the existence of extremophiles adapted to high UV radiation conditions has been reported. In this way, to discern the evolutionary mechanisms that underlie this extreme adaptation is a question remaining in evolutionary biology. It has recently been proposed that hybridization may be a useful process to colonize new ecological niches. In an interspecific hybridization event, alterations at the nucleotidic and chromosomal levels contribute to a high genome plasticity that could be key for adaptation in stressful environments. The present project aims to discover if hybridization promotes adaptive evolution under extreme conditions of UV radiation. The Saccharomyces interspecific hybrids are optimal model for the study of the adaptation of yeasts to stressful conditions by using the experimental evolution under UV conditions. Therefore, it will be possible to determine if after multiple generations there is a more marked increase in resistance to UV radiation in hybrids in comparison with the parental lines. In addition, following sequencing of the genome, it will be possible to elucidate what genomic changes occurred during evolution: mutations, copy number variations and chromosomal alterations. Altogether, this project will give us a general idea of the extent to which hybridization participates in adaptation to extreme conditions, contributing to important fields such as evolutionary biology and astrobiology.

Identification and characterization of a novel mitotic target site for Haspin on Histone H2B


Ibrahim Alharbi1,2,3, SABINE ELOWE1,2,4
1Université Laval 2Reproduction, santé de la mère et de l'enfant, Centre de, Recherche du CHU de Québec, Centre Hospitalier de l'Université Laval (CHUL) 3Programme in Cellular and Molecular Biology, faculté de Médecine, Université Laval. 4Department de Pédiatrie, Faculté de Médicine, Université Laval.

Background: Haspin is a conserved serine/threonine kinase that has a crucial catalytic role in achieving accurate kinetochore-microtubule attachment during mitosis. In particular, Haspin promotes a sufficient localization of the chromosomal passenger complex (CPC) to the centromere. This occurs by phosphorylating the threonine 3 of histone H3 (H3T3), which functions as a centromeric adaptor for CPC components. The CPC catalytic subunit (the Ser/Thr kinase Aurora B) phosphorylates several substrates at the kinetochore to destabilize and correct the kinetochore-microtubule mis-attachments. However, histone H3T3 phosphorylation does not seem to be the only substrate for Haspin during mitosis because evidence suggests a CPC-independent function for Haspin in protecting sister chromatids cohesion. The stable interactions between sister chromatids is also important to achieve accurate kinetochore-microtubule attachment for proper chromosome segregation. Here, we show that Haspin can also phosphorylate Histone H2B at threonine-119 during mitosis.  We hypothesize that Haspin may contribute to sister chromatid cohesion protection by a novel mechanism involving Histone H2B. Thus, we are investigating H2BT119 phosphorylation during mitosis.

MATERIALS AND METHODS: Combinations of cell biology- and biochemistry-based approaches were used to investigate the regulation and function of the novel Haspin phosphorylation site. We generated a phospho-antibody for H2BpT119 to explore this phosphorylation in vivo and in vitro. We used Hela S3 cell that is arrested in mitosis by nocodazole to verify H2BpT119 signal by western blot and immunofluorescence microscopy. Also, we used cytological approaches to investigate H2BpT119 localization and its involvement in the protection of sister chromatid cohesion.

RESULTS: Bioinformatics analysis showed that threonine 119 in H2B is a conserved site that can be phosphorylated by Serine/threonine kinases and, more importantly, it is a Haspin target site. Indeed, the in vitro kinase assay confirmed Histone H2B phosphorylation by Haspin. The H2B phospho-threonine 119 (H2BpT119) was observed by western blot to occur particularly in the cells that arrested in mitosis by nocodazole, but not in the interphase cells that blocked by thymidine at G1/S. Interestingly, inhibiting Haspin by the 5-iodotubercidin (5-Itu) or silencing Haspin expression by siRNA were associated with a robust reduction in H2BpT119 signal. The immunofluorescence staining revealed that H2BpT119 signal appears and localizes at the centromere region during prophase and persists until it totally disappears in anaphase. Furthermore, transfection of mutant H2B-mcherry (threonine-119 to alanine (T119A)) in Hela S3 cells causes an early loss in the sister chromatid cohesion compared to the wild-type (WT) and the phosphomimetic (threonine-119 to aspartic acid (T119D)).

CONCLUSION: This discovery may lead to finding a novel mitotic pathway that can be an important potential regulator for proper chromosome segregation.

Identification of a molecular hinge controlling the amyloid self-assembly and cytotoxicity of islet amyloid polypeptide


Elizabeth Godin1, Phuong Trang Nguyen2, Ximena Zottig3, Steve Bourgault4
1Université du Québec à Montréal 2Université du Québec à Montréal 3Université du Québec à Montréal 4Université du Québec à Montréal

Numerous endogenous proteins are known for their propensity to misfold and self-assemble into amorphous aggregates, oligomers and/or amyloid fibrils. Amyloids are highly organized β-sheet-rich protein assemblies that are associated with many diseases, such as Alzheimer’s disease, Parkinson’s disease and type II diabetes. Islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone that is co-secreted with insulin from pancreatic β-cells. Tissue deposition of IAPP in Langerhans’s pancreatic islets into amyloid fibrils is associated with the progression of type II diabetes. However, the mechanisms by which this peptide self-assembles and mediates it’s cytotoxicity are still not well-understood. It has been reported that asparagine residues are important in IAPP self-assembly. Specifically, asparagine-21, located in the amyloidogenic region, appears to play a key role in self-assembly. Therefore, we investigated the role of this residue by modifying its side chain and peptide backbone to better understand the specific interactions involved in amyloid formation. Rodent IAPP (rIAPP) was used as a negative control since it is known to be resistant to amyloidogenesis and to be non-toxic. Peptide assemblies were analysed by circular dichroism spectroscopy, fluorescence spectroscopy with thioflavin-T (ThT) and 8-anilino-1-naphthalenesulfonic acid (ANS), atomic force microscopy and transmission electron microscopy. In addition, cytotoxicity was evaluated using rat pancreatic cells (INS-1E) with resazurin reduction. Our results showed that the mutation N21F maintains IAPP assemblies into a highly toxic quaternary conformation. Besides, substitutions N21n and N21P were found to accelerate amyloid formation while most other mutations inhibited amyloid formation. This study indicates that specific side-chain interactions involving asparagine-21 are essential in amyloid formation and that β-sheet destabilizing mutations in this hinge region modulate amyloidogenicity and cytotoxicity.

Identification of structural determinants of biased signaling of the apelin receptor


Laurent Bruneau Cossette1, Éric Marsault1, Philippe Sarret1, Pierre Lavigne1
1Université de Sherbrooke 2Université de Sherbrooke 3Université de Sherbrooke 4Unibersité de Sherbrooke

G protein-coupled receptors (GPCRs) are transmembrane proteins responsible for the conversion of extracellular signals into intracellular signals, a process called signal transduction. They do so upon binding of an extracellular compound - the ligand - by coupling with and activating intracellular effector proteins, G proteins and arrestins being the main ones. A phenomenon knows as biased signaling - that is, the ability of the GPCR to couple preferentially with either of its effector proteins - is currently under investigation by both academia and industry for its potential to limit or prevent side effect of pharmaceutical compounds that target GPCRs. Although a lot of experimental data is now available on this phenomenon and what structural components of the protein are involved in the process of GPCR signaling, the dynamic processes by which these structural components enable the binding of effector proteins remain poorly understood. The apelin receptor (APJ) is a receptor involved in various cardiovascular processes, the most interesting being vasodilation and cardioprotection for their application to heart failure and hypertension. Because it has been relatively not much studied, little is known about whether it is possible to produce targeted effects (e.g. cardioprotection without vasodilation) because little is known on its ability to produce signaling that is biased towards effector proteins other than Gi.

In a bid to understand the dynamic processes involved in APJ biased signaling, a two-pronged approach, using molecular dynamics for structural data and Bioluminescence Resonance Energy Transfer (BRET) on several APJ mutants for signaling data, has been used. Molecular systems containing APJ with apelin-13 - one of its ligands - and APJ without apelin-13, stemming from an X-ray structure of an active-like APJ with a peptidic macrocyclic agonist (Ma Y et al. 2017), were simulated using Gromacs (GROMOS 54a7 forcefield) over 10-16 µs. By using the combination of a time-lagged Independent Component Analysys (tICA) and a Hidden Markov Model (HMM) for simulation analysis, it has been found that two structural rearrangements could constitute early activation events: a diminution of the angle between transmembrane domains (TM) 3 and 6, likely caused by residues W261 (6.48) and F257 (6.44), as well as the formation of a kink in TM7 around the NPxxY motif. Using simulation data on which residues and structural regions are involved, as well as data from existing literature, a set of 23 APJ mutants was created and analyzed for apelin-13 binding, cell surface expression and signaling for both Gi and beta-arrestin-2 pathways using BRET. Mutant N112G was identified as constitutively active on both Gi and beta-arrestin-2 pathways, and mutants D126V and V254L were identified as constitutively active only for the Gi pathway. Several other mutants also exhibited various degrees of constitutive activity, but were redundant in behaviour and were thus excluded from further study. Subsequent simulations on N112G molecular systems equivalent to those of WT APJ confirm that both structural rearrangements that were observed in WT simulations also occur in N112G APJ, this time with or without apelin. More data is currently required for D126V and V254L to obtain accurate information on their simulation data. This study provides tremendous insight on the dynamics of APJ signaling, is consistent with existing structural data on other GPCRs and led to the discovery of one mutant with apparently indiscriminate constitutive activity, and two mutants with a Gi-exclusive constitutive activity. These mutants can be used for further studies on the signaling properties of both APJ and its various ligands to better understand their behavior.

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


Sara Banerjee1,2,3, Kévin Jacquet1,2,3, Nicolas Bisson1,2,3,4
1Centre de recherche sur le cancer de l'Université Laval 2Regroupement stratégique PROTEO 3Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec 4Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Université Laval

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 EphRs 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 dépendants des EphRs. basés soit sur la répulsion ou la ségrégation cellulaire

RÉSULTATS: Nous avons obtenu un réseau protéique pour les 4 EphRs 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 commun aux 4 EphRs. Nos résultats d’essais biologiques ont démontré que la délétion de certains de ces partenaires bloque la ségrégation cellulaire EphB2 dépendante.

