Des peptoïdes perméants comme transporteurs de molécules imperméables à travers la membrane cellulaire.
Andréanne Laniel
15N-Heteronuclear Single Quantum Coherence (HSQC) titration of Galectin 7: comparison between two ligands.
Carolina Perusquía Hernández
A Controlled Method to Immobilize Antibodies for the Surface Modification of Vascular Stent
Omar S. Bashth
A phage protein impedes bacterial resistance to phage infection
Marie-Laurence Lemay
Analyse des interactions médiées par les domaines SRC homology 3 (SH3) de SLA1 en les substituant par CRISPR-Cas9
Émilie Bourgault
Analysis of the Impact of Conformational Entropy on the Accuracy of the Molecular Docking Software FlexAID in Binding Mode Prediction
Louis-Philippe Morency
Brighter red fluorescent proteins display reduced structural dynamics
Adam M. Damry
Brighter Red Fluorescent Proteins through Improved Chromophore Packing
Sandrine Legault
Caractérisation de la liaison membranaire de l’arrestine
Marc-Antoine Millette
Caractérisation des mécanismes moléculaires de la liaison des doigts de zinc 10 à 12 de Miz-1 à l’ADN par RMN en solution
Olivier Boisvert
Caractérisation fonctionnelle et structurale des transporteurs de nickel chez Helicobacter pylori
Mariem Chalbi
Cell-based fluorescent assay for screening ligand-gated ion channel function
Mykhaylo Slobodyanyuk
CHARACTERIZATION OF THE TDP-L-VANCOSAMINE BIOSYNTHETIC PATHWAY FROM TDP-4-KETO-6-DEOXY-D-GLUCOSE
Lan Huong Thi Nguyen
Clonage, surexpression et purification de la RDH8 en fusion avec plusieurs étiquettes dans le but de mesurer son activité enzymatique
Charlotte Lemay-Lefebvre
Computational modeling of the death complex between glyceraldehyde-3-phosphate dehydrogenase and seven-in-absentia homolog 1 (GAPDH-Siah1)
Ritu Arora
Computationally Designed Grafting of a Ru Mediator to Improve Redox Potential of a Laccase
Ferran Sancho
Conformational landscape of homologous enzymes with distinct biological functions
Chitra Narayanan
Conformational space derived from normal mode analysis: a dynamical metric for scoring 3D predictions of RNA, proteins and their complexes
Olivier Mailhot
DESIGNER BIOSENSORS FOR ENGINEERED METABOLIC PATHWAY OPTIMIZATION
Mohamed Nasr
Developing a family specific molecular docking energy scoring function to accelerate drug discovery
I.B. Matala
Development of a high-throughput assay to detect fatty acid decarboxylase activity
Jama Hagi-Yusuf
Development of a versatile vaccination platform based on papaya mosaic virus (PapMV) nanoparticles
Denis Leclerc
DEVELOPMENT OF CD125-TARGETED ENGINEERED ANTIBODY FRAGMENTS FOR USE IN MUSCELE-INVASIVE BLADDER CANCER THERAPY
Olga Bednova
Development of sulfahydantoin compound as potential antibiotics and β-lactamase inhibitors.
Pierre-Alexandre Paquet-Côté
Devrait-on utiliser la souris comme modèle animal pour caractériser la lécithine rétinol acyltransférase humaine?
Sarah Roy
Direct phosphorylation of SH3 domains by tyrosine kinase receptors disassembles ligand-induced signaling networks
Ugo Dionne
Effect of spinning rate on the molecular structure and amino acid dynamics in native and supercontracted spider dragline silk: a solid-state NMR and Raman spectroscopic study
Jane Gagné
Efficient Site-Specific Antibody-Drug Conjugation by Engineering of Different Recognition Motifs for Microbial Transglutaminase
Lukas Deweid
Evolution des complexes protéiques après hybridation entre espèces
Caroline Berger
Evolutionary divergence of conformational exchange phenomena in a host defense enzyme family
David N. Bernard
Ex situ and in cell 13C solid-state NMR characterization of starch and glycoprotein-rich cell-wall in microalgae
Alexandre POULHAZAN
Exposing Small-molecule Nano-entities By An NMR Relaxation Assay
Yann Ayotte
Expression and purification of CsgA, a functional amyloid composing bacterial biofilm
Dominic Arpin
Function and engineering of enzymes involved in the glycosylation of natural products
Fathima Mohideen
Gesicles: a promising nucleic acid delivery tool.
Mathias Mangion
GFP-based Biosensor to Detect Transiently Expressed Proteins
Matthew G. Eason
HOMODIMER INTERFACE MUTATIONS OF HUMAN GALECTIN-7 ALTER ITS BIOLOGICAL ACTIVITY
Ngoc Thu Hang PHAM
Identification de biomarqueurs en relation avec PACE4 pour le cancer de la prostate
Amanda Toupin
Identification des protéines TAM dépendantes chez Pseudomonas aeruginosa
Jihen Ati
Identification of promising angiogenin allosteric modulators by screening of a chemical compound library
Myriam Létourneau
Immune Response of Small Drug-like Molecules: Influence of Self-aggregation
FATMA SHAHOUT
Implication des chaines de glycosaminoglycanes membranaires dans la toxicité et l’auto-assemblage d’un peptide amyloïdogénique
Mathilde Fortier
Importance of the β5−β6 Loop for the Structure, Catalytic Effi-ciency, and Stability of the Carbapenem-Hydrolyzing Class D beta-lactamase Subfamily OXA-143
Denize Favaro
Increasing the genome-targeting scope of base editing using Streptococcus thermophilus CRISPR1-Cas9
Minja Velimirovic
Influence of nucleotide modifications at the C2′ position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA
William Copp
Inhibition and Activation of Kinases by ADP, Revealed by Isothermal Titration Calorimetry (ITC)
yun wang
Investigation of the functional specificity of adaptor proteins NCK1 & NCK2
François Chartier
Investigation phytochimique de lichens nordiques Cladonia Stellaris et Mitis
meggan beaudoin
Is hybridization an adaptive force in response to DNA damage?
Carla Bautista Rodríguez
Kinetically Programmed, One-Pot DNA Reactions for Molecular Detection Directly in Whole Blood
Guichi Zhu
La liaison membranaire de la protéine S100A10 et du peptide d’AHNAK intervenant dans la réparation membranaire
Xiaolin YAN
Laccase identification from the native ligninolytic basidiomycete Dictyopanus pusillus
Andres Rueda
Le complexe du pore nucléaire de la levure comme système modèle pour l'étude des déterminants de la trajectoire évolutive suivie par les gènes dupliqués
Simon Aubé
Le complexe TAM et son rôle dans la biogenèse des protéines membranaires chez les bactéries Gram-négatif
Jihen Ati
Les récepteurs Eph régulent la morphogénèse épithéliale
Noémie Lavoie
Lobaric acid and pseudodepsidones from the lichen Stereocaulon paschale inhibits NF-κB signaling pathway
Claudia Carpentier
Mapping Eph receptor signaling networks via proximity-dependent biotinylation
Sara Banerjee
Modulation de la multicellularité bactérienne via différents polysaccharides sécrétés.
fares saidi
Molecular fingerprints determining the toxicity-functionality equilibrium of protein amyloid assemblies
Phuong Trang Nguyen
Molecular insights into the bacterial acetylcholinesterase ChoE from Pseudomonas aeruginosa
VAN DUNG PHAM
Molecular Insights of Val430Ile Mutation Impact on Influenza Neuraminidase Resistance to Zanamivir
Gabriel Bégin
Monitoring Protein Function using Fluorescent Nanoantennas
Scott Harroun
New developments of flow biocatalysis systems: Synthesis of indigo and raspberry ketone using cytochrome P450 enzymes.
Ali Fendri
Optimization of a production and purification process for VSVg pseudotyped gesicles
Juliette Champeil
Optimization of matriptase-2 inhibitors
Méryl-Farelle Oye mintsa mi-mba
Origin of Dynamics in a Small Globular Protein
Mayer Marc
Peptide assemblies as self-adjuvanted platform for nanovaccine design
Ximena Zottig
Peptide-based Drug Discovery: Artificial Selection of Genetically Encoded TB Drugs
Trisha Ghosh
Perturbation de la structure tridimensionnelle du mutant S175R de la lécithine rétinol acyltransférase tronquée par le SDS : une étude par résonance magnétique nucléaire
Marie-Eve Gauthier
Phase separation and conformational conversion processes drive the self-assembly of non-pathological amyloid in the presence of linear polyanions
Mathew Sebastiao
Phenolic acid decarboxylase structures lead to new insights in its decarboxylation mechanism
Marie-Ève Picard
Positive epistasis towards cefotaxime resistance is maintained in highly dynamic, engineered ß-lactamases
Adem H.-Parisien
Programming complex regulation mechanisms through simple molecular assembly
Dominic Lauzon
Protein engineering and immobilization of LipA from Pseudomonas aeruginosa to broaden its industrial applications
Ingrid Yamile Pulido
Region-focused protein engineering in Cal-A lipase reveals triglyceride-binding hotspot
Lorea Alejaldre
Rôle des protéines S100A16 et Annexine A4 dans le maintien de l’intégrité membranaire
Francis Noël
Sequential coselections for CRISPR-driven genome editing in human cells
Sébastien Levesque
Structural changes along the evolutionary trajectory of a de novo designed enzyme
Rakotoharisoa R
Structure et mode d’action des transporteurs membranaires du nickel chez Helicobacter pylori
Imène Kouidmi
Structure-self-assembly relationships study of the islet amyloid polypeptide
Elizabeth Godin
Surexpression, purification, caractérisation et liaison membranaire 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
Thomas Tremblay
Synthèse de nouveaux glucoses trifluorés et analyse de leur lipophilie
Megan Bouchard
Synthesis and Modify of Sialyl Lewis X Enable It Attach to Cell Surface ex vivo
Haoyu Wu
Synthesis and NMR-Screening of a Fluorinated Library to be Used in FBDD via 19-F NMR
David Bendahan
Systematic perturbation of the yeast essential proteome using base editing
Philippe C Després
Systematic perturbation of yeast essential genes using base editing
Philippe C Després
Teaching an old dog a new trick: Oxime resin as versatile solid-support towards various cyclic peptide scaffolds
Christopher Bérubé
The Recruitment of Endothelial Progenitor Cells on Bio-memetic Functionalized Surfaces
Mohamed Elkhodiry
The role of structural pleiotropy and regulatory evolution in the retention of heteromers of paralogs
Angel Fernando Cisneros Caballero
The role of the RhoGEF ARHGEF17 (TEM4) in Mps1 function during mitosis
DIOGJENA PRIFTI
Towards the structural characterization of the abortive infection (Abi) system protein AbiV
Xiaojun Zhu
Usefulness of Recoverin to express and purify visual proteins
Line Cantin
Utilisation d’un support solide pour le développement d’une méthodologie de synthèse de glycopeptides
Gabrielle Robert-Scott
Vibrational Circular Dichroism Reveals Supramolecular Chirality Inversion of α‑Synuclein Peptide Assemblies upon Interactions with Anionic Membranes
Benjamin Martial
Visualizing the heme loading of a protein in live cells using green fluorescent protein
Samaneh Dastpeyman

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


Andréanne Laniel1, Claire McCartney2, Étienne Marouseau1, Christine Lavoie1, Éric Marsault1
1Université de Sherbrooke 2Université Bishop's

Les peptides perméants ou Cell penetrating peptide (CPP) sont des véhicules permettant le transport de médicaments en raison de leur capacité à induire une absorption cellulaire efficace de molécules qui autrement ne pénètrent pas dans les cellules. Parmi la classe de peptides perméants cationique, les transporteurs riches en guanidines (TRG) se sont révélé être des agents d’administration efficaces compte tenu de leur stabilité plasmatique accrue. Nous avons récemment synthétisé de nouveaux TRGs avec un squelette guanidine à conformation restreinte, afin d’échantillonner comment cette variation structurelle améliore la pénétration et sélectivité cellulaire. Dans cette étude, nous avons comparé l'efficacité et la spécificité de pénétration cellulaire, les voies d’endocytoses et l’évasion endosomale de ces TGRs à la nona-arginine (R9) (un CPP bien caractérisé), dans deux lignées de cellules cancéreuses HeLa et MCF7. La cytométrie de flux a été utilisée pour tester la pénétration cellulaire des TRGs et de R9 en mesurant la fluorescence moyenne de leur cargo, la fluorescéine. Les résultats ont montré que les TRGs commençaient à entrer dans les cellules HeLa à partir de 6 guanidines et avaient un taux de pénétration optimal de 80% de la référence R9 soit 8 guanidines (nommé PGua4). Ce niveau d'absorption est plus bas dans les cellules MCF7 où PGua4 n'a qu'un taux de pénétration cellulaire de 30%.

 

La voie d’endocytose spécifique à PGua4 a ensuite été caractérisée par microscopie confocale sur cellules vivantes utilisant divers inhibiteurs d'internalisation et des marqueurs sélectionnés pour les compartiments intracellulaires, soit les protéines Rab. Semblable à R9, PGua4 est partiellement internalisé par une voie énergie dépendante et se retrouve également dans les endosomes précoces (Rab5), tardifs (Rab7) et de recyclage (Rab11) ainsi que dans le cytoplasme et le noyau. Les données préliminaires suggèrent que PGua4, contrairement à R9, s'évade des endosomes pour se retrouver dans le cytoplasme, ce qui permettrait aux cargos d'atteindre leurs cibles thérapeutiques. Nous quantifions actuellement le niveau d’évasion endosomale de ces composés. En conclusion, nous avons identifié et caractérisé un nouveau CPP potentiellement efficace pour administrer des composés thérapeutiques à leur cible intracellulaire.

 

15N-Heteronuclear Single Quantum Coherence (HSQC) titration of Galectin 7: comparison between two ligands.


Carolina Perusquía Hernández1, Myriam Létourneau1, David Bernard1, Nicolas Doucet1,2
1INRS - University of Quebec 2PROTEO

Galectins can act as cell regulators by binding glycan motifs on membrane receptors. These proteins are involved in eukaryotic cell adhesion and signaling networks through their ß-galactosyl-binding properties. A plethora of side effects have been described for galectin’s increased expression, which was observed in neoplasms, such as promoting angiogenesis and inducing apoptosis of T cells, both of which are involved in tumor progression.

 

Several studies have led to the development of inhibitors, mainly compounds precluding binding involving the glycan binding site (GBS). Even though amino acid sequence identity is rather low among protein family members, galectins share the same three-dimensional fold. Thus, GBS inhibitors generally lack specificity, although some have shown high affinity. A better understanding of ligand binding dynamics will help improve design of ligands with higher specificity and affinity. As a result, molecular dynamics simulations and Nuclear Magnetic Resonance (NMR) experiments aiming at deciphering dynamic networks in three different prototype galectins, i.e.galectin-7, -10 and -13, will be carried out in the presence and absence of ligands. A correlation between these networks and biological activity, evaluated using T-cell apoptosis and hemagglutination assays, will then be attempted in order to point out possible intramolecular communications governing prototype galectin ß-galactosyl binding and activities.

 

A first set of NMR experiments were carried out using galectin 7. In the present work, results of 15N-HSQC titration of galectin 7 with lactose are compared with those obtained with N-acetyl lactosamine.

A Controlled Method to Immobilize Antibodies for the Surface Modification of Vascular Stent


Omar S. Bashth1, Mohamed A. Elkhodiry1, Gaétan Laroche2, Corinne A. Hoesli1
11. Department of Chemical Engineering, McGill University, Montréal, Québec, Canada. 22. Centre de Recherche du CHU de Québec, Département de Génie des Mines, des Matériaux et de la Métallurgie, Université Laval, QC

To treat patients with coronary artery disease, stent implantation is considered the most common medical intervention. This approach might cause restenosis – a renarrowing of blood vessels – leading to several complications. Naturally, in events of inflammation and endothelial dysfunction, circulating endothelial progenitor cells [EPCs] can differentiate into endothelial cells [ECs] forming a new healthy layer of endothelium. The long-term goal of this project is to design a biomimetic stent that would capture EPCs and facilitate reendothelialization on the surface. To capture the circulating EPCs, several antibodies recognizing endothelial cell surface markers such as anti-CD144 (anti-VE-cadherin) were immobilized onto commercially‑available aminated polystyrene surfaces. The surface grafting method consisted in (1) activation using an amine-to-thiol linking arm; (2) grafting of antibody-binding peptides via a thiol group; (3) incubation with antibodies followed by detection using fluorophore-labelled antibodies. When the antibody-binding peptides were spotted onto surfaces, fluorescent signal was only detected on the regions with peptide spots, suggesting that the peptide/antibody interactions were highly specific. To test the stability of the immobilized antibodies, surfaces were incubated at different aqueous conditions in static and under flow at a wall shear stress of 1.5 dyn/cm2 for one hour. The conditions range from phosphate buffer saline (PBS), 10% and 70% of fetal bovine serum (FBS) in endothelial cell growth medium. Detected by fluorophore-labelled antibodies, the fluorescent signal of antibodies reacted on spots of peptide-conjugated regions was significantly higher compared to background and antibodies reacted on spots of peptide-adsorbed regions proving that the antibody immobilization strategy was more stable on the conjugation method. In future work, the capability of the immobilized antibodies to capture EPCs will be assessed by flowing endothelial colony forming cells (ECFCs) that were successfully isolated from peripheral blood mononuclear cells (PBMCs, which contain EPCs). The antibody immobilization method developed in this project could be applied to specifically capture a variety of cell types on functionalized surfaces for biomedical applications ranging from cell isolation to biomaterials engineering.

A phage protein impedes bacterial resistance to phage infection


Marie-Laurence Lemay1, Sandra Maaß2, Andreas Otto2, Jérémie Hamel1, Geneviève M. Rousseau1, Denise M. Tremblay1, Rong Shi1, Stéphane M. Gagné1, Dörte Becher2, Sylvain Moineau1
1Université Laval 2University of Greifswald

The unequalled abundance and diversity of bacterial viruses (phages) partly explain why so many of the deduced proteins encoded by phage genomes have no known function and no homologue in public databases. While structural proteins are found in the virion particles, non-structural phage proteins are produced inside the bacterial host where it is presumed that they play a role in hijacking the cellular machinery for viral production. Virulent lactococcal phages belonging to the Skunavirus genus (Siphoviridae family) are by far the most endemic and problematic in the dairy industry worldwide. Phage p2 is a model for this viral genus and it infects Lactococcus lactis MG1363, the international reference strain for lactococcal research. Phage p2 structural proteins have been analyzed in great details, but most of its non-structural proteins are still uncharacterized. To study these proteins, a multidisciplinary approach is required.

 

Here, we made use of structural biology, genomics, physiology, and proteomics to provide insights into the function of phage p2 protein ORF47, the most conserved non-structural protein of unknown function among members of the Skunavirus genus. We solved the protein structure through circular dichroism and nuclear magnetic resonance. Using CRISPR-Cas9, we knocked out orf47 from phage p2 genome and confirmed gene disruption in a recombinant phage (p2∆47) by whole genome sequencing. The lack of ORF47 did not affect the duration of the phage lytic cycle, but the number of infective particles released per infected bacterium was significantly lower with p2∆47 yielding a burst size of 80 ± 7 in comparison to 129 ± 17 for p2. Moreover, we made use of label-free quantitative proteomics to compare the proteotypes of L. lactis MG1363 infected by phage p2∆47 or phage p2. Infection by p2 resulted in an increase of most metabolic pathways while a global decrease was observed during the infection by p2∆47. Our data showed that ORF47 is inducing the expression of many genes that are not functionally related and that without this protein, the phage induces a dormancy-like state in the bacterial host. Most interestingly, we found that ORF47 hinders L. lactis MG1363 resistance to phage infection.

Analyse des interactions médiées par les domaines SRC homology 3 (SH3) de SLA1 en les substituant par CRISPR-Cas9


Émilie Bourgault1,2, Ugo Dionne2,3,4, Alexandre Dubé1,2,5, Philippe Despré1,2, Nicolas Bisson2,3,4,6, Christian Landry1,2,5
1Département de Biochimie, Microbiologie et Bio-Informatique, Université Laval, Québec, QC, Canada. 2PROTEO-regroupement québécois de recherche sur la fonction, l'ingénierie et les applications des protéines. 3Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Québec, QC, Canada. 4Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada. 5Département de Biologie, Université Laval, Québec, QC, Canada. 6Département de biologie moléculaire, de biochimie médicale et pathologie, Université Laval, Québec, Canada.

Les cellules s’adaptent à leur environnement en détectant des signaux extracellulaires et en changeant leur comportement. L’intégration de ces signaux est souvent médiée par des protéines cytoplasmiques constituées de domaines d’interactions protéines-protéines comme les domaines SRC Homology 3 (SH3). Ces domaines hydrophobes lient principalement des motifs d’acides aminés PXXP sur leurs partenaires. Leur structure ainsi que leurs fonctions sont conservées à travers l’évolution. Chez Saccharomyces cerevisiae, il existe 27 domaines SH3 présents sur 23 protéines. Ils sont majoritairement impliqués dans le réarrangement du cytosquelette d’actine et dans l’endocytose, comme pour la protéine SLA1 qui est constituée de plusieurs domaines dont trois SH3. Des études in vitro ont permis d’identifier les différents motifs reconnus par les domaines SH3 de la levure. Cependant, les interactions SH3-dépendantes ainsi que leurs fonctions in vivo sont toujours peu connues. Nous avons développé une stratégie combinant la Protein Complementation Assay (PCA) avec CRISPR-Cas9 qui permet d’étudier les interactions SH3-dépendantes endogènes in vivo chez la levure. D’abord, les domaines SH3 de SLA1 ont été remplacés par de courtes séquences polyglycines (stuffers) par CRISPR-Cas9. Les interactions qui dépendent de ces domaines ont été déterminées par PCA en comparant l’interactome de SLA1 sauvage avec ceux de la protéine sans ses domaines SH3. Cela a permis de conclure que la majorité des interactions de SLA1 sont médiées par ses domaines localisés dans la partie N-terminale. Ensuite, les séquences des stuffers ont été ciblées par CRISPR-Cas9 à l’aide d’un ARN guide spécifique afin de les remplacer par différents domaines SH3. Cette stratégie nous a donc permis de changer la position des domaines SH3 de SLA1 ou de les remplacer par celui provenant d’une autre protéine, ABP1. Les différences dans les réseaux d’interactions ont de nouveau été détectées par PCA. Ces expériences ont mis en évidence l’influence du contexte protéique sur les interactions médiées par les domaines SH3, principalement pour le troisième domaine de SLA1. Finalement, l’impact de ces échanges de domaines dans SLA1 a été analysé en mesurant la croissance des différentes souches dans plusieurs conditions de stress. Notre stratégie combinant la PCA et CRISPR-Cas9 nous a permis de déterminer les interactions SH3-dépendante de SLA1 au niveau endogène in vivo et de mettre en évidence l’importance du contexte protéique de ces domaines. En perspective, les effets de ces changements de position seront investigués au niveau de l’endocytose.

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


Louis-Philippe Morency1,2, Rafael Najmanovich1,2
1Université de Montréal 2PROTÉO

INTRODUCTION

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.

 

METHODS

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 along thermodynamics parameters. (PDB : 3b7e Influenza virus neuraminidase in complex with Zanamivir predicted at 1.60Å RMSD from the crystal pose)

 

RESULTS & CONCLUSIONS

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 Set4 and the HAP25 dataset. We show that FlexAID outperforms other open-source docking methods when molecular flexibility is critical. We believe that its higher accuracy in complex scenarios, 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. Furthermore, FlexAID now outputs statistical thermodynamic parameters, i.e. ∆G, ∆H and -T∆S, as well as multiple conformations for each predicted binding modes, two unique features useful in the visualisation of results, in a more thorough binding energy analysis between different ligands and for the analysis of entropic contributions to the energy of formation of a complex of interest.

 

REFERENCES

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

Brighter red fluorescent proteins display reduced structural dynamics


Adam M. Damry1, Serena E. Hunt1, 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 can provide brighter FPs, mutations proximal to the chromophore often cause unwanted hypsochromic shifts in emission wavelength. Distal sites provide the possibility of rigidifying the chromophore through trickle-down dynamics without directly affecting its electrostatic environment, but the magnitude and extent of their contributions to dynamics has never been systematically evaluated. Here, we study this relationship using nuclear magnetic resonance (NMR) spectroscopy of a family of related monomeric RFPs with a range of QY between 0.02 and 0.70. A residue-by-residue comparison using 1H–15N HSQC spectra showed line-width broadening correlating with QY in roughly 12% 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 6% of backbone amide peaks. While many positions selected by these experiments are dispersed throughout the RFP scaffold, the β strand 7­–10 region shows a cluster of residues whose dynamics correlate with QY. This indicates a potential link between dynamics in this region and chromophore flexibility and brightness. To further probe this relationship, we saturated six sites along this face in mPlum-E16P (QY = 0.14), resulting in the production of mutants displaying QY enhanced by up to 35% without significant alteration of their emission wavelength. Our results show that RFP QY can be increased through mutagenesis of sites distal to the chromophore identified by NMR, opening the door to the rational design of more rigid, brighter RFPs.

Brighter Red Fluorescent Proteins through Improved Chromophore Packing


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

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 deeper tissue penetration. However, RFPs generally display lower brightness relative to other widely used fluorescent proteins, emphasizing the need for the development of brighter RFP variants. Previously, we demonstrated that RFP brightness can be enhanced by rational design of triple-decker motifs of aromatic rings formed by the chromophore and neighboring residues. Here, we explore two additional approaches based on improved chromophore packing to enhance RFP brightness. The first approach is based on the creation of a “super-decker” motif of five aromatic residues stacked with the chromophore, which we hypothesize will force a parallel π-stacking arrangement of aromatic rings instead of the edge-to-face orientation observed in the triple-decker and thereby result in improved packing. The second approach aims to optimize chromophore packing using aliphatic instead of aromatic amino acids. Our results show that brightness can be improved using both approaches, as the “super-decker” mutants, mRojo-SD1 and mRojo-SD2, and the aliphatic packing variant, L1-IM, display brightness increases of 150% or 550%, respectively. Interestingly, the L1-IM mutant has a similar emission wavelength and is 1.5-fold brighter than the most widely used monomeric RFP, mCherry. Altogether, our findings open the door to the design of brighter RFPs.

Caractérisation de la liaison membranaire de l’arrestine


Marc-Antoine Millette1,2,3, Sergey Vishnivetskiy4, Vsevolod V. Gurevich4, 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 Saint-Sacrement, CHU de Québec-Université Laval 3Regroupement stratégique PROTEO, Université Laval 4Department of Pharmacology, Vanderbilt University, États-Unis

Objectif : Les récepteurs couplés aux protéines G (GPCR) forment une grande famille de récepteurs permettant l’intégration de signaux extracellulaires. La famille des arrestines joue un rôle critique dans l’inactivation des GPCR. La rhodopsine est le modèle le plus étudié des GPCR. Elle est constituée de 7 hélices alpha transmembranaires localisées dans la membrane des disques des bâtonnets. La rhodopsine est activée par l’absorption d’un photon, ce qui marque le début de la cascade de phototransduction visuelle. L’inactivation de la phototransduction débute par la désactivation de la rhodopsine qui se déroule en deux étapes : 1) la phosphorylation de la rhodopsine catalysée par la rhodopsine kinase et 2) la liaison de l’arrestine-1 à la rhodopsine phosphorylée. Il a été postulé que l’arrestine-1 pourrait lier les membranes, ce qui contribuerait à sa liaison à la rhodopsine et à la stabilité du complexe. L’objectif de ces travaux consiste donc à caractériser la liaison membranaire de l’arrestine-1.

Méthodologie : Les membranes des disques des bâtonnets ont une composition en phospholipides très particulière. En effet, les phospholipides les plus fréquemment retrouvés incluent la phosphoéthanolamine (PE, 44%), la phosphocholine (PC, 41%) et la phosphosérine (PS, 15%). Parmi les chaînes acyles les plus abondantes de ces phospholipides, on note celles de type stéaroyl (C18:0, 19%), oléoyl (C18:1, 3%) et docosahexaénoyl (C22:6, 45%). Ces phospholipides ont été utilisés pour caractériser la liaison membranaire de l’arrestine-1. Le modèle des monocouches de Langmuir permet de mimer l’un des feuillets membranaires en utilisant un bain de monocouches. Une sonde au milieu du puits où est formée la monocouche permet de mesurer la pression de surface en fonction du temps. L’injection de l’arrestine-1 en concentration saturante dans la sous-phase mène à une hausse de la pression de surface, ce qui permet de caractériser l’interaction de la protéine avec la monocouche lipidique. Ces mesures permettent de déterminer différents paramètres, soit la pression d’insertion maximale (PIM) et la synergie. La PIM correspond à la pression au-delà de laquelle la protéine n’arrive plus à lier la monocouche et la synergie est une mesure de l’affinité de la protéine pour la monocouche.

Résultats :  La pression latérale estimée des membranes est d’environ 30 mN/m. Une PIM supérieure à cette valeur suggère une liaison membranaire de la protéine. Parmi les valeurs de PIM de l’arrestine-1 en présence de monocouches de C18:0-PC, C18:0-PE, C18:0-PS, C18:1-PC, C18:1-PE, C18:1-PS, C22:6-PC, C22:6-PS, C18:0-22:6-PC, C18:0-22:6-PE, seule la valeur de PIM du C18:0-PE excède la pression latérale estimée des membranes. Les valeurs de synergie de l’arrestine-1 en présence de monocouches de C18:0-PE, C18:0-PS, C18:1-PC, C18:1-PE, C18:1-PS, C18:0-22:6-PC, C18:0-22:6-PE sont positives, ce qui indique une interaction favorable entre l’arrestine-1 et ces phospholipides. La synergie de l’arrestine-1 en présence de la monocouche de C18:0-PC est nulle, ce qui suggère une interaction ni favorable, ni défavorable. La synergie de l’arrestine-1 en présence de la monocouche de C22:6-PS est légèrement négative, ce qui suggère une répulsion entre la protéine et cette monocouche.

Conclusion : La valeur de PIM élevée obtenue en présence d’une monocouche de C18:0-PE est cohérente avec la localisation de l’arrestine-1 sur le feuillet externe des disques des bâtonnets qui contient 60% de PE. Des mesures de spectroscopie infrarouge à modulation de polarisation de l’arrestine-1 en présence d’une monocouche de phospholipide permettront de déterminer l’orientation membranaire de cette protéine et de comparer ces données avec les modèles proposés dans la littérature.

 

Caractérisation des mécanismes moléculaires de la liaison des doigts de zinc 10 à 12 de Miz-1 à l’ADN par RMN en solution


Olivier Boisvert1, Jean-Michel Moreau1, Patrick Delattre1, Danny Letourneau1, Martin Montagne1, Pierre Lavigne1
1Université de Sherbrooke

La protéine Miz-1 (Myc Interacting Zinc Finger Protein-1) est un facteur de transcription de type BTB/POZ. Cette protéine possède 13 doigts de zinc de type C2H2 (ZF). Miz-1 lient majoritairement les «Transcriptional Start Sites » d’une multitude de gènes via une séquence de reconnaissance consensus récemment découverte.  Les ZFs sont des modules ou domaines protéiques avec un repliement ββα. Notre groupe de recherche se penche sur l’élucidation des ZFs de Miz-1 permettant la reconnaissance de l’ADN. Plusieurs ZFs de cette protéine présentent des divergences au niveau de la structure primaire consensus et des « linkers » qui les relient ensemble. En effet, la présence d’une histidine au début du premier brin-β diverge du consensus (i.e. tyrosine) et cause une dynamique moléculaire marquée sur l’échelle de la μs-ms dans les ZF 1, 6 et 11. Afin de mieux comprendre les mécanismes de la liaison et la reconnaissance de la séquence d’ADN consensus, nous avons caractérisé la dynamique moléculaire des ZFs 10-12 par RMN en solution et relaxation des 15N (« relaxation dispersion »). Nous avons aussi évalué l’effet de la mutation H586Y dans le ZF11 sur la dynamique moléculaire et l’affinité à l’ADN des ZFs 10-12 par anisotropie de fluorescence. Nos résultats montrent que la mutation diminue la dynamique moléculaire sur l’échelle de la µs-ms et augmente l’affinité pour la séquence consensus. Pris dans un contexte plus général, nos résultats suggèrent que ces divergences servent à moduler, à la baisse, l’affinité des protéines poly-ZFs pour l’ADN. De plus, certaines protéines poly-ZFs, (e.g. YY1 et CTCF) possédant également ces éléments structuraux non-canoniques émergent comme des protéines pouvant contrôler la topologie, la structure 3D de la chromatine et la transcription. Nous présentons un modèle selon lequel Miz-1 pourrait jouer un rôle similaire.

Caractérisation fonctionnelle et structurale des transporteurs de nickel chez Helicobacter pylori


Mariem Chalbi1, Michel Lê1, Imène Kouidmi1, Charles Calmettes1
1Institut National de la Recherche Scientifiques, Institut Armand-Frappier, Laval, QC

 L’acquisition des métaux est indispensable à la survie et à la virulence de plusieurs bactéries notamment chez Helicobacter pylori. Il s’agit du seul pathogène capable de coloniser la niche gastrique, infectant la moitié de la population humaine, et causant diverses pathologies telles que les ulcères et les cancers gastriques. Le traitement de l’infection par H. pylori est une combinaison d’antibiotiques et de sels de bismuth.

 

L’un des plus importants ions métallique chez H.pylori est le nickel qui est considéré comme un déterminant de virulence chez ce pathogène. En effet, cette bactérie nécessite le nickel comme co-facteur catalytique de deux enzymes clefs de son adaptation à la niche gastrique qui sont l’uréase et l’hydrogénase. Cependant, le nickel est un élément rare dans le corps humain, H. pylori nécessite donc des mécanismes d'acquisition hautement spécifiques et efficaces.

 

Nous visons à étudier, par des approches biochimiques et des études structurales, le système de transport de nickel NiuBDE chez H. pylori et plus particulièrement les navettes périplasmiques NiuBs. Mes travaux visent à caractériser la structure des transporteurs, NiuB1 et NiuB2, et à déterminer leur rôle dans le transport du nickel et des sels de bismuth par des études structurales (cristallographie aux rayons-X).

Cell-based fluorescent assay for screening ligand-gated ion channel function


Mykhaylo Slobodyanyuk1, Roberto Chica1, Corrie daCosta1
1University of Ottawa

Ligand-gated ion channels (LGIC) are integral membrane proteins that allow for the highly selective and rapid flow of ions across a cell membrane upon ligand binding. These ion channels are essential for cellular communication, and their misregulation leads to a plethora of neurological disorders such as schizophrenia, Alzheimer's, and Parkinson's disease. Patch clamping methods are the gold standard for studying structure-function relationships and ligand-induced responses of LGIC. However, these methods are low-throughput and labour-intensive. To screen for channel function when dealing with large mutant and/or ligand libraries, a high-throughput technique is required. In this study, we demonstrate that a cell-based, fluorescent screening assay can be used to detect an agonist-induced response in two different LGICs: human α7 nicotinic acetylcholine receptor (nAChR) and Erwinia ligand-gated ion channel (ELIC), both expressed in a human kidney BOSC23 cell line. This assay utilizes genetically-encoded calcium indicators of the GCaMP family to detect LGIC activation by producing a fluorescence signal in response to ion influx. Addition of the natural ligands for human α7 nAChR or ELIC produced a two to four-fold increase in GCaMP fluorescence intensity within 20 seconds or 130 seconds, respectively, making this assay effective for screening channel function within cells. Future work will be focused on optimizing the assay for use in fluorescence-activated cell sorting (FACS) to rapidly identify functionally important residues and ligands for further analysis using patch clamping methods.

CHARACTERIZATION OF THE TDP-L-VANCOSAMINE BIOSYNTHETIC PATHWAY FROM TDP-4-KETO-6-DEOXY-D-GLUCOSE


Lan Huong Thi Nguyen1, David Kwan2
1Concordia University 2Concordia University

Due to the large structure diversity of deoxysugars, and their critical importance for biological activities, much attention has been given to elucidate their biosynthetic pathways. Recent progress in understanding these pathways has proven that an array of glycosylated natural products can be generated through enzymatic glycodiversification. However, the successful application of this concepts requires an in-depth knowledge of various deoxysugar biosynthetic pathway enzymes including their catalytic activities, the preferred substrates, and their flexibility towards unnatural substrates.

 

Although gene sequences for dozens of GTs are known, only a marginal number of these enzymes have been fully characterized. To understand the biosynthesis of TDP-L-vancosamine, five putative genes, EvaA, DnmJ, EvaC, DnmU, and DnmV encoding the deoxysugar, have been expressed heterologously in E.coli, and were identified through in vitro assays and one-pot enzymatic synthesis. The results presented here now set the stage to produce TDP-L-vancosamine routinely for future combinatorial biosynthesis for glycosylation studies.

Clonage, surexpression et purification de la RDH8 en fusion avec plusieurs étiquettes dans le but de mesurer son activité enzymatique


Charlotte Lemay-Lefebvre1,2,3, Line Cantin1,2,3, Camille Bérubé1,2,3, Christian Salesse1,2,3
1Département d'ophtalmologie, Faculté de médecine, Université Laval 2CUO-recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement 3Regroupement stratégique PROTEO, Université Laval

La rétinol déshydrogénase 8 (RDH8) est la première enzyme impliquée dans le cycle visuel. Son rôle est de réduire le tout-trans rétinal en tout-trans rétinol, ce qui prévient l’accumulation de lipofuscine (A2E), une molécule toxique pour les photorécepteurs et l’épithélium pigmentaire rétinien, ce qui peut mener à la dégénérescence maculaire liée à l’âge. Il y a cependant une accumulation légère d’A2E dans les photorécepteurs et importante dans l’EPR, même en présence de RDH8, ce qui suscite des interrogations au niveau de son efficacité enzymatique. La RDH8 est insoluble dans le lysat bactérien suite à sa surexpression dans un système E. coli. Il est cependant possible de la solubiliser en utilisant un détergent, ce qui cependant empêche la caractérisation de son activité enzymatique. L’objectif de ces travaux consistait donc à utiliser différentes étiquettes de purification/solubilisation ainsi que son ligand ou son cofacteur pour solubiliser la RDH8 dans le lysat bactérien et ainsi rendre possible la caractérisation de son activité enzymatique. Le niveau d’expression ainsi que la solubilité, la purification et la possibilité de cliver les étiquettes de solubilisation/purification ont donc étudiés dans le but de caractériser l’activité enzymatique de la RDH8.

 

Plusieurs protéines de fusion de la RDH8 ont été clonées puis surexprimées dans le système d’expression E. coli (souche BL21 DE3 RIPL). Les étiquettes de purification/solubilisation étudiées incluent le His10, la MBP, la TagR-TagR, la SUMO, la NusA ou une combinaison de plusieurs de ces étiquettes. Le tout-trans rétinal, le tout-trans rétinol (ligands) ou le NADPH (cofacteur) ont aussi été ajoutés lors de la surexpression et de la lyse cellulaire de la protéine de fusion His10-RDH8 pour favoriser la surexpression d’une forme soluble de la RDH8. Les différents tests de purification ont été faits par chromatographie avec une colonne de nickel lorsque la protéine de fusion contient l’étiquette His10 et une colonne hydrophobe lorsqu’elle contient le TagR-TagR. La RDH8 a aussi été surexprimée dans le système d’expression Lactocuccus lactis permettant la sécrétion de la protéine dans le milieu de culture afin d’éviter l’utilisation d’un détergent pour solubiliser la protéine dans le lysat bactérien.

 

Mes travaux ont montré que l’ajout du ligand ou du cofacteur de la RDH8 ne permet pas de solubiliser la protéine de fusion His10-RDH8 dans le lysat bactérien. Les différentes étiquettes de purification/solubilisation utilisées mènent à des résultats variables mais dont le niveau de surexpression et de solubilisation des protéines de fusion est généralement faible. Par ailleurs, les protéines de fusion utilisées ne permettent pas l’obtention de la protéine pure en quantité suffisante pour effectuer des mesures d’activité enzymatique. De plus, suite au clivage de différentes étiquettes de purification/solubilisation, il a été impossible de récupérer la RDH8 purifiée. Le système d’expression L. lactis n’a pas permis la surexpression de la RDH8.

 

La détermination de l’activité enzymatique de la RDH8 est une tâche ardue car elle nécessite l’obtention d’une protéine soluble avec un haut niveau de pureté sans utiliser de détergent, ce qui est impossible jusqu’à maintenant en utilisant les technologies les plus avancées de production de protéines de fusion. L’utilisation de la levure Pichia pastoris est maintenant envisagée pour la surexpression de la RDH8 en fusion avec différentes étiquettes.

Computational modeling of the death complex between glyceraldehyde-3-phosphate dehydrogenase and seven-in-absentia homolog 1 (GAPDH-Siah1)


Ritu Arora1, Vinod Parmar1, Ann M. English1, Gilles H. Peslherbe1
1Concordia University

Tetrameric GAPDH is a well-known glycolytic enzyme with many moonlighting functions. Monomeric and dimeric forms of GAPDH also have been identified in cells and are implicated in many of its physiological and pathophysiological functions. The focus of this investigation is on GAPDH’s role in apoptosis, which is linked to its binding to seven-in-absentia homolog 1 (Siah1), an E3 ubiquitin-protein ligase [1,2]. GAPDH forms a complex with Siah1 that is translocated to the nucleus and initiates a cascade that triggers apoptosis [1]. Despite this death complex being of vital biological importance, very little is known about its structure. Computational modeling of the association of tetrameric GAPDH with four Siah1 dimers has been performed [3]. However, our electrostatic surface analyses reveal that the GAPDH monomer, but not the tetramer, has a large complementary electrostatic surface area for Siah1 binding. Also, molecular docking analyses based on interaction-refined shape complementarity principles confirm strong complexation between monomeric GAPDH and Siah1. In particular, the most favorable docking poses of the complex disclose that the S-loop of the GAPDH monomer becomes less disordered upon interaction with Siah1, thereby promoting complexation. We propose that stabilization of the S-loop drives binding between the GAPDH monomer and Siah1. To test our hypothesis, molecular dynamics simulations on the best docked complexes are ongoing.

 

 

References

  1. Hara, M. R.; et al. Nat. Cell Biol. 2005, 7, 665–674.
  2. Hara, M. R.; et al. Biochim. Biophys. Acta - Mol. Basis Dis. 2006, 1762, 502–509.
  3. Jenkins, J. L.; Tanner, J. J. D. Biol. Crystallogr. 2006, 62, 290–301.

Computationally Designed Grafting of a Ru Mediator to Improve Redox Potential of a Laccase


Viviane Robert1, Emanuele Monza2, Lionel Tarrago1, Ferran Sancho2, Anna De Falco1, Ludovic Schneider1, Eloïne Npetgat Ngoutane1, Yasmina Mekmouche1, Pierre Rousselot Pailley1, Jalila Simaan1, Victor Guallar2,3, Thierry Tron1
1Aix Marseille Université 2Barcelona Supercomputing Center 3ICREA

Laccases are blue multicopper oxidases that can oxidize a wide range of substrates in mild conditions. The use of molecular oxygen as the final electron acceptor makes water the only byproduct, which is ideal for green chemistry. Therefore, laccases are of great interest in different industrial applications from organic synthesis to biomedical applications1,2.

Despite being widely applied, the redox potential of these oxidation reactions is a limiting factor, and increase it by mutating residues near the electron-accepting copper centers is challenging. This problem can be overcome by using a photosensitizer as a mediator3: under visible light, photosensitizers can reduce laccases, allowing oxidation of otherwise inert compounds by the virtual increase of the redox potential, with correspondingly improved kinetic constants.

Toward this end, an improved laccase was designed by adding a Ru complex that can be excited with visible light. After grafting the mediator to the protein, experimental results did not show the expected improvement in efficiency, suggesting a problem with the electron back-transfer after oxidation. A computational rationalization is presented here, by means of our in-house Protein Energy Landscape Exploration (PELE) algorithm and Pathways plugin from VMD. We further designed new positions to graft the complex based on our computational analysis, leading to promising variants to be tested in the lab that show up to 100-fold in silico improvement in electron coupling.

 

References

1. Bauer, C. G. et al. New enzyme sensors for morphine and codeine based on morphine dehydrogenase and laccase. Fresenius J. Anal. Chem. 364, 179–183 (1999).

2. Milligan, C. & Ghindilis, A. Laccase Based Sandwich Scheme Immunosensor Employing Mediatorless Electrocatalysis. Electroanalysis 14, 415–419 (2002).

3. Robert, V. et al. Probing the Surface of a Laccase for Clues towards the Design of Chemo-Enzymatic Catalysts. ChemPlusChem 82, 607–614 (2017).

Conformational landscape of homologous enzymes with distinct biological functions


Chitra Narayanan1, Pratul K. Agarwal2, Nicolas Doucet1,3
1INRS – Centre Armand-Frappier Santé Biotechnologie, Université du Québec, Laval, QC 2Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 3PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications, Université Laval, Québec, QC

Human eosinophil derived neurotoxin (HsR2) and eosinophil cationic protein (HsR3) are rapidly evolving members of the pancreatic ribonuclease superfamily in humans. Phylogenetic analysis of members of this superfamily showed that HsR2 and HsR3 sequences arose as a result of a gene duplication event from a common ancestor. The rapid divergence of the two enzymes is indicative of a response to evolutionary pressure. Despite a large sequence similarity of 76 %, the two enzymes display significant differences in their catalytic rates and biological functions. In previous studies, we showed differences in the dynamical properties on the functionally relevant millisecond timescale. However, the potential effect of these differences on the distinctly different biochemical and biological functions in these two enzymes is not well understood. In this study, we perform a systematic characterization of the structural and dynamical properties of HsR2 and HsR3 in the free and ligand-bound states. We combine observations from NMR with computational analyses to identify conformational properties influencing the observed differences in their biochemical and biological functions. We implement a variety of computational approaches to characterize the range and timescale of motions on the MD timescale. Our results will provide important insights on the atomistic changes that influence the distinctly different biochemical/biological functions of these two enzymes, in addition to providing a novel framework for analyzing the conformational landscape of enzymes.

Conformational space derived from normal mode analysis: a dynamical metric for scoring 3D predictions of RNA, proteins and their complexes


Olivier Mailhot1, Vincent Frappier1, François Major1, Rafael Najmanovich1
1Université de Montréal

All the metrics currently used in the scoring of predictions in the RNA-Puzzles are static: they consider only one solved structure. In reality, these RNA molecules are occupying vast conformational landscape in vivo due to thermal fluctuations and the interaction with partner macromolecules. Normal mode analysis (NMA) is a fast and robust way to explore the most energetically favored conformational transitions of macromolecules, in contrast to molecular dynamics simulations which are more precise, but computationally costly and very sensitive to initial conditions, thus hard to reproduce independently. Here, we present a metric using the most global motions obtained from NMA to rescore predicted 3D structures with regards to the experimentally resolved structure. Interestingly, this metric can change the ranking of the predictions and in some cases even the winner of a particular RNA-Puzzles contest. The motions which favor the new winners are realistic, for example the slight elongation of a double-helix or the bending of a hinge-like junction. This metric is implemented in an open-source, user-friendly software package and will be submitted soon for publication.

DESIGNER BIOSENSORS FOR ENGINEERED METABOLIC PATHWAY OPTIMIZATION


Mohamed Nasr1, 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 value-added 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 laborious 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.

Transcriptional repressors bind specific effectors or effector families, which limits their usability as biosensors in many engineered pathways. This project aims 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.

Developing a family specific molecular docking energy scoring function to accelerate drug discovery


I.B. Matala1, L.-P. Morency1, F. Gaudreault2, R. Najmanovich1
1Department of Pharmacology and Physiology, Université de Montréal 2Department of Biochemistry, University of Sherbrooke

The interactions between cognate ligands or drugs and macromolecules, such as protein, is critical for modulating biological activity. Molecular recognition is a term that describes all the factors that affect the specificity and selectivity of such interactions.  The three-dimensional structure of macromolecule-ligand complex offers key insights when studying molecular recognition as well as in the rational design of drugs. In the absence of an experimentally determined structure, computational docking offers the possibility to predict the 3D structure of the complex. Additionally, molecular docking is also used in virtual high-throughput screening for new candidate drugs targeting a protein of interest. It is a highly cost-effective and time-saving solution for the initial steps in drug discovery compared to experimental alternatives. Docking is an optimization problem which requires evaluating computationally a large number of possible solutions to find the best one as defined by the scoring function utilized. Previously, the Najmanovich Research Group (NRG) developed a docking algorithm called FlexAID, which stands for Flexible Artificial Intelligence Docking. As FlexAID was originally designed to be a tool applicable in principle to any protein and ligand interaction, the atoms pairwise interaction parameters were derived using machine learning techniques and varied dataset of protein-ligand complexes. However, there can be significant differences in the distribution of atoms types among proteins families and the type of ligands that they bind. As such, the development of pairwise interaction parameters for specific protein families and the ligands they bind may lead to considerable improvements in accuracy. The goal of this project is to curate and utilize data for specific protein families specifically, human kinases and WD40 domains, to generate such focused scoring functions and compare these to the existing generalist scoring function. Additionally, we will further improve the NRGsuite FlexAID graphical user interface, that exists to facilitate the visualization and use of the FlexAID docking algorithm by non-experts.

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


David Kwan1, Jama Hagi-Yusuf1
1Concordia University 2Centre for Applied Synthetic Biology

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 focusing on an ethanol-dependent fuel source, to more complex and energy rich hydrocarbons, similar to those found in petroleum. Recently, a heme-dependent P450 decarboxylase enzyme, OleTJE, was discovered in the bacterium Jeotgalicoccus sp, which was determined via in vitro and in vivo studies to be capable of catalyzing the decarboxylation of long-chain fatty acids, producing terminal alkenes and CO2, thus making OleTJE an ideal candidate for biofuel production. The development of a high-throughput assay is important in engineering and studying the protein. OleTJE activity can be determined by detecting CO2, a byproduct of the OleTJE-catalyzed conversion of fatty acids into terminal alkenes. Here we adapted an existing coupled-enzymatic assay to detect OleTJE activity for further research. In this assay, CO2 is 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 versatile vaccination platform based on papaya mosaic virus (PapMV) nanoparticles


Denis Leclerc1, Marilène Bolduc1, Marie-Eve Laliberté-Gagné1, Pierre Savard2
1CHU de Québec/U Laval 2Neurosciences, CHU de Québec, U Laval

Nanoparticles made of the papaya mosaic virus (PapMV) coat protein (CP) and a ssRNA have been showed to be a strong immune modulator through the activation of the toll like receptor (TLR) 7/8. Therefore, it is an ideal platform for the development of vaccines to non-immunogenic peptide antigens. The attachment of peptide antigens, like the universal influenza antigen M2e, to the nanoparticle is mediated by a bacterial transaminase named sortase A (SrtA). The decorated nanoparticle can induce a very strong immune response leading to a protection against a infection with influenza in vaccinated animals. In this study, we have engineered the PapMV vaccine platform to either attach the antigens to the N or the C-terminus of the C,P that makes 95% of the nanoparticles. The impact of the position of attachment as well as the density of the peptides onto the vaccine platform were analysed based on their ability to elicit an humoral or a cellular mediated immune response to the antigen. We will discuss the advantages of these approaches in relation to the type of immune protection that need to be elicited and the future perspective of protein engineering of this innovative tool.

DEVELOPMENT OF CD125-TARGETED ENGINEERED ANTIBODY FRAGMENTS FOR USE IN MUSCELE-INVASIVE BLADDER CANCER THERAPY


Olga Bednova1, Tim Hercus2, Angel Lopez2, Jeffrey V. Leyton1
1Department of Nuclear Medicine and Radiobiology Faculty of Medicine and Health Sciences Université de Sherbrooke – CHUS, Sherbrooke, QC, Canada 2Centre for Cancer Biology, University of South Australia, North Terrace, Adelaide, SA, Australia

Muscle-invasive bladder cancer (MIBC) is most often treated by radical cystectomy and supported by chemotherapy, which have demonstrated promising results in decreasing mortality rate.  However, this treatment is associated with high rates of tumor recurrence and treatment-related mortality.

Antibody–drug conjugates (ADCs) are novel drugs that provide selective delivery of toxins to tumour cells while sparing healthy cells. Current ADCs have prolonged circulation that can increase nonspecific toxicity. Antibody engineering allows for antibodies to maintain desired properties such as tumor uptake while increasing blood clearance to minimize exposure to healthy tissue.

CD125 is a novel receptor specifically overexpressed on MIBC cells but not on superficial bladder tumor or healthy urothelial cells. RT-PCR amplification was performed on hybridoma cells expressing the monoclonal antibody A14 that is specific for CD125 to obtain the cDNA sequence. We engineered a coding sequence of the A14 single-chain variable fragment (scFv), which is a fusion protein of the variable regions of the heavy and light chains of A14. The scFv sequence was cloned into a mammalian plasmid vector containing an IL-2 signal sequence and enhanced green fluorescent protein (EGFP) to evaluate and optimize cloning and transfection conditions. HEK293T cells were analyzed 48 hours after transfection and the protein products in the conditioned media and cell lysates were analyzed by Western Blot. An abundant amount of A14 scFv-EGFP was found in cell lysates but not in media. The scFv-EGFP product migrated by SDS-PAGE at ~57 kDa. Higher molecular weight oligmeration was observed as well as expression of EGFP not fused to A14 scFv. This preliminary data demonstrates that the chosen vector provides high expression level of the desirable protein; however, the secretion needs improvement for current A14 scFv.

Development of sulfahydantoin compound as potential antibiotics and β-lactamase inhibitors.


Pierre-Alexandre Paquet-Côté1,2, Rosalie Lamoureux1,2, Camille Lapointe Verreault1,2, Laurie Bédard1,2, Normand Voyer1,2
1Université Laval 2PROTEO

One of the most widely used antibiotics family is the β-lactams. However, the increased bacterial resistance greatly impairs their medical use. The main resistance mechanism against these antibiotics is the production of the enzyme family know as β-lactamases. This resistance can be overcome by developing β-lactamase inhibitors. One unexplored class of compounds with a potential activity is the sulfahydantoins.

 

The sulfahydantoin moiety is a structural analog of the lactivicin1, an antibiotic acting using the same mechanism as the β-lactam family without the β-lactam heterocycle. In addition, sulfahydantoin compounds have already shown inhibition of human elastase2, a serine protease using a similar active site than β-lactamases. Moreover, the sulfahydantoin cycle is a dipeptide mimic3, allowing an convenient synthesis starting from different amino acids.

 

In this presentation, the synthetic approach to make a library of sulfahydantoin analogs will be presented. Furthermore, results of antibacterial assays and the inhibition of relevant β-lactamases will be discussed.

 

  1. Natsugari, H.; Kawano, Y.; Morimoto, A.; Yoshioka, K.; Ochiai, M. J. Chem. Soc. Chem. Comm. 1987, 62-63.
  2. Groutas, W. C.; Huang, W. J.; Yamamoto, Y.; Li, Y.; Dou, D. F.; Alliston, K. R.; Hanzlik, R. P.; Williams, T. D. J Med Chem 2008, 51, 2003
  3. Boudjabi, S.; Dewynter, G.; Voyer, N.; Toupet, L.; Montero, J.-L. Eur J Org Chem 1999, 1999, 2275-2283.

Devrait-on utiliser la souris comme modèle animal pour caractériser la lécithine rétinol acyltransférase humaine?


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

INTRODUCTION : La lécithine rétinol acyltransférase (LRAT) est une enzyme qui est impliquée dans le cycle visuel, qui sert à régénérer le chromophore du pigment visuel. Elle catalyse l’estérification du tout-trans rétinol (tt-ROH) en tout-trans rétinyl ester (tt-RE). Les souris sont utilisées abondamment pour obtenir de l’information sur la LRAT. La séquence protéique de la LRAT murine (mLRAT) a 80 % d’identité et 90 % de similitude avec celle de la LRAT humaine (hLRAT). Cependant, une arginine est présente en position 173 chez la mLRAT, tandis qu’une proline est trouvée à cette position chez la hLRAT. En plus de la grande différence structurelle et chimique entre ces deux acides aminés, la mutation de la P173 pour une leucine (P173L) chez la hLRAT mène à la dégénérescence des photorécepteurs. Nous avons montré que cette mutation de la hLRAT tronquée (htLRAT) cause une perte de son activité. L’activité de la mLRAT pourrait donc différer significativement de celle de la hLRAT. L’objectif de ces travaux consistait donc à déterminer si la mLRAT est un bon modèle pour obtenir de l’information sur la hLRAT en raison de la présence de R173 dans la mLRAT au lieu de P173.

MATÉRIEL ET MÉTHODES : Les protéines tLRAT ont été surexprimées dans des cellules de E. coli et purifiées par chromatographie d’affinité. L’activité enzymatique de la htLRAT, mtLRAT, P173L-htLRAT, P173R-htLRAT et R173P-mtLRAT a été comparé en mesurant la formation du tt-RE. La structure secondaire et la stabilité thermique de ces protéines ont été étudiées par dichroïsme circulaire (CD). Des informations sur la structure des protéines tLRAT et de leur liaison au tt-ROH ont été obtenues par spectrofluorimétrie.

RÉSULTATS : L’activité de la mtLRAT est 2,7 fois plus faible que celle de la htLRAT (1 998 ± 43 moles de tt-RE/min par mole d’enzyme pour la htLRAT par rapport à 747 ± 10 moles de tt-RE/min par mole d’enzyme pour la mtLRAT). Cependant, seulement une légère modification de l’activité a été observée pour la P173R-htLRAT (1 856 ± 26 moles de tt-RE/min par mole d’enzyme) par rapport à la htLRAT ainsi que pour la R173P-mtLRAT (891 ± 12 moles tt-RE/min par mole d’enzyme) comparativement à la mtLRAT. En revanche, une perte d’activité importante est observée avec la P173L-htLRAT (319 ± 10 moles de tt-RE/min par mole d’enzyme, 6,3 fois moins élevée que celle de la htLRAT). Des effets très différents sur l’activité enzymatique sont donc observés lors du changement de P173 pour une arginine ou une leucine. Les mesures de CD montrent des spectres très similaires pour toutes les protéines, révélant une structure secondaire composée majoritairement d’hélices alpha. Une plus grande similitude peut cependant être observée entre les protéines de la même espèce à l’exception de la P173R-htLRAT. Les mesures de stabilité thermique montrent toutefois que les protéines murines sont moins thermostables que les protéines humaines, ce qui suggère la présence de différences structurelles significatives entre les protéines murines et humaines. Les mesures de fluorescence montrent que le site actif de la htLRAT est plus rigide que celui de la mtLRAT, mais qu’ils ont une accessibilité similaire au solvant. Cependant, la htLRAT a une affinité pour le tt-ROH beaucoup plus élevée que dans le cas de la mtLRAT (33 ± 4 nM pour la htLRAT par rapport à 128 ± 14 nM pour la mtLRAT).

CONCLUSIONS : Ces travaux montrent que la souris n’est pas un très bon modèle animal pour obtenir de l’information sur la hLRAT. Il est donc nécessaire d’éviter toute affirmation spéculative sur la hLRAT basée sur des études avec la mLRAT. Cette étude nous a également permis de mieux comprendre comment la mutation P173L affecte l’activité de la hLRAT et l’importance de ce résidu chez la hLRAT.

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


Ugo Dionne1,2,3, François J. M. Chartier1,2,3, Kévin Jacquet1,2,3, Christian R. Landry3,5,6, Nicolas Bisson1,2,3,4
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 4Department of Molecular Biology, Medical Biochemistry and Pathology, Université Laval, Québec, Canada. 5Department of Biology, Université Laval, Québec, QC, Canada 6Department of Biochemistry, microbiology and Bioinformatics, Université Laval, Québec, Québec, 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 and GRB2, 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 indicates that this mechanism is common and that it may entail signaling specificity. For example, the first SH3 domain of GRB2 is phosphorylated by the RTKs MER and FGFR but not by MET. We postulate that the specificity of the RTKs phosphorylation on SH3 domains sculpt the composition of signalling complexes and could help understand how different receptors use the same effectors but yield different outcomes. 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.

Effect of spinning rate on the molecular structure and amino acid dynamics in native and supercontracted spider dragline silk: a solid-state NMR and Raman spectroscopic study


Jane Gagné1, Koralie Mélançon1, Thierry Lefèvre1, Michèle Auger1, Normand Voyer1
1Université Laval

Spider silk is an exceptional natural protein fiber. Its mechanical properties exceed those of almost all known materials, both natural and synthetic. Spider silk is known to have a high tenacity, good mechanical resistance while remaining extensible. It is therefore very interesting for various spheres of applications such as medicine and textiles. Its molecular structure and properties are influenced by external factors such as relative humidity and spinning speed. The dragline fiber, for example, shrinks by almost half its length when exposed to high humidity, a phenomenon called supercontraction. Despite various works surrounding spider dragline silk, no systematic study of the impact of spinning speed and relative humidity on the quantification of molecular orientation and secondary structure has been reported.

 

We characterized fibers by Raman spectromicroscopy and solid-state nuclear magnetic resonance (SS-NMR). The former technique allows to quantify the orientation of the protein chains and to characterize their secondary structure. SS-NMR is used to determine the secondary structure and dynamics of amino acids. Both techniques are therefore complementary and provide a global vision and a better understanding of the impact of spinning speed and supercontraction on the structure of spider silk. The silk of two species of spiders, Nephila clavipes and Araneus diadematus, are obtained by forced spinning at different speeds (0.3 to 20 cm/s) and subjected to a relative humidity level higher than 90%. The silk of two groups of spiders were isotopically labeled with 1-13C-gly and 1-13C- ala for the purpose of analyzing the structure and dynamics of particularly important amino acid carbonyl groups.

 

Raman spectromicroscopy shows that molecular orientation is maximum at 1 cm/s and decreases at lower and higher spinning rates. Similar results have already been observed in the past. The study of native silk dynamics by NMR indicates that the carbonyl groups of the alanine are mainly in the form of b-sheet (which gives rigidity to the fiber) and that those of the glycine are essentially present under two distinct secondary structures the more present being the 31-helix (which confer extensibility to the fiber).

 

We have shown the secondary structure of the carbonyl groups of the two amino acids most present in spider silk. Our results are consistent with the model of spider silk reported in the literature. For NMR studies, supercontracted silk will be analyzed shortly and compared to native fiber data. Raman spectromicroscopy revealed the orientation of the proteins as a function of the spinning speed and estimated a favorable speed for a better orientation of these in the axis of the fiber. Our work should help understanding the molecular origin of an “optimal” molecular orientation in dragline silk and its biological advantage.

Efficient Site-Specific Antibody-Drug Conjugation by Engineering of Different Recognition Motifs for Microbial Transglutaminase


Lukas Deweid1,3,4,5, Aileen Ebenig1, Norbert Egon Juettner2, Kiana Lafontaine3,4,5, Olga Avrutina1, Hans-Lothar Fuchsbauer2, Joelle Pelletier3,4,5, Harald Kolmar1
1Clemens-Schoepf Institute for Organic Chemistry and Biochemistry, TU Darmstadt 2University of Applied Sciences, Darmstadt 3Département de chimie, Université de Montréal, Montréal, Canada 4PROTEO Network, Université Laval, Québec, Canada 5Center for Green Chemistry and Catalysis (CGCC), Montréal, Canada

Cancer costs millions of lives each year and challenges researchers of different fields to develop more efficient and patient-friendly therapies. Among innovative strategies to fight this fickle disease, antibody-drug conjugates (ADCs) have evoked rapidly growing interest. These macromolecular constructs combine the exquisite target specificity of monoclonal antibodies and the toxicity of small organic compounds by covalent linkage of both molecules, resulting in a widened therapeutic window.1 To that end, multiple chemical and enzymatic strategies have been developed. Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines enabling efficient site-specific conjugation of molecular architectures.2 Unfortunately, monoclonal antibodies lack native transamidation sites. This limitation can be bypassed via incorporation of specific peptide recognition sequences.3

 

Herein we present a rapid and efficient mTG-catalyzed bioconjugation that relies on a novel recognition motif derived from its native substrate, Streptomyces papain inhibitor (SPIP). Improved reaction kinetics compared to commonly applied sequences was demonstrated for model peptides and for biotinylation of Her2-targeting antibody trastuzumab variants. Moreover, an antibody-drug conjugate assembled from trastuzumab that was C-terminally tagged with the novel recognition sequence revealed a >10-fold improved cytotoxicity on Her2-positive breast cancer cells due to higher payload density. Our results successfully demonstrate that recognition motifs known to mediate highly efficient labelling by mTG with intrinsic substrates can be grafted to allow posttranslational modification of therapeutic antibodies.

 

Though peptidic recognition tags promote rapid labeling by mTG, incorporation of decameric sequences into full-length antibodies may lead to undesired disadvantages such as poor folding or decreased stability. Consequently, usage of larger tags is often limited to terminal sites of the antibody chains. In this work, we aim to overcome this limitation by incorporating solitary glutamine residues into surface-exposed regions of a human Fc-fragment to identify positions that are efficiently modified by mTG. To that end, a strategy for the recombinant expression of an Fc in E. coli cells was established. Upon purification of the respective Fc variants, reactivity of the enzyme towards the substituted residues will be investigated using a rapid fluorescent screening assay. 4

 

(1) Deweid, L., Avrutina, O., and Kolmar, H. (2019) Microbial transglutaminase for biotechnological and biomedical engineering. Biol Chem 400 (3), 257-274.

(2) Deweid, L., Neureiter, L., Englert, S., Schneider, H., Deweid, J., Yanakieva, D., Sturm, J., Bitsch, S., Christmann, A., Avrutina, O., Fuchsbauer, H. L., and Kolmar, H. (2018) Directed Evolution of a Bond-Forming Enzyme: Ultrahigh-Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display. Chem. Eur. J. 24 (57), 15195-15200.

(3) Siegmund, V., Schmelz, S., Dickgiesser, S., Beck, J., Ebenig, A., Fittler, H., Frauendorf, H., Piater, B., Betz, U. A., Avrutina, O., Scrima, A., Fuchsbauer, H. L., and Kolmar, H. (2015) Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation. Angew. Chem. Int. Ed. 54 (45), 13420-13424.

(4) Rachel, N. M., Quaglia, D., Levesque, E., Charette, A. B., and Pelletier, J. N. (2017) Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements. Protein Sci 26 (11), 2268-2279.

Evolution des complexes protéiques après hybridation entre espèces


Caroline Berger1, Rohan Dandage1, Isabelle Gagnon-Arsenault1, Kyung-Mee Moon2, Richard Greg Stacey2, Léonard J. Foster2, Christian 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.

En nature, les phénotypes des espèces hybrides diffèrent souvent des phénotypes parentaux. De nouvelles propriétés telles que l’incompatibilité des hybrides (phénotype des hybrides inférieur à celui des parents) ou l’hétérosis (phénotype des hybrides supérieur à celui des parents) expliquent en général ces caractères (Darwin. 1876, Shull. 1908). Toutefois, les bases moléculaires à l’origine de ces nouveaux phénotypes restent à explorer (Bar-Zvi et al. 2017). Parmi les mécanismes moléculaires possibles, les complexes protéiques jouent un rôle central dans la cellule par la réalisation de nombreuses fonctions (transcription, traduction, dégradation, transport, etc.) qui ont un impact direct sur le phénotype des organismes. Dans cette étude, nous nous intéressons aux changements potentiels qui peuvent survenir dans l’organisation des complexes protéiques chez les hybrides. Pour répondre à cette question, nous utilisons une approche protéomique qui permet d’étudier l’ensemble des complexes protéiques de la cellule (Kristensen et al. 2012). Des hybrides de levures ont été générés au laboratoire, et la méthode SEC-PCP-SILAC a été utilisée pour comparer les complexes protéiques des hybrides par rapport aux complexes parentaux. Nos premières analyses ont mené à l’identification de 600-700 protéines et 200-300 complexes protéiques. Nos résultats démontrent que les complexes parentaux ont tendance à être maintenus chez les hybrides, ce qui suggère la robustesse générale des complexes protéiques suite à l’hybridation. Malgré cette tendance générale, certaines protéines, telles que les protéines ribosomiques, semblent fréquemment modifiées (gain ou perte) chez les hybrides par rapport aux espèces parentales. Les observations faites avec la méthode SEC-PCP-SILAC sont actuellement confirmées par une stratégie alternative d’étude des interactions protéiques (PCA Protein-fragment Complementation Assay).

Evolutionary divergence of conformational exchange phenomena in a host defense enzyme family


David N. Bernard1, Chitra Narayanan1, Tim Hempel2, Myriam Letourneau1, Purva Prashant Bhojane3, Khushboo Bafna3,4, Marie-Christine Groleau1, Eric Déziel1, Elizabeth E. Howell3, Pratul Agarwal4, Nicolas Doucet1,5
1Centre Armand-Frappier Santé Biotechnologie - INRS 2Freie Universität Berlin 3University of Tennessee, Knoxville, TN, USA 4Oak Ridge National Laboratory 5PROTEO

Recent advancements in protein engineering have shown the importance of conformational exchange in the emergence of new functions. However, knowledge of conformational exchange is limited to a finite number of proteins, which are typically functionally and structurally distinct from each other. To better understand structural exchange phenomena, our group sought to perform extensive characterization of these types of events in the ribonuclease A (RNase A) superfamily. Previous results from our laboratory have highlighted large differences between the concerted atomistic behavior of RNases from various subfamilies displaying distinct biological roles, but few studies were devoted to proteins exhibiting similar functions. We thus sought to understand the conformational flexibility within a single RNase subfamily, i.e. hominidae-exclusive RNases from subfamily 3. These enzymes 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 gene duplication about 50 million years ago, resulting in two distinct enzyme families exhibiting distinct biological functions. To better understand how the biological roles of host defense RNase subfamilies 2 and 3 came to diverge, we have characterized the biological activities of various subfamily members, in addition to monitoring their conformational exchange behavior. We also performed enzymatic assays, antibacterial assays against E. coli and S. aureus, cytotoxicity assays against human HeLa cells, and characterized their conformational exchange on various timescales using NMR relaxation experiments (CPMG, CEST) and bioinformatic analyses (MD simulations, Markov State Model analyses). Despite conservation of their biological roles, we show that these enzymes can either display similar or distinct conformational exchange profiles, thus hinting at divergence rather than conservation of flexibility. Comparing progressively more distant enzymes to their human counterpart should provide information on how these enzymes may evolve new functions and/or alter their behavior on a molecular level.

Ex situ and in cell 13C solid-state NMR characterization of starch and glycoprotein-rich cell-wall in microalgae


Alexandre POULHAZAN1, Alexandre Arnold1, Dror WARSCHAWSKI1, Isabelle Marcotte1
1Université du Québec à Montréal

Human and industrial activities are associated with fast global population growth, with potentially harmful repercussions on the environment. Microalgae deserve careful study since they support the aquatic food chain. Pollutants can bio-accumulate into the cell and be transferred to higher organisms, with potential consequences on aquaculture and human health. Alternatively, they can cross the microalgal cell wall to exert an adverse effect on internal sites. It is thus important to provide tools to study interactions and effects with the various microalgal cell constituents. The objective of this work was to characterize the cell wall and starch grains of Chlamydomonas reinhardtii, - a model microalga used in a wide range of applications. To do so, high-resolution two-dimensional (2D) 13C solid-state NMR experiments were carried out on 13C-labelled microalgae. Isolated constituents were also used to facilitate resonance assignments.

Chlamydomonas reinhardtii is a green microalga protected by a cell wall which protein composition and structure have common features with higher plants and the mammalian extracellular matrix. Indeed, its cell wall is mainly composed of hydroxyprolines-rich glycoproteins. Using 2D-INADEQUATE and 2D-TOBSY experiments, we were able to assign several amino acids and identify saccharide links. Moreover, highly crystalline, amorphous, and hydrated regions in the cell wall could also be distinguished. Altogether, our solid-state NMR approach allowed revealing both the molecular and dynamic complexity of the cell wall.

Chlamydomonas reinhardtii uses starch grains as energy reserves. Starch is the most important energy storage molecule in plants and is, with cellulose, the most abundant polysaccharide in nature. It is made of amorphous and crystalline domains that can be mainly found into A and B-type structures. Using complementary high-resolution 2D 13C solid-state NMR, we established complete and unambiguous assignments for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. We were able to assign unreported non-reducing end groups, and to assess starch chain length, crystallinity, structure, hydration, and amylose content. Information on the dynamics and conformational disorder, in particular in the amorphous state, was also obtained.

This work illustrates how high-resolution solid-state NMR enables the detection and identification of starch and cell wall constituents in situ in intact cells, thus eliminating time consuming and potentially altering purification steps. We thus provide a solid basis for the NMR study of starch and cell-wall structures, and their chemical modifications or biosynthesis in living microorganisms, making in situ solid-state NMR a powerful tool to study molecules directly in the cell.

Exposing Small-molecule Nano-entities By An NMR Relaxation Assay


Yann Ayotte1, Victoria Marando2, Louis Vaillancourt2, Patricia Bouchard2, Gregory Heffron3, Paul W. Coote2,3, Sacha T. Larda2, Steven R. LaPlante1
1INRS - Institut Armand-Frappier 2NMX Research and Solution 3Harvard Medical School

Small-molecules can self-assemble in aqueous solution into a wide range of nano-entity types and sizes (dimers, n-mers, micelles, colloids, etc), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to non-specific binding, off-target effects, and false-positives and -negatives. Here, we demonstrate the use of the T2-CPMG relaxation NMR experiment which is sensitive to molecular tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone tumbling molecules versus nano-entities of various sizes. The technique is highly sensitive to chemical exchange between single-molecule and aggregate states, and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in solution behavior for compounds within structurally related series’, demonstrating structure–nano-entity-relationships. This practical experiment should be a valuable tool to support drug discovery efforts.

Expression and purification of CsgA, a functional amyloid composing bacterial biofilm


Dominic Arpin1, Steve Bourgault1, Denis Archambault1
1Université du Québec à Montréal

Amyloids are protein assemblies that were historically associated with degenerative diseases such as Alzheimer’s and Parkinson’s. However, an increasingly growing number of amyloids are not disease-associated and have beneficial biological functions in host organisms, including melanin biosynthesis in humans. Functional amyloids are the result of various pathways allowing controlled aggregation of monomers into fibrils with typical cross β-sheet quaternary structure. In bacteria, curli is a functional amyloid found in the extracellular matrix and is the main structural component of the biofilm found in many colony-forming species. In this study, we aim to harness the unique auto-assembly properties and structural stability of CsgA, a functional amyloid in the biofilm of E. coli, to form amyloid fibrils that would serve as a proteic scaffold, or carrier, for proteins with potential biomedical applications. Recombinant chimeric proteins containing CsgA were produced in E. coli. Expression yields were optimized through an extensive screening of growth conditions. To obtain the highest purity, a two-step affinity chromatography approach was used with an engineered E. coli strain. Pure CsgA monomers spontaneously form fibrils in aqueous buffer. This assembly process was monitored with circular dichroism spectroscopy, thioflavin T (ThT) fluorescence and atomic force microscopy. In vitro cell-viability assays were performed with freshly purified monomers and mature fibrils. The results revealed a structural transition from a random coil to β-sheet secondary structure with a ThT fluorescence plateau reached after 24 hours of incubation. In contrast with pathological amyloids, both CsgA monomers and fibrils show weak toxicity on J774A.1 cells.

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


Fathima Mohideen1, Joel Richard1, Nathalia Kravchenko1, David Kwan1
1Concordia University

Natural products and their derivatives play a major role in pharmaceutical industry. Many of these small molecule natural products produced by plants and bacteria contain sugar moieties that have a great significance upon their biological activity. An example of one class of such molecules are the anthracyclines which include the anticancer doxorubicin and its semisynthetic derivative epirubicin. These natural product glycosides are biosynthesized by action of glycosyltransferases (GTs). To modify or improve the bioactivity of these molecules by altering glycosylation, enzymatic engineering, in vivo and in vitro enzymatic methods could provide an attractive alternative to multistep, labor-intensive derivatization by organic synthesis. This aim would facilitate by screening and engineering GTs to produce modified glycosides. Thus, we 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.

Gesicles: a promising nucleic acid delivery tool.


Mathias Mangion1,2,3, Alexandre Audy1,2,3, Igor Slivac1,2,3, Jacques Tremblay3,4, Rénald Gilbert5, Bruno Gaillet1,2,3
1Université Laval 2PROTEO 3ThéCell: FRQS Cell and Tissue Therapy Network 4Laboratory of Human Genetic, Unit CHUL-CHUQ 5Human Health Therapeutics Portfolio, National Research Council Canada, Montréal, Canada

Gesicles are a promising tool to deliver DNA and RNA into primary cells. Gesicles are small cellular vesicles containing, across their membrane, the envelope glycoprotein of the vesicular stomatitis virus (VSV-G). In presence of polybrene, Gesicles were able to deliver plasmids in several animal cells. Unfortunately, their production, characterization and use for nucleic acid delivery are poorly documented. First, best parameters to transfect the producer cells for VSV-G expression were identified in static culture. Then, a time course of Gesicles production was assessed. Interestingly, we demonstrated that specific mutations in the VSV-G sequence allow a Gesicle production. Transmission electron microscopy of Gesicles revealed spherical particles with membrane mimicking enveloped viruses. Immuno-gold staining confirmed that Gesicles contain VSV-G. Dynamic light scattering analysis reported that Gesicle production have a mean size of 140 nm in a PBS solution. Gesicle proteomic analysis identified 877 human proteins originating from the producer cells. These proteins are mainly represented by enzymes (52%), cytoskeleton (19%), receptors (15%) and ribosomal (12%). The analysis also identified 692 bovine proteins from the serum used during cell culture process. Then, parameters involved in the DNA-Gesicles delivery such as component concentrations, incubation time, assembling order, volume and nature of solutions were studied to determine the simplest transfection method. Stability assays also demonstrated robustness of Gesicles to several freezing and thawing cycles and long term storage at +4°C, -20°C and -80°C. Finally, transfections experiments delivered plasmids in 70% of HEK293 cells, 55% of HeLa cells and 22% of human myoblasts without cytotoxicity. Gesicles also delivered large plasmids. Furthermore, siRNA-induced gene silencing was successfully achieved by a 58%-inhibition of over-expressed reporter gene. In conclusion, Gesicles are a promising tool for nucleic acid delivery under further optimization which could be useful for several applications oriented toward cell and gene therapies.

GFP-based Biosensor to Detect Transiently Expressed Proteins


Matthew G. Eason1, Antonia T. Pandelieva1, Safwat T. Khan1, Guido F. Calderini1, Hernan G. Garcia2, Roberto A. Chica1
1Department of Chemistry and Biomolecular Sciences, University of Ottawa 2Department of Molecular & Cell Biology, UC Berkeley

Green fluorescent protein (GFP) fusion tags are commonly used to study protein expression and cellular localization in vivo. However, GFP must undergo an autocatalytic post-translational modification, known as chromophore maturation, to become fluorescent, a process that can have a half-time longer than 30 minutes inside research model animals. The timescale of chromophore maturation in GFP is thus slower than many key biological processes, limiting its usefulness in measuring those processes. To address this issue, we have developed a biosensor based on a fully matured but dim GFP. Upon specific binding of a small (15.5 kDa) protein to the sensor, full fluorescence is restored. Thus, by genetically fusing this small protein to a protein of interest, it can be detected by our biosensor as soon as folding is complete, without any maturation delay. Our sensor has been validated in vitro and by flow cytometry in E. coli cells, and we have found that the fluorescence signal increases four-fold upon binding, with a Kd of 170 ± 30 nM and a kon of roughly 0.3 µM-1s-1, allowing detection of the protein of interest on the sub-second timescale. We have also created a yellow version of the biosensor, and are working on developing orthogonal binding pairs, as well as additional colours of sensors, to allow them to be used in multiplex experiments. Our biosensor opens the door to the study of short-timescale processes in research model animals, such as Drosophila embryogenesis, which we are currently investigating.

HOMODIMER INTERFACE MUTATIONS OF HUMAN GALECTIN-7 ALTER ITS BIOLOGICAL ACTIVITY


Ngoc Thu Hang PHAM1, Myriam Létourneau1, Marlène Fortier1, Carolina Perusquía Hernández1, Marie-Aude Pinoteau1, Jacinthe Gagnon1, Philippe Egesborg1, David Chatenet1, Yves St-Pierre1, Charles Calmettes1,2, Nicolas Doucet1,2
1Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique (INRS), Université du Québec, 531 Boul. des Prairies, Laval, QC, H7V 1B7, Canada. 2PROTEO, le regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines, Université Laval, 1045 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada.

Human galectins are b-galactoside binding proteins subdivided into three groups in accordance with their structural organization: tandem repeat, chimera, and prototype. Among these galectins, prototype galectin-7 (GAL-7), characterized by a homodimeric molecular organization of its carbohydrate recognition domain (CRD), is involved in different types of cancer, including carcinomas, lymphomas and melanomas. Its overexpression in tumor cells not only confers resistance to cell death stimuli, but extracellular GAL-7 also induces apoptosis of lymphocytes, abrogating immune system response against tumor antigens. Consequently, GAL-7 is a promising target for cancer therapy. To this day, the development of GAL-7 modulators has almost exclusively focused on small-molecule Glycan Binding Site (GBS) inhibitors aimed at perturbation of glycoreceptor interactions. However, due to high GBS similarity among different galectin homologs, this remains a high risk strategy because of unwanted off-target effects on other beneficial anti-tumor galectins. Furthermore, GBS inhibitors are ineffective at targeting glycan-independent function of GAL-7. New approaches are thus required to develop effective and highly specific GAL-7 inhibitors. Prior structural investigations of ancestral galectins have suggested that stabilization of their oligomeric state through evolutionary pressure improves ligand affinity and biological function. Since destabilization of GAL-7 architecture could potentially alter its affinity towards glycoproteins and biological function, our main research objective is to dissect the molecular importance of GAL-7 homodimer formation in cellular function. In this study, we will present the impact of homodimer interface mutations on protein stability and induction of Jurkat T-cell apoptosis.

Identification de biomarqueurs en relation avec PACE4 pour le cancer de la prostate


Amanda Toupin1, Roxane Desjardins1, Anna Kwiatkowska1, Robert Day1
1Institut de pharmacologie de Sherbrooke (IPS), département de chirurgie/service d’urologie, Faculté de médecine et des sciences de la santé (FMSS), Université de Sherbrooke, Canada

Touchant 21 300 hommes par année au Canada, le cancer de la prostate est classé comme étant le troisième des plus mortels. La proprotéine convertase PACE4 s’est avérée être une cible thérapeutique intéressante pour inhiber la prolifération du cancer de la prostate. Notre étude vise à découvrir des biomarqueurs dans le sang ou l’urine pour suivre l’activité de PACE4 qui n’est quantifiable que dans les tissus prostatiques. Ces biomarqueurs permettraient d’identifier les patients qui auront une récurrence biochimique, soit une augmentation de l’antigène prostatique spécifique (APS), représentant 40% des cas à la suite d'un traitement par prostatectomie. Dans 50% des cas de récurrence biochimique, le cancer passera d’une phase hormono-sensible à une phase résistante à la castration (CPCR) menant à la formation de métastases dans 60% des cas. Ces biomarqueurs reflétant l’activité de PACE4 permettront de prédire quel CPCR mènera à la formation de métastase. La découverte de tels biomarqueurs permettrait d’appliquer une médecine personnalisée dès l’apparition du cancer de la prostate.  

 

Une approche par spectrométrie de masse sera utilisée pour déterminer une liste de biomarqueurs potentiels. Deux lignées cellulaires caractéristiques au cancer de la prostate soit les LNCaP et Du145 seront inhibées au niveau de la PACE4 pour distinguer les protéines ayant une importante variation d’expression en réponse à cette altération. L’inhibition de PACE4 sera effectuée en traitant les cellules avec des shARN, des siARN ou par le traitement au C23 ou encore au multi-leu qui sont tous deux des inhibiteurs peptidiques développés par notre laboratoire. Ces différentes conditions seront analysées par spectrométrie de masse en utilisant l’acquisition SWATH qui enregistre les spectres MS/MS de tous les peptides présents dans les échantillons. Les multiples fenêtres d’acquisition permettent de sélectionner les ions compris dans une gamme de rapport masse sur charge (m/z) précis pour être fragmentés et quantifiés. Une déconvolution des rapports m/z identifiera quels ions produits proviennent de quels ions parents. Ce mode d’acquisition permet de réduire la complexité des échantillons et de limiter l’interférence des protéines plus abondantes et qui pourraient cacher les signaux plus faibles. En conservant seulement les protéines communes aux quatre différentes techniques d’inhibition, il sera possible d’éliminer une grande proportion de résultats faussement positifs et donc de ne conserver que celles dont la variation sera causée par l’inhibition de la PACE4.

 

Puisqu’il y a un grand nombre de molécules à analyser, il est important d’avoir une procédure de sélection stricte et rigoureuse. Une fois les analyses par spectrométrie de masse réalisées, il est essentiel d’effectuer des tests statistiques pour observer les variations d’expression des cibles potentielles. Les molécules ayant une variation de quatre fois ou plus sont examinées sur des banques de données, ce qui permet de choisir les molécules ayant les caractéristiques les plus avantageuses comme biomarqueurs reliés à la PACE4. À long terme, ces biomarqueurs potentiels pourront être validés dans l’urine, le sang et les tissus de patients atteints de différents stades du cancer de la prostate.

Identification des protéines TAM dépendantes chez Pseudomonas aeruginosa


Hamid Zeneba1, Jihen Ati1, Marie-Christine Groleau1, Éric Déziel1
1INRS-Institut Armand-Frappier

La membrane externe participe à de nombreuses fonctions importantes aux cellules bactériennes et joue un rôle majeur dans les interactions hôte-pathogène. En effet, les protéines de la membrane externe participent à de nombreuses fonctions telles que l’adhésion, le transport des nutriments et des macromolécules, la communication, la pathogenèse bactérienne, et la division cellulaire. La plupart des protéines de la membrane externe sont synthétisées dans le cytoplasme, puis transférées vers le périplasme via la membrane interne avant d’être redirigées vers la membrane externe où elles sont reconnues, assemblées et insérées dans la bicouche lipidique. L’assemblage et l’insertion de ces protéines nécessitent la machinerie d’assemblage des tonneaux beta (BAM). Cependant, un second complexe d’assemblage des protéines transmembranaires appelé module de translocation et d’assemblage (TAM) a été récemment identifié chez de nombreuses protéobactéries. Il est composé de deux sous-unités TamA et TamB formant un complexe trans-enveloppe. TamA est une protéine de la famille des Omp85 ancrée dans la membrane externe, et est homologue à la protéine BamA. TamB est ancrée à la membrane interne par une hélice N-terminale transmembranaire, et ne possède pas d’homologue connu. L’association de TamA et TamB permet le repliement et l’assemblage spécifique de certaines protéines de la membrane externe, cependant la gamme de protéines TAM-dépendantes demeure inconnue. La délétion du complexe TAM chez les bactéries Salmonella enterica, Klebsiella pneumonia, Proteus mirabilis et Vibrio fischeri entraine un défaut de colonisation et une perte de virulence, suggérant que le complexe TAM est un élément important dans la virulence de plusieurs bactéries pathogènes à Gram négatif. Nos travaux préliminaires réalisés chez Pseudomonas aeruginosa, un important pathogène impliqué dans les infections nosocomiales, montre qu’une mutation par insertion de transposons inactivant le gène tamA ou tamB entraine un défaut de formation de biofilm, un trait de virulence majeur de la bactérie. Le but de ce projet est d’identifier les protéines sécrétées et insérées dans la membrane externe par le module TAM, et de définir leurs fonctions moléculaires. Les protéines requérant TAM pour leur assemblage pourraient être d’importants facteurs de virulence et de ce fait constituer des cibles potentielles pour de nouveaux traitements antimicrobiens contre les infections à P. aeruginosa.

Identification of promising angiogenin allosteric modulators by screening of a chemical compound library


Marie-Aude Pinoteau1, Myriam Létourneau1, Steven R.LaPlante1,2, Nicolas Doucet1,2
1INRS - University of Quebec 2PROTEO

Angiogenin (ANG) is a pancreatic-type ribonuclease that has been shown to regulate various biological processes, including cell growth, survival and angiogenesis. High tissue levels of ANG have been linked to cancer progression and poor survival prognosis. Like other family members, ANG (or RNase 5) catalyzes RNA cleavage with an enzymatic activity that appears to be essential for promoting angiogenesis. Interestingly, ANG is the only family member exhibiting angiogenic activity and the only angiogenic factor displaying ribonucleolytic activity. Although targeting its active site could be an attractive strategy for drug development, obtaining highly specific ANG inhibitors without off-target effects for the highly similar binding pockets of closely related family members remains an important challenge. To overcome this limitation, allosteric modulation has been considered as a promising option since allosteric inhibitors can trigger long-range functional alteration of enzyme activity with high specificity afforded by targeting less conserved protein regions. This study aimed at finding small-molecule binders of ANG by screening a chemical compound library using NMR and to evaluate candidates for their ability to modulate the biological activity of ANG. From a small fragment library of 1640 compounds, 4 hits showed clear differences in 1H NMR spectra in the presence of ANG. Binding affinity was determined by NMR titration of 15N-labelled ANG using 1H-15N HSQC spectra, revealing Kd values in the low mM range. The 4 compounds were then tested for their ability to modulate ANG stability, ribonucleolytic activity and cell growth. A protein thermal shift assay revealed that ANG was not destabilized in the presence of these compounds. Moreover, these 4 hits did not inhibit tRNA cleavage but modulated HeLa (human epithelial carcinoma) and SH-SY5Y (human neuroblastoma) cell proliferation, supporting binding outside the catalytic pocket of the enzyme. Although binding affinity is low, these 4 compounds could serve as scaffolds for the development of allosteric modulators of ANG activity.

Immune Response of Small Drug-like Molecules: Influence of Self-aggregation


FATMA SHAHOUT1, Steven laplante2
1INRS university 2INRS-IAF

Many small drug-like molecule currently used to treat cancer can spontaneously self-assemble into aggregates in aqueous solution at micromolar concentrations. These small-molecule aggregators can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immune response of small-molecule aggregators is, therefore, an important issue. There is an evidence that aggregation of biotherapeutic proteins and nanoparticles has the potential induce an immunogenic response; however, immunogenicity of small molecule aggregators has not been yet reported. Here, we show that colloidal anticancer drugs, formed at 37 °C, can enhance the in vitro immune response of a murine macrophage (RAW 264.7) cells and human peripheral blood mononuclear cells (PBMCs) compared to non-colloidal forming drugs and inherent immunogenicity of the monomer form. This response depended on the aggregate type and size of colloids. We propose a cytokine signature including IL-6 and TNF-α as a potential biomarker of the in vitro murine macrophage and human (PBMCs) cells response to aggregates. Large colloidal aggregates including lapatinib and erlotinib induced the highest response compared with intermediate colloidal aggregates and non-colloidal and monomer forms. With an increased understanding of small-molecule aggregation enhanced immune responses, it may be possible to develop improved manufacturing and screening processes to avoid.

Implication des chaines de glycosaminoglycanes membranaires dans la toxicité et l’auto-assemblage d’un peptide amyloïdogénique


Mathilde Fortier1,2, Noé Quittot1,2, Mathew Sebastiao1,2, Phuong Trang1,2, Steve Bourgault1,2
1Department of Chemistry, CERMO-FC, Université du Québec à Montréal, Montreal OC, Canada, H3C 3P8 2Quebec Network for Research on Protein Function, Engineering and Applications, PROTEO

Un grand nombre de peptides et de protéines sont reconnus pour leur capacité à s'auto-assembler en fibres amyloïdes, dont leur déposition tissulaire est associée à des maladies dégénératives, telles que le diabète de type II (T2D) et la maladie d'Alzheimer (AD). Communément à ces pathologies, la mort cellulaire et la dégénérescence tissulaire seraient principalement induites par des intermédiaires de la cascade amyloïdogénique et non par la déposition des fibres amyloïdes. Ceci est connu sous l’hypothèse des oligomères toxiques. En outre, de nombreuses macromolécules biologiques tels que les lipides, d’autres protéines ainsi que les glycosaminoglycanes (GAGs) peuvent moduler l’auto-assemblage des polypeptides amyloïdogéniques, et pourraient ultimement, modifier leur toxicité. Par exemple, les GAGs sont universellement associés aux dépôts amyloïdes pathologiques et accélèrent in vitro l’auto-assemblage des polypeptides amyloïdogéniques. Cependant, leurs rôles dans les mécanismes de cytotoxicité associées à l’auto-assemblage de polypeptides amyloïdogéniques restent à élucider. Dans ce contexte, ce projet vise à étudier si la présence des GAGs dans un environnement complexe membranaire a un impact sur l’auto-assemblage et la cytotoxicité de polypetides amyloïdogéniques. Le peptide islet amyloid polypeptide (IAPP), dont le dépôt pancréatique est associé au diabète de type II, a été utilisé comme polypeptide amyloïdogénique modèle. Dans un premier temps, nous avons évalués l’implication des GAGs dans la cytotoxicité associée à l’auto-assemblage de l’IAPP à l’aide de deux modèles cellulaires, l’un présentant des GAGs à la surface membranaire et l’autre en étant dépourvu. Dans un second temps, pour évaluer biophysiquement la contribution des GAGs dans l’auto-assemblage de l’IAPP en environnement biologique complexe, des vésicules géantes de membrane plasmique (VGMP) ont été formées à partir des deux types cellulaires. Ainsi, les cinétiques d’auto-assemblage de l’IAPP en présence des deux types de VGMP ont été évaluées via des essais de fluorescence. Parallèlement, la perturbation membranaire des VGMP en présence de l’IAPP a été déterminé par microscopie confocale. Les différentes approches développées dans ce projet permettent de mettre en évidence que la présence de GAGs dans environnement biologique complexe a un impact sur la cytotoxicité et l’auto-assemblage de l’IAPP. Finalement, ces approches pourraient être transposées à de nombreux polypeptides amyloidogéniques et permettre une meilleure compréhension en général des mécanismes de toxicité des maladies dite amyloïdes.

Importance of the β5−β6 Loop for the Structure, Catalytic Effi-ciency, and Stability of the Carbapenem-Hydrolyzing Class D beta-lactamase Subfamily OXA-143


Denize Favaro1,8, Víctor U. Antunes1, Edgar E. Llontop3, Ronaldo J. Oliveira4, Fernanda N. da Costa Vasconcelos5, Yossef López de los Santos6, Nicolas Doucet6, Anthony Mittermaier8
1University of Campinas 2University of Campinas 3University of Sao Paulo 4Universidade Federal do Triângulo Mineiro, 5University of Florida 6INRS-Institut Armand-Frappier 7INRS-Institut Armand-Frappier 8McGill University

The presence of Carbapenem-hydrolyzing Class D ß-lactamases (CHDLs) is frequently associated with outbreaks of the clinically challenging Gram-negative ROD genus Acinetobacter baumannii.CHDLs ß-lactamases, which are also known as OXA-enzymes due to their preferential hydrolysis of oxacillin, are serine nucleophile-based catalysts which have an unusual carboxylated lysine that acts as a general base in a covalent catalysis mechanism.  OXA enzymes share highly conserved motifs (STFK and SAV) and structural elements: the P-loop (residues 95−119); the Ω-loop (residues 154−174), and the β5−β6 loop (residues 221−230). The three loops are located near the antibiotic binding pocket and influence substrate specificity. To investigate the importance of the β5−β6 loop to the subfamily OXA-143, we describe here an investigation of the enzymatic activities of these three enzymes toward the antibiotics: ampicillin (AMP), cloxacillin (CLO), meropenem (MER), aztreonam (AZT) and ceftazidime (CTZ). In addition, we also report the biophysical and biochemical parameters associated with the observed differences. The most important outcomes of our results are the following: i) crystallographic structure of the enzyme OXA-231; ii) thermal stability of OXA-143, OXA-231, and OXA-143(P227S) in the presence and absence of bicarbonate; iii) complete catalytic parameters of the three enzymes towards ampicillin, cloxacillin, meropenem and ceftazidime; iv) Molecular Docking showing the most important residues for the complexes stabilization, and v) Molecular Dynamics describing the effect of the mutations for the electrostatic interactions and dynamics of the enzymes. 

Increasing the genome-targeting scope of base editing using Streptococcus thermophilus CRISPR1-Cas9


Minja Velimirovic1, Daniel Agudelo1, Yannick Doyon1
1Université Laval

Majority of known human genetic variants associated with disease are due to point mutations. Using CRISPR-Cas technology to correct point mutations cleanly and efficiently has shown to be difficult because of the low homology-directed repair efficiencies (HDR) and genotoxicity due to double-strand break (DSB) induction. Alternatively, base editors enable the programmable conversion of one base pair into another without induction of DSB, excess stochastic insertions and deletions, or dependence on HDR.

A limitation of the current base editing technology is that the PAM must be appropriately positioned relative to the target base to ensure efficient editing. Thus, there is a need to develop base editors with additional PAM compatibilities to increase the number of targetable bases in a genome.

Here we report the development of cytosine base editors (CBEs) based on Streptococcus thermophilus Cas9 (St1Cas9) with the aim of expanding the choice of sequences that can be targeted. We characterized functional PAM sequences (NNAGAA and NNGGAA) for efficient base editing in human cells with St1Cas9 derived from the LMD-9 strain. In addition, we identified St1Cas9 variants isolated from different strains of S. thermophilus, that display unique PAM specificities. These variants are also active when converted to CBEs and enable robust editing of endogenous sites in human cells not currently targetable by existing base editors. By expanding the repertoire of natural Cas nucleases with different PAM requirements the desirability of base editor variants with narrower activity window increases and reduces the bystander editing.

Another step toward overcoming the current Cas9 targeting range restrictions that we describe here is characterization of St1ABEmax variants, a series of adenine base editors (ABEs) with distinct PAM specifities.

Undoubtedly, these base-editing approaches expand the molecular toolbox to potentially treat human genetic diseases that arise due to single-base alterations.

Influence of nucleotide modifications at the C2′ position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA


William Copp1, Alexey Denisov1, Jingwei Xie2, Anne M. Noronha1, Kalle Gehring2, Christopher Wilds1
1Concordia University 2McGill University

Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2′-deoxyribose, 2′-O-methyl-ribose, 2′-deoxy-2′-fluoro-ribose, arabinose and 2′-deoxy-2′-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with DNA poly(A) oligomers. Arabinose and 2′-deoxy-2′-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2’-O-methyl and 2′-deoxy-2′-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions, respectively. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of RNA poly(A) duplexes with a single DNA residue by NMR spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts, and changes in hydration determine duplex stability.

 

References: [1] Rich, A.; Davies, D. R.; Crick, F. H. C.; Watson, J. D., J. Mol. Biol. 1961, 3, 71–86. [2] Safaee, N.; Noronha, A. M.; Rodionov, D.; Kozlov, G.; Wilds, C. J.; Sheldrick, G. M.; Gehring, K., Angew. Chem. Int. Ed. 2013, 52, 10370–10373. [3] Copp, W.; Denisov, A. Y.; Xie, J.; Noronha, A. M.; Liczner, C.; Safaee, N.; Wilds, C. J.; Gehring, K., Nucleic Acids Res. 2017, 45, 10321–10331.

Inhibition and Activation of Kinases by ADP, Revealed by Isothermal Titration Calorimetry (ITC)


yun wang1, Anthony Mittermaier2
1McGill University 2McGill University

Kinases are widely distributed in nature and are implicated in many human diseases, thus an understanding of their activity and regulation is of fundamental importance. It has been previously reported that several kinases are inhibited by ADP, however thorough investigation of this phenomenon is hampered by the lack of a simple and effective assay for studying enzyme/ADP interactions.  Here we show that isothermal titration calorimetry has great potential as a general approach for measuring the effects of ADP on kinase activity. In applications to an aminoglycoside phosphotransferase (APH(3’)-IIIa) and pantothenate kinases from E. coli (EcPanK) and P. aeruginosa (PaPanK), we found ADP to be an efficient inhibitor, with Ki values similar to or lower than the Km values of ATP. For EcPanK, ADP was an activator at low concentrations and an inhibitor at high concentrations. This unusual effect was quantitatively modelled in terms of cooperative interactions between the two subunits of the dimeric enzyme. At typical bacterial intracellular concentrations of ATP and ADP, all three kinases are partially inhibited by ADP, an effect which could allow their activities to respond to changes in the levels of both metabolites.

Investigation of the functional specificity of adaptor proteins NCK1 & NCK2


Kevin Jacquet1,2, François Chartier1, Sara Banerjee1,2, Nicolas Bisson1,3
1CHU de Québec 2Université Laval 3Université Laval

Adaptor proteins NCK1 and NCK2 (NCKs) relay signals from cell surface receptors and serve as hubs to assemble the specific protein complexes that are required to activate intracellular signalling pathways. Although NCK1/2 functions are often considered redundant, we hypothesized that each displays functional specificity. We used AP-MS and BioID proximity labelling to delineate NCK1/2-specific signalling networks. The combination of these approaches allowed the identification of 98 interactions for both NCKs. Strikingly, we found 30 proteins restricted to NCK1 and 28 specific to NCK2. Bioinformatics analyzes of the functions of the NCK-associated proteins highlighted that NCK2-restricted partners are more specifically involved in the regulation of the actin cytoskeleton organization. Investigation of this distinctive feature using cell division assays revealed a previously uncharacterized and exclusive function for NCK2 in cell abscission during cytokinesis. We further characterized the molecular determinants that generate NCK1/2 selectivity toward specific targets. We determined that a subset of these targets including PKP4, a key regulator of cytokinesis, were able to bind directly and exclusively to NCK2. We showed that PKP4 association with NCK2 is dependent on a functional Src-Homology (SH)2 domain on the latter. Strikingly, this stays true only in the context of full-length adaptors, as isolated NCK1/2 SH2 domains are both able to bind to PKP4. Our preliminary experiments suggest that the selectivity of the association originates from low homology inter-SH3/ SH2 domains regions. This finding highlights how protein context affects NCK1/2 adaptors SH2 function and may contribute to explaining their specific interaction profiles and non-redundant functions.

Investigation phytochimique de lichens nordiques Cladonia Stellaris et Mitis


meggan beaudoin1,2, Normand Voyer1,2
1Université Laval 2PROTEO

Identification et caractérisation de l'activité biologique de produits naturels de lichens nordiques

 

Les organismes vivants des écosystèmes du Grand Nord du Québec subissent des stress uniques. En sus, les changements climatiques menacent certaines espèces. Par exemple, certains lichens peuvent disparaitre par l'action d'espèces arbustives qui poussent de manière abusive. Il y a donc urgence d'étudier la composition chimique d'organismes nordiques pour découvrir des composés bioactifs originaux qui pourraient éventuellement servir pour le développement de nouveaux médicaments.

 

Ce projet vise à effectuer une investigation phytochimiques des lichens Cladonia Stellaris et Cladonia mitis dans le but d'identifier leurs métabolites secondaires et ainsi de valoriser la flore nordique. La méthodologie utilisée pour l’isolement, la purification et la caractérisation des produits sera présentée. De même, nous présenterons les résultats de tests biologiques visant à déterminer l'activité anti-oxydante, anti-tyrosinase et anti-glucosidase des extraits et des métabolites secondaires purifiés. Les résultats attendus de ce projet sont la découverte de nouveaux produits naturels bioactifs et la détermination du profil en métabolites secondaires de chacun des lichens étudiés.

Is hybridization an adaptive force in response to DNA damage?


Carla Bautista Rodríguez1,2,3, Souhir Marsit1,2,3, Christian R Landry1,2,3
1Institut de Biologie Intégrative et des Systèmes 2PROTEO 3Université Laval

The environment is heterogeneous and includes extreme conditions such as high ultraviolet (UV) radiation. High UV radiation represents a particular challenge in terms of adaptability because it affects the viability of organisms by directly damaging the genome (Torres et al., 2013) while also challenging future generations by increasing mutation rate. Extremophiles thriving in high UV radiation conditions have been reported (Lidzbarsky et al., 2009), suggesting that adaptation to these conditions is possible. It has recently been proposed that hybridization may facilitate the colonization of new ecological niches and increase the probability of evolutionary rescue. This could be caused by increased genome plasticity in hybrids, as reflected by alterations at the nucleotide and chromosomal levels, which could boost adaptability in stressful environments (Lopandic, 2017). We directly test if hybridization promotes adaptive evolution in response to DNA damage, here represented by conditions that mimic UV radiation using Saccharomyces yeast hybrids as models. We exposed hybrid and parental strains to UV-mimetic conditions for 200 generations and measured growth continuously. Although we found that adaptation occurs in most strains, contrary to our expectations, yeast hybrids achieved a similar adaptation potential than the two parental lines. We hypothesize that this result is caused by increased genome instability in hybrids that is further enhanced by the UV conditions, preventing adaptation. We are currently examining the instability of the hybrids by measuring ploidy changes along the genome. Altogether, our results show that hybridization may reduce adaptive potential in extreme conditions if these conditions accelerate genome evolution.

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


Guichi Zhu1, Carl Prévost-Tremblay2, Dominic Lauzon3, Alexis Vallée-Bélisle2
1Université de Montréal 2Université de Montréal 3University of Montreal

Nature has developed many one-pot reactions where multiple reactions are sequentially programmed to achieve complex synthesis within a single location and condition. Here, 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 detection time. Using this strategy in an electrochemical format, we demonstrate the one-step detection of small molecule and protein in less than 5 minutes directly in whole blood.

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


Xiaolin YAN1,2, Marie-France Lebel-Beaucage3, Samuel Tremblay1,2, Gary Shaw4, Dror Warschawski5, Élodie Boisselier1,2
1Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval 2CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec 3Département de chimie, biochimie et physique, Faculté des sciences, Université du Québec à Trois-Rivières 4Département de biochimie, Faculté de RMN biomoléculaire, Université de Western Ontario 5Département de chimie, Faculté de sciences, Université de Québec à Montréal

CONTEXTE ET OBJECTIF : La protéine S100A10 pourrait être un biomarqueur précoce du développement du diabète, conduisant à la rétinopathie diabétique. Le complexe protéique S100A10/annexine A2 permettrait le recrutement 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 disponible sur la liaison membranaire de la S100A10 et du peptide du C-terminal d’AHNAK de ce complexe. Nous étudions leur liaison membranaire 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. La stabilité de sa structure secondaire a été analysée par dichroïsme circulaire. Également, un peptide du C-terminal d’AHNAK a été synthétisé. Ensuite, le modèle des monocouches de Langmuir a été utilisé pour mimer les membranes cellulaires et caractériser l’interaction des protéines avec différents phospholipides les composant. L’affinité de ces protéines pour les différentes têtes polaires a été étudiée par la RMN solide en collaboration avec le Dr Dror Warschawski. La profondeur d’insertion des protéines dans des monocouches lipidiques sera déterminée par ellipsométrie. 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.  RÉSULTATS : L’optimisation de la surexpression et la purification de la S100A10 a été effectuée. L’identification de la protéine et des études de stabilité ont aussi été complétées. 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. Les résultats préliminaires de RMN solide confirment cette préférence du peptide d’AHNAK à 37 °C. L’étude par ellipsométrie du peptide d’AHNAK est actuellement en cours. CONCLUSION : Ce projet permettra de développer les connaissances 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 aidera à mieux déterminer leurs rôles dans la réparation membranaire ainsi que dans les autres mécanismes physiologiques auxquels ces protéines participent.

Laccase identification from the native ligninolytic basidiomycete Dictyopanus pusillus


Andres Rueda1, Yossef Lopez de los Santos1, Myriam Letourneau1, Antony Vincent1, Clara Sánchez3, Daniel Molina3, Sonia Ospina2, Nicolas Doucet1
1INRS - University of Quebec 2Universidad Nacional de Colombia 3Universidad Industrial de Santander

Basidiomycete fungi and their enzymes can oxidize lignin from wood, offering an environmental-friendly alternative for the pretreatment and exploitation of lignocellulosic biomass for second-generation bioethanol production. The use of ligninolytic enzymes in this context is still underrepresented due to enzyme production costs and low thermal and pH stability. Consequently, bioprospection of native basidiomycetes would be key to finding new and robust ligninolytic enzymes to be used in the industrial pretreatment of lignocellulose for cellulosic ethanol production. Here, we performed bioprospection of native Colombian basidiomycetes exhibiting ligninolytic activity and explored their genome to identify laccase enzymes. Selection of ligninolytic activity was achieved using solid-state fermentation and delignification capacity of enzymatic extracts was evaluated against lignocellulose from oil palm. Isolation of native Dictyopanus pusillus exhibiting relevant laccase activity was also achieved. Crude enzymatic extracts from D. pusillus displaying laccase activity were used to synthesize reducing sugars from lignocellulose pretreatment. Genomic exploration of D. pusillus revealed the existence of 13 potential laccase genes. To the best of our knowledge, this work represents the first genomic exploration of the Dictyopanus genera, further emphasizing the importance of Dictyopanus pusillus as a promising new ligninolytic species. These results also illustrate that laccases are promising green alternatives for the pretreatment of lignocellulose biomass.

Le complexe du pore nucléaire de la levure comme système modèle pour l'étude des déterminants de la trajectoire évolutive suivie par les gènes dupliqués


Simon Aubé1,3,4, Axelle Marchant2,3,4, Alexandre Dubé1,2,3,4, François Rouleau1,3,4, Isabelle Gagnon-Arsenault1,2,3,4, Diana Ascencio2,3,4, Philippe Després1,3,4, Christian Landry1,2,3,4
1Département de biochimie, de microbiologie et de bio-informatique, Université Laval 2Département de biologie, Université Laval 3Institut de biologie intégrative et des systèmes, Université Laval 4Regroupement PROTEO

La duplication de gènes est un évènement fréquent au cours de l’évolution. Par le fait même, il s’agit aussi d’une des principales sources de nouvelles protéines. Selon le modèle traditionnel, le maintien des gènes dupliqués implique habituellement une divergence fonctionnelle, que ce soit par l’acquisition de nouvelles fonctions ou par la partition des fonctions ancestrales. Cependant, il semble que les contraintes de dosage pourraient aussi avoir une influence importante sur la trajectoire évolutive suivie par les duplicats. Il a en effet été postulé qu’à la suite d’un évènement de duplication du génome complet (WGD), la rétention des deux copies d’un gène soit favorisée jusqu’à ce que l’expression de l’une d’elle ait suffisamment diminué pour que sa perte en soit neutre. Afin de contribuer à élucider les déterminants de la rétention et de la perte des gènes dupliqués, nous avons combiné l’édition génétique CRISPR-Cas9 et le criblage d’interactions protéine-protéine dans le but de reconstruire l’histoire évolutive de nucléoporines paralogues du complexe du pore nucléaire (NPC) de la levure Saccharomyces cerevisiae. Nos résultats préliminaires sur le couple NUP53/NUP59 montrent que la majeure partie des changements fonctionnels sont survenus symétriquement entre les deux copies et suggèrent que la divergence régulatrice a joué un rôle important dans le maintien de ces deux duplicats.   

Le complexe TAM et son rôle dans la biogenèse des protéines membranaires chez les bactéries Gram-négatif


Jihen Ati1, Zeneba Hamid1, Charles Calmettes1
1INRS Institut Armand Frappier


La membrane externe (ME) est l’une des caractéristiques les plus distinctives et importantes des bactéries Gram-négatives. Elle est extrêmement riche en protéines de type tonneaux β, ainsi que des protéines à ancre lipidique. Ces dernières sont impliquées dans plusieurs mécanismes biologiques tels que la protection contre l’entrée de molécules toxiques, le transport de nutriments et de protéines, la communication cellulaire, ainsi que la survie et la pathogénicité des bactéries. De ce fait, l’assemblage et l’insertion des protéines membranaires intégrales est considéré comme un processus fondamental et hautement important. Cependant, très peu est connu sur ce phénomène. Ce processus requiert le fonctionnement d’une machinerie d’assemblage des tonneaux-β (BAM) qui assure le bon repliement et l’insertion de la majorité des protéines provenant du cytoplasme en passant par la membrane interne. Cependant un sous-ensemble mineur de protéines requiert un facteur supplémentaire identifié comme module de translocation et d'assemblage (TAM). Dans cette étude, nous nous intéressons au complexe TAM qui comprend deux protéines, TamA et TamB. Ce module forme un complexe trans-enveloppe et est conservé chez le protéobactéries. Des études antérieures basées sur la suppression des gènes TamA et TamB ont démontré la réduction de la virulence de différents agents pathogènes bactériens. De ce fait, ce système est considéré comme une cible anti-infectieuse prometteuse. Afin d’élucider le mécanisme fonctionnel du complexe TAM, certains objectifs ont été fixés. Dans un premier temps, nous chercherons à mettre en évidence le lien direct que joue TamA, dont la structure est connue, dans l’assemblage et l’insertion des protéines au sein de la bicouche asymétrique de la membrane externe. Pour cela, des membranes artificielles sous forme de liposomes seront assemblées et l’insertion de deux protéines membranaires dénaturés, OMP-A et OMP-X sera évaluée en présence et absence de la protéine TamA. Cette même méthodologie a permis de mettre en évidence la fonction de son homologue BamA, qui dans notre étude sera considéré comme témoin positif. Dans un deuxième temps, nous nous intéresserons à la protéine TamB, dont la structure et la fonction restent méconnus. Résoudre la structure de cette protéine est considéré comme un grand défi due à sa taille gigantesque de 140 kDa. Afin de réaliser ces études structurales, les différents domaines de TamB seront clonés et exprimés séparément, suivi de multiples essais de cristallisations. En troisième temps, nous nous intéresserons à la structure de l’hetero-complexe TamA/TamB afin d’élucider la régulation et le mécanisme d’interaction de ce complexe. Ce projet ouvrira la voie à une meilleure compréhension du mécanisme d’assemblage des protéines de la membrane externe chez les bactéries pathogènes, et offrira de nouvelles perspectives pour trouver des cibles thérapeutiques innovantes. 
 

Les récepteurs Eph régulent la morphogénèse épithéliale


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 comme, par exemple, les récepteurs tyrosine kinase (RTK). La plus grande famille de RTK est celle des récepteurs Eph (EphR), qui sont activés suite au contact de leurs ligands membranaires nommés éphrines. La signalisation EphR/éphrines est notamment impliquée dans la régulation de la morphologie et de la motilité cellulaires. Nous avons récemment découvert par spectrométrie de masse que les EphR s’associent à des protéines requises pour l’établissement et le maintien de la polarité des cellules épithéliales. L’objectif du projet est donc d’explorer le rôle des EphR dans ces processus. Nous avons d’abord montré que cinq des quatorze EphR sont exprimés dans les cellules épithéliales Caco-2. Par la suite, nous avons optimisé une méthode de culture en trois dimensions (sphéroïdes) pour ces cellules. Nous étudions maintenant les effets de la surexpression et de la déplétion de ces récepteurs sur la morphogénèse de sphéroïdes de Caco-2. Nos résultats préliminaires suggèrent que la déplétion des récepteurs EphA1 et EphB4 perturbe la formation de sphéroïdes. Dans l’ensemble, ces études permettront de décrire un rôle pour les EphR dans la polarité des cellules épithéliales. Ceci contribuera aux connaissances sur la morphogenèse et la physiologie des tissus épithéliaux. La polarité des cellules étant perdue dans les cellules cancéreuses, nos travaux seront importants pour comprendre comment ces récepteurs contribuent aux phénotypes cancéreux.

Lobaric acid and pseudodepsidones from the lichen Stereocaulon paschale inhibits NF-κB signaling pathway


Claudia Carpentier1, Xavier Barbeau2, Jabrane Azelmat3, Daniel Grenier3, Patrick Lague2, Normand Voyer1
1Département de Chimie and PROTEO, Université Laval 2Département de Biochimie and PROTEO, Université Laval 3Faculté de Médecine Dentaire, Université Laval

Lichens produce a vast diversity of highly bioactive defence compounds in response to environmental stress to protect the symbiotic partners.1 Therefore, lichens of Northen Quebec represent a source of potential bioactive natural products due to the extreme growing conditions in the Nunavik region.

 

Chemical investigation of the lichen Stereocaulon paschale has led to the isolation and identification of two new dibenzofurans and 11 knowns lichen metabolites.2 Six pseudodepsidone-type metabolites were identified and derived from the cleavage of the depsidone linkage of lobaric acid, the major compound of the crude extract. Lobaric acid and the pseudodepsidones metabolites demonstrated significant in vitro inhibitory activity against major pro-inflammatory targets (NF-κB, TNF-α and IL-1β). Docking simulations were performed to investigate the mechanism involved. To further investigate their anti-inflammatory potency, we have developed a synthetic methodology to give access to a variety of lichens metabolites. Phytochemical investigation, inhibitory activity against pro-inflammatory targets and the synthetic methodology will be presented.


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

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

Mapping Eph receptor signaling networks via proximity-dependent biotinylation


Sara Banerjee1,2,3, Kévin Jacquet1,2,3, François Chartier1,2,3, Nicolas Bisson1,2,3,4
1Centre de Recherche sur le Cancer de Université Laval 2PROTEO 3Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec 4Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Université Laval

The Eph family of receptor tyrosine kinases (RTK) is the largest in humans. In contrast to other RTKs, Eph receptors (EphRs) cognate ligands, ephrins, are tethered to the cell surface. This results in EphRs-ephrin signaling being mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration and tissue boundary formation. Although EphRs functions have been broadly studied, the molecular mechanisms by which they mediate these processes are far from being understood.

To address this question, we sought to identify new downstream effector proteins for EphRs and to determine their requirement for EphR-regulated functions.

To unravel EphR-associated signaling complexes under native conditions, we applied a mass spectrometry-based approach, called BioID. We obtained a composite signaling network for four EphRs, namely EphA4, -B2, -B3 and -B4, which contains 188 proteins, most of which not previously linked to Eph signaling. We examined the function of several candidates in EphR-controlled biological processes by exploring the effect of a loss-of-function in a cell sorting assay. We showed that depletion of some of the candidate proteins blocks Eph-dependent cell sorting, suggesting that their function is required to transmit EphR signals. In conclusion, the definition of EphR signaling networks shed light on Eph-centered signaling events. Overall, this research will lead to a better understanding of the mechanisms by which EphRs signal at the membrane and will give insight into how a deregulation of these pathways contributes to boundary disruption in disease states.

Modulation de la multicellularité bactérienne via différents polysaccharides sécrétés.


fares saidi1, Israel Vergara2, Emilia Mauriello2, Salim Timo Islam1
1INRS- Centre Armand-Frappier 2CNRS – Laboratoire de chimie bactérienne, Marseille, France

Le développement de la multicellularité est une transition évolutive indispensable permettant une différenciation des fonctions physiologiques au sein d'une même population cellulaire, conférant des avantages en termes de survie. Chez les bactéries unicellulaires, cela peut conduire à des comportements développementaux complexes et à la formation de structures communautaires supérieures. Cependant, les connaissances concernant les déterminants de la multicellularité bactérienne sont limitées. Ici, nous démontrons chez une δ-protéobactérie Myxococcus xanthus, bactérie sociale, la sécrétion d’un nouveau polysaccharide aux propriétés biosurfactante (BPS) par un complexe protéique nouvellement identifié joue un rôle dans la régulation de migration d'essaims et de prédation d'autres bactéries, ainsi que la formation de biofilms multicellulaires et de la formation des spores. Il a été démontré que la biosynthèse du BPS titrait la propriété adhésive de l’exopolysaccharide (EPS) à la surface des cellules végétatives intervenant dans les différents types de motilité des M. xanthus via une augmentation de l’hydrophobicité. Le BPS et EPS sont produits et sécrétés via des voies protéiques Wzx/Wzy-dépendants distinct de celle responsable de l’assemblage de la couche de spores. Ces travaux montrent l’importance centrale des polysaccharides sécrétés dans des comportements complexes qui coordonnent la multicellularité bactérienne.

Molecular fingerprints determining the toxicity-functionality equilibrium of protein amyloid assemblies


Phuong Trang Nguyen1,2, Elizabeth Godin1,2, Ximena Zottig1,2, Mathew Sebastiao1,2, Steve Bourgault1,2
1Department of Chemistry, CERMO-FC, Université du Québec à Montréal, Montreal, QC, Canada, H3C 3P8 2Quebec Network for Research on Protein Function, Engineering and Applications, PROTEO

Amyloid formation and tissue deposition from endogenous proteins are implicated in a broad range of human diseases including the Alzheimer's disease, type II diabetes (DM-2) and systemic amyloidosis. Although amyloid deposition has been characterized more than a century ago, the structural nature of protein species causing cell death and tissue degeneration remains unknown. In this context, using islet amyloid polypeptide (IAPP) as a model amyloidogenic peptide, we investigated the structure-self-assembly relationships in order to elucidate the molecular mechanisms of amyloidogenesis and to better define the conformational nature of the cytotoxic species. The deposition of IAPP into the pancreatic islets, as insoluble amyloid fibrils correlates proportionally with the progression of DM-2. We observed that the Asn-21 residue plays a critical role in the self-assembly of IAPP. This position acts as a molecular hinge that controls the structural conversion between cytotoxic oligomers and non-toxic mature amyloid fibers. In addition, we identified a single N-to-Q mutation (N21Q) makesfibrillar IAPP highly toxic to pancreatic cells, inducing apoptosis and membrane perturbation. 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, dot blot immunoassay, 8-anilino-1-naphtalenesulfonic acid fluorescenceand thioflavin T fluorescence revealed that [N21Q] IAPP and IAPP fibrils were structurally distinct. These results contribute to a better understanding of the relationship between amyloid deposits and the cellular degeneration observed in amyloid-related diseasesand open up new therapeutic approaches targeting specific assembly conformations.

Molecular insights into the bacterial acetylcholinesterase ChoE from Pseudomonas aeruginosa


VAN DUNG PHAM1, Tuan Anh To1, Cynthia Gagné-Thivierge1, Deqiang Yao3, Marie-Ève Picard1, Manon Couture1, Roger Levesque2, Steve Charette1, Rong Shi1
1Département de biochimie, de microbiologie et de bio-informatiqu, Université Laval 2Institut de biologie intégrative et des systèmes, Université Laval 3Shanghai synchrotron radiation facility

Mammalian acetylcholinesterase (AChE), one of the most efficient enzymes of nervous system, hydrolyses the neurotransmitter acetylcholine into choline and acetate and plays a pivotal role in cholinergic neurotransmission. 

Acetylcholine hydrolyzing activity has also been recognized in prokaryotes although they lack a nervous system. This was especially the case in the bacterial Pseudomonasgenus, which are able to inhabit a large range of environments with many species like Pseudomonas aeruginosa being pathogens for many different hosts. It was reported that P. aeruginosa is able to use acetylcholine as a carbon and nitrogen source. In this work, we have structurally characterized the bacterial acetylcholinesterase ChoE at 1.85Å. Site-directed mutagenesis of ChoE followed by activity assays has confirmed the role of Ser38, Asp285, and His288 in the catalytic triad and Asn147 of oxyanion hole. Furthermore, a PAO1 mutant lacking this ChoE gene was investigated in various media, confirming that the mutant strain is unable to use acetylcholine as a carbon and nitrogen source as the wild-type strain. This study reveals the first structural insight into the bacterial acetylcholinesterase, indicating the important role of ChoE in the metabolic pathways of P. aeruginosa. Furthermore, our analysis also provides an overview of biological evolution of acetylcholinesterases of both prokaryotic and eukaryotic origins. 

Molecular Insights of Val430Ile Mutation Impact on Influenza Neuraminidase Resistance to Zanamivir


Gabriel Bégin1,2,3, Patrick Lague1,2,3
1PROTEO 2Université Laval 3IBIS

The treatment of Influenza infection with antiviral compounds has become a common approach in the past few years. Various inhibitors as Oseltamivir, Zanamivir, Paramivir, and Laninamivir were designed to bind to Neuraminidase, an Influenza protein involved in the replication process. A recent clinical surveillance report has identified a Neuraminidase mutation located outside of the Zanamivir binding site. According to experimental data, the mutation Val430Ile has shown a reduction of affinity between the Zanamivir and the Neuraminidase. The present study has used molecular dynamic simulations with a homologous structure of Neuraminidase to predict the molecular details of this mutation. An analysis based on residue-residue contact frequency has revealed a reorganization of the H-bond network at the vicinity of the mutated residue. These simulations have provided structural insights into the understanding of this resistance to Zanamivir.

Monitoring Protein Function using Fluorescent Nanoantennas


Scott Harroun1, Alexis Vallée-Bélisle2
1Université de Montréal 2Université de Montréal

To measure enzymatic activity, one may need a different assay for each reaction 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- and PEG-based molecular probes to drive enzyme-dye interaction via a streptavidin platform in order to measure enzymatic activity. We demonstrate that our assay only reports signal change due to a local effect during catalysis, possibly from structural destabilisation too small to measure by FRET. Our approach correlates well with traditional measurements of enzymatic activity (i.e. KM), thereby confirming its validity to measure kinetics. We optimised the attachment strategy, DNA composition and length, the effect of steric hindrance, the ratio of DNA and enzyme added, and the choice of dye. 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 may be possible to probe various regions of the enzyme surface during catalysis.

New developments of flow biocatalysis systems: Synthesis of indigo and raspberry ketone using cytochrome P450 enzymes.


Ali Fendri1,2,3, Adem H.-Parisien1,3,4, Joelle Pelletier1,2,3,4
1PROTEO Network, Université Laval, Québec, Canada 2Département de chimie, Université de Montréal, Montréal, Canada 3Center for Green Chemistry and Catalysis (CCVC), Montréal, Canada 4Département de biochimie, Université de Montréal, Montréal, Canada

 

Flow chemistry is the process of carrying out chemical reactions in a flow reactor, such as a capillary or micro-structured device, where the residence time determines the reaction time. The nature of the flow chemistry makes it possible to better control the parameters of the reaction, such as mixture efficiency, reagents addition, the temperature or the reaction time control [1]. This means that we can develop more productive, more selective and cleaner reactions. It is an approach that offers better reaction control and reduces side reactions and is therefore radically different from performing reactions in traditional reactors.

 Recently in the Pelletier laboratory, directed evolution of a cytochrome P450 oxidase from Bacillus megaterium, BM3, led to the discovery of variants that show an increased production of indigo (a dye used in several industrial fields) and raspberry ketone (a high-value aroma in the food industry) [2]. In order to better exploit the industrial potential of these discoveries, optimization of the production yield and process efficiency are necessary.

Taking advantage of the infrastructure available at the Université de Montréal flow chemistry laboratory, we propose to develop flow biocatalytic methods to this end. Particular attention will be given to NADPH cofactor recycling, where classical enzymatic regeneration will be compared to the substitution of NADPH with a photocatalytically-regenerated electron system. The feasibility and effectiveness of various photochemical montages have been demonstrated in the work of the Collins laboratory at Université de Montréal [3]. Whole cells of E. coli overexpressing the P450 variants of interest were immobilized and trapped between membranes in a capillary reactor. Suitable reagents for the synthesis of indigo or raspberry ketone are flowed in and product formation will be monitored by matching efficient mixing systems to the appropriate detectors, to optimize the biocatalysis conditions and the production rate of products with high added value.  

 

1. Britton, J., S. Majumdar, and G.A. Weiss, Continuous flow biocatalysis. Chem Soc Rev, 2018. 47(15): p. 5891-5918.

2. Rousseau, O., et al., Indigo is a robust predictor of raspberry ketone production by engineered cytochrome P450 BM3 variants. ChemCatChem, 2018., Revisions requested.

3. Parisien-Collette, S. and S.K. Collins, Exploiting Photochemical Processes in Multi-Step Continuous Flow: Derivatization of the Natural Product Clausine C. ChemPhotoChem, 2018. 2(10): p. 855-859.

Optimization 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
1Chemical Engineering Department, Université Laval, Quebec, QC, Canada. 2Thécell : FRQS Cell and Tissue Therapy Network 3PROTÉO : The Quebec Network for Research on Protein Function, Structure, and Engineering 4Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada

One of the bottlenecks in cell therapy is to successfully introduce a DNA inside cell, especially in cells known as difficult to transfect. Nowadays, different technics exist but none are fully satisfying. Therefore, virus-like particles have been developed and proven to efficiently transfect cells. This study is focusing on the development of a production and purification process for virus-like particles pseudotyped by the VSVg protein (gesicles). Those particles are produced with suspension cells HEK 293 F in a serum-free media. Three production 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 exchange anion chromatography with a NaCl elution gradient, has shown to induce a loss in gesicles efficiency to transfect cells. Therefore, a new way to purify the gesicles, using polybrene, was investigated. This cationic polymer snatches the gesicles from the anionic resin thanks to its positive charges and induces the gesicles’ elution. The gesicles efficiency to deliver DNA was proven using a cell transfection efficiency assays and western blot. Even though this method needs to be optimized, we are now able to purify the gesicles. 

Optimization of matriptase-2 inhibitors


Méryl-Farelle Oye mintsa mi-mba1,2, Pierre-Luc Boudreault1,2, Antoine Désilets1,2, Richard Leduc1,2, Éric Marsault1,2
1Department of pharmacology-physiology, Faculty of medicine and health sciences 2Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke

Matriptase 2 (MAT-2) is a type II transmembrane serine protease located on the surface of hepatocytes. Several studies have shown that MAT-2 acts as a regulator of iron homeostasis : it negatively regulates hepcidin levels by cleaving hemojuvelin, which promotes the release of iron stored in cells into the circulation. Inhibition of this enzyme paves the way for the treatment of iron overload-related diseases such as beta-thalassemia and juvenile hemochromatosis. Our laboratory has previously designed and synthesized peptidomimetic MAT-2 inhibitors. The objectives of this project are, first, to optimize these compounds to improve their inhibition and selectively towards MAT-2, then in parallel to develop a prodrug on an inhibitor with high selectivity for MAT-2. 

 

The design of the inhibitors is based on molecular modeling based on the structure of the catalytic protease pouches, which are visualized using the MOE software. The compounds consist of a sequence of three amino acids followed by a serine trap (warhead) allowing the inhibition of the enzyme. These peptides are synthesized in two steps : the tripeptides will be synthesized on a solid phase, while the warhead will be synthesized in liquid phase. This is followed by the synthesis of the tetrapeptide in the liquid phase. In order to characterize the enzyme/inhibitor interaction, the determination of the dissociation constant (Ki) is performed using a fluorogenic substrate. The synthesis of prodrug consists of similar steps : the synthesis of tripeptides in solid phase, and that of warhead in liquid phase. A double carbamate group will be added to the arginine guanidine ; then the reactive group will be added to the same arginine. This new reactive group will be coupled to the tripeptide (H)F-hF-(Allo)T in order to generate the prodrug. A protocol has been established to test the latter in pharmacokinetics. 

 

The results obtained by modeling allowed the design of a library of compounds with inhibitory potential that was synthesized and tested in bioassays to validate molecular coupling hypotheses. The synthesis of prodrug offers a new approach to the plasma stability of the compound known for its selectivity to MAT-2.

Origin of Dynamics in a Small Globular Protein


Mayer Marc1, Adam M. Damry1, Aron Broom1, Natalie Goto1, Roberto A. Chica1
1University of Ottawa

Proteins are molecular machines that carry out many of the intricate biological processes required to sustain life. These processes often involve protein motions, with dynamics known to play a vital role in enzyme catalysis, allosteric regulation, and molecular recognition. Despite the demonstrated importance of dynamics to protein function, the underlying mechanisms of how sequence changes can lead to novel modes of conformational exchange in globular proteins remains elusive. Previously, we used computational design to create a series of streptococcal protein G domain β1 (Gβ1) variants termed DANCERs that undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. DANCERs are unique in that the designed Trp43 conformational exchange did not result from dynamic enrichment of low-occupancy sub-states present in the parent and therefore comprise an ideal system to study how conformational exchange can appear in a globular protein. Here, we evaluate the impact of individual substitutions of core residues in a representative DANCER on Trp43 conformational exchange in order to understand the role of each residue in the designed mode of conformational exchange. Using solution NMR, we found that only two mutations were necessary to convert the static wild-type Gβ1 into a DANCER capable of undergoing Trp43 conformational exchange. Through molecular dynamics simulations, these mutations were shown to work synergistically, with one enabling escape from the energy well that defines the native Gβ1 structure, while the other guides the escape towards a region of the protein energy landscape conducive to the designed dynamic motions. Our results thus provide a model for the origin of conformational exchange in globular proteins.

Peptide assemblies as self-adjuvanted platform for nanovaccine design


Ximena Zottig1,2, Al Halifa Soultan1,2, Mélanie Côté-Cyr1,2, Margaryta Babych1,2, Laurie Gauthier1,2, Jessica Dion1,2, Denis Archambault1, Steve Bourgault1,2
1Université du Québec à Montréal 2PROTEO

Peptide self-assembly has recently gained increasing attention for the design and the medical applications of nanoparticles and soft functional biomaterials. Among peptide-based nanostructures, cross-ß-sheet assemblies (CBSA) are particularly appealing owing to their mechanical strength, resistance to enzymatic degradation and immunomodulatory effect. In this regard, we are currently developing CBSA as a novel nanovaccine platform. First, we demonstrated that a cross-ß-sheet self-assembling sequence (I10) can be functionalized with an immunogenic epitope (M2e) and self-assemble into filaments. The M2e epitope derived from the extracellular domain of influenza A ion channel membrane matrix protein was attached to β-sheet forming sequence via a flexible spacer by standard solid-phase synthesis strategies. The resulting epitope-functionalized filaments elicited a robust immune response against M2e epitope in mice. Nevertheless, the polymorphism and polydispersity associated with the process of CBSA formation could be ultimately detrimental for their clinical translation as the immune response is sensitive to the nanoparticle properties. Therefore, we implemented an innovative approach to engineer CBSA nanoparticles with specific sizes, shapes and morphologies. Among these assemblies, nanorods were particularly interesting because of their unique morphology and their remarkable low polydispersity. The resulting M2e-functionalized nanorods were cytocompatible and elicited a strong immune response against the M2e epitope in mice even without the addition of adjuvants. Protection and survival of mice infected with the influenza A virus are being investigated. This study highlights that CBSA constitute a promising platform for the design of synthetic adjuvant-free nanovaccines, uncovering a new avenue for traditionnal vaccination and activation immunotherapies.

Peptide-based Drug Discovery: Artificial Selection of Genetically Encoded TB Drugs


Trisha Ghosh1, Lan Huong Thi Nguyen1, David Kwan1, Christopher Hipolito2
1Concordia University 2University of Tsukuba, Ibaraki, Japan

Mycobacterium tuberculosis is a causative agent of tuberculosis (TB) in humans. Conforming to the data from the World Health Organization (WHO), TB is one of the leading causes of death worldwide. Currently, multidrug-resistant (MDR) strains have made it difficult for establishing a course of treatment, limiting the use of existing antibiotics against TB. Cell wall biosynthesis has become a target for developing new drugs. Mycobacterium cell wall is composed of a complex, formed by peptidoglycan, lipid mycolic acid, and polysaccharide arabinogalactan. Arabinogalactan (AG) is a heteropolysaccharide that comprises of arabinose and galactose sugars that are present in the furanoid ring form, D-galactofuranosyl (Galf) and D-arabinofuranosyl (Araf). UDP-Galactopyranose mutase (UGM) catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). It plays a key role in the biosynthesis of the galactofuran. In this study, we will focus on UGM as our molecular target. The aim of this research project is to identify potent inhibitory molecules that restrict cell wall biosynthesis by binding to UGM and inhibiting its activity. mRNA display technique will be applied to identify inhibitory molecules from a library of non-standard macrocyclic peptides. Macrocyclic structure of peptidic natural products play critical roles in their biological activity. Study on protein engineering and development of high-throughput assay will provide new insights for developing a novel drug against TB.

Perturbation de la structure tridimensionnelle du mutant S175R de la lécithine rétinol acyltransférase tronquée par le SDS : une étude par résonance magnétique nucléaire


Marie-Eve Gauthier1,2,3,4,5, Line Cantin1,2,3, Stéphane Gagné3,4,5, Christian Salesse1,2,3
1CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement, CHU de Québec-Université Laval 21Département d’ophtalmologie, Faculté de médecine, 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 détaillé de l’activité enzymatique de la LRAT est inconnu. Cependant, quatre résidus ont été identifiés comme étant essentiels à la catalyse (H60, H72, Y154 et C161). Il est donc important de déterminer la structure tridimensionnelle de la LRAT afin d’améliorer notre compréhension de son activité enzymatique ainsi que l’effet des mutations sur ce processus en utilisant une forme tronquée de la LRAT (tLRAT, LRAT sans ses segments hydrophobes en N- et C-terminal). Dans le cadre de ce projet, la structure tridimensionnelle du mutant tLRAT-S175R a été déterminée par résonance magnétique nucléaire (RMN). La mutation S175R mène à une perte complète de l’activité enzymatique de la LRAT, contrairement aux autres mutants où une activité résiduelle est observée. Notre mutant tLRAT-S175R comprend aussi les mutations C161S et C168S de la LRAT (tLRAT-C161S/C168S/S175R) pour rendre notre échantillon plus homogène. MATÉRIEL ET MÉTHODES : La tLRAT-C161S/C168S/S175R marquée au 15N et 13C a été purifiée par chromatographie d’affinité en présence du détergent sodium dodécyl sulfate (SDS). Les spectres RMN ont été mesurés avec des spectromètres de 600 (Université Laval) et 800 MHz (Université McGill). Les spectres 15N-HSQC, HNCO, HN(CA)CO, HN(CO)CA, HNCA, CBCA(CO)NH, HNCACB, CCONH, HCONH et 3D 13C-HSQC NOESY ont été enregistrés et chaque pic a été attribué à des résidus spécifiques de la protéine. RÉSULTATS : La structure tridimensionnelle (3D) obtenue par RMN est composée d’hélices-α et de pelote statistique. Cependant, contrairement aux modèles de prédiction de la structure du mutant tLRAT-C161S/C168S/S175R, aucun feuillet-β n’est présent dans notre structure RMN de cette protéine. Des analyses détaillées ont cependant permis d’observer une valeur de RMSD (root mean square deviation) de 14,8 ± 2,0 Å entre les 20 structures obtenues. Cette valeur élevée de RMSD a donc soulevé des questions à propos de la fiabilité de ces structures. Des analyses de notre échantillon RMN de tLRAT-C161S/C168S/S175R ont permis de montrer que la concentration de SDS augmentait au cours de la purification ainsi que des étapes visant à concentrer notre échantillon pour les mesures RMN pour atteindre des valeurs > 100 mM, ce qui représente une concentration très élevée comparativement à la valeur initiale de 1,7 mM de SDS. Par la suite, des mesures d’activité enzymatique de la tLRAT en présence de diverses concentrations de SDS ont permis de démontrer que notre échantillon RMN de tLRAT ne présentait aucune activité. CONCLUSION : La structure 3D de la tLRAT-C161S/C168S/S175R n’est donc pas fiable, vraisemblablement en raison de la présence de micelles de SDS, qui est un détergent connu pour favoriser la formation d’hélices-α, ce qui résulterait en la production d’une une forme non-native et inactive de la protéine. Des travaux sont en cours pour identifier un autre type de détergent qui permettra de solubiliser la tLRAT, de la purifier et d’obtenir une concentration suffisamment élevée d’une forme active de cette protéine pour déterminer sa structure par RMN.

Phase separation and conformational conversion processes drive the self-assembly of non-pathological amyloid in the presence of linear polyanions


Mathew Sebastiao1, Noé Quittot1, Isabelle Marcotte1,2, Steve Bourgault1,2
1Université du Québec à Montréal 2PROTEO

Amyloids are supramolecular polypeptide assemblies that have been historically associated with various degenerative and debilitating pathological conditions. Nonetheless, recent studies have identified many instances where amyloid and amyloid-like structures accomplish vital physiological functions. Interestingly, amyloid fibrils, either pathological or functional, have been reported to be consistently associated with linear polyanions such as non-coding RNA and glycosaminoglycans (GAGs). These biomolecules have also demonstrated an inherent ability to accelerate or to promote amyloid formation in vitro. GAGs, including heparan sulfate, are highly charged polysaccharides that may have essential roles in the storage of peptide hormones in the form of amyloids within secretory granules. In this study, we evaluated the ability of sulfated GAGs to promote the self-assembly of the peptide (neuro)hormone PACAP27 and investigated both secondary and quaternary conformational transitions associated with the amyloid self-assembly process. On its own, PACAP27 remained disordered and soluble yet readily self-assembled into insoluble, α-helix-rich globular particulates in the presence of sulfated GAGs. These particulates gradually condensed and disappeared as non-toxic β-sheet-rich amyloid fibrils were formed, with the condensed particulates acting as nucleation sites. The proposed mechanism of GAG-induced self-assembly originating from insoluble particulates appears to be fundamentally different than usual amyloidogenic systems, which commonly implicates the formation of soluble pre-fibrillar proteospecies (e.g. oligomers, protofibrils, etc.). Overall, this study provides new insights into the mechanistic details involved in the formation of functional amyloids catalyzed by polyanions, such as the amyloidogenic storage of peptide hormones and the formation of recently described RNA-induced amyloid bodies in the cell nucleus.

Phenolic acid decarboxylase structures lead to new insights in its decarboxylation mechanism


Marie-Ève Picard1,2, Rong Shi1,2
1Université Laval 2PROTEO

Phenolic acid decarboxylase (PAD) is an enzyme present in bacteria, notably Bacillus pumilus. This protein is characterized by a ten strand beta-barrel adopting a lipocalin-like fold flanked with three alpha helices. This fold is mainly known for binding small hydrophobic molecules. PAD shares a high similarity with the Ferulic Acid Decarboxylase (FAD), where both are involved in detoxification processes through the decarboxylation of small aromatic molecules of the hydroxycinnamic acids family such as ferulic, p-coumaric and caffeic acids.

 

In organic chemistry, decarboxylation is an important process for the elimination of carboxylic acids, mostly achieved through the use of progressive heating up to 200 degrees C. While this process is simple, high temperature may be detrimental for more complex molecules. Achieving decarboxylation through biocatalysis is thus an interesting goal. The bioconversion of ferulic acid, the most abundant hydroxycinnamic acid found in plants leads to the formation of many value-added products such as flavoring agents vinyl guaiacol and vanillin, and biopolymers.

 

An engineered PAD protein, designated SAD for sinapic acid decarboxylase, was previously developed in order to use sinapic acid as a substrate to produce the antioxidant canolol [Green Chem., 2013, 15, 3312]. Further development of a larger variety of substrates relies on an ultimate understanding of the enzymatic mechanism.

 

Here, we report the crystal structures of wild-type and mutants SAD with substrate analogues present in the catalytic site. Interestingly, its binding mode in the pocket is different from those previously reported in the literature. Based on these structural results as well as mutagenesis assays data, we hereby suggest an alternative mechanism for the decarboxylation reaction by phenolic acid decarboxylases.

Positive epistasis towards cefotaxime resistance is maintained in highly dynamic, engineered ß-lactamases


Lorea Alejaldre1,2,3, Ferran Sancho Jodar1,2,4, Claudèle Lemay-St-Denis1,2,3, Adem H.-Parisien1,2,3, Joelle Pelletier1,2,3,5
1PROTEO Network, Université Laval, Québec, Canada 2Département de biochimie, Université de Montréal, Montréal, Canada 3Center for Green Chemistry and Catalysis (CGCC), Montréal, Canada 4Barcelona Supercomputing Center, Barcelona, Spain 5Département de chimie, Université de Montréal, Montréal, Canada

TEM-1 β-lactamase confers bacterial resistance to antibiotics such as penicillin and its derivatives upon hydrolyzing them. In nature, TEM-1 β-lactamase evolves at an impressive speed due to the massive use of β-lactam antibiotics, readily adapting to newly developed molecules. Understanding the evolutionary mechanisms of β-lactamases is essential for the development of future drugs. Although the links between structure and function of enzymes are fairly well established, the involvement of protein dynamics in their evolution of new protein function is still poorly understood. Two variants of TEM-1 ß-lactamase are studied in this project. Each displays a breadth of motions on the time-scale of catalysis (μs to ms) that does not exist in the native TEM-1 ß-lactamase (1). Here, we verify whether those new dynamics alter the effect of mutations that are known to confer high clinical resistance to cefotaxime. This antibiotic is poorly hydrolysed by the native TEM-1 ß-lactamase, but inclusion of mutations E104K and G238S increase catalytic efficiency by more than 250-fold (2).

Those mutations were introduced into the dynamic variants.  The mutations resulted in similar kinetics for cefotaxime hydrolysis to TEM-1 ß-lactamase, with different trends on thermostability and in vivo resistance. Specifically, the dynamic changes observed in the TEM-1 ß-lactamase variants did not alter the gain of catalytic activity caused by these mutations, yet they mitigated the loss of thermostability in some new variants. Ongoing computational work will allow rationalizing the impact of these mutations on the reaction mechanism of the dynamic variants.

 

1.         S. Gobeil, M. E., J. Park, S. Gagné, N. Doucet, A. Berghuis, J. Pleiss & J. Pelletier (2019). Scientific Reports. doi: 10.1038/s41598-019-42866-8

2.         Wang, X., Minasov, G., & Shoichet, B. K. (2002). J Mol Biol, 320(1), 85-95. doi: 10.1016/s0022-2836(02)00400-x

Programming complex regulation mechanisms through simple molecular assembly


Dominic Lauzon1, Alexis Vallée-Bélisle1
1Université de Montréal

Self-assembled molecular systems, or nanosystems, are at the basis of life and are becoming increasingly important in nanotechnology.(1) Assembling these nanosystems typically involve multiple non-covalent interactions either through intramolecular folding or by association with one or multiple partners. To accomplish their function, these nanosystems require a precise fine tuning of their regulation as well as a high level of programmability in order to be use in many different situations. In living organisms, a myriad of monomeric, dimeric or multimeric self-regulated nanomachines have been created to enable life at the molecular level. For exemple, human enzymes, are built either using one “foldable” molecular component (23%) or by association of two (44%) or multiple (33%) molecular components.(2) This leeds to this simple question : What are the advantages and limitations of constructing self-regulated molecular systems using one, two or multiple molecular components? This question becomes essential as chemists and engineers attempt to develop novel self-regulated nanosystems for multiple applications (e.g. biosensing, drug delivery, smart materials…).(3-5)

 

With this poster, I present some answers to this fundamental question by explaining the advantages and limitations of building nanosystems using 1, 2 or 3 molecular components. I do so by building three molecular systems based on DNA chemistry containing 1, 2 or 3 DNA strands. I first look at their thermodynamic stability and their rate of assembly/disassembly and find a surprising correlation with the number of components involved. Then, I focus on the regulation of the 2- and 3-components systems. I characterize the regulation potential of these nanosystems with two parameters: 1) [A]50%, the concentration at which 50% of the system is assembled; and 2) the cooperativity (or dynamic range) of the assembly process, which can be defined as the variation of [A] required to provide a change in  assembly from 10% to 90% (D.R. = [A]90%/[A]10%). All experiments indicate a higher level of regulation for the 3-components system over the 2-components system. I also introduce the new parameter ΔΔG (ΔGTri - ΔGDim) to describe the complex regulation profiles of the 3-components system thus enabling us to explore all the possibility of regulation of the 3-components system. However, I find a kinetic limitation for the 3-components system which has been mechanistically studied and interpret as a direct consequence of having more possibility of regulation.

 

In summary, these experiments reveal the thermodynamic and kinetic rules that guide the complex regulation of simple molecular assembly made of only 1, 2 or 3 components. This provides a toolbox for chemists and engineers in order to build more efficient, programmable self-assembled molecular systems which can be finely tuned for a specific application. I validate this by rationally engineering a new nature-inspired regulation mechanism on an artificially synthesized DNAzyme system.(6)  

 

 

(1) Grzybowski, B.A., Huck, W.T.S., Nat. Nanotechnol. 2016, 11, 585.

(2) BRENDA, The Comprehensive Enzyme Information System, http://www.brenda-enzymes.org/

(3) Cheng, C., McGonigal, P.R., Schneebeli, S.T., Li, H., Vermeulen, N.A., Ke, C., Stoddart, J.F. Nat. Nanotechnol. 2015, 10, 547.

(4) Douglas, S.M., Bachelet, I., Church, G.M., Science 2012, 335, 831.

(5) Webber, M.J., Langer, R. Chem. Soc. Rev.2017, 46, 6600.

(6) Torabi, S.F., Wu, P., McGhee, C.E., Chen, L., Hwang, K., Zheng, N., Cheng, J., Lu, L. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 5903.

Protein engineering and immobilization of LipA from Pseudomonas aeruginosa to broaden its industrial applications


Ingrid Yamile Pulido1, Lorea Alejaldre2, Charlotte-Skye Fullerton2, Rosa E. Prieto1, Carlos A. Jiménez1, Joelle N. Pelletier2
1Universidad de La Sabana 2Université de Montréal

Lipases comprise a heterogeneous group of enzymes in high demand for biotechnological processes [1]. Lipase A of Pseudomonas aeruginosa (LipA) exhibits enantioselectivity, activity over a broad range of substrates and high tolerance to organic solvents,  features that are potentially useful in synthetic reactions [2]. However, other qualities such as stability and activity towards diverse triacylglycerols need to be engineered in this enzyme to broaden its industrial use.

Here we present two approaches to increase the biotechnological value of LipA from P. aeruginosa. First, we immobilized the enzyme, using a matrix of hydrophobic polypropylene powder. Enzymes that are adsorbed to the surface of this polymer may be resistant to denaturation due to high temperature and solvents [3]. Using crude lysates of recombinant Escherichia coli, we demonstrate that LipA is stable upon incubation at 50oC for 24 hours and displays increased stability to ethanol, confirming the benefits of immobilization.

Secondly, we seek to engineer the lipase to increase its activity towards short chain triglycerides because of their importance in the food and fragrance sectors. We established conditions to maximize soluble expression of LipA by evaluating expression conditions and E. coli strains. The lipolytic activity was verified by the formation of halos upon plating cells on tributyrin-containing agar [4], demonstrating the production of soluble and active lipase. Random mutagenesis was then applied to generate a library of LipA variants. Library screening toward hydrolysis of short-chain (tributyrin) and long-chain (olive oil) triglycerides will allow the identification of variants with higher activity (more rapid halo formation) and chain-length selectivity [4]. This halo-screening methodology and the selected variants could be further used to boost other properties of the enzyme, such as the thermostability and selectivity towards other substrates.

[1]       G. M. Borrelli and D. Trono, Int. J. Mol. Sci., vol. 16, no. 9, pp. 20774–20840, 2015.

[2]       A. Kumar, K. Dhar, S. S. Kanwar, and P. K. Arora, Biol. Proced. Online, vol. 18, no. 1, 2016.

[3]       S. Cesarini, B. Infanzón, F. I. J. Pastor, and P. Diaz, BMC Biotechnol., vol. 14, no. 1, p. 27, Jan. 2014.

[4]       D. Quaglia, M. C. C. J. C. Ebert, P. F. Mugford, and J. N. Pelletier, PLoS One, vol. 12, no. 2, p. e0171741, 2017.

Region-focused protein engineering in Cal-A lipase reveals triglyceride-binding hotspot


Daniela Quaglia1,3,4, Lorea Alejaldre2,3,4, Sara Ouadhi1,3,4, Olivier Rousseau1,3,4, Joelle Pelletier1,2,3,4
1Chemistry Department, Université de Montréal 2Biochemistry Department, Université de Montréal 3Centre for Green Chemistry and Catalysis 4PROTEO

Protein engineering is widely used to modify industrially relevant enzymes into better catalysts for improved activity, stability, selectivity or even novel activities. In order to maximize the efficiency of protein engineering, strategies that provide a higher ratio of active variants per variant screened are of high value. One popular approach involves performing site-saturation mutagenesis guided by structural analysis.

As an alternative, we present a region-focused approach combined with random mutagenesis to identify mutational hotspots. This approach is demonstrated using the enzyme Candida antarctica lipase A (Cal-A). Lipases are of special relevance in the food industry as tools to create food additives that show health benefits, such as milk-fat products enriched with short-chain diglycerides2. Cal-A is of particular interest due to its high stability, substrate promiscuity and simple interfacial activation mechanism compared to other lipases.

In our engineering approach, the Cal-A gene was divided into three fragments which were independently mutated by site-directed mutagenesis or random mutagenesis. Recombination of the newly obtained mutated fragments with the wild-type parts to regenerate the full gene was done seamlessly using a Golden-gate based assembly1. The focused libraries obtained were subsequently screened with an in vivo high-throughput method to detect Cal-A variants able to discriminate between hydrolysis of long- and short-chain triglycerides. As representative substrates, two triglycerides were selected: short-chained tributyrin (C4) and long-chained olive oil (mainly C18). Variants exhibiting differential hydrolysis of short- vs. long-chain triglycerides were sequenced to reveal the mutations that modified the enzymatic activity. Analysis of the mutational patterns using an in-house script allowed identification of predominant mutations and interactions between mutations, as well as mutational hotspots3.

Using this focused approach, we identified key regions and novel improved variants that allow for improved discrimination of short- vs. long-chain triglycerides. In addition, our results improve our understanding of Cal-A lipase to direct future engineering efforts and widen its application as a biocatalyst.

 

1.         Quaglia, D., Ebert, M. C. C. J. C., Mugford, P. F. & Pelletier, J. N. Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. PLOS ONE 12, e0171741 (2017).

2.         Nyyssölä, A. et al. Treatment of milk fat with sn-2 specific Pseudozyma antarctica lipase A for targeted hydrolysis of saturated medium and long-chain fatty acids. Int. Dairy J. 41, 16–22 (2015).

3.         Quaglia, D., Alejaldre, L., Ouadhi, S., Rousseau, O. & Pelletier, J. N. Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot. PLOS ONE 14, e0210100 (2019).

 

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


Francis Noël1,2,3, Xiaolin YAN1,2,3, Stefan Vetter4, Elodie Boisselier1,2,3
1Département d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval 2CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec-Université Laval 3PROTEO 4École de pharmacie, Université d’État du Dakota du Nord, États-Unis

CONTEXTE ET OBJECTIFS : 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 (ANXA4) 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 consiste à étudier les interactions membranaires des protéines S100A16 et ANXA4 afin de mieux comprendre leurs fonctions dans le maintien de l’intégrité membranaire. Les objectifs spécifiques sont : i) de réaliser la purification de ces protéines, ii) d’obtenir des informations sur leurs interactions membranaires, et iii) d’étudier l’influence du calcium sur ces interactions.

 

MATÉRIEL ET MÉTHODES : La protéine S100A16 est obtenue par clivage suivie d’une purification sur une colonne His-Trap. Les interactions membranaires sont étudiées avec le modèle des monocouches de Langmuir. Après mesure de la concentration saturante, 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 S100A16 a été obtenue avec une pureté supérieure à 99%. L’obtention de ANXA4 est en cours. La concentration saturante déterminée pour la protéine S100A16 est de 0,5µM. Des études biophysiques avec différents phospholipides en monocouches sont actuellement en cours de réalisation. Les résultats préliminaires suggèrent que la protéine S100A16 interagit préférentiellement avec les lipides saturés composés de chaînes acyles courtes. 

 

CONCLUSION : L’obtention de la protéine S100A16 pure permet de poursuivre les études biophysiques. La purification de ANXA4 et l’étude biophysique 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.

Sequential coselections for CRISPR-driven genome editing in human cells


Sébastien Levesque1, Eva Bouchard1, Daniel Agudelo1, Yannick Doyon1
1Centre de recherche du CHU de Québec - Université Laval

Achieving high gene editing efficiency via homology-directed repair is no easy task. The development of flexible methods for the selection of engineered human cells provides crucial tools for fundamental and medical research. We previously reported a simple and robust coselection strategy for enrichment of edited cells based on the selection of dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain, a plant-derived inhibitor of the pump.

 

To extend the applications of this coselection strategy, we identified point mutations that confer different levels of resistance to ouabain. Combining these mutations allows sequential coselections to be performed by progressively increasing the dose of ouabain at each step. Hence, the ATP1A1 locus can be used repetitively to facilitate the isolation of cells with multiple editing events.

 

To demonstrate this, we tagged two endogenous genes (LMNA and NPM1) with fluorescent proteins via two steps of coselection. This approach allowed the enrichment of up to 15.2% of cells with both fluorescent tags. We are currently adapting this strategy to enable three sequential rounds of coselection. We also demonstrate that fluorescent protein expression cassettes can be sequentially targeted to the ATP1A1 locus itself to allow marker-free selection of up to 92.9% of cells expressing both transgenes.

 

We foresee that this approach could be useful to tag endogenous genes and then create variants to study their function. In addition, this would allow the restoration of an original sequence, abolishing a previous editing event. Our work should facilitate human cell lines engineering to study protein functions and interactions.

Structural changes along the evolutionary trajectory of a de novo designed enzyme


Broom A1, Rakotoharisoa R1, Thompson EM2, Fraser JS2, Chica RA1
1Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie-Curie, K1N 6N5, Ottawa, Ontario, Canada 2Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, California 94158, United States

Enzymes are outstanding catalysts increasingly used in therapeutic and industrial applications. However, natural enzymes perform only a small fraction of commercially or medicinally valuable reactions. As such, the design of artificial enzymes able to perform any desired reaction has been a key goal of Computational Protein Design (CPD) for the past decade.  Although several de novo enzymes have been successfully designed, these have failed to reach the efficiency of natural enzymes, requiring multiple rounds of laborious directed evolution to improve their activity. To gain insights into the design of highly active de novo enzymes, we used room-temperature x-ray crystallography to examine the structural changes that occurred during directed evolution of a de novo designed and poorly active Kemp eliminase (HG3, kcat/KM = 423 M–1 s–1) to one with native-like activity (HG3.17, kcat/KM = 230,000 M–1 s–1). We observed that over the course of 17 rounds of evolution, the catalytic residues were rigidified through improved packing, the active site was pre-organized to favor productive binding of the substrate, and the active-site entrance was widened by mutation of bulky amino acids to smaller ones. Additionally, we solved the room-temperature crystal structure of a highly active variant that contains all active-site mutations found in HG3.17 but not distal ones (HG3.17-core, kcat/KM = 98,000 M–1 s–1). Using CPD, we demonstrate that HG3.17-core can be successfully designed but only when an ensemble of backbone templates is used instead of the single crystal structure that served as a template during the design of HG3. Overall, our data pave the way to the rational design of more efficient de novo enzymes.

Structure et mode d’action des transporteurs membranaires du nickel chez Helicobacter pylori


Imène Kouidmi1, Ines Feriel1, Mariem Chalbi1, Charles Calmettes1
1INRS-IAF

Helicobacter pylori est un pathogène humain qui colonise l’estomac et provoque plusieurs maladies dont les cancers gastriques. Plus de la moitié de la population mondiale est infectée par H. pylori, on estime que 1 à 3% des personnes infectées développeront un cancer gastrique. Pour résister à l’acidité de l’estomac, H. pylori possède une enzyme indispensable, l’uréase. L’activité de ce facteur de virulence est étroitement dépendante d’ions nickel qui ne sont présents qu’à très faible concentration chez l’humain. Pour ce fait, H. pylori possède des mécanismes d’acquisitions du nickel hautement efficaces pour assurer sa survie chez l’hôte. Plusieurs transporteurs de nickel ont été identifié chez H. pylori; on retrouve les transporteurs de la membarne externe, FrpB4 et FecA3, de la membrane interne NixA et NiuDE, et les protéines périplasmiques NiuB. Cependant, la structure et mode d’action de ces protéines ne sont pas encore connus. Le but de ce travail est de caractériser la structure des transporteurs membranaires du nickel et de déterminer les mécanismes moléculaires par lesquels le nickel est transporté. De plus, une étude récente a démontré que les sels de bismuth, un complexe organométallique utilisé dans le traitement des infections à H. pylori, empreinte la voie de transport du nickel pour pénétrer à l’intérieur de la bactérie. Ainsi, l’étude de la structure de ses transporteurs permettrait de mieux comprendre la spécificité des sels bactéricides de bismuth contre ce pathogène, et pourrait conduire à la conception d’une stratégie visant à améliorer davantage l’efficacité du traitement des infections à H. pylori.

Structure-self-assembly relationships study of the islet amyloid polypeptide


Elizabeth Godin1, Phuong Trang Nguyen1, Ximena Zottig1, Steve Bourgault1
1Université du Québec à Montréal

The islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone whose deposition as amyloid fibrils in the pancreatic islets is associated with type 2 diabetes. Previous studies have suggested that residue Asn-21 plays a critical role in the in vitro self-assembly of IAPP. Herein, we studied structure-self-assembly relationships focusing on position 21 to gain detailed insights into the molecular mechanisms of IAPP self-assembly and to probe the conformational nature of the toxic assemblies associated with β-cell death. Thioflavin T (ThT) fluorescence, CD spectroscopy and transmission EM analysis revealed that the Asn-21 amide side chain is not required for IAPP nucleation and amyloid elongation, as N21A and N21G variants assembled into prototypical fibrils. In contrast, Asn-21 substitution with the conformationally constrained and turn-inducing residue Pro accelerated IAPP self-assembly. Successive substitutions with hydrophobic residues led to the formation of ThT-negative β-sheet-rich aggregates having high surface hydrophobicity. Cell-based assays revealed no direct correlation between the in vitro amyloidogenicity of these variants and their toxicity. In contrast, leakage of anionic lipid vesicles disclosed that membrane disruption is closely associated with cytotoxicity. We observed that N21F variant self-assembles into worm-like aggregates, causing loss of lipid membrane structural integrity and inducing β-cells apoptosis. These results indicate that specific intra and inter-molecular interactions involving Asn-21 promote IAPP primary nucleation events by modulating the conformational conversion of the oligomeric intermediates into amyloid fibrils. Our study identifies position 21 as a hinge residue that modulates IAPP amyloidogenicity and cytotoxicity

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


Alexandre Vaillancourt1,2,3,4, Line Cantin1,2,3,4, Christian Salesse1,2,3,4
1Université Laval 2Département d'ophtalmologie et d'ORL-CCF, Faculté de médecine, Univertisé Laval 3CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec-Université Laval 4Regroupement stratégique PROTEO, Université Laval

CONTEXTE ET OBJECTIFS : L’absorption de la lumière par la rhodopsine, une protéine transmembranaire présente dans les bâtonnets, provoque l’isomérisation de son chromophore et provoque un changement conformationnel se traduisant par 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 permet l’échange du GDP pour le GTP, ce qui mène à la dissociation de la sous-unité alpha et à la cascade réactionnelle de la phototransduction visuelle. Les sous-unités alpha et gamma possèdent des acylations et sont responsables de la liaison membranaire. La contribution de la sous-unité gamma de la transducine (Gγt) à ce processus est encore inconnue : il est proposé de surexprimer et purifier la Gγt avec et sans acylation afin de caractériser sa liaison membranaire. MATÉRIEL ET MÉTHODES : La Gγt a été surexprimée en fusion avec des étiquettes de purification ainsi qu’un site de reconnaissance de la protéase du virus de la gravure du tabac. Ces étiquettes sont ensuite clivées, ce qui a permis la purification de la Gγt avec un niveau de pureté supérieur à 99%. RÉSULTATS : Les analyses de la structure secondaire par dichroïsme circulaire et spectroscopie infrarouge ont permis de confirmer la structure secondaire de la Gγt. Des mesures de stabilité thermique ont permis de déterminer les conditions optimales de son utilisation et son stockage. Des analyses en monocouche de Langmuir, de PM-IRRAS et de fluorescence ont permis de déterminer la liaison membranaire de la Gγt non-acylée. CONCLUSIONS : Il sera nécessaire de procéder à la production et la purification de la Gγt acylée. Par la suite, nos travaux sur la forme non-acylée et acylée permettront de caractériser les propriétés de la Gγt ainsi que d’analyser sa liaison membranaire.

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


Thomas Tremblay1, Vincent Denavit1, Denis Giguere1
1Université Laval

Plusieurs polysaccharides microbiens et certains glycopeptides sont des antigènes pertinents dans le domaine médical. La préparation de ces antigènes glycosidiques synthétiques peuvent aider au développement d’outils dans le diagnostic de maladies ou pourraient également être utilisés 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 bien définis 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 utilisées 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 décalage 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.

 

 

                                                  

Synthèse de nouveaux glucoses trifluorés et analyse de leur lipophilie


Megan Bouchard1, Jacob St-Gelais1, Vincent Denavit1, Denis Giguere1
1Université Laval

Les glucides jouent plusieurs rôles importants dans différents processus biologiques et la synthèse de leurs dérivés permet d’étudier certains mécanismes biologiques. La substitution de groupements hydroxyle par l’atome de fluor, qui est de plus en plus utilisé en chimie médicinale, peut avoir des effets bénéfiques comme l’augmentation de la stabilité métabolique des médicaments ou encore la présence d’interactions lectine-glucide favorables. Il y a donc un grand intérêt envers la préparation rapide de ces glucides fluorés pouvant conduire à la découverte de nouvelles applications biologiques.

 

La voie de synthèse employée pour la préparation d’un éventail de glucides polyfluorés utilise le lévoglucosane (1,6-anhydro-β-ᴅ-glucopyranose) comme produit de départ, qui est peu dispendieux et possédant une structure qui permet facilement de différencier les groupements hydroxyles et d’éviter la protection prématurée de la position O-6 et anomérique. Différents isomères de glucoses polyfluorés ont ainsi été préparés via des protocoles expérimentaux optimisés et leur lipophilie a été mesurée à l’aide de la résonnance magnétique nucléaire du fluor 19.

Synthesis and Modify of Sialyl Lewis X Enable It Attach to Cell Surface ex vivo


Haoyu Wu1, David Kwan1
1Concordia University

 Stem cell-based therapies show a large opportunity to cure patients who are suffering from different kinds of diseases and disorders especially in regenerative medicine, the most recent and emerging branch of medical science. Stem cell therapy can deal with functional restoration of specific tissues and/or organs of the patients suffering with severe injuries or chronic disease conditions. Our bodies own regenerative responses but usually these do not suffice to cure these conditions. In the present condition, donated tissues and organs cannot meet the transplantation demands of patients. Thus, we must find alternatives. Stem cells are thought having indefinite cell division potential and they can differentiate into other types of cells Stem cells have become the hottest object of study in regenerative medicine, particular in the repair of tissues and organs anomalies occurring due to congenital defects, disease, and age associated effects. A member of stem cell family is Adult mesenchymal stem cells (MSCs), which are the progenitor cells for most of tissues, are in pre-clinical and clinical trials to prove that they have the function in treatment of different diseases. Infused MSCs are thought have the capability to home and engraft (Homing is the phenomenon whereby cells migrate to the organ of their origin), but homing of MSCs is at a very low efficiency, typically less than I % of the infused cells reach the targeted site.         

  Recent researches point out that some cell surface structure may be a solution of their poor efficiency. Here we find sialyl Lewis X (SLeX), a tetrasaccharide carbohydrate which is usually attached to O-glycans on the surface of cells. SLeX is known to play a vital role in cell-to-cell recognition processes. These processes are also how an egg attracts sperm. There is also some report said the lack of some surface targeting agents like SLeX is responsible for MSCs its poor engraftment ability. But there is still no relevant research about how to use SLeX to modify cells, especially in vitro. Since the great therapeutic potential of MSCs depends on their efficiency of home and engraftment, it is worth for us to improve stem cells’ homing ability.

We decide to synthesis SLeX by several glycosylation steps and attach it on cell surface in vitro.  After we get our SLeX structure, click chemistry will be used to link a chemical handle to it by the catalysis of cupric ion, which will enable us to attach it to cell surface proteins by covalent interaction.  Then we will test the local rate of our SLeX installing stem cells with boyden-chamber assay. If our work is successful, this synthesis pathway can exploit the medical potential of stem cells and very hopefully be turned into applications for stem cell therapy.

Synthesis and NMR-Screening of a Fluorinated Library to be Used in FBDD via 19-F NMR


David Bendahan1,2, Pat Forgione2, Steven laplante1
1INRS-IAF 2Concordia University

The discovery of biologically active drugs has played a crucial role in modern medicine and has resulted in an increase to the human lifespan and quality of life. Nonetheless, there is an urgent need for the discovery of new small molecule drugs given global health issues. Classical methods of identifying the seeds for new drugs have notoriously low success rates, tend to be expensive, are time consuming, and require large teams of talented scientists. Fortunately, the relatively recent approach of fragment-based drug discovery (FBDD) holds promise for overcoming many of the traditional hurdles. In short, rather than the high-throughput strategy of screening millions of compounds to find a unique lead, FBDD screens a smaller library of molecular fragments from which a potent lead is built up by growing and/or merging different binding fragments. This bottom-up approach has its advantages, but many methods need to be developed to maximize its successes. Nuclear magnetic resonance (NMR) spectroscopy has proven to be a valuable biophysical technique for binding detection in FBDD and 19F NMR provides added advantages over 1H NMR for drug discovery.  The present research will revolve around the use of 19F NMR spectroscopy and the synthesis of a fluorinated, thiophene based, fragment library to be screened against several drug targets.

Systematic perturbation of the yeast essential proteome using base editing


Philippe C Després1,2,3,4, Alexandre K Dubé1,2,3,4,5, Maria Isabel Acosta1,2,3,4,5, Motoaki Seki6,7,8,9, Nozomu Yachie6,7,8,9, Christian R Landry1,2,3,4,5
1Département de biochimie, microbiologie et bio-informatique, Université Laval 2Institut de biologie intégrative et des systèmes, Université Laval 3PROTEO 4CRDM, Université Laval 5Département de biologie, Université Laval 6RCAST, University of Tokyo 7Department of biological sciences, graduate school of science, the University of Tokyo 8Institute for advanced biosciences, Keio University 9PRESTO

Investigating the link between genotype and phenotype is one of the greatest challenges in proteomics. Yeast is a powerful model to investigate this question. Recent developments in genome editing technologies have allowed for the investigation of this question at high resolution using perturbation approaches. Methods based on the CRISPR-Cas9 genome editing technology have facilitated high throughput variant strain engineering but may not be appropriate for all organisms or experiments. Base editors, engineered from an inactivated Cas9 and a DNA editing enzyme such as a cytidine deaminases, offer an efficient alternative to double-stranded break based genome modification approaches, but have yet to be used at the genome scale. Previous research has determined that a specific set of yeast genes are essential for cell growth. The proteins encoded by these genes tend to be more highly conserved and are known to be hubs in protein-protein and genetic interaction networks. Understanding how and when cells can buffer mutations in these highly important genes could offer insights on the molecular basis of cellular robustness. Here, we use the Target-AID base editor to systematically perturb yeast essential genes by creating point mutations in the genome, allowing us to assess the effect over 17,000 mutations simultaneously. We identify over 1100 guides that generate mutations with a significant impact on fitness. We then use an orthogonal approach for essential gene variant strain testing to confirm observed effects and investigate site specific sensitivities. By using a modified protein fragment tag, we can then easily quantify the mutational effects on protein abundance and interactions. The interesting perturbations we investigate include a highly conserved residue in GLN4 where substitutions have a high lethality rate and residues in the master regulator RAP1. We also use this set of randomly chosen mutations to benchmark different mutation effect prediction software and highlight discrepancies linked to the integration of evolutionary information in the algorithm. Finally, using a set of stop codon generating guides, we find new rules for effective base editing guide design based on sequence and target properties. This model can then be used to optimize library design in future base editing experiments. Our results show that base editing is amenable to large-scale screening experiments and is a powerful alternative to Cas9-based approaches for residue-level precision mutagenesis.

Systematic perturbation of yeast essential genes using base editing


Philippe C Després1,2,3,4, Alexandre K Dubé1,2,4,5, Maria Isabel Acosta1,2,5, Motoaki Seki6, Nozomu Yachie6, Christian R Landry1,2,3,4,5
1Département de Biochimie, Microbiologie et Bio-informatique, Université Laval 2IBIS, Université Laval 3CRDM, Université Laval 4PROTEO 5Département de Biologie, Université Laval 6RCAST, University of Tokyo

Investigating the link between genotype and phenotype is one of the greatest challenges in proteomics, and yeast is a powerful model to investigate this question. Recent developments in genome editing technologies have allowed for the upscaling of experimental approaches that rely on protein sequence perturbation approaches. Methods based on the CRISPR-Cas9 genome editing technology have facilitated high throughput variant engineering but may not be appropriate for all organisms or experiments. Base editors, engineered from an inactivated Cas9 and a DNA editing enzyme such as a cytidine deaminase offer an efficient alternative to double-stranded break based genome modification approaches, but have yet to be used at the genome scale. Here, we use the Target-AID base editor to systematically perturb the yeast proteome, with a focus on essential genes. We use our approach to test the effect of over 17 000 mutations simultaneously and identify over a thousand guides which generate non-synonymous mutants with significant growth defects. Our method allows us to gain new biological insights on protein function: for example, we identify a highly sensitive phosphorylation site in the RAP1 essential transcription factor. We also explore the importance of sequence conservation on site sensitivity and use our data to find rules for effective base editing guide design. Our results show that base editing is amenable to large-scale screening experiments and is a powerful alternative to Cas9-based approaches for residue-level precision mutagenesis.

Teaching an old dog a new trick: Oxime resin as versatile solid-support towards various cyclic peptide scaffolds


Christopher Bérubé3, Alexandre Borgia1,2, Normand Voyer1,2
1Université Laval 2PROTEO 3Université Laval and PROTEO

Discovered by Kaiser and DeGrado in the early 80’s, Kaiser oxime resin possesses a unique labile oxime ester link that can be cleaved by aminolysis to release the peptide.1As oxime ester linkage is stable to acidic, as well as to non nucleophilic basic conditions, anchoring linear peptides as an oxime ester bond allows peptide elongation on solid-support using Boc strategy. After N-Boc removal, the susceptibility of oxime resin to nucleophilic attack of the free amino terminal group allows the release of the peptide via acid-catalyzed N-terminal macrocyclization. Based on groundwork from our lab towards large peptide macrocycles,2-5we will report our recent results on the chemical reactivity of the Kaiser oxime resin to prepare naturally occuring macrocyclic peptides, from pentapeptides to decapeptides, in a head-to-tail fashion.

 

We will report also the first on-resin head-to-side chain concomitant cyclization/cleavage using orthogonally protected amino acids (Boc/Cbz) to prepare natural pseudacyclins A-E as well as schizopeptin 791 and anabaenopeptin NZ825 macrocyclic peptide phytochemicals.3-4

 

1.  DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 198045, 1295-1300.

2.  Bérubé, C.; Borgia, A.; Simon, G.; Grenier, D.; Voyer, N. Phytochem. Lett. 201826, 101-105.

3. Bérubé, C.; Borgia, A.; Voyer, N. Tetrahedron Lett. 201859, 4176-4179.

4. Bérubé, C.; Borgia, A.; Voyer, N. Org. Biomol. Chem. 201816, 9117-9123.

5. Bérubé, C.; Gagnon, D.; Borgia, A.; Richard, D.; Voyer, N. submitted to Chemical Communications

The Recruitment of Endothelial Progenitor Cells on Bio-memetic Functionalized Surfaces


Mohamed Elkhodiry1, Omar Bashth1, Gaétan Laroche2, Jean-François Tanguay3, Corinne Hoesli4
1McGill 2Université Laval 3Montreal Heart Institute 4McGill

Endothelial progenitor cells (EPCs) are bone marrow-derived circulating cells that contribute to vascular regeneration in ischemic tissue throughout the body. Their high proliferative capacity makes them an ideal target for enhancing the re-endothelialization of vascular substitutes and contributing to vascular regenerative medicine. Yet, little is known about EPC adhesion and overall surface interactions.  The objective of the current study was to evaluate the potential of the bio-mimetic surface functionalization strategies consisting of extracellular matrix (ECM)-derived peptides and capture antibodies in promoting the adhesion of EPC-derived endothelial colony forming cells (ECFCs).

 

Peripheral blood from 22 adult human donors was used to obtain ECFCs on polystyrene culture surfaces that are modified via covalent conjugation of a fluorophore-labeled RGD peptide (CGK(PEG3-TAMRA)GGRGDS, referred to as “RGD TAMRA”), surface adsorbed with rat tail collagen (positive control), or left unmodified (negative control). After 4 weeks of culture, formation of colonies of ECFCs was observed and their endothelial hallmarks were confirmed using phenotypic and functional assays. Clonal expansion of the expanded ECFCs on the peptide-modified surfaces was studied by single cell sorting them in modified 96 well plates and quantifying the colony sizes 10 days after seeding. To study the adhesion of expanded ECFCs on different functionalized surfaces, aminated culture surfaces were modified by either the covalent conjugation with RGD-TAMRA, immobilization of CD144 (VE-Cadherin) antibody, or both processes together, and unmodified surfaces were used as a negative control.  ECFCs were seeded on the four surfaces for 3 hours of adhesion before they were fixed for immunocytochemistry.

 

RGD-TAMRA-modified surfaces promoted the formation of colonies of ECFCs from human peripheral blood at a rate that is not statistically different from conventionally used collagen-coated surfaces. Endothelial surface markers were similarly expressed on ECFCs from both the RGD‑TAMRA and collagen surfaces while hematopoietic markers were absent in cells from both surfaces. ECFCs from both surfaces formed tubular networks upon seeding on Matrigel® and cellular and actin filament alignment in the direction of the flow was observed on ECFCs from both surfaces, after 6 hours of exposure to 25 dyn/cm2 of wall shear stress. Clonal expansion using single cell sorting highlighted a significant increase in the size of ECFC colonies formed on the RGD‑TAMRA surfaces after 10 days of seeding compared to collagen-coated surfaces and the negative control. Surfaces immobilized with anti-CD144 antibodies displayed a significantly enhanced ECFC capturing potential compared to RGD-TAMRA and unmodified controls as quantified by the number of adhered cells. In contrast, average ECFC spreading was significantly increased on RGD-TAMRA surfaces compared to the anti-CD144 and unmodified controls. Yet, maximum total ECFC surface coverage was displayed by the combinatorial strategy, consisting of both the peptide and the antibody, that seemed to combine the capturing ability of the antibody and the cell spreading ability of the peptide.

 

To our knowledge, this is the first study showcasing the ability of an extracellular-derived peptide to promote the adhesion and ex vivo expansion of ECFCs derived from adult peripheral blood. The enhanced ECFC clonal expansion on RGD-TAMRA surfaces is a particularly interesting finding due to its important implication on re‑endothelialization applications. Our results also display a clear distinction between the mechanisms by which human-derived-ECFCs react to immobilized capture antibodies and conjugated ECM-peptides. The work also introduces novel combinatorial surface modifications that target sequential steps of EPC trafficking to mimic endogenous processes of vascular healing.

The role of structural pleiotropy and regulatory evolution in the retention of heteromers of paralogs


Axelle Marchant1, Angel Fernando Cisneros Caballero1, Alexandre Dubé1, Isabelle Gagnon-Arsenault1, Diana Ascencio1, Honey A. Jain2, Simon Aubé1, Chris Eberlein1, Daniel Evans-Yamamoto3, Nozomu Yachie3, Christian Landry1
1Laval University 2Birla Institute of Technology and Sciences - Pilani 3University of Tokyo

Paralogous proteins often arise from the duplication of genes encoding homomeric proteins. Such events lead to the formation of homomers and heteromers, thus creating new complexes after a single duplication event. We exhaustively characterize this phenomenon using the budding yeast protein-protein interaction network. We observe that heteromerizing paralogs are very frequent and less functionally diverged than non-heteromerizing ones, raising the possibility that heteromerization prevents functional divergence. Using in silico evolution, we show that for homomers and heteromers that share binding interfaces, mutations in one paralog have pleiotropic effects on the homomer and the heteromer, affecting both paralogous proteins at the same time and resulting in highly correlated responses to selection. As a result, heteromerization could be preserved indirectly due to negative selection for the maintenance of homomers. By integrating data on gene expression and protein localization, we find that paralogs can overcome the obstacle of structural pleiotropy and develop functional divergence through regulatory evolution.

The role of the RhoGEF ARHGEF17 (TEM4) in Mps1 function during mitosis


DIOGJENA PRIFTI1,2, Guillaume Combes1,2, Chantal Garand1,2, SABINE ELOWE1
1Centre de recherche CHU, CHUL 2Université Laval

Chromosome segregation is a crucial process which ensures genome integrity. To achieve that and minimize aneuploidy a crucial surveillance machine, the spindle assembly checkpoint (SAC) is activated during mitosis. The main role of the SAC is to inhibit the anaphase promoting complex/cyclosome (APC/C) in order to prevent mitotic exit through a complex kinase-phosphatase signaling cascade until all chromosomes are properly aligned. Defective SAC signaling leads to premature sister chromatid separation and aneuploid daughter cells. The correct kinetochore localization of SAC proteins is a prerequisite for robust SAC signaling and, hence, accurate chromosome segregation. Mps1 is a mitotic kinase with major role in SAC signaling which is recruited to unattached kinetochores (KT) in early mitosis and phosphorylates its substrates to activate the SAC. Mps1 has a highly conserved C-terminal kinase domain responsible for SAC activation and a N- terminal domain critical for its localization. Mps1 is subjected to auto trans-phosphorylation following dimerization. Even though for the moment, the exact mechanism of Mps1 activation and localization at KT is not clear, dimerization, oligomerization or clustering of this protein facilitates its activation and its localization. Studies indicate that the N-Terminal region of Mps1 which includes the N-terminal Extension (NTE) and tetratricopeptide repeat (TPR), play an important role in the regulation of its localization and its kinase activity.  Both regions, NTE and TPR have been shown to have an affinity for binding to the KT, but NTE is the dominant player of its localization. A recent study suggested that ARHGEF17 (TEM4), a Rho family GTPase exchange factor protein, is essential for the SAC through targeting Mps1 to mitotic KTs independently of its Rho GEF catalytic activity. According to that model TEM4 acts as a timer for retention of Mps1 at kinetochores. While attractive, this current model does not explain why a kinase dead Mps1 is localized more than the WT Mps1 at KT, neither explains how TEM4 is recruited at KT even in the absence of Mps1 according to their results. We have confirmed the interaction between Mps1 and TEM4. Using co-immunoprecipitation and a panel of Mps1 mutants, we are exploring the interaction between Mps1 and TEM4. In addition, we detected TEM4 localization at the spindle and the cytoplasm but not at the KT. Based on our results, binding of TEM4 to Mps1 requires a fully active Mps1, as Mps1 kinase dead, is not able to bind TEM4. TEM4 localizes at the spindle in the presence of Taxol (a drug that hyperstabilizes the spindle) and Reversine (an Mps1 inhibitor) is able to shift its localization from the cytoplasm to the spindle. Our preliminary results suggest that KT proteins involved in microtubule (MT) binding regulate TEM4 protein levels. These preliminary results highlighted the importance of the Mps1-TEM4 interaction. We speculate that TEM4 competes with MTs for binding at KT, favorizing Mps1 binding while SAC is activated, in order to promote proper chromosome alignment. In this project, we seek to determine the mechanism by which Mps1 and TEM4 interacts as well as the role of TEM4 in mitosis. This study will directly contribute to a better understanding of how Mps1 activity is regulated to ensure timely activation and silencing of the SAC and therefore chromosome fidelity, and to explore novel approaches and therapeutic strategies against cancer.

Towards the structural characterization of the abortive infection (Abi) system protein AbiV


Xiaojun Zhu1,2, Carlee Morency1,3, Geneviève Rousseau1,3, Marie-Ève Picard1,2, Sylvain Moineau1,3, Rong Shi1,2
1Université Laval 2Institut de Biologie Intégrative et des Systèmes, Université Laval 3Centre de référence pour virus bactériens Félix d'Hérelle

Bacteria develop their own super weapons to combat aganist phage infection. The abortive infection (Abi) systems in bacteria are post-infection resistance mechanisms that are crucial for phage resistance, which functions after the phage DNA ejection and before the cell lysis, interferes with phage multiplication ans causes premature bacterial cell death. So far, twenty three Abi systems have been isolated from Lactococcus lactis, and functions of some of these Abi systems have been characterized. However, the molecular mechanism of their activity is not well understood. The proteins in Abi family have a very loe homology to the proteins of known structures. Among the Abi proteins, AbiV was reported to inhinit the lytic cycle through its interaction with the phage p2 Sav. We will present here our preliminary results on the structural characterization of this protein.

Usefulness of Recoverin to express and purify visual proteins


Line Cantin1, Christian Salesse1
1Université Laval

The expression, solubility and purification of recombinant proteins are still challenging in spite of the array of available fusion tags on the market. Recoverin, a protein involved in the phototransduction cascade, has properties typical of solubility-enhancing and purification tags. Indeed, it is a highly soluble protein and it can be purified with high purity in a single step on the basis of the properties of its calcium-myristoyl switch.Given that only a few tags are available and that proteins can hardly be produced and purified on the basis of their intrinsic properties, this work was aiming to find out whether visual Recoverin could be used as a solubility-enhancing and purification tagto express and purify photoreceptor and visual cycle proteins.Recoverin and 2XRecoverin were individually cloned in fusion with Retinitis Pigmentosa 2 (RP2), RGS9-1-Anchor-Protein (R9AP)and truncated Lecithin retinol acyltransferase (tLRAT).These fusion proteins have then been expressed in E. coli. Their level of expression and solubility has been evaluated by polyacrylamide gel electrophoresis and compared to that of two well-known solubility-enhancing and purification tags: Maltose-Binding protein (MBP) and Glutathione-S-Transferase (GST). The enzymatic activity of tLRAT has been measured in fusion with Recoverin and after its cleavage.The expression level of the Recoverin-RP2 and 2XRecoverin-RP2 fusion proteins was a little smaller to that of MBP and GST. However, the solubility of RP2 in fusion with the Recoverin tags was much higher than when RP2 was in fusion with MBP and GST. In addition, the level of expression of the R9AP fusion proteins was as follows: GST-R9AP > Recoverin-R9AP > 2XRecoverin-R9AP = MBP-R9AP. R9AP was however most soluble in fusion with MBP and 2XRecoverin. In contrast, although a larger level of expression was observed for GST-tLRAT and MBP-tLRAT, these proteins were poorly soluble. High solubility was however achieved with Recoverin-tLRAT and 2XRecoverin-tLRAT. The enzymatic activity of tLRAT from the Recoverin-tLRAT fusion protein was found to be similar to that previously reported. Recoverin can therefore be successfully used to express and solubilize the photoreceptor and visual cycle proteins assayed, as efficiently as well-known solubility-enhancing and purification tags.

Utilisation d’un support solide pour le développement d’une méthodologie de synthèse de glycopeptides


Gabrielle Robert-Scott1, Thomas Tremblay1, Antoine Carpentier1, Christopher Bérubé1, Normand Voyer1, Denis Giguere1
1Université Laval

Certains glycopeptides possèdent des applications biologiques et thérapeutiques intéressantes, mais la synthèse de ceux-ci peut s’avérer de vrais défis synthétiques. En effet la formation de liens peptidiques en solution présente plusieurs désavantages comme de faibles rendements ainsi que des problèmes lors des purifications. L’objectif principal du projet est donc de développer une nouvelle méthode de couplage peptidique entre un glucide possédant une fonction amine et un peptide installé sur résine oxime. Cette méthode permet efficacement et rapidement de synthétiser plusieurs glycopeptides ce qui a été démontré avec plus de 30 exemples.

 

Les conditions de couplage peptidique ont tout d’abord été optimisées à l’aide d’un composé modèle. Par la suite, différents analogues ont été synthétisés afin de tester la robustesse et la flexibilité de cette nouvelle méthode. Différents groupements protecteurs des glucides ont permis de vérifier l’effet d’encombrement de ceux-ci et l’effet de leur rigidité. L’effet de la position de l’amine a ensuite été testé. De plus, quatre dipeptides de séquence d’acides aminés variables ont été testés. Ces peptides ont permis de confirmer l’effet d’encombrement et stéréoisomérie des acides aminés au C-terminal. D’autre part, afin d’évaluer l’impact de la chaîne peptidique, de plus longs peptides ont été clivés de la résine oxime. 

 

Finalement, avec cette nouvelle méthodologie, des sucres aminés ont été utilisés afin de synthétiser plusieurs glycopeptides d’intérêts. Ces exemples démontrent la possibilité d’utiliser la résine oxime afin de générer de nouveaux dérivés glycopeptidiques pour des applications biologiques en chimie médicinale.


Vibrational Circular Dichroism Reveals Supramolecular Chirality Inversion of α‑Synuclein Peptide Assemblies upon Interactions with Anionic Membranes


Benjamin Martial1, Thierry Lefèvre1, thierry buffeteau2, Michèle Auger1
1Université Laval 2Université Bordeaux 1

Parkinson’s disease is an incurable neurodegenerative disorder caused by the aggregation of α-synuclein (AS). This amyloid protein contains a 12-residue-long segment, AS71-82, that triggers AS pathological aggregation [1]. This peptide is then essential to better understand the polymorphism and the dynamics of formation of AS fibrillar structures. In this work, vibrational circular dichroism showed that AS71-82 is random coil in solution and forms parallel β-sheet fibrillar aggregates in the presence of anionic vesicles. Vibrational circular dichroism, with transmission electronic microscopy, revealed that the fibrillar structures exhibit a nanoscale tape-like morphology with a preferential supramolecular helicity. Whereas the structure handedness of some other amyloid peptides has been shown to be driven by pH, that of AS71-82 is controlled by peptide concentration and peptide-to-lipid (P:L) molar ratio. At low concentrations and low P:L molar ratios, AS71-82 assemblies have a left-twisted handedness, while at high concentrations and high P:L ratios, a right-twisted handedness is adopted. Left-twisted assemblies interconvert into right-twisted ones with time, suggesting a maturation of the amyloid structures [2]. Since fibril species with two chiralities have also been reported previously in Parkinson’s disease Lewy bodies and fibrils, the present results seem relevant to better understand AS amyloid assembly and fibrillization in vivo. From a diagnosis or therapeutic point of view, it becomes essential that future fibril probes, inhibitors or breakers target pathological assemblies with specific chirality and morphology, in particular because they may change with the stage of the disease.

 

 

References

[1] B. Giasson et al., J. Biol. Chem. 276, 2380-2386 (2001)

[2] B. Martial et al., ACS Nano, 13, 3232-3242 (2019)

Visualizing the heme loading of a protein in live cells using green fluorescent protein


Samaneh Dastpeyman1, Gonzalo Cosa2, Ann English1
1Concordia University 2McGill University

Heme proteins are involved in numerous biological processes, including aerobic respiration, cell signaling, antioxidant stress and xenobiotic metabolism. Despite its role as an essential prosthetic group, we do not known how heme is distributed from the mitochondrial matrix, where it is synthesized, to the proteins that need it. We do know, however, that apocatalase A (Cta1) maturation involves recruitment of heme from cytochrome c peroxidase (Ccp1) in yeast mitochondria. Thus, tracking Ccp1’s heme loading in live cells is of interest but this has not been achieved for a heme protein to date. Heme is a highly efficient quencher of green fluorescent protein (GFP) so we chose to use yeast chromosomally expressing the Ccp1-GFP fusion under the native Ccp1 promotor to monitor the heme status of Ccp1 in live cells. We first characterized the time-resolved fluorescence of recombinant apo- and holoCcp1-GFP in vitro. ApoCcp1-GFP exhibits a fluorescence lifetime of 2.86 ns whereas holoCcp1-GFP decays with two lifetimes, 0.96 ns and 2.45 ns. The fractional amplitude of the 0.96-ns lifetime increases linearly with heme loading of apoCcp1-GFP. With this knowledge, we followed by fluorescence lifetime imaging microscopy (FLIM) the heme loading of Ccp1 in the live yeast cells expressing Ccp1-GFP. From the lifetime amplitudes of GFP fluorescence, we find that Ccp1-GFP is ~ 90% heme loaded and resides in the mitochondria of 2-day cells. In contrast, in 7-day cells, half of Ccp1-GFP is extra-mitochondrial and 100% heme-free. Overall, our unprecedented study reveals the power of FLIM in monitoring both the heme loading and location of a GFP-fusion protein in live cells. We also have enlarged the toolkit for monitoring intracellular heme trafficking as well as expanding the functional repertoire of genetically encoded fluorescent probes.