A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells
Razan Sheta
A SECOND PROTEIN ENCODED BY THE MIEF1/SMCR7L/MID51 GENE INDUCES MITOCHONDRIAL FISSION.
Maxime Beaudoin
ANALYSE PROTÉOMIQUE DE LA SPÉCIFICITÉ DES PROTÉINES ADAPTATRICES NCK1 ET NCK2
Kévin Jacquet
Assessing the evolutionary potential of an antibiotic resistance enzyme
Lorea Alejaldre
Catching de novo proteins as they arise in natural populations
Eléonore Durand
Challenges related to the expression and solubilization of membrane-associated proteins
Sarah Bernier
Characterization of a fluorinated peptide: towards the development of a novel ion channel
Maud AUGER
Characterization of a putative laccase gene from Dictyopanus pusillus
Andres Rueda
Comparaison entre la structure et l’activité catalytique de la lécithine rétinol acyltransférase sauvage et de son mutant R173P chez la souris ainsi que des mutants P173L et P173R chez l’humain.
Marie-Ève Gauthier
Comparaison structurale et fonctionnelle de deux homologues fonctionnels de la superfamille des RNases pancréatiques retrouvés chez l’humain
Jacinthe Gagnon
Computational Design of Dynamics into a Stable Globular Protein
Adam Damry
Computational Design of Multi-Substrate Enzyme Specificity
Antony St-Jacques
Computational evidence for the evolution of P450 monooxygenases towards the exclusion of non-productive gases
Maximilian C.C.J.C. Ebert
Contributions of glycosaminoglycans to membrane adsorption and conformational conversion of the peptide hormone secretin
Noé Quittot
Contributions of tyrosine nitration and histidine carbonylation in the amyloid assembly of immunoglobulin light chains
Ximena Zottig
Correlating functional divergence and dynamical variations in the pancreatic-type ribonuclease superfamily
Chitra Narayanan
Crystallization and Structure Determination of PieE: A Flavin-binding Hydroxylase
Mahder Manenda
Deep functional proteomic identification of alternative proteins gives insight into their functions
Vivian Delcourt
Deep transcriptome annotation provides functional insights for thousands of novel proteins
Sondos Samandi
Délivrance quantitative de médicament par effet de tampon
Arnaud Desrosiers
Dengue fusion peptide: determination of its binding parameters upon insertion into Langmuir monolayers mimicking cell membranes
Thaís F. Schmidt
Developing a screening platform by Surface Plasmon Resonance (SPR) for the characterization and discovery of enzyme inhibitors
Sarah Melissa Jane Abraham
Development of a SPR immunosensor for monitoring silent hypersensitivity to L-asparaginase during chemotherapy for leukemia
David M. Charbonneau
Development of a whole cell high-throughput screening of P450 BM3 for the generation of efficient and reliable computational training sets in a mutation prediction algorithm for desired substrate conversion
Olivier Rousseau
Development of new β-lactamases inhibitors using a sulfahydantoin scaffold
Pierre-Alexandre Paquet-Côté
Développement d’un procédé en deux étapes pour la purification de vésicules pseudotypées avec le VSV-G
Juliette Champeil
Discovery and Structure-Activity Relationship of a Bioactive Fragment of ELABELA that Modulates Vascular and Cardiac Functions
Alexandre Murza
Dissecting the functional role of the non-conserved N-terminal region of Drosophila melanogaster Small Heat Shock Protein DmHSP27.
Mohamed Taha MOUTAOUFIK
Diversity among five newly isolated phages infecting Citrobacter freundii
Sana Hamdi
Échange conformationnel dans les doigts de Zinc de Miz-1 : Implication de résidus His non-conservés
Jean-Michel Moreau
Élucidation du mécanisme d’activité enzymatique de la lécithine rétinol acyltransférase par des études structurales en résonance magnétique nucléaire
Sarah Roy
Enzyme-Operated DNA-Based Nanodevices
Erica Del Grosso
Étude des réseaux signalétiques de GRB2 dans la signalisation des récepteurs de la famille HER
Alice Beigbeder
Fluorinated NMR Probes for the Study of Membrane Topology: Monofluorinated Dimyristoylphosphatidylcholine and Dimyristoylphosphatidylglycerol Lipid Membranes
Marie-Claude Gagnon
Functionalized Low Generation Dendrimers Modulate Islet Amyloid Polypeptide Self-Assembly and Cytotoxicity
Phuong Trang Nguyen
Générer des mutants par sauvetage évolutif afin de mieux comprendre les bases moléculaires de la réponse au stress
Clara Bleuven
Glycosaminoglycan-mediated amyloid assembly of the neuropeptide hormone PACAP
Mathew Sebastiao
Identification and optimization of inhibitors of type II dihydrofolate reductases, trimethoprim-resistant enzymes
Jacynthe Toulouse
Identification de nouveaux acteurs de la signalisation dépendante du récepteur aux androgènes par approches combinées de protéomique
Lauriane Vélot
Identification de nouvelles protéines effectrices dans la signalisation des récepteurs Eph
Sara Banerjee
Impact de la charge des lipides et de la fluidité membranaire, ainsi que du calcium sur la recoverine, une neuroprotéine des photorécepteurs sensible au calcium
Kim Potvin-Fournier
Incorporation of Crown Ether Peptides in Model Membranes : A Study Using Synchrotron Radiation-Based Oriented Circular Dichroism Spectroscopy
Jean-Daniel Savoie
Influence de la composition chimique de la membrane plasmique du spermatozoïde sur l'action de la protéine BSPA1-A2 chez les bovins