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

Impact of the incorporation of a monofluoroalkene moiety on the hydrophobicity of small peptides


José Laxio Arenas1, Myriam Drouin1, Jean-François Paquin1
1Université Laval

Due to its unique properties, the incorporation of one or many fluorine atoms into a bioactive compound has become a leading strategy for drug design. Fluorine is frequently employed to modify the biologically relevant properties of a molecule, such as lipophilicity.1 In the case of proteins, as the hydrophobicity of amino acids has an important impact on its folding and/or biological activity,2 fluorinated amino acids, such as ones bearing a trifluoromethyl group on the lateral chain, can be used to modulate these features.3 Computational chemistry, partition coefficient or reverse-phase HPLC (RP-HPLC) are common tools to estimate the effect of an amino acid on the hydrophobicity of a peptide. RP-HPLC is the most direct technique to obtain the hydrophobicity index of small peptides.4

 

On the other hand, the monofluoroalkene moiety is as a non-hydrolyzable peptide bond isostere.5 Nevertheless, its influence upon the hydrophobicity of a peptide in which it is incorporated has not been thoroughly demonstrated.6 Thus, we report a study of the hydrophobicity of short peptides containing a monofluoroalkene. The synthesis of the monofluoroalkene-based dipeptide isostere will be discussed, such as the preliminary results towards the solid phase synthesis of longer peptides. Finally, the initial work of RP-HPLC to obtain hydrophobic indexes will be presented.

 

 

 

 

 

References

 

(1)       D. O’Hagan, Chem. Soc. Rev., 2008, 37, 308.

(2)       C. N. Pace, J. M. Scholtz and G. R. Grimsley, FEBS Letters, 2014, 588, 2177.

(3)       Q. A. Huchet, B. Kuhn, B. Wagner, H. Fischer, M. Kansy, D. Zimmerli, E. M. Carreira and K. Müller, J. Fluorine Chem., 2013, 152, 119.

(4)       (a) A. J. Leo, Pharmacochem. Libr., 1991, 16, 349. (b) D. D. Desmarteau and C. Lu, J. Fluorine Chem., 2007, 128, 1326. (c) C. Gadais, E. Devillers, V. Gasparik, E. Chelain, J. Pytkowicz and T. Brigaud, ChemBioChem, 2018,
DOI: 10.1002/cbic.201800088.

(5)       M. Drouin and J.-F. Paquin, Beilstein J. Org. Chem., 2018, 13, 2637.

(6)       A. Choudhary and R. T. Raines, ChemBioChem, 2011, 12, 1801.

Inhibition and activation mechanism study of the kinase by isothermal titration calorimetry (ITC)


yun wang1, Jinming Guan1, Justin M. Di Trani1, Karine Auclair1, Anthony Mittermaier*1
1McGill University

Kinase is a large family of enzymes that catalyze phosphorylation of ATP to generate phosphorylated product and ADP. More than 30% of the human protein can be phosphorylated by the kinase activity, and it plays a critical role such as transmit signal and regulated the complex cell process. Aberrant activity of the kinase usually alters the function of target substrate or protein and results in cancer, neurodegeneration and immune disorders, etc. Therefore, kinase becomes one of the major drug targets. The most common strategy for the kinase inhibitor is by targeting ATP binding site to form a competitive inhibitor. Therefore, understanding the kinetic and regulation of the kinase, especially the ATP binding site, is very important. Here we developed a new para technique using Isothermal titration calorimetry (ITC) to study the kinetic mechanism of kinase. Three kinases, including APH(3’),PaPank and EcPanK, are studied in this research. The results showed that the product ADP can significantly inhibit the kinase activity, especially for the APH(3’). We also demonstrate that ADP can activate EcPanK activity at low ATP concentration.

Interactions of amyloid peptide AS71-82 with model membranes: structural and morphological study via FTIR and ssNMR


Benjamin Martial1,2, Thierry Lefèvre1,2, Gabrielle Raiche-Marcoux1,2, Michèle Auger1,2
1Université Laval 2Département de chimie, PROTEO, CERMA, CQMF, Université Laval

a-Synuclein (AS) is an amyloid protein involved in Parkinson’s disease. In pathological cases, aggregates of this protein form in the dopaminergic neuronal network, leading to its progressive degeneration accompanied with a dramatic decrease in dopamine levels. Under physiological conditions, AS is disordered in solution or weakly bound to neuronal membranes, via the formation of a-helices. The triggers and steps underlying the formation of insoluble β-sheet rich fibrils are still unclear. In our work, we focus on a central 12 amino acids segment of AS in the amyloidogenic part of the protein that is believed to be responsible for the fibrillization of the whole protein: AS71-82.

Interactions between AS and neuronal membranes are thought to be the starting point of the fibrillization process. In order to investigate and probe the mechanisms responsible for this fibrillization, model membranes composed of different ratios of zwitterionic (PC) and anionic (PG) phospholipids were used in our work. Infrared spectroscopy allowed the identification of irreversible changes in the b-sheet structure of AS71-82 upon the gel→fluid phase transition of the lipids, underlining the critical role of peptide/membrane interactions. Furthermore, we studied via the 31P 2D solid-state NMR pulse sequence PROCSA the impact of AS71-82 on phospholipid headgroups, in order to identify the preferentially interacting phospholipid in membranes composed of a PC-PG mixture.

Investigations phytochimiques du Bouleau glanduleux et isolation d’actifs cosméceutiques


Claudia Carpentier1,2, Meggan Beaudoin1, Gaëlle Simon1, François Béland2, Maxim Maheux3, Normand Voyer1
1Université Laval 2Silicycle 3TransBio Tech

Le bouleau glanduleux est un arbuste de la famille des Betulaceae, qui est très abondant au cœur de la flore nordique du Québec. Sa croissance rapide depuis les années 1990, est une conséquence directe des changements climatiques. L’expansion de cet arbuste est un problème préoccupant puisqu’elle menace la biodiversité en altérant tant l’environnement biotique qu’abiotique des écosystèmes nordiques. Afin de maintenir l’intégrité des écosystèmes nordiques, l’utilisation durable du bouleau glanduleux comme source naturelle d’ingrédients actifs en cosméceutique pourrait servir de stratégie de mitigation. Les feuilles des Betulaceae contiennent une variété de composés phénoliques (anthocyanes, flavonoïdes, acides phénoliques, stilbènes, tanins, etc.). Les polyphénols sont maintenant bien connus pour leurs propriétés antioxydantes et anti-inflammatoires ce qui en fait une source très intéressante en cosméceutique étant donné qu’ils aident à limiter le photoviellissement de la peau. L’isolation ciblée des composés phénoliques actifs des feuilles du bouleau glanduleux a été réalisée en effectuant un fractionnement bio guidé (anti-tyrosinase, anti-élastase et antioxydante). La purification et l’identification des composés bioactifs seront également présentées.

Kinetically Programmed, One-Pot DNA Reactions for Molecular Detection Directly in Whole Blood


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

Human designed 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 sequentially programmed to achieve complex synthesis within a single location and condition. In this paper we report a versatile DNA-based one-pot, three reactions, that can be used to detect any specific molecules directly in complex biological samples. We show that careful programming of the kinetic of these different reactions enable to significantly increase the gain of the sensing mechanism and decrease the reaction time. Using this strategy in an electrochemical format, we demonstrate the multiplexed, quantitative, one-step detection of two models 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 and, more generally, the importance of programming the kinetic of one-pot reactions in order to achieve high efficiency in a timely manner.

La liaison du tout-trans rétinol avec la lécithine rétinol acyltransférase tronquée et ses mutants n’explique pas la faible activité enzymatique des mutants


Sarah Roy1,2,3,4,5, Ana Coutinho6, Line Cantin1,3,5, Marie-Eve Gauthier1,2,3,4,5, Manuel Prieto6, Stéphane M. Gagné2,4,5, Christian Salesse1,3,5
1Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval 22Département 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 6Instituto Superior Técnico, Universidade Lisboa, Lisboa, Portugal

CONTEXTE ET OBJECTIFS : Le pigment visuel des photorécepteurs est composé d’une protéine et d’un chromophore, le 11-cis rétinal. L’absorption de la lumière par le pigment visuel mène à l’isomérisation de son chromophore en tout-trans rétinal, qui est par la suite 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). Des mutations de la LRAT mènent à la dégénérescence des photorécepteurs et à une perte de la vision. Nous avons montré auparavant que les mutations délétères de la LRAT tronquée (tLRAT) mènent à une perte presque complète de son activité. Par contre, nous avons montré par résonance magnétique nucléaire que ces mutations n’ont pas d’effet sur le repliement global de cette 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. L’objectif de ces travaux consiste donc à comparer la liaison du tout-trans rétinol par la tLRAT et ses mutants. MATÉRIEL ET MÉTHODES : La tLRAT et ses mutants ont été purifiées par chromatographie d’affinité. La liaison du tout-trans rétinol par la tLRAT a été mesurée par spectrofluorimétrie. RÉSULTATS : Une augmentation importante de la fluorescence du tout-trans rétinol a été observée suite à sa liaison à la tLRAT. Le lissage du graphique de l’intensité de fluorescence en fonction de la concentration en rétinol avec un modèle mathématique a permis de calculer la constante de dissociation (KD) du tout-trans rétinol. Une valeur de KD de 34 ± 12 nM a été obtenue pour la tLRAT native. Étonnamment, seulement le mutant naturel P173L‑tLRAT a une valeur de KD (250 ± 70 nM) qui diffère significativement de celle de la tLRAT native. CONCLUSIONS : Ces données suggèrent que tous les mutants lient aussi efficacement le tout‑trans rétinol que la tLRAT native sauf le mutant P173L-tLRAT. Cette information permet de mieux comprendre l’effet des mutations de la LRAT sur son activité.

La liaison membranaire de la protéine S100A10 et du peptide d’AHNAK intervenant dans la réparation membranaire


Xiaolin Yan1,2,3, Marie-France Lebel-Beaucage4, Samuel Tremblay1,3, Gary Shaw5, Élodie Boisselier1,3
1Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval 2Département de biochimie microbiologie et bio-informatique, Faculté de sciences et génie, Université Laval 3CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec 4Département de chimie, biochimie et physique, Faculté des sciences, Université du Québec à Trois-Rivières 5Département de biochimie, Faculté de RMN biomoléculaire, Université de Western Ontario

CONTEXTE ET OBJECTIF : Les protéines de la famille S100 et les annexines interviennent dans des mécanismes membranaires vitaux.  En effet, le complexe protéique S100A10/annexine A2 permettrait le recrutement d’une partie du C-terminal de la protéine AHNAK à la membrane en présence de calcium, puis formerait une plateforme initiant la réparation membranaire. Cependant, aucune donnée moléculaire n’est présentement disponible sur la liaison membranaire de la S100A10 et du peptide du C-terminal d’AHNAK de ce complexe. Ces travaux visent donc à étudier la liaison membranaire de la protéine S100A10 et du peptide d’AHNAK pour mieux comprendre leurs rôles lors du processus de réparation membranaire.

 

MATÉRIEL ET MÉTHODES : La protéine S100A10 a été surexprimée et purifiée. Puis, elle a été identifiée par spectrométrie de masse et sera confirmée par Western blot. La stabilité de sa structure secondaire a été analysée par dichroïsme circulaire. Également, nous nous sommes procuré un peptide du C-terminal d’AHNAK synthétisé. Ensuite, le modèle des monocouches de Langmuir a été utilisé pour mimer les membranes cellulaires et ainsi caractériser l’interaction des protéines avec différents phospholipides les composant. Leur structure secondaire, orientation et organisation membranaire seront étudiées par spectroscopie infrarouge de réflexion-absorption par modulation de la polarisation. Leur localisation sera déterminée par microscopie de fluorescence grâce à l’étude de l’influence de leur liaison sur l’état physique des phospholipides. Enfin, l’étude par RMN solide de l’affinité de ces protéines pour les différentes têtes polaires aura lieu en collaboration avec le Dr Dror Warschawski.

 

RÉSULTATS : L’optimisation de la purification de la S100A10 par chromatographie d’affinité a permis d’obtenir une pureté de 96 %. L’identification de la protéine et des études de stabilité ont déjà été effectuées. Des analyses par Western blot suivront. Les études de la liaison membranaire de la S100A10 seront mises en place avec le modèle des monocouches de Langmuir. L’étude de la liaison membranaire du peptide d’AHNAK avec cette méthode a permis d’identifier une préférence de liaison avec les lipides composés de chaînes acyles insaturées et d’une tête polaire phosphaditylsérine.

 

CONCLUSION : Ce projet permettra de développer les connaissances actuelles sur la liaison membranaire des protéines S100A10 et du peptide d’AHNAK. 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.

Le complexe du pore nucléaire de la levure comme système-modèle pour l'étude de la rétention des gènes dupliqués


Simon Aubé1,2,3,4, Axelle Marchant1,2,3,4, Alexandre Dubé1,2,3,4, Isabelle Gagnon-Arsenault1,2,3,4, Philippe Després1,3,4, Christian Landry1,2,3,4
1Institut de Biologie Intégrative et des Systèmes 2Département de biologie, Université Laval 3Département de biochimie, de microbiologie et de bioinformatique, Université Laval 4Regroupement PROTEO

La duplication de gènes serait une des principales sources de nouvelles protéines et même de nouvelles fonctions. Les duplicats résultants se retrouveraient en effet libérés de plusieurs contraintes évolutives, permettant leur divergence par sous-fonctionnalisation (répartition des fonctions ancestrales) ou par néo-fonctionnalisation (acquisition de nouvelles fonctions). Les évènements de duplication génique ont joué un rôle dans l’évolution de plusieurs complexes protéiques des cellules eucaryotes. Chez la levure, c’est notamment le cas pour le complexe du pore nucléaire (NPC) — cet assemblage de centaines de protéines contrôlant le passage de macromolécules de part et d’autre de l’enveloppe nucléaire. Le NPC de Saccharomyces cerevisiae compte trois couples de paralogues issus d’un évènement de duplication complète du génome (WGD). Les membres de chacun d’eux semblent avoir des fonctions distinctes. Néanmoins, les contributions respectives de la néo-fonctionnalisation et de la sous-fonctionnalisation dans ces différences restent à éclaircir. La combinaison des méthodes modernes d’édition génétique et du criblage à haut débit des interactions protéine-protéine offre l’opportunité de reconstituer le pore nucléaire ancestral et d’ainsi répondre à cette question. De même, une telle démarche peut contribuer à élucider le rôle de l’innovation dans le maintien des gènes dupliqués. Le système d’édition génétique CRISPR-Cas9 a été utilisé afin de remplacer les paralogues NUP53 et NUP59 chez S. cerevisiae par leurs orthologues issus d’espèces de levures ayant divergé avant ainsi qu’après l’évènement de WGD. Les interactomes de chacun de ces orthologues au sein du pore nucléaire de S. cerevisiae ont été établis par des essais de complémentation protéique à la dihydrofolate réductase (DHFR-PCA) et comparés avec ceux des nucléoporines natives. Ces manipulations montrent que l’histoire évolutive des paralogues étudiés a bien été marquée par une divergence de fonction.

Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η


Christopher J. Wilds1, Derek K. O'Flaherty1, Amritraj Patra2, Yan Su2, F. Peter Guengerich2, Martin Egli2
1Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec H4B1R6, Canada 2Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA

Alkylation of DNA can result from exposure to endogenous or exogenous agents to the cell. Lesions which are formed can have adverse effects including DNA polymerase (Pol) blockage and nucleotide misincorporation. Lesions that evade DNA repair can undergo translesion synthesis (TLS) by Y-family DNA Pols with bypass of the modifications occurring in an error-free or error-prone manner. Alkylation of the O4-atom of thymidine produces a lesion that results in high fidelity replicative DNA Pol misinserting dGTP. The present study examines the influence of O4-alkyl lesion orientation on human Y-family Pol η (hPol η) bypass processivity with oligonucleotides containing analogs with the C5 and O4 atoms connected by a dimethylene (DFP) or trimethylene (TPP) linkage where the O4-methylene group is limited to adopt an anti-conformation. Single nucleotide incorporation studies demonstrated increased selectivity towards dATP over dGTP for these analogs. Primer extension reactions in the presence of all four dNTPs revealed proficient nucleotide incorporation by hPol η across and past the modified site. hPol η preferentially incorporated dATP followed by dGTP for these analogs with differences observed relative to O4-methyl and O4-ethyl thymidine demonstrating that restriction of the O4-methylene group to an anti-conformation influences dNTP incorporation. The crystal structures of ternary hPol η•DNA•dATP and hPol η•DNA•dGTP complexes with the template strands containing O4-methyl thymidine reveal that the lesion stacks atop a tryptophan residue near the ceiling of the active site instead of pairing with the incoming nucleotide. Together, these results provide mechanistic insights on the mutagenicity of O4-alkylated thymidine when acted upon by hPol η.

Linear and cyclic peptides as green catalysts for chiral epoxidations


Christopher Bérubé1, Xavier Barbeau1,2, Patrick Lague1,3, Normand Voyer1,4
1Université Laval and PROTEO 2Laval University 3Université Laval 4Université Laval

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 and Colonna, is the asymmetric epoxidation of electron deficient enones using polypeptides, which lead to epoxyketones with high yields and optical purities.

Inspired by those results, we developed a novel ecofriendly way for the chiral epoxidation of a,b-unsaturated ketones in water without any co-solvent. Our strategy is based on the use of linear oligopeptides or cyclic peptides as chiral catalysts. In the case of linear oligopeptides, conformational and molecular modelling studies demonstrated the importance of both the a-helical structure and their hydrophobic nature for reactivity and selectivity. Poly-L-Leucine (PLL) efficiently catalyzes the epoxidation of a wide variety of electron-deficient enones, leading to high enantioselectivities.1This new process in water opens the way to easily and rapidly produce a variety of chiral epoxyketones through environmentally benign enantioselective transformations.

Along the same line, we also synthesized cyclic peptides and evaluated their efficacy as catalysts in the asymmetric epoxidation of trans-chalcone.2-4Highly variable results were obtained and the presence of a co-solvent is essential for yields and enantioselectivities. Structure-activity and computational studies provided insights into the mechanism of the triphasic process. The cyclic peptides act as catalyst by templating a supramolecular arrangement at the aqueous-organic interface required for an efficient transformation to occur.

 

 

 

 

 

Measuring covalent inhibitor kinetics using isothermal titration calorimetry


Caroline Dufresne1, David Nie1, Nicolas Moitessier1, Anthony Mittermaier1
1McGill University

Covalent drugs have rarely been intentionally designed in drug discovery  programs, even though they are a significant part of the drug market. With their increasing popularity, one problem emerges, which is their lack of characterization. The classical assays with IC50 and Ki are of very little significance in their case, while kinetic parameters, such as association and dissociation rates and residence time, are much more representative of their inhibitory activity. Isothermal Titration Calorimetry (ITC) is a quite powerful technique entirely in solution to achieve these rates, which doesn’t need spectroscopically active solutions, or high concentrations. Historically, ITC was more of a thermodynamic technique, and the development of kinetic experiments is still rather at its beginning. We developed new ITC kinetic experiments to characterize quick covalent inhibitors of prolyl oligopeptidase (POP), an enzyme involved in many types of cancer.

Measuring Enzyme Kinetics Using Isothermal Titration Calorimetry


Justin Di Trani1, Anthony Mittermaier2
1McGill University 2McGill University

Isothermal titration calorimetry (ITC) is a powerful tool for acquiring both thermodynamic and kinetic data for biological systems. ITC offers several advantages over other experimental kinetics methods as it can be performed entirely in solution under physiological conditions, does not require spectroscopically-active (eg. fluorescent) molecules, it is compatible with spectroscopically opaque solutions, and can be applied to relatively dilute samples. Despite its long history and technical advantages, kinetic applications of ITC remain fairly rare. In order to expand the use of ITC kinetics we have developed several techniques in order to measure physical properties of enzymes and enzyme inhibitors. These techniques allow us to measure both michaelis-menten and non-michaelis menten properties of rapidly evolving enzyme reactions, measurement of the mode and strength of enzyme inhibitors in a single experiment and, lastly, permits the direct measurement of the association and dissociation rates of enzyme inhibitors. These experiments are simple and allow for rapid and complete characterization of enzymes and enzyme inhibitors and will broaden the overall applicability of ITC.

Optimisation de l’activité et de la sélectivité d’agonistes des récepteurs neurotensinergiques​


Michael Desgagné1, Marc Sousbie1, Philipe Saret1, Éric Marsault2
1Université de Sherbrooke 2Université de Sherbrooke

La neurotensine est un neuropeptide. c'est le ligand endogène de deux membres de la superfamille des récepteurs couplés aux protéines G, les récepteurs NTS1 et NTS2. L'activation de ces récepteurs par ce ligand est connue pour provoquer un puissant effet analgésique. Le système neurotensinergique est donc une cible de choix pour le développement des composés analgésiques. Afin d'explorer et faire avancer les connaissances de relations structure-activité entre la neurotensine et ses récepteurs, nous avons utilisé une approche de conception rationnelle d'analogues du ligand endogène. En nous basant sur les informations disponibles dans la littérature scientifique, nous avons macrocyclisé ce peptide afin de contraindre sa conformation. Nous avons aussi évalué l'effet de la macrocyclisation sur la stabilité des anaogues ainsi produits

 

La première étape a consisté à trouver une manière de macrocycliser qui favorise la liaison au récepteur. Ceci a été accompli après de nombreux essais infructueux et a été facilité par la structure cristallographique du récepteur NTS1.

 

L'étape suivant a consisté à optimiser le macrocycle obtenu, tant au niveau de la liaison/activation du récepteur qu'au niveau de la résistance aux protéases. Deux macrocycles ont ainsi été sélectionnés pour être caractérisés dans des essais in vivo, l'un avec la meilleure affinité et une stabilité modérée, l'autre avec une excellente stabilité mais une affinité modérée. Ceux-ci ont démontré leur capacité à induire un puissant effet analgésique dans deux modèles de douleur.

 

Enfin, la dernière étape a été d'explorer comment des macrocycles sélectifs envers NTS2 peuvent être obtenus dans le but de produire des composés présentant des moins d'effets indésirables.

Palladium-Catalyzed Synthesis of Functionalized Monofluoroalkenes


Myriam Drouin1, Sébastien Tremblay1, Jean-François Paquin1
1Université Laval

The monofluoroalkene is an interesting moiety in medicinal and agricultural chemistry, as it is a good mimic of the amide bond or the enol ether. Thus, this functional group can be used as a non-hydrolyzable peptide bond surrogate. Hence, many synthetic approaches have been elaborated in the last decades.1 Our research group is interested in the development of new methodologies to synthesize monofluoroalkenes starting from 3,3-difluoropropenes.2 In particular, the palladium-catalyzed addition of malonates to obtain monofluoroalkenes was studied. The use of different nucleophiles and starting substrates will be presented, as well as the derivatization of the obtained products. An asymmetric variant was also developed to obtain β-amino acid derivatives. This transformation and its scope will also be discussed.3

 

 

 

 

 

 

 

1 Drouin, M.; Hamel, J.-D.; Paquin, J.-F Synthesis 2018, 55, 881.

2 Drouin, M.; Hamel, J.-D.; Paquin, J.-F. Synlett 2016, 27, 821.

3 Drouin, M.; Tremblay, S.; Paquin, J.-F. Org. Biomol. Chem. 2017, 15, 2379.

Plasma membrane vesicles derived from mammalian cells to study the perturbative nature of amyloid fibril assembly


Mathew Sebastiao1,2, Noé Quittot1,2, Dror WARSCHAWSKI1,3, Mathilde Fortier1,2, Isabelle Marcotte1,2, Steve Bourgault1,2
1Université du Québec à Montréal 2PROTEO 3Centre National de Recherche Scientifique (CNRS) UMR7099, Institut de Biologie Physico-Chimique, University Paris Diderot (Paris 7), Paris, France

The accumulation of proteins as amyloid fibrils is associated with a variety of degenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes (TIID). While the causative agent of these conditions was traditionally assumed to be the amyloid fibrils themselves, emerging evidence suggests that pre-fibrillar species (oligomers) are the most culprit proteospecies. The mechanisms of cytotoxicity in these diseases are thought to arise from the generation of membrane perturbations. These hypotheses have been so far developed using synthetic model membranes such as micelles, small unilemellar vesicles, and large unilemellar vesicles. Model membranes such as these have been essential tools to simulate the perturbative and cytotoxic effects of amyloidogenic proteins on living cells, though do not fully represent the complexity of biological membranes. The goal of this project is to observe the perturbative effects of the islet amyloid polypeptide (IAPP) as a model peptide on membranes derived from eukaryotic cells, retaining as much of the native membrane composition and membrane-associated structures as possible. Membranes were prepared using red blood cells (RBCs) and giant plasma membrane vesicles (GPMVs) and incubated in the presence of monomeric hIAPP. Membrane perturbation and permeability were evaluated at different time intervals by means of fluorescence spectroscopy, confocal microscopy, nuclear magnetic resonance, and UV-vis assay. IAPP was able to demonstrate sufficient membrane perturbations in RBCs to cause total hemolysis as detected by UV-vis assay. Additionally, it was observed by confocal microscopy that IAPP distort GPMVs. These results demonstrate that it is possible to use vesicles originating from eukaryotic membranes and may lead to an improved understanding of the plasma membrane perturbations associated with amyloid-related diseases.

Préparation de peptides macrocycliques sur résine oxime


Alexandre Borgia1,2, Christopher Bérubé3, Gaëlle Simon3, Daniel Grenier4, Normand Voyer3,4
1Université Laval 2PROTEO 3Université Laval and PROTEO 4Université Laval

Les macrocycles peptidiques constituent une catégorie de molécules d’intérêt pour l’élaboration de nouveaux agents thérapeutiques. À cet égard, plusieurs d’entre eux possèdent un fort potentiel d’activité biologique. En revanche, la conception de nouveaux peptides cycliques naturels peut s’avérer une tâche fastidieuse. C’est pourquoi peu de méthodes rapides, efficaces et générales sont actuellement disponibles pour la préparation de larges macrocycles.

 

Parmi une multitude de macrocycles peptidiques existant dans l’environnement, treize macrocycles, dont l’isolation est rapportée dans la littérature, ont été ciblés par le groupe de recherche afin d’en effectuer la synthèse. Chacun d’entre eux représente un défi synthétique majeur en raison de leurs structures uniques dont la séquence d’acides aminés varie, mais également au niveau de la construction efficace de larges cycles constitués de 15 à 30 atomes. 

 

L’approche mise au point par le laboratoire fait appelle aux propriétés uniques de la résine oxime1afin de créer un point d’ancrage temporaire du peptide, ce qui permet la construction et l’allongement du squelette peptidique sous une forme linéaire. Par la suite, une étape concomitante de cyclisation/clivage intramoléculaire permet à la fois de libérer le peptide de son support solide, ce qui donne accès à la macrocyclisation de type tête-queue.

 

Nous présenterons la première synthèse totale, la purification et la caractérisation de ces macrocycles peptidiques naturels obtenus par l’application de la méthodologie de synthèse peptidique sur résine oxime. Pour ce faire, nous nous appuierons principalement sur les résultats de la Crotogossamide, un nonapeptide macrocyclique issu du latex de Croton Gossypifolius initialement ciblé par notre groupe. 2,3

 

Références

 

1.     DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1980, 45, 1295-1300.

             

2.     Bérubé, Borgia, Simon, Grenier, Voyer, 2018. Soumis à Phytochemistry Letters.  

3.     Quintyne-Walcott, S.; Maxwell, A. R.; Reynolds, W. F. J. Nat. Prod. 2007,70, 1374-1376. 

Proteomic analysis of NCK1/2 adaptors reveals a new NCK2-specific role in cell abscission during cytokinesis.


Kévin Jacquet1,2,3, François Chartier1,2,3, Sara L. Banerjee1,2,3, Sabine Elowe2,3,4, Nicolas Bisson1,2,3
1Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Axe Oncologie 2PROTEO 3Centre de recherche sur le cancer de l’Université Laval 4Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Axe Reproduction, santé de la mère et de l’enfant

Signals from cell surface receptors are often relayed via adaptor proteins. NCK1 and NCK2 are Src-Homology (SH) 2 and 3 domain adaptors that regulate processes requiring a remodelling of the actin cytoskeleton. Evidence from gene inactivation in mouse suggests that NCK1 and NCK2 are functionally redundant, although recent reports support the idea of unique functions for NCK1 and NCK2. We sought to examine this question further by delineating NCK1- and NCK2-specific signalling networks. We used both affinity purification-mass spectrometry and BioID proximity labelling to identify NCK1/2 signalling networks comprised of 98 proteins. Strikingly, we found 30 proteins restricted to NCK1 and 28 proteins specifically associated with NCK2, suggesting differences in their function. We report that Nck2-/-, but not Nck1-/- mouse embryo fibroblasts (MEFs) are multi-nucleated and display extended protrusions reminiscent of intercellular bridges, which correlate with an extended time spent in cytokinesis as well as a failure of a significant proportion of cells to complete abscission. Our data also show that the midbody of NCK2-deficient cells is not only increased in length, but also altered in composition, as judged by the mislocalization of AURKB, PLK1 and ECT2. Finally, we show that NCK2 function during cytokinesis requires its SH2 domain. Taken together, our data delineate the first high-confidence interactome for NCK1/2 adaptors and highlight a number of proteins specifically associated with either protein. Thus, contrary to what is generally accepted, we demonstrate that NCK1 and NCK2 are not completely redundant, and shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division.

Règles thermodynamiques et cinétiques pour l'assemblage et la régulation de nanomachines polymoléculaires à base d’ADN


Dominic Lauzon1, Alexis Vallée-Bélisle2
1University of Montreal 2Université de Montréal

Les nanomachines sont des assemblages nanométriques de molécules (ex. ADN, ARN, protéine) qui produisent un mouvement quasi-mécanique (ouput) en présence de stimulus spécifique (input).[1] Ces nanomachines servent autant pour le développement de stratégie en délivrance de médicament, que pour la détection de marqueur de maladies ou pour déclencher des évènements en bio-informatique. Étant donné sa grande programmabilité, l’ADN est une biomolécule de choix pour la création de nanomachine. Toutefois, avec l’augmentation rapide de la complexité des nano-assemblages d’ADN, il devient maintenant crucial d’optimiser le rendement de formation et la régulation de ces nanomachines afin de rendre plus accessible leur application à grande échelle. (ex. imagerie in vivo et production de masse de transporteurs moléculaires).[2]

 

Pour répondre à ce problème, il est important de mieux comprendre la thermodynamique et la cinétique d’assemblage de ces nanomachines. Pour ce faire, j’ai utilisé une approche de simulation numérique afin d’étudier plusieurs scénarios d’auto-assemblage en modifiant les constantes d’équilibre et les constantes de vitesses du système. Ces simulations numériques ont permis de déterminer des conditions expérimentales intéressantes qui ont ensuite été validé en laboratoire avec une nanomachine d’ADN contenant un, deux ou trois brins d’ADN. En incorporant des nucléotides modifiés avec un fluorophore et un quencher dans les brins d’ADN, il est possible de suivre la formation de la nanomachine dans différentes conditions par spectroscopie de fluorescence ou par électrophorèse sur gel. D’ailleurs, en modifiant la teneur en pair AT/GC, la longueur du linker ou la température, il est aussi possible de modifier de façon contrôlé les constantes thermodynamiques et la vitesse d’hybridation ou de déshybridation de l’ADN. Les résultats expérimentaux illustrent pourquoi certaines nanomachines d’ADN ne peuvent pas s’assembler avec un rendement quantitatif. De plus, les résultats démontrent qu’un bon contrôle sur l’auto-assemblage permet de réguler l’activité de cette dernière en introduisant un mécanisme de rétro-inhibition par exemple.

 

Dans le futur, ces travaux serviront de base théorique pour le développement d’un biocapteur polymoléculaire inspiré de l’hémoglobine. J’utiliserai aussi ces observations afin d’introduire un niveau de régulation supplémentaire à l’activité d’un aptazyme qui catalyse le clivage d’un lien phosphodiester d’un ribonucléotide. Le tout pave la voie vers un meilleur contrôle dans l’assemblage et la régulation de nanomachines à base d’ADN.

 

[1] Ballardini, R. et al. Acc. Chem. Res. 2001, 34, 445-455.

[2] Tørring, T. et Gothelf, K.V. F1000Prime Reports 2013, 5, 14.

Rôle des protéines S100A16 et Annexine A4 dans le maintien de l’intégrité membranaire


Francis Noël1,2, Xiaolin YAN1,2, Stefan Vetter3, Elodie Boisselier2,4
1Département de biochimie, microbiologie et bio-informatique, Faculté de sciences et génie, Université Laval 2CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec 3School of pharmacy, North Dakota State University 4Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval

Problématique : Le maintien de l’intégrité structurale et fonctionnelle des membranes est essentiel au bon fonctionnement des cellules. Une étude protéomique récente suggère que la protéine S100A16 et l’annexine A4 participent au maintien de l’intégrité membranaire dans le segment externe des photorécepteurs de l’œil. La protéine S100A16, récemment découverte, fait partie des protéines de la famille S100 pour laquelle aucune interaction protéique et membranaire n’a encore été identifiée. De plus, le maintien de l’intégrité membranaire est un processus sensible au calcium.

L’objectif général de mes recherches consiste à étudier les interactions membranaires des protéines S100A16 et annexine A4 afin de mieux comprendre leurs fonctions dans le maintien de l’intégrité membranaire. Les objectifs spécifiques sont : i) de réaliser la purification des protéines S100A16 et annexine A4, ii) d’obtenir des informations sur leurs interactions membranaires, et iii) d’étudier l’influence du calcium sur ces interactions.

Méthodologie : La protéine S100A16 est obtenue par clivage d’une protéine de fusion GST-HisTag-S100A16 avec la protéase TEV. La protéine S100A16 est ensuite purifiée sur une colonne His-Trap. Les interactions membranaires sont étudiées avec le modèle des monocouches de Langmuir. La concentration saturante en protéine est dans un premier temps déterminée et sera utilisée pour les expériences subséquentes. Les paramètres de liaison des protéines, pression d’insertion maximale et synergie, seront ensuite déterminés en présence de plusieurs phospholipides représentatifs des membranes.

Résultats : La protéine de fusion GST-HisTag-S100A16 a été clivée avec succès avec la protéase TEV en présence du β-mercaptoéthanol, et la protéine S100A16 a été isolée avec une pureté supérieure à 99%. La concentration saturante déterminée en présence de plusieurs concentrations de protéine pure est de 0.5µM. Des études biophysiques avec différents phospholipides en monocouches sont actuellement en cours de réalisation.

Conclusion : L’obtention de la protéine S100A16 pure permet de poursuivre les études biophysiques pour comprendre ses interactions membranaires. La purification de l’annexine A4 et l’étude biophysique avec différents phospholipides de cette protéine seule et en complexe avec la protéine S100A16 permettront d’avoir une meilleure compréhension du comportement membranaire de ces protéines et de leurs rôles dans le maintien de l’intégrité membranaire.

Rôle des récepteurs Eph dans l’établissement 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 Centre Hospitalier Universitaire (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, 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 tyrosine kinase (RTK). La plus grande famille de RTK sont les récepteurs Eph (EphR), qui sont activés suite au contact de leurs ligands membranaires, les éphrines. La signalisation EphR/éphrines est notamment impliquée dans la régulation de la morphologie et de la motilité cellulaires. Nos travaux récents ont montré pour la première fois que les EphR s’associent à des protéines impliquées dans la polarité épithéliale. Cela soulève l’hypothèse que les EphR jouent un rôle central dans la coordination de la polarisation cellulaire. L’objectif principal du projet est d’explorer le rôle des EphR dans l’établissement de la polarité des cellules épithéliales.

L’expression et la localisation des récepteurs Eph dans les cellules ont été confirmées par immunoburvardage et immunofluorescence. Nous avons d’abord montré qu’au moins six des quatorze EphR sont exprimés dans les cellules épithéliales Caco-2 et MDCK-2. Suite à la mise au point d’une méthode de culture 3D de sphéroïdes pour ces cellules, nous avons aussi observé par immunofluorescence la compartimentalisation 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 EphR dans l’établissement de la polarité des cellules épithéliales.

Self-assembled fibrillar nanostructures for vaccine development


Margaryta Babych1,2,3, Geneviève Bertheau-Mailhot3, Laurie Gauthier1,2,3, Ximena Zottig1,2, Soultan Al-Halifa1,2, Denis Archambault3, Steve Bourgault1,2
1Department of Chemistry, Université du Québec à Montréal, Montréal, Qc, CANADA 2Quebec Network for Research on Protein Function, Engineering, and Applications, PROTEO 3Department of Biological Sciences, Université du Québec à Montréal, Montréal, Qc, CANADA

Recent progress in the manipulation and understanding of self-assembly has highlighted potential applications for nanovaccine design. Self-assembly refers to a phenomenon in which basic constituents, such as proteins or peptides, associate spontaneously into a well-ordered structure. Virus-like particles, which result from the self-assembly of viral capsid proteins, are a good example of the usage of this property for vaccine development. In order to reconstruct a pathogen-like structure, such as Tobacco mosaic virus, one can consider using self-assembling peptides as building block units to create fibrillar nanostructures for vaccination. Utilization of self-assembling peptides has several advantages such as ease of production by chemical synthesis, biodegradability, low cytotoxicity, control of size and the possibility of surface functionalization. Ideally, self-assembly provides the flexibility of developing novel particles with tailored morphologies and desired functions by the modulation of sequences. In this work, we investigated the usage of a synthetic β-sheet-rich nanofibrilar vaccination platform based on short amyloidogenic peptide I10, in which the N- or C-terminal group can be used as a fusion site for functional epitopes. The nanofibrils take the form of a cross-β-sheet quaternary conformation as observed in amyloids, which results in nanoassemblies with high density of antigen distributed on the surface of the amyloid core. The fibrils decorated with the linear epitope were characterized by biophysical assays such as circular dichroism spectroscopy, atomic force microscopy and transmission electronic microscopy. To evaluate the potential of the I10-derivative vaccine, immunizations were carried out using mice and the IgG responses to the epitope were measured. In vitro and in vivo studies suggest that the β-sheet-rich nanofibrilar vaccination platform I10 allows the presentation of multiple copies of the antigen, enhances antigen stability and immunogenicity, and constitutes an effective way to address the limitations of traditional vaccines.

Solid-state NMR study of the microalga Chlamydomonas reinhardtii and its constituents


Alexandre POULHAZAN1, Alexandre A Arnold1, Jean-Philippe Bourgouin 2, Dror E Warschawski3, Isabelle Marcotte3
1Université du Québec à Montréal 2Université du Québec à Montréal 3Université du Québec à Montréal

Chlamydomonas reinhardtii is a model unicellular microalga used for the study of functions such as photosynthesis, cell-division, or mitochondrial function. This micro-organism could also potentially be used as an environmental sensor by monitoring how it is affected by the external medium. Solid-State NMR is non-destructive and can be applied to the in vivo study of micro-organisms. We herein explore SS-NMR strategies to study this organism in conditions as native as possible.

In a first step, intact fully 13C labelled algae were studied. Whole cell spectra are simplified by applying excitation schemes (ultrashort CP, NOE and INEPT) which differentially excite rigid, semi-rigid and mobile constituents. Resolution is further enhanced by dipolar (DARR or PDSD) or J-mediated 2D polarization transfers. We thus characterized the highly crystalline starch reserves and assigned the highly mobile galactolipid head groups. The complex dynamics of cell-wall forming glycoproteins is also evidenced.

In order to refine the assignment and characterization of some constituents, it appears necessary to isolate them from the microalga. Due to the importance of starch - the most used polysaccharide in the food industry - we first concentrate on its detailed characterization after extraction from C. reinhardtii and compare it to the in situ case. The identification of all starch resonances in situ enables a functional understanding of starch formation in vivo.

            The NMR strategies herein described not only provide an extensive characterization of whole microalgae but are also applicable to the study of other micro-organisms in vivo and the potential valorization of C. reinhardtii.

Structural and (supra)molecular basis of the cellular toxicity of amyloid fibrils


Phuong Trang Nguyen1,2, Elizabeth Godin1,2, Ximena Zottig1,2, Noe Quittot1,2, Mathew Sebastiao1,2, Steve Bourgault1,2
1Department of Chemistry, Pharmaqam, University of Québec in Montreal, Montreal, QC, Canada, H3C 3P8 2Quebec Network for Research on Protein Function, Engineering and Applications, PROTEO

The self-assembly of many endogenous proteins into amyloid fibrils is linked with numerous diseases including the Alzheimer's disease, type II diabetes (DM-2) and systemic amyloidosis. The close association between the deposition of amyloids and tissue degeneration has initially led to the hypothesis that amyloid fibrils are causing cell death. However, recent and compelling evidences have suggested that pre-fibrillar proteospecies and/or the amyloidogenic process itself mediate degeneration whereas amyloid fibrils are poorly cytotoxic. Nevertheless, the exact conformational nature of the toxic proteospecies and the molecular mechanisms leading to cytotoxicity remain ambiguous. In this study, by using the islet amyloid polypeptide (IAPP), we investigated the relation between the supramolecular morphology of amyloids and their cytotoxicity. The deposition of IAPP, into the pancreatic islets, as insoluble amyloid fibrils correlates proportionally with the progression of DM-2. We recently identified a single N-to-Q mutation at position 21 that controls the toxicity of IAPP amyloid fibrils. IAPP and [N21Q] IAPP self-assembled into amyloid fibrils, but with slight distinctive structural properties leading to major differences of toxicity. [N21Q] IAPP fibrils were toxic to pancreatic β-cells whereas IAPP fibrils were not, as demonstrated by caspase-3 activation, metabolic assay and oxidative stress. The toxicity correlated with membrane leakage of large unilamellar vesicles and erythrocyte. A combination of atomic force microscopy, transmission electronic microscopy, X-ray diffraction, Fourrier transformed infrared, 8-anilino-1-naphtalenesulfonic acid fluorescence and thioflavin T fluorescence revealed that [N21Q] IAPP and IAPP fibrils were structurally distinct. This study highlights that the frontier between toxic and non-toxic amyloid fibrils is incredibly thin, as revealed by the fact that the introduction of a single methyl moiety (-CH2-) modulates cytotoxicity. Understanding the (supra)molecular basis of fibril toxicity is critical for the development of efficient diagnostic and therapeutic strategies against amyloid-related diseases.

STRUCTURAL AND BIOPHYSICAL CHARACTERIZATION OF THE HOMODIMERIC INTERFACE OF HUMAN GALECTIN-7


Myriam Letourneau1, Nhung Nguyen-Thi1, Louise Roux1, Donald Gagné1, Nicolas Doucet1
1INRS - University of Quebec

Xylanases catalyze the hydrolysis of glycosidic bonds in heteroxylan, a biopolymer with significant value in the fields of industrial biomass degradation and renewable energy. This enzyme family has been used in the pulp and paper and agro-food industries as an ecofriendly alternative for pre-bleaching processes and for the clarification of fruit juices. A number of studies have been devoted to the catalytic improvement of xylanases but success remains limited due to generally poor understanding of their molecular properties. The typical xylanase b-jellyroll architecture preserves a closed right-handed structure with 2 b-sheets and an a-helix posing as fingers and palm, in addition to 2 functionally important loop regions forming a ‘thumb-loop’ and a cord. Previous studies have suggested that thumb-loop motions could be involved in substrate binding, catalysis, and/or product release. To better characterize potentially conserved atomic-scale residue motions among structural homologs, we performed NMR titration and relaxation dispersion experiments (15N-CPMG) to probe conformational exchange occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and xylanase C (XlnC) from Streptomyces lividans. Although both enzymes share 75% sequence homology and similar catalytic efficiency, significant differences in their atomic-scale flexibility is observed on the investigated time frame. Indeed, the apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft, while apo XlnC is devoid of important structural flexibility on this time-scale. We also observe that ligand binding induces conformational exchange in XlnC and enhances millisecond motions of the fingers and thumb-loop regions in XlnB2. While these results emphasize the potential role of coordinated residue motions in ligand recognition, positioning, and/or stabilization, they also illustrate a potentially distinct evolutionary trajectory in conformational exchange to achieve similar catalytic function. In addition to offering new insights into the potential role of conformational exchange in GH11 xylanases, we hope this work will provide essential dynamic information to help improve protein engineering and design applications of this industrially-relevant enzyme family.

Structural and dynamic characterization of UbKEKS, a newly identified ubiquitin encoded in a pseudogene


Patrick Delattre1
1Université de Sherbrooke

The research team of Pr. Xavier Roucou developed a new database which considers the non-coding regions of the genome. This new database led to the discovery of multiple new proteins from overlooked regions, namely 5’ & 3’ UTR, alternate reading frames, and pseudogenes. This new database, coupled with the re-analysis of high through-put mass spectrometry, led to the discovery of UbKEKS, a ubiquitin isoform encoded in the pseudogene UBBP4. Until now, only one ubiquitin was known and expressed in every cellular type.

Ubiquitin is a 76 amino acid protein use as a post-translational modification by covalently linking the c-terminal glycine to the amine group of a lysine. This protein serves as a signal in a variety of cellular pathways, notably the TGFβ, MAP kinase and degradation pathway. The regulation of these pathways being affected in most cancers, their study is of the utmost importance.

Preliminary results suggest that this new ubiquitin act as a post-translational modification but doesn’t interact with the proteasome as opposed to its canonical counterpart.

In this study, we will investigate the structural and dynamic properties of this newly identified ubiquitin and eventually characterize its interaction with different ubiquitin binding domains.

Structural determinants of conformational exchange in GB1 DANCERs


Mayer Marc1, Adam M. Damry1, Roberto A. Chica1
1University of Ottawa

Proteins are intrinsically dynamic molecules that perform a large number of functions in living organisms. Their biological function is primarily dictated by their 3D structure and dynamic properties that permit exchange between multiple conformational states. Understanding the key elements that regulate dynamics is crucial in the field of protein design. The Chica Lab recently developed meta-MSD, a computational method allowing for the rational design of protein dynamics. Using this method, we designed DANCERs, streptococcal protein G domain β1 (GB1) mutants that undergo a specific mode of exchange between two target conformational states. Here we studied the structural determinants of dynamics in DANCER-3 by individually reverting its five mutations and characterizing the stability, structure and dynamics of the resulting five mutants. All mutants adopt the GB1 fold and are stable at room temperature. NMR chemical shift displacement and ZZ-exchange experiments revealed that two mutations, A34F and V39L are essential to the designed conformational exchange in DANCER-3. These results demonstrate that the designed mode of conformational exchange is not caused by a single mutation, but rather by several interacting mutations, highlighting the need for methods like meta-MSD when designing complex protein functions such as catalysis, allostery, and more.

Structure et liaison membranaire de la R9AP, une protéine impliquée dans la phototransduction visuelle


Sarah Bernier1,2,3, Marc-Antoine Millette1,2,3, Sarah Roy1,2,3, Line Cantin1,2,3, Christian Salesse1,2,3
1Université Laval 2CUO-recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement, CHU de Québec-Université Laval 3Regroupement stratégique PROTEO, Université Laval

CONTEXTE ET OBJECTIFS : La cascade de phototransduction visuelle implique plusieurs protéines servant à convertir le signal lumineux en un signal électrique. Des mutations au niveau de la séquence de la R9AP (RGS9-1-Anchor Protein) mènent à une maladie appelée bradyopsie qui se caractérise notamment par une photophobie et une difficulté à suivre les objets en mouvement. La R9AP est une protéine qui permet l’ancrage d’un complexe protéique à la membrane des disques des photorécepteurs via son segment C-terminal hydrophobe. Ce complexe joue un rôle dans l’inactivation de la phosphodiésterase et met fin à la phototransduction visuelle. Nous proposons de déterminer l’importance du segment C-terminal de la R9AP sur sa liaison membranaire ainsi que son implication dans la bradyopsie. Les objectifs de ce projet consistent donc à cloner, surexprimer et purifier la R9AP avec et sans segment C-terminal (∆TM) ainsi que le segment C-terminal seul, afin de caractériser leurs propriétés structurales et de liaison membranaire. 

 

MATÉRIEL ET MÉTHODES : La R9AP complète et la R9AP∆TM ont été surexprimées en fusion avec l’étiquette de purification GST (glutathione S-transférase) qui a ensuite été clivée. La structure des différentes formes de la R9AP a été évaluée par dichroïsme circulaire, spectroscopie infrarouge et spectroscopie de fluorescence. Les monocouches de Langmuir ont été utilisées pour caractériser la liaison membranaire de la R9AP. 

 

RÉSULTATS: Les mesures de solubilité montrent que la protéine complète est beaucoup moins soluble que la R9AP∆TM, ce qui suggère un rôle important du segment C-terminal hydrophobe dans la solubilité de cette protéine. La purification de la R9AP∆TM a été optimisée alors que celle de la R9AP complète est présentement en cours. Les mesures par dichroïsme circulaire et spectroscopie infrarouge ont montré que la R9AP∆TM ainsi que le peptide C-terminal sont majoritairement constitués d’hélices alpha. Les mesures de liaison membranaire ont montré que, parmi les phospholipides des photorécepteurs, la R9AP∆TM possède affinité particulière pour ceux qui sont chargés négativement, alors que le segment C-terminal seul possède une plus forte affinité pour la majorité de ces phospholipides. 

 

CONCLUSIONS : Ces travaux montrent un rôle important du segment C-terminal de la R9AP pour sa liaison membranaire et sa solubilité. Aussi, les analyses structurales des différentes formes de la R9AP sont en accord avec le modèle de prédiction de la structure de cette protéine. Ultimement, ces travaux permettront d’aider à comprendre le rôle du segment transmembranaire et des mutations de la R9AP dans la bradyopsie.

Surexpression et purification de la sous-unité gamma de la transducine, une protéine de la phototransduction visuelle


Alexandre Vaillancourt1,2,3, Line Cantin1,2,3,4, Christian Salesse1,2,3,4
1Département d'ophtalmologie, 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, Université Laval 4Université Laval

CONTEXTE ET OBJECTIFS : La phototransduction visuelle implique plusieurs protéines nécessaires à la conversion du signal lumineux en un signal électrique. La rhodopsine, une protéine transmembranaire présente dans les bâtonnets, est stimulée par la lumière, ce qui provoque l’isomérisation de son chromophore, le 11-cis rétinal, en tout-trans rétinal. Cette isomérisation mène à un changement conformationnel de la rhodopsine et une augmentation importante de son affinité pour la protéine G appelée transducine. La transducine est constituée de trois sous-unités (alpha, bêta et gamma). La liaison de la rhodopsine avec la transducine a pour effet de permettre l’échange du GDP de la transducine pour le GTP, ce qui mène à la dissociation de la sous-unité alpha de la transducine et à l’activation subséquente de la phosphodiestérase et l’hyperpolarisation des photorécepteurs. Les sous-unités alpha et gamma sont responsables de la liaison membranaire de la transducine. La contribution de la sous-unité gamma de la transducine (Tg) à ce processus est encore inconnue. Il est donc proposé de surexprimer et purifier la Tg afin de caractériser sa structure secondaire et sa liaison membranaire. MATÉRIEL ET MÉTHODES : La sous-unité gamma de la transducine a été surexprimée en fusion avec deux étiquettes de purification, la MBP (maltose-binding protein) (MBP-Tg) et une queue de dix histidines (PolyHis-Tg). Ces étiquettes ont ensuite été clivées grâce à l’ajout d’un site de reconnaissance de la protéase TEV (protéase du virus de la gravure du tabac), ce qui devrait en principe permettre la purification de la Tg. Il est prévu ensuite de procéder à des mesures de dichroïsme circulaire et de spectroscopie infrarouge ainsi que de liaison membranaire. RÉSULTATS : Des logiciels de modélisation ont permis de prévoir que la Tg devrait être très soluble grâce à ses hélices alpha contenant beaucoup de résidus chargés. Nous avons observé respectivement une faible et une très forte surexpression des protéines de fusion PolyHis-Tg et MBP-Tg. Le clivage de la MBP-Tg sur la colonne d’amylose est très efficace mais rend difficile l’obtention de la Tg pure. CONCLUSIONS : Il sera nécessaire d’optimiser la purification de la Tg. Par la suite, nos travaux sur la structure secondaire et la liaison membranaire de la Tg devraient permettre d’améliorer notre compréhension du rôle de cette sous-unité dans la phototransduction visuelle.

Synthèse de glycopeptides comme outils immunogéniques dans la recherche antifongique et antitumorale


Tremblay Thomas1, Vincent Denavit2, Denis Giguere3
1Université Laval 2Université Laval 3Université Laval

Plusieurs polysaccharides microbiens et certains glycopeptides sont des antigènes pertinents dans le domaine médical. La préparation de ces antigènes glycosidiques synthétiques peut aider au développement d’outils dans le diagnostic de maladies ou même dans la formation de vaccins.

 

Les infections fongiques sont l’une des principales causes de mortalité chez les patients présentant une déficience immunitaire sous-jacente et chez les personnes souffrant de maladies pulmonaires chroniques. La majorité des traitements antifongiques se sont révélés inefficaces au fil du temps, l’enjeu est encore plus important. Les champignons sont caractérisés par une paroi cellulaire riche en polysaccharides qui joue un rôle central dans les interactions hôte-champignon. Presque toutes les parois cellulaires fongiques contiennent de la chitine, un polymère de N-acétylglucosamine (GlcNAcβ1-4)n. La chitine est partiellement désacétylée à des degrés divers par des désacétylases fongiques pour produire du chitosane (GlcNβ1-4)n. Le premier objectif est de synthétiser des antigènes de chitosane et de chitine conjugués avec une chaîne latérale de polyglycine. Cette fonction d'ancrage sera utilisée pour être liée chimiquement à une protéine immunogène: la protéine d'enveloppe du virus de la mosaïque du papayer (PapMV). Les oligosaccharides conjugués à la protéine PapMV pourraient donc être utilisés comme de nouvelles nanoparticules vaccinales pouvant déclencher une forte réponse immunitaire humorale ou être utilisés comme outils de diagnostic.

 

Dans le même ordre d'idées, nous avons synthétisé des antigènes glycosidiques associés aux tumeurs: l'antigène Tn (N-acétylgalactosamine) et l’antigène TF qui sont attachés à une fonction d'ancrage qui permettra la conjugaison avec la surface de nanoparticules d'argent ou sur une plateforme SPR dont la surface est un mince film d’or. La détection de l'interaction spécifique entre les anticorps ciblés dans différents fluides corporels et les antigènes sera réalisée en mesurant le déplacement spectral du pic plasmonique de nanoparticules fonctionnalisées par les antigènes glycosidiques. Notre objectif à long terme est de développer une plate-forme de détection analytique robuste pour détecter les biomarqueurs du cancer.

 

 

Synthesis of poly-fluorinated glucopyranose derivatives from levoglucosan


Megan Bouchard1, Jacob St-Gelais1, Denis Giguère1
1Département de Chimie, Université Laval, PROTEO, Québec, Qc, Canada G1V 0A6

Medicinal chemistry is increasingly exploiting fluorine atoms. Substitution of a hydroxyl function by a fluorine atom can provide beneficial effects such as an increase of drugs metabolic stability or enable favorable interactions with a target protein. 

Carbohydrates bearing a fluorine atom in place of a hydroxyl group have found applications in biochemical investigations as mechanistic probes or to study lectin−carbohydrate interactions. Thus, the rapid synthesis of fluorinated carbohydrates could lead to the discovery of glycomimetic with novel biological activities.

As part of our ongoing program related to the preparation of a vast pattern of fluorinated carbohydrates, we investigated the rapid synthesis of polyfluorinated glucopyranoses starting from levoglucosan (1,6-anhydro-β-ᴅ-glucopyranose). This inexpensive starting material allowed us to use the 1,6-anhydro core to avoid the preliminary protection of the O-6 and anomeric positions and could easily afford scalable fluorinated carbohydrates via simple experimental protocols. Thus, the synthesis of heavily fluorinated glucose was achieved. This strategy also allowed us to prepare various orthogonally protected fluorinated sugars, useful intermediates for the preparation of complex oligosaccharide derivatives.

 

Systematic perturbation of yeast essential genes using base editing


Philippe Després1,2, Alexandre Dubé1,2, Nozomu Yachie3, Christian Landry1,2
1IBIS, Université Laval 2Université Laval 3RCAST, the University of Tokyo 4ULAVAL

Cell function relies on many cellular networks that show remarkable robustness to perturbations. In yeast a significant fraction of genes are known to be essential to cell survival. Many of those genes are conserved in humans, and the function of many of these genes is still unknown. Furthermore, their essentialness makes their robustness to different mutations difficult to evaluate using current techniques. In recent years, genome editing using the CRISPR-Cas9 toolkit and the development of base editors has led to many new techniques for pinpoint genetic perturbation in many cellular contexts. We recently developed double selection, a method for yeast genome editing that can allow mutagenesis of target nucleotides at a 100% rate using the Target-AID base editor. To show the power of the new method, we developed a library of ~40 000 guides to perform systematic perturbation of the yeast essential gene set by base editing. By using barcode sequencing, we can track simultaneously the effect on fitness of these mutations in different environmental contexts. This new tool will allow us to precisely measure the robustness of these genes to mutations, as well as provide new insights on gene function.

The bacterial protein Curli: expression and characterization for the biomedical application of functional amyloid assemblies


Dominic Arpin1, Ximena Zottig1, Geneviève Bertheau-Mailhot1, Steve Bourgault1, Denis Archambault1
1Université du Québec à Montréal 2Université du Québec à Montréal

The deposition and accumulation of amyloid fibrils are the hallmark of numerous human diseases. In recent years, a growing number of amyloid assemblies have been found to play essential physiological roles in almost all living organisms, from bacteria to mammals, and have been termed ‘’functional’’ amyloids. The study of functional amyloids is promising not only to provide insights into the molecular mechanisms of protein self-assembly, but also for a wide range of potential biomedical and nanomaterial applications. A functional amyloid in E.coli is curli-specific gene A (CsgA), a key protein of the extracellular matrix that is essential for the formation of biofilm. CsgA is a 14kDa protein secreted in the extracellular medium as a monomer. CsgA then polymerizes in a nucleation-dependent manner using the protein CsgB as an initial scaffold, a lipid-anchored protein on the outer membrane, to form long fibers with crossed β-sheet structure. Previous studies have shown that CsgA alone can polymerize into fibers in vitro. In this study, we aim to harness the unique auto-assembly properties of CsgA to form amyloid fibrils that would serve as a proteic scaffold, or carrier, for proteins with potential applications in order to develop functional nanomaterials. Precisely, a recombinant protein containing CsgA was produced in E.coli. Optimal conditions for CsgA expression are 4 hours at 25°C with 1mM IPTG. Purification steps are done in 6M urea to prevent amyloid assembly. The highest level of purity has been attained with 5mM imidazole when binding proteins to Ni-NTA resin and subsequent washes.

The crystal structure of the Cdc5-Dbf4 complex provides insight into Polo-box domain substrate recognition.


Ahmad Almawi1, Stephen Boulton1, Giuseppe Melacini1, Alba Guarné2
1McMaster University 2McMaster University & McGill University

Polo-like kinases (Plks) are a conserved family of kinases that drive cell cycle events, primarily during mitosis. Beyond their kinase domain, Plks contain a Polo-box domain, which has a conserved phosphoSerine/Threonine(pS/T)-binding pocket involved in substrate targeting (Elia et al., (2003) Cell). Polo-box domains can also mediate phosphorylation-independent interactions (Archambault et al., (2008) Genes Dev; Rahal and Amon (2008) Cell Cycle; Chen and Weinreich (2010) JBC). One of these is with the regulatory subunit of the Dbf4-dependent kinase (DDK) complex. DDK is required for initiating eukaryotic DNA replication, as well as regulating mitosis and meiosis I (Jackson et al., (1993) Mol Cell Biol; Dowell et al., (1994) Science; Matos et al., (2008) Cell; Miller et al., (2009) PLoS Genet). Its role outside replication requires the interaction between Dbf4 and Cdc5 (the only Plk in budding yeast). Intriguingly, DDK can both inhibit and promote Cdc5 function and it remains unclear whether Dbf4 prevents binding of Cdc5 to its substrates or limits Cdc5 binding to a subset of targets (Miller et al., (2009) PLoS Genet; Chen and Weinreich, (2010) JBC; Princz et al., (2016) EMBO J; Argunhan et al., (2017) EMBO J). To understand this further, we solved the crystal structures of the Polo-box domain of Cdc5 alone and bound to peptides derived from Dbf4 and a canonical phosphorylated-substrate (the spindle-pole body protein Spc72). We found that Cdc5 recognizes phosphorylated-substrates similarly to other Plks, and that the Dbf4-binding surface is located opposite to the pS/T-binding pocket. We confirmed that mutating the Dbf4-binding surface abrogates the Cdc5-Dbf4 interaction in vitro, and we are currently assessing the phenotype of the Cdc5 variants to delineate how Dbf4 affects Cdc5 functions in vivo. Using isothermal calorimetry and NMR, we showed that Cdc5 interacts with Dbf4 and Spc72 simultaneously and non-competitively. However, Dbf4-binding triggers a series of minor conformational changes within the domain that propagate toward the pS/T-binding pocket. We propose that these conformational changes may modulate Cdc5 binding to certain phosphorylated substrates.

The first crystal structure of a bacterial acetylcholinesterase


Van Dung Pham1, Deqiang Yao2, Roger Levesque1,3, Steve Charette1, Rong Shi1
1Université Laval 2Shanghai Synchrotron Radiation Facility 3IBIS

The PA4921 gene of Pseudomonas aeruginosa PAO1codes for a cholinesterase (ChoE), which belongs to the SGNH hydrolase family that can catalyze the hydrolysis of a broad range of substrates. The ChoE enzyme plays a critical role in corneal infection and it acts coordinately with two other enzymes: acid phosphatase and hemolytic phospholipase C as a system under a tight regulation mode to provide the metabolic needs of the pathogen.Numerous structures of eukaryotic acetylcholinesterase have been reported because of its important roles in mammals. We report here the first structure of a bacterial acetylcholinesterase at 2.1A. 


 

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


Louis-Philippe Morency1, Rafael Najmanovich1
1Université de Montréal

Molecular docking aims to predict the experimentally observed binding mode between a target, i.e. a polymer of amino or nucleic acids, and a ligand— e.g. small molecules, peptides or nucleic acids. Historically, our understanding of these association events has been constrained by quite a static view of molecular structures shown through crystallography. Hence, actual molecular docking methods are optimized to evaluate the molecular interactions of a single conformation and they are trained to estimate the enthalpic fraction (∆H) of the binding free energy (∆G). Consequently, most current molecular docking methods fail to model the entropic contributions (-T∆S), especially those of conformational nature, who are fundamental in molecular recognition events. Our research group develops FlexAID1, an accessible and competitive ligand and biomolecule molecular docking software whose focus is on molecular flexibility. Here we present the impact of FlexAID’s new feature, the integration of conformational entropy, on its precision in binding mode prediction using three increasingly complex popular molecular docking benchmarking datasets.

 

FlexAID’s new scoring function allows its genetic algorithm to select less favourable, but frequently observed binding modes, with a probability following a Boltzmann distribution. This implementation allows it to consider conformational entropy of the complexes during molecular docking simulations. The core of this implementation within FlexAID resides in a density-based clustering algorithm charged to group similar poses together, thus allowing its scoring function to estimate the conformational entropy by replacing the static binding mode usually predicted in molecular docking by a dynamic collection of similar poses evaluated altogether (Figure 1).

 

 

 

 

 

 

 

 

 

 

Figure 1. Visual representation of a dynamic binding mode output by FlexAID (PDB : 3b7e Influenza virus neuraminidase in complex with Zanamivir predicted at 1.60Å RMSD from the crystal pose).

 

We present the impact of FlexAID’s new feature on its accuracy in binding mode prediction using three increasingly complex scenarios: the Astex Diverse Set3, the Astex Non Native Set4and the HAP25dataset. We show that FlexAID outperforms other open-source docking methods when molecular flexibility is critical. Furthermore, FlexAID now outputs thermodynamic parameters, i.e. ∆G, ∆H and -T∆S, as well as multiple conformations that are computed for each predicted binding modes. We believe that its higher accuracy in complex scenarios, the addition of novel features, e.g. the conformational entropy and the added output of thermodynamic parameters, its accessibility and its easy-to-use graphical user interface, the NRGsuite2, put FlexAID in an interesting position to tackle biologically challenging and pharmacologically relevant situations currently ignored by other molecular docking methods.

 

References 

1J. Chem. Inf. Model. 55: 1323–1336 (2015); 2Bioinformatics btv458 (2015); 3J. Med. Chem. 50, 726–741 (2007); 4J. Chem. Inf. Model. 48, 2214–2225 (2008); 5Bioinformatics 28, i423–i430 (2012).

The periplasmic reductase DsbG has a chaperone activity in the elyC mutant of Escherichia coli


Imène Kouidmi1, Laura Alvarez2, Jean François Collet3, Felipe Cava2, Catherine Paradis-Bleau1
1Department of Microbiology, Infectiology and Immunology, Université de Montreal, Montreal, Quebec, Canada 2Laboratory for Molecular Infection Medecine Sweden, Department of Molecular Biology, Umeå Center for Microbial Research, Umeå, Sweden 3De Duve Institute, Université catholique de Louvain, Av. Hippocrate 75, Brussels, Belgium

The Escherichia coli factor ElyC is important for peptidoglycan biosynthesis at low temperatures. The ΔelyC mutant presents a PG biosynthesis arrest followed by bacterial cell lysis at the end of the logarithmic phase of growth at 21°C. In this study, we showed that the overexpression of the periplasmic reductase DsbG corrected the peptidoglycan defect and suppressed the ΔelyC mutant cell lysis phenotype. DsbG is known to reduce oxidized single cysteine residues that form in periplasmic L,D-transpeptidases under oxidative conditions. Interestingly, we discovered that DsbG acts independently of the L,D-transpeptidases and of its reductase activity to suppress the ΔelyC mutant cell lysis. We hypothesized that DsbG acts by a novel activity, which is likely a chaperone activity. We identified the periplasmic chaperone Spy as another multi-copy suppressor of the ΔelyC cell lysis, which supports our model that DsbG acts by a chaperone activity. Furthermore, we showed DsbG, like Spy, reduced the amount of protein aggregates in the ΔelyC mutant. In fact, the amount of protein aggregates was found to be two folds higher in the mutant as compared to the wild-type. Altogether, our results reveal for the first that that the periplasmic protein DsbG possesses a chaperone activity in vivo.

THE SYNTHESIS OF KERATAN SULFATE GLYCOSAMINOGLYCANS BY A GLYCOSYNTHASE APPROACH


Xiaohua Zhang1, Gautier Bailleul1, Peter Pawelek2, David Kwan1
1Concordia University 2Concordia University

Glycosaminoglycans serve an important role in cell communication. Keratan sulfate is an example of a glycosaminoglycan that exhibits varied biological function in important 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 mutated, but the mutant Keratanase II variants did not show glycosynthase activity. Preliminary crystallization trials were attempted for wild-type enzyme and conditions were optimize to obtain single crystals suitable for collecting diffraction data. Once the enzyme structural information is available, several mutant variants will be generated with the aim of identifying glycosynthase activity. We will also perform crystallization trials on mutant enzymes—suitable crystals will be soaked with synthetic oxazoline intermediate to obtain structural data on the complex. We anticipate that one of the mutant enzymes will function as a glycosynthase enzyme capable of synthesizing homogeneous keratan sulfate oligo- and polysaccharides of defined length and sulfation pattern so that 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.

Unraveling the controversial role of the pseudokinase domain of BUBR1 in mitosis


Luciano Gama Braga1, Philippe Thebault1, Michelle Mathieu1, SABINE ELOWE2
1Université Laval 2Université Laval

Aneuploidy is defined as an abnormal complement of chromosomes for a particular organism and is a hallmark of most solid tumours, where it contributes to and drives their development. Whole chromosome aneuploidy is caused by abnormal chromosome segregation during mitosis and cell division. The cellular control mechanism that monitors the fidelity of chromosome segregation, and therefore a major protective mechanism against aneuploidy, is the Spindle Assembly Checkpoint (SAC), a signalling pathway responsible for delaying sister chromatid segregation until all chromosomes are correctly attached in a bioriented manner. The SAC relies on essential proteins for its activity, including the pseudokinase BUBR1. This protein is overexpressed in oral cancer, lung adenocarcinoma, and gastric cancer, among others, and its overexpression correlates with poor patient prognosis. Localization of BUBR1 and other SAC proteins to kinetochores, macromolecular structures that assemble onto centromeric DNA during mitosis, activates the SAC, which results in chromosome segregation delay. When all the chromosomes are properly attached, the SAC signal is silenced, resulting in the segregation of sister chromatids and anaphase onset.

BUBR1 is a conserved pseudokinase that has a crucial role in regulating the SAC and chromosome attachments through its recruitment of other checkpoint proteins, such as the protein phosphatase PP2A-B56, which interacts with the phosphorylated BUBR1 Kinase-Attachment-Regulatory-Domain (KARD). PP2A-B56 recruitment to kinetochores is proposed to initiate a PP2A and PP1 dephosphorylation cascade, culminating in SAC inactivation. This model, however, does not account for regulation of BUBR1 KARD phosphorylation. Our analysis shows a possible evolutionary correlation between species that retained the pseudokinase domain and the KARD. Furthermore, we demonstrated that the BUBR1 pseudokinase domain regulates KARD domain phosphorylation, thus potentially modulating SAC silencing and chromosome congression. We show that truncation or mutation of the BUBR1 pseudokinase domain results in a hypophosphorylated protein less likely to associate with PP2A-B56. Cells expressing BUBR1 pseudokinase domain mutants display defects in SAC silencing and chromosome alignment. We propose that this pseudokinase domain is functionally relevant, explaining why it was retained throughout evolution, despite loss of kinase catalytic function in higher eukaryotes.

In conclusion, this study aims to elucidate major BUBR1 activities that remain completely unexplored. Our research will directly contribute to a better understanding of the contribution of the SAC to whole chromosome aneuploidy and to novel approaches for the development of rational therapeutic strategies to treat cancer.

Valorisation des huiles usagées à moteur


manel Ghribi1,3,4, Fatma Meddeb2,3,4, Marc Beauregard2,3,4
1UQTR 2UQTR 3CRML 4PROTÉO

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. Il est alors primordial de continuer la recherche sur des méthodes de traitement ou de valorisation et pourquoi pas, 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.

 

La microbiologie industrielle représente aujourd’hui un secteur phare des biotechnologies, particulièrement dans l’innovation, la bio-valorisation et la bio-décontamination des sols. Il serait alors intéressant d’utiliser ces technologies vertes en impliquant des bactéries productrices d’enzymes pertinentes. Les échantillons environnementaux, surtout contaminés par les huiles de moteurs étant 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 visait en premier lieu 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). Le criblage des microorganismes a été effectué dans des milieux de culture contenant 10% d’huile de moteur usagée. Plusieurs dizaines de souches bactériennes ont été sélectionnées, purifiés et caractérisées sur la base de leur capacité à croitre et à se développer sur les huiles usées de moteur. L’identification génétique avec le séquençage de l’ADNr16S ainsi que la caractérisation biochimique de ces microorganismes ont été effectués et des tests de production de bioplastique de type PHA (Polyhydroxyalkanoate) sur milieu ont été réalisé et complété par une étude bio-informatique ainsi qu’une détection par PCR des gènes responsables de la production du PHA De plus une méthode quantitative de PHA a été mise au point pour sélectionner les meilleurs producteurs bactériens de ce bioplastique. Outre la capacité de ces bactéries à produire du bioplastique et/ou à dégrader des composés toxiques, nous avons identifié un nouveau consortium bactérien capable de produire un élastomère de type EPDM à partir de 1 % des huiles usées de moteur (identifie par FTIR).

Vers le développement d’une nouvelle génération d’inhibiteurs de l’oncoprotéine c-Myc


Jean-Michel Moreau1, Danny Létourneau1, Martin Montagne1, Pierre Lavigne1
1Université de Sherbrooke

La protéine Miz-1 est un facteur de transcription de type BTB/POZ et possède 13 doigts de zinc (ZF) de type C2H2. Miz-1 active la transcription de gènes impliqués dans plusieurs aspects de la biologie de la cellule tel que des gènes cytostatiques (e.g. p15, p21, p27 et p57, des inhibiteurs de CDK). . Une région de Miz-1 attire notre attention : Mid2 (c-Myc interacting domain). c-Myc est un facteur de transcription de type b-HLH-LZ dérégulé dans la majorité des cancers humains (Peukert et al. 1997). Une partie de MID2 aurait une propensité à former une hélice alpha et cette région en particulier serait impliquée dans la liaison de Miz-1 avec c-Myc (Peukert et al. 1997, Bédard et al. 2016).

Les objectifs de ce projet sont de caractériser l’interaction c-Myc – Miz-1 via Mid2 à l’aide de techniques structurales telles que le dichroïsme circulaire et la RMN afin d’élucider une structure 3D de c-Myc monomérique. Cela nous permettra de produire un pharmacomimétique inhibiteur de c-Myc et qui pourrait avoir d’éventuelles débouchées dans le traitement du cancer.