Dominic Lauzon
Investigating the kinetics of hypoxia-inducible factors heterodimerization by surface plasmon resonance
Billel Djerir
Investigation of the mechanism of action of synthetic peptides displaying antimicrobial potential
Matthieu Fillion
IsoMif - Detection of binding site molecular interaction field similarities
Matthieu Chartier
La caspase-7 a un faible pour PARP-1
Alexandre Desroches
La fonction des protéines S100A10 et annexine A2 dans la réparation membranaire
Xiaolin YAN
La modification du métabolisme des adipocytes humain pour la prévention du diabète de type 2.
Thierry Chénard
Les liaisons dangereuses de la caspase-7 et de p23.
Cyrielle Martini
Ligand Binding Induces Enhanced Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans
Louise Roux
Macrocycle mimics active conformation of apelin-13
Kien Tran
Measuring protein-protein assemblies with a molecular ruler in living cells
Andrée-Ève Chrétien
Measuring the temperature around an enzyme using DNA thermometers
Scott Harroun
Modeling the GAPDH – SIAH1 Death Complex
Vinod Parmar
Modelling conformational entropy in the scoring function of the molecular docking software FlexAID
Louis-Philippe Morency
Molecular insights into the interplay between metal coordination and DNA binding by the Campylobacter jejuni Ferric Uptake Regulator (CjFur)
Sabina Sarvan
New insight on experimental data using a streamlined, naive protocol for molecular dynamics analysis
Laurent Bruneau Cossette
Nouveau protocole de recherche d’antibiotiques appliqué à Clostridium difficile.
Mathieu Larocque
Optimisation d’un milieu de culture défini pour la culture de myoblastes humains
Alexandre Côté
Optimization and characterization of synthetic antimicrobial peptides
Nicolas Poulin
Peptides macrocycliques pour l'exploration conformationnelle de l'interaction Neurotensine-NTS1
Marc Sousbie
Phytochemical investigation of northern Quebec lichens
Claudia Carpentier
Prédiction d'un site d'inhibition allostérique spécifique à une ectonucléotide pyrophosphatase/phosphodiestérase 1 humaine (ENPP1) par modélisation moléculaire
Xavier Barbeau
Prédiction de structure et de fonction de toutes les protéines du phage Lactococcus lactis P680
Wiem Kristou
Preparation of Various 2,3,3-triarylacrylic Acid Esters, a Particular Class of 1,2,2-triarylethene Compounds, using Suzuki-Miyaura Coupling Reactions
Sébastien Cardinal
Protein engineering of the CalB lipase to synthesize methyl salicylate
Ying Chew Fajardo
Protein-protein interactions of a thermosensitive allele in different environments
Véronique Hamel
Proteins and oligopeptides as green catalysts for chiral epoxidations
Christopher Bérubé
Recréer et comprendre les mécanismes de reconnaissance moléculaire à l'aide d'interrupteurs d'ADN
Carl Prévost-Tremblay
Régulation par phosphorylation de la fonction des domaines SH3 de NCK1/2
Ugo Dionne
Selective binding of functionalized porphyrin compounds at the homodimeric interface of human galectin-7
Philippe Egesborg
Semi-rational evolution of the RhlA enzyme from Pseudomonas aeruginosa for the synthesis of industrially relevant rhamnolipids.
Carlos Eduardo DulceyJordan
Similarities Between Conformational Exchange Patterns in Members of the Ribonuclease 3 Subfamily
David N. Bernard
Smart mutational exploration of the CalB lipase active site using a combination of virtual docking and iterative saturation mutagenesis
Yossef Lopez de los Santos
Spatial distribution of an anti-cancer drug in mouse skin studied by infrared microspectroscopy.
Quoc Chon Le
Spectroscopic Investigation of α-Synuclein 71-82, a Peptide Derived from a Protein Involved in Parkinson’s Disease
Benjamin Martial
Structural insights into the assembly of the Ash2L/DPY-30 heterotrimer
John Haddad
Structural Investigations of Supercontracted Spider Dragline Silk
Justine Dionne
Structure prediction of a conserved membrane peptide from Lactococcus lactis phages from molecular dynamics simulations
Christine J. LeBlanc
Structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes
Sophie Gobeil
Study of the interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact gram+ and gram - bacteria by solid-state NMR
Marwa Laadhari
STUDY OF THE STRUCTURE AND FUNCTION OF THREE DROSOPHILA MELANOGASTER HSP22 ARGININE MUTANTS
Afrooz Dabbaghizadeh
Substrate recycling in secondary metabolism: a smart design in microbes
Marie-Ève Picard
Sulfur-Aromatic Interactions: Modeling Cysteine and Methionine Binding to Tyrosinate and Histidinium Ions
Esam Orabi
SYNTHÈSE ET ACTIVITÉ IN VITRO D’INHIBITEURS D’UNE ECTONUCLÉOTIDE PYROPHOSPHATASE / PHOSPHODIESTERASE DE TYPE 1 (ENPP1)
Elsa Forcellini
The BDKRB2 Gene Encodes Two Distinct Proteins: The Bradykinin B2 Receptor (B2R) and a Regulator of the B2R Signalling
Maxime Gagnon
The binding affinity of StarD6 with different ligands
Patrick Delattre
The TagR as a New and Versatile Fusion Protein Tool to Improve Solubility and Purification of Proteins
Line Cantin
Une approche de biologie des systèmes appliquée au domaine acéricole mène à l’élaboration d’un nouvel outil pour le contrôle de la qualité de la sève d’érable
Marie Filteau
VESICULAR STOMATITIS VIRUS-BASED VESICLES USED FOR NUCLEIC ACIDS DELIVERY.
Mathias Mangion

A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells


Razan Sheta1,2, Christina M. Woo3, Florence Roux-Dalvai4, Frédéric Fournier4, Sylvie Bourassa4, Arnaud Droit1,4, Carolyn R. Bertozzi3,5, Dimcho Bachvarov1,2
1Department of Molecular Medicine, Laval University, Québec PQ, Canada 2Centre de recherche du CHU de Québec, L’Hôtel-Dieu de Québec, Québec PQ, Canada 3Department of Chemistry, Stanford University, Stanford, CA, USA 4Centre de recherche du CHU de Québec, CHUL, Québec PQ, Canada 5Howard Hughes Medical Institute, Stanford University, Stanford, CA USA

Epithelial ovarian cancer (EOC) is a disease that is responsible for more cancer deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of the mechanisms involved in EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade (HG) serous EOC tumors, compared to low malignant potential (LMP) and normal ovarian tissues. This data implicated GALNT3 in the aberrant glycosylation of the MUC1 oncogene, as well as other glycoproteins, in EOC cells. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry- based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most of the identified glycoproteins are involved in mechanisms of cellular metabolic functions, post-translational modifications; and some have been reported to be implicated in EOC etiology.

The GALNT3-dependent glycoproteins revealed by this metabolic labeling approach confirm the oncogenic role of GALNT3 in EOC dissemination and may be pursued as a novel EOC biomarker and/or therapeutic target.

A SECOND PROTEIN ENCODED BY THE MIEF1/SMCR7L/MID51 GENE INDUCES MITOCHONDRIAL FISSION.


Maxime Beaudoin1, Annie.V.Roy 1, Sondos Samandi1, Jean-François Jacques1, Xavier Roucou1
1Université de sherbrooke

INTRODUCTION:

The human proteome has more than 50, 000 proteins encoded in specific coding sequences (CDS) of mature mRNAs. This simplistic view is however challenged by recent evidence of the human mRNA multicoding potential. Novel proteins are translated from alternative open reading frames (AltORFs) located either in 5’UTRs or 3’UTRs, normally considered as untranslated regions, or AltORFs overlapping the CDS in the non-canonical +2 and +3 reading frames. The MIEF1/SMCR7L/MID51 gene encodes the Mitochondrial Dynamics Protein of 51kDa (MiD51), a mitochondrial fission receptor. We’ve previously shown that a 70 amino acids alternative protein termed AltMID51 is translated from an altORF in the 5’UTR of MIEF1/SMCR7L/MID51 mRNA. We show here that AltMiD51 is a mitochondrial matrix protein with a conserved LYR superfamily domain with a mitochondrial fission activity similar to MID51.

 

OBJECTIVE:

1-To determine the region of the protein involved in its trafficking and fission activity.

 

2-To elucidate AltMID51 interactome.

 

MÉTHODS:

Objective 1: AltMiD51 deletion mutants were used to determine the localization of the mitochondrial targeting sequence the protein is responsible of the mitochondrial signal and fission activity. Next steps is to mutate precisely some amino acids to establish which of those are implicated in these functions. Those experiences are performed by immunofluorescence and visualise by confocal microscopy.

Objective 2: We used mass spectrometry-based immunoprecipitation proteomics to characterize altMID51 interactors.  A specific interaction was confirmed in vivo by bimolecular fluorescence complementation and GST Pull down assays.

 

RESULTS:

We show that the LYR domain of AltMid51 is essential for the fission activity. The N-terminal 23 amino acid residues contain both the mitochondrial localisation signal and the fission activity. Furthermore, we show that AltMiD51 homodimerises and interacts directly with the Mitochondrial Acyl carrier protein (ACPM). Our results indicate that AltMiD51 is a novel mitochondrial protein implicated in mitochondrial fission.

 

CONCLUSION:

MID51 is a dual coding gene expressing two completely different proteins involved in mitochondrial fission. We will next explore whether the complex AltMID51/ACPM is necessary for mitochondrial fission. Our results suggest that alternative proteins are functional proteins.

ANALYSE PROTÉOMIQUE DE LA SPÉCIFICITÉ DES PROTÉINES ADAPTATRICES NCK1 ET NCK2


Kévin Jacquet1,2,4, Ugo Dionne1,2,4, Nicolas Bisson1,2,3,4
1Centre de Recherche sur le Cancer 2PROTEO 3Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Université Laval 4Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec

Plusieurs réponses cellulaires aux stimuli extracellulaires sont transmises par des voies de signalisation qui agissent en aval de récepteurs membranaires tels les récepteurs tyrosine kinase (RTK). Les signaux provenant des RTK sont souvent relayés via des protéines adaptatrices. Parmi celles-ci, notre laboratoire étudie plus particulièrement les protéines NCK1 (Non-Catalytic region of tyrosine Kinase 1) et NCK2 dont les fonctions sont considérées redondantes et indissociables. Cependant, il a été suggéré que NCK1 et NCK2 pourraient avoir des cibles cellulaires spécifiques et des fonctions uniques. L’objectif de mon projet est d’analyser la spécificité des protéines NCK1/2 en identifiant pour chacune des cibles uniques, pour ensuite caractériser ce qui génère cette spécificité tout en définissant la fonction de ces interactions. En premier lieu, nous avons utilisé des purifications d’affinité suivies d’analyses en spectrométrie de masse (AP-MS) pour identifier près d’une centaine de partenaires spécifiques à NCK1/2 dans différentes lignées cellulaires. Par la suite, en utilisant une technique de marquage de proximité (BioID), nous avons tenté de confirmer ces interactions tout en détectant plus de 100 nouveaux partenaires plus transitoires non détectés lors de nos approches d’AP-MS standard. Sur la base de ces résultats et des données disponibles dans la littérature, nous avons sélectionné 27 partenaires pour confirmer par une méthode orthogonale leur interaction avec NCK1/2. Grâce à des tests de liaison in vitro nous avons pu déterminer que 2 de ces partenaires (PKP4 et CPSF6) lient directement et spécifiquement NCK2 grâce à leurs régions N-terminales. Enfin, en inversant les régions interdomaines de NCK1 avec celles de NCK2, nous avons pu montrer que ces dernières ne sont pas impliquées dans la spécificité des NCK. Les données obtenues rendent maintenant possible l’identification d’un groupe de résidus au sein des domaines de chaque NCK pouvant expliquer la spécificité de ces protéines

Assessing the evolutionary potential of an antibiotic resistance enzyme


Lorea Alejaldre1,2, Joelle Pelletier1,2,3
1Université de Montréal 2PROTEO: The Quebec Network for Research on Protein Function, Engineering and Applications 3Centre in Green Chemistry and Catalysis (CGCC)

The rise of antibiotic resistance is an emergent health crisis due to the speed at which it is developing and its economical and clinical repercussions. The appearance of new enzymatic activities within bacterial cells is one of the most common causes of antibiotic resistance. Therefore, assessing the capacity of an enzyme to evolve towards new activities, its innovability, is important to understand and counteract this issue.

In the present study, we aim to explore the capacity of the primitive enzyme R67 dihydrofolate reductase (R67 DHFR), that confers resistance to the commonly prescribed antibiotic trimethoprim, to develop new activities.

A site-directed saturation mutagenesis library in the residues involved in binding and catalysis in the enzyme R67 DHFR has been screened against several types of antibiotics. Differential survival of clones has allowed to identify a variant which confers weak resistance to an antibiotic chemically unrelated to trimethoprim: tetracycline.

Characterization of this new variant will help to understand the evolution of this primitive protein and its potential as a multi-drug resistant source. 

 

 

Catching de novo proteins as they arise in natural populations


Eléonore Durand1, Isabelle Gagnon-Arsenault1, Lou Nielly-Thibault1, Guillaume Charron1, Isabelle Hatin2, Olivier Namy2, Christian R. Landry1
1Institut de biologie intégrative des systèmes (IBIS), PROTEO, Département de biologie, Université Laval 2Institut de Biologie Intégrative de la Cellule (I2BC), CEA, CNRS, Université Paris-Sud, Orsay, France.

The emergence of new proteins is a driving engine for the acquisition of adaptive innovations. New proteins may arise de novo from previously non-genic regions or from pre-existing gene structures such as gene duplication. The de novo origination is the initial source of genetic novelties and comparative studies between and within species brought to light the potential contribution of intergenic regions in this process. De novo protein origination involves the acquisition of an intergenic ORF by mutations conferring a gain of in frame start and stop codons, and regulatory sites to allow its transcription and translation. Here we explore the dynamics of recently emerging de novo proteins in natural Saccharomyces paradoxus populations. We first characterized intergenic ORF diversity in 24 S. paradoxus strains, sampled in North America, that are structured in 3 lineages. These lineages are used to investigate an on ongoing speciation event, making them a perfect model in the context of recently emerging proteins and their role in adaptation and potentially speciation [1]. We annotated intergenic ORFs and classified them according to the conservation of their positions. In total, 73,657 ORF families were identified: some (2%) are conserved between S. cerevisiae and S. paradoxus while 28 and 8% are fixed and specific to S. cerevisiae or S. paradoxus respectively. Interestingly, the remaining 62% of intergenic ORFs still segregate within and among the S. paradoxus lineages. This diversity illustrates the power of using population data to investigate the emergence of de novo proteins from scratch, which is expected to be rare. We are now characterizing the coding potential of intergenic ORFs at the transcriptional and translational levels to compare the rate of transcription/translation gain or loss versus ORF birth and death. Our results show that intergenic regions contain a large pool of ORFs readily available for natural selection to draw from during species formation. 

 

1. Leducq JB. et al. (2016) Speciation driven by hybridization and chromosomal plasticity in a wild yeast. Nature Microbiol. 1: 15003. doi:10.1038/nmicrobiol.2015.3.

Challenges related to the expression and solubilization of membrane-associated proteins


Sarah Bernier1,2,3, Line Cantin1,2,3, Habib Horchani 1,2,3, Christian Salesse1,2,3
1Université Laval 2CUO-recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement 3PROTEO

Membrane-associated proteins typically have a low solubility and are very difficult to express as recombinant proteins in prokaryotic cells. Different expression systems and tags can be assayed but finding the proper combination can be very tedious. We have expressed several different membrane-associated proteins in prokaryotic cells over the years and producing such purified proteins is always very challenging. In addition, the removal of the protein tag used to improve the solubility (and to allow purification) of the protein of interest may result in the loss of the solubility. In the present work, we are showing data to illustrate these challenges using the membrane-associated proteins « lecithin retinol acyltransferase (LRAT) » and « RGS9-1 anchor protein (R9AP) ». LRAT is involved in the visual cycle whereas R9AP plays an important role in visual phototransduction. LRAT and R9AP are associated to membranes likely via alpha helical segments located both or either in their N- and C-terminal. They however likely also bear additional sites involved in their membrane binding which further complicates their manipulation. Truncated proteins without their membrane-associated alpha helical segments have been used until now because full-length proteins are both difficult to express and to solubilize.

The expression of truncated LRAT (tLRAT) resulted in a protein that could solely be solubilized using a low concentration of the detergent sodium dodecyl sulfate (SDS). This is also true for the truncated human and bovine R9AP (tR9AP) except for the glutathion-S-transferase (GST)-tagged bovine protein. The role of the tag on protein expression, solubility and purification has then been assayed in details using different constructions of the bovine and human tR9AP (b-tR9AP and h-tR9AP). Despite the high sequence identity between b-tR9AP and h-tR9AP (79% using global alignment), the N-terminal GST-tagged bovine protein was highly soluble whereas SDS was necessary to achieve solubilization of the human protein. Structural analyses using I-Tasser models of these proteins allowed to provide tentative explanations for this different behavior. However, a lower molecular mass protein labeled with the anti-GST and anti-R9AP antibodies could be observed by SDS-PAGE and western blot after purification of the GST-tagged b-tR9AP. An additional tag (polyHis) was thus added at the C-terminal of the human and bovine proteins to find out where proteolysis was taking place. The lower molecular mass protein was also purified using His-trap column with this construction (GST-tR9AP-polyHis), thus suggesting that some type of proteolysis takes place at the N-terminal although the affinity of GST for glutathion remains unchanged. It is noteworthy that the addition of the C-terminal polyHis tag resulted in a complete loss of the solubility of the GST-tagged bovine protein that could solely be recovered using SDS such as the human protein (with only a GST-tag). In addition, it is interesting to mention that the bovine GST-tR9AP-polyHis was soluble using both SDS and sodium cholate whereas the human protein was only soluble in SDS.

SDS is required for the solubilization of the human R9AP, regardless of the tag used. Moreover, this detergent prevents proper binding of GST-tagged proteins. An additional construction was thus also prepared (polyHis-GST-tR9AP) which could allow to achieve R9AP purification, in addition to the GST-tR9AP-polyHis construction which however resulted in proteolysis, as mentioned above. In conclusion, although expression and purification is easily achieved for some membrane-associated proteins, the challenge of expressing and purifying this type of proteins is generally very difficult. 

Characterization of a fluorinated peptide: towards the development of a novel ion channel


Maud AUGER1, Raphaël Godbout1, Normand Voyer1, Michèle Auger1
1Université Laval 2PROTEO 3CQMF 4CERMA

Peptides play major roles in cells. Among others, they can act as chemical messenger, hormone, antimicrobial agents, and ion channels. Peptide can adopt various functional conformations. The group of DeGrado has demonstrated that a α-helical peptide with 21 residues constituted only of leucine and serine residues can form an ion channel1. We have substituted the serine residues by trifluoro-alanines to obtain a more stable peptide assembly due to inter-helix fluorine interactions.

 

Molecular modelization suggests that incorporation of fluorine atoms in the sequence of the peptide promotes a stable alpha-helical conformation. In the presence of lipid membranes, the peptide LX2 is predicted to auto-assemble into tri- and tetramer ion channels in which the fluorine atoms are located in the center of the channel. This template peptide could be modified to develop novel nanostructures having chemotherapeutic activity. The aim of this study is to investigate the interactions between LX2 and model lipid membranes and to examine experimentally the presence of tetrameric LX2 ion channels.


The synthesis and characterization of a peptide containing 21 amino acids made of 15 leucines and six trifluoro-alanines has been carried out2. We will present a structural analysis of the LX2 peptide by circular dichroism and infrared spectroscopy. Ongoing biophysical studies on peptide-membrane interactions will also be presented.

 

Références:

1) DeGrado, W.F.; Wasserman, Z.R.; Lear, J.D. Science. 1989, 243, 622-628.
2) Lear, J.D.; Wasserman, Z.R.; DeGrado, W.F. Science. 1988, 1177-1181.

Characterization of a putative laccase gene from Dictyopanus pusillus


Andres Rueda1,3,4, Nicolas Doucet2, Clara Sánchez4, Sonia Ospina3, Daniel Molina4
1INRS-Institut Armand-Frappier 2INRS-Université du Québec 3Instituto de Biotecnología-Universidad Nacional de Colombia 4Universidad Industrial de Santander-Colombia

Lignolytic enzymes are a group of biocatalysts with potential applications in delignification and bioremediation. The enzymatic delignification process is a green chemistry alternative for the pretreatment of lignocellulosic material, providing a means for the efficient removal of lignin and the synthesis of biologically active compounds such as monolignols. Laccases (EC 1.10.3.2) are the most studied enzymes in delignification processes and Basidiomycete fungi are the main source. The aim of this study was to identify the gene sequence and protein structure of a protein band identified with a laccase activity from an enzymatic extract obtained by solid-state fermentation (SSF) of Dictyopanus pusillus. The enzymatic extract from D. pusillus was concentrated and purified by fast protein liquid chromatography (FPLC) using an anion exchanger. SDS-PAGE and native PAGE were used to determine the molecular weight and activity of the bands obtained. Tryptic digestion and Micro-HPLC-MS analyses were performed to identify peptides belonging to the protein band identified with laccase activity. From those peptides, degenerate primers were designed to amplify the coding gene sequence from D. pusillus. Three DNA sequences with high identity were obtained and have been used to elucidate the putative laccase gene. These results confirm the expression of a new laccase in D. pusillus, further allowing overproduction of this enzyme in a heterologous system.

Comparaison entre la structure et l’activité catalytique de la lécithine rétinol acyltransférase sauvage et de son mutant R173P chez la souris ainsi que des mutants P173L et P173R chez l’humain.


Marie-Ève Gauthier1,2,3, Line Cantin1,2,3, Christian Salesse1,2,3
1Département d’ophtalmologie, Faculté de médecine, Université Laval 2CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement 3Regroupement stratégique PROTEO

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 qui permet la régénération du pigment visuel. Chez la LRAT humaine (hLRAT), des mutations de substitution mènent à la dégénérescence des photorécepteurs. Une de ces mutations implique le changement de la proline 173 pour une leucine (P173L). Des études précédentes ont démontré que l’activité catalytique de cette enzyme, sans ses segments hydrophobes en N- et C-terminal (htLRAT), est grandement diminuée avec la mutation P173L. Il a donc été postulé que cet acide aminé est important pour la catalyse. De plus, cette proline est très conservée chez les mammifères. Toutefois elle est remplacée par une arginine chez la souris. L’hypothèse de ce projet de recherche est donc que l’activité catalytique de la LRAT de la souris (mLRAT) est plus faible que celle de la hLRAT et que, par conséquent, l’activité du mutant R173P-mLRAT est similaire à celle de la hLRAT alors que celle du mutant P173R-hLRAT est similaire à celle du mutant P173L-hLRAT. Les objectifs de ce projet consistaient donc à 1) cloner, surexprimer et purifier la mtLRAT, son mutant R173P ainsi que le mutant P173R-htLRAT et 2) comparer leur activité enzymatique et leur structure secondaire avec celle de la hLRAT sauvage et de son mutant P173L-htLRAT. MATÉRIEL ET MÉTHODES : L’ARN a été extrait du foie de souris et l’ADNc de la mtLRAT a été amplifié par RT-PCR et inséré dans le vecteur pET11a qui a servi à surexprimer la mtLRAT. Les mutants R173P-mtLRAT et P173R-htLRAT ont été préparés par mutagenèse dirigée. La protéine htLRAT et le mutant P173L-htLRAT avaient été préparés auparavant au laboratoire. Les différentes tLRAT ont été purifiées par chromatographie d’affinité. Leur activité enzymatique a été déterminée par HPLC et leur structure secondaire par dichroïsme circulaire. RÉSULTATS : Nos résultats préliminaires ont démontré que l’activité enzymatique de la mtLRAT est similaire à celle de la htLRAT. Les mutations R173P-mtLRAT et P173R-htLRAT n’ont pas eu d’effet drastique sur l’activité enzymatique en comparaison avec celle de la protéine sauvage sauf dans le cas de la P173L-htLRAT. De plus, la structure secondaire de ces protéines est très similaire. CONCLUSION : Même si nos données suggèrent que la proline 173 est très importante pour le maintien de l’activité enzymatique de la tLRAT humaine et murine, la modification de cet acide aminé pour une arginine ne semble pas avoir d’effet drastique sur ses propriétés. Il sera donc nécessaire de caractériser l’impact des mutations de la tLRAT sur son activité catalytique  par la détermination de la structure tridimensionnelle de ces différents mutants.

Comparaison structurale et fonctionnelle de deux homologues fonctionnels de la superfamille des RNases pancréatiques retrouvés chez l’humain


Jacinthe Gagnon1, Sabina Sarvan2, Donald Gagné1, Jean-François Couture2, Nicolas Doucet1
1INRS-Université du Québec 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.

Les membres de la superfamille de la ribonucléase (RNase) A ont été associés à une grande variété de fonctions biologiques, en plus de leurs activités ribonucléolytiques fortement conservées. Par exemple, certaines RNases ont des propriétés antibactériennes, cytotoxiques, angiogéniques, immunosuppressives, anti-tumorales et/ou antivirales. Il existe 8 membres de la famille des RNases A chez l’être humain. Ces enzymes ont évolué rapidement et possèdent divers degrés de d’homologie et d’activité enzymatique. La première structure cristalline de la RNase 6 (ou RNase k6) n’a été que récemment résolue en présence d’anions de sulfate, qui se fixent à deux sites distincts sur l’enzyme. Nous avons cristallisé la RNase 6 en présence d’anions de phosphate, démontrant également deux sites de liaison distincts avec le phosphate, dont l’un étant situé dans la boucle 4 et n’ayant jamais été identifié chez aucun autre membre des RNases humaines. Les propriétés biophysiques des RNases A, 4 et 6 ont également été analysées par titrage en résonance magnétique nucléaire (RMN) et par titrage calorimétrique isotherme (TCI) avec deux ligands : 3´-UMP et 5´-AMP. Les similarités et différences entre ces analyses seront présentées. La structure cristalline de la RNase 6 sera également comparée avec celles des RNase A et RNase 4. Finalement, les différences structurales pouvant partiellement expliquer leur identité fonctionnelle seront également discutées, offrant plusieurs indices indispensables à la compréhension de leur fonction biologique.

Computational Design of Dynamics into a Stable Globular Protein


Adam Damry1, James Davey1, Natalie Goto1, Roberto Chica1
1University of Ottawa

Proteins are widely used in research, industry, and medicine for their ability to carry out complex molecular processes with high precision and efficiency. It is generally thought that the three-dimensional structure of proteins dictates their function, but increasing evidence demonstrates that complex functions are equally mediated by dynamics. Traditional rational design methodologies however do not consider this important property when engineering proteins. This is in part due to the lack of a framework for the structure-based rational design of protein dynamics. To address this issue, we have developed a methodology based on multistate design (MSD), an emerging methodology in computational protein design that optimizes sequences in the context of multiple structural states. As a proof-of-concept for our framework, we predicted and experimentally validated sequences that stabilize and facilitate exchange between two non-native conformations of Trp43 in the streptococcal protein G domain β1 (Gβ1) fold. Four candidate sequences predicted to exchange between core and solvent-exposed conformations of Trp43 were identified by MSD across a sequence space of 1296 possible mutants evaluated on an ensemble of 12,648 unique Gβ1 backbones. 15N-HSQC and ZZ-exchange NMR spectroscopy confirmed that all four candidate sequences are dynamic, with two of them exchanging between two distinct conformations on the 10 to 100 millisecond time scale. Solution structures of two Gβ1 mutants displaying decreased dynamics, whose 15N-HSQC spectra showed peaks with highly similar chemical shifts to those from a single conformer of each dynamic variant, were solved. The structures of these mutants, coupled with an analysis of NNOE correlations from the Trp43 Hε1, confirmed that Trp43 adopts conformations corresponding to those designed in the exchanging mutants. Overall, the successful application of our MSD strategy paves the way to the rational design of protein dynamics as we move towards increasingly complex protein functions.

Computational Design of Multi-Substrate Enzyme Specificity


Antony St-Jacques1, Roberto A. Chica1
1Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada

The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes require multiple substrates to complete their catalytic cycle. Thus, the design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple states (i.e., multiple substrates) because mutations designed to change specificity for one substrate may be detrimental to the binding of a second substrate. This design objective can be tackled using multistate design (MSD), an emerging methodology in CPD that allows sequence selection to be driven by the energetic contributions of multiple states simultaneously. Herein, we report the development and validation of a MSD procedure to enable the rational design of multi-substrate enzyme specificity. As a case study, we used our MSD methodology to redesign E. coli branched-chain amino acid aminotransferase (BCAT) to catalyze the transamination of α-ketoglutarate with the non-native substrate L-His. Using our approach, we obtained BCAT mutants displaying up to 10-fold increased kcat/KM for transamination of α-ketoglutarate with L-His relative to the wild type. Additionally, we developed a negative MSD approach to identify BCAT mutants displaying improved activity towards the desired L-His substrate (i.e., the positive state) while simultaneously displaying decreased activity towards the undesired native substrate L-Leu (i.e., the negative state). Receiver operating characteristic analysis of predicted sequences demonstrates that consideration of the negative state during calculation results in improved predictions of substrate specificity. Overall, our approach opens the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

Computational evidence for the evolution of P450 monooxygenases towards the exclusion of non-productive gases


Maximilian C.C.J.C. Ebert1,4,5, Simon L. Dürr1,2,4,5, Armande A. Houle1,4,5, Guillaume Lamoureux3,4, Joelle N. Pelletier1,4,5
1Université de Montréal 2University of Konstanz 3Concordia University 4PROTEO 5CGCC

Cytochrome P450s catalyze the oxidation of non-activated carbon atoms. There is increasing industrial interest in engineering P450s to catalyze the formation of new products. The main focus of engineering is to modify the P450s substrate preference or the chemistry it catalyzes, while oxygen gas migration is poorly understood. Enzymes within this super family contain an unusually high number of channels. Substrates, products, solvents and gases are speculated to share the same channels to access and exit the active site, with their chemical properties dictating which channel is preferential. We present a computational analysis of the channel networks of two representative bacterial P450s, CYP102A1 (BM3) and CYP102A5. Using Implicit Ligand Sampling and free MD calculations, we successfully identified and characterized gas-conveying channels. Free energy calculations of O2, CO and N2 clearly identified preferred gas migration channels, which are different from preferred substrate/product egress channels. Our results contradict previous speculations of mutual preference of gas and substrates for the same channels. Additionally, calculation of the S2 order parameters that describe the ps-ns time scale dynamics allowed us to reveal the transient nature of substrate and product channels in these P450s. Combining the protein dynamics data and free energy calculations, this suggests evidence of evolution towards O2 binding, and protection against inhibitory CO and exclusion of N2 gas. Our approach could serve to provide a clear map of gas binding sites for homologs including human P450s, and support the development of new classes of inhibitors targeting O2 accessibility.

Contributions of glycosaminoglycans to membrane adsorption and conformational conversion of the peptide hormone secretin


Noé Quittot1, Armelle Tchoumi Nerée1, Phuong Trang Nguyen1, Steve Bourgault1
1Université Québec à Montréal

The membrane catalysis hypothesis advocates that a peptide hormone initially interacts with the plasma membrane and this interaction increases the local concentration at the vicinity of its cognate receptor. Besides, this model proposes that upon membrane adsorption, the peptide hormone undergoes a structural rearrangement into its bioactive conformation to promote receptor binding. However, this membrane catalysis hypothesis doesn't consider the complexity of the membrane environment. Indeed, the cell surface is composed of numerous macromolecules, including glycosaminoglycans (GAGs). These long polysulfated heteropolysaccharide chains have be shown to participate in the recruitment of chemokines, mediating their biological activity. Nonetheless, no study has specifically addressed the roles of GAGs in the membrane adsorption of cationic peptidic hormones. In this study, by combining biological and biophysical approaches we evaluated the contributions of GAGs in the membrane adsorption of secretin. Secretin is a 27-residue peptide, secreted by the S cells of duodenum and known for regulating water homeostasis. To investigate the role of GAGs in the cell surface adsorption of secretin, we used CHO pgs-A-745 cells, which lack the first enzyme for GAGs biosynthesis, and their wild type counterpart, CHO K1. By confocal microscopy and flow cytometry, we observed that CHO pgs A-745 cells absorbed less fluorescein-labelled peptides than CHO-K1 cells. Then, we characterized the interaction between GAGs and secretin by affinity chromatography, isothermal titration calorimetry and surface plasmon resonance. A strong interaction between the peptide and the sulfated GAG was observed with a Kd in the low M. Interestingly, the interaction between secretin and heparin stabilized the helical conformation. By designing secretin derivatives with a restricted conformational ensemble, we then probed the role of the spatial distribution of cationic residues for GAGs recognition. Overall, this study redefines the membrane catalysis hypothesis and strongly suggests that cationic peptide hormones initially bind to cell surface GAGs and this interaction could play a key role in receptor binding.

Contributions of tyrosine nitration and histidine carbonylation in the amyloid assembly of immunoglobulin light chains


Ximena Zottig1,2, Mathieu Laporte Wolwertza1,2, Makan Golizeha1, Leanne Ohlunda1, Steve Bourgault1,2
1UQAM 2PROTEO

Light chain amyloidosis (AL) is the most common form of systemic amyloidosis, which originates from plasma cell over proliferation. This lethal disease is primarily characterized by an overproduction of immunoglobulin light chains (LC) and followed by pathological deposition of amyloid fibrils in the extracellular space of vital organs causing organ dysfunction. Non-enzymatic post-translational modifications (PTMs) can profoundly affect protein properties and have been shown to contribute to the pathogenesis of several protein misfolding diseases. However, few is known about PTMs effects on LC amyloidogenicity. Here, we investigated the impact of oxidative PTMs, particularly carbonylation by hydroxynonenal (HNE), oxidation and nitration, on the structure, thermodynamic stability and aggregation of Wil, a LC variable domain of the λ6 germline. In order to achieve this, we initially identified the residues that are prone to oxidative chemical modifications by LC-MS/MS analysis performed after pepsin digestion. Subsequently, we noted that HNE-carbonylation at specific His residues and nitration of precise Tyr side chains modulate Wil propensity to self-assemble and to form ThT-positive fibrillar aggregates. Nitration appears to accelerate the formation of aggregates with low cross-b-sheets quaternary structure. This effect has been associated with a decrease in thermodynamic stability. In contrast, HNE-conjugation on specific His imidazole group did not affect the structural stability although it altered the conformational conversion driving the aggregation process. No effect on LC Wil aggregation and structural stability has been noted for oxidation Wil PTMs. Thus, both the thermodynamic stability and the physicochemical and structural properties have to be considered concomitantly when evaluating the amyloidogenic propensity of a LC variable domain in the context of AL.

Correlating functional divergence and dynamical variations in the pancreatic-type ribonuclease superfamily


Chitra Narayanan1, David Bernard1, Khushboo Bafna2, Om P. Choudhary3, Chakra S. Chennubhotla3, Pratul K. Agarwal4, Nicolas Doucet1,5,6
1INRS-Université du Québec 2Genome Science and Technology, University of Tennessee, Knoxville, TN 37996, USA 3Department of Computational & Systems Biology, University of Pittsburgh Medical Center, Pittsburgh, PA 15260, USA. 4Computational Biology Institute and Computer Science and Mathematics Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37830, USA. 5PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications, 1045 Avenue de la Médecine, Université Laval, Québec, QC, G1V 0A6, Canada. 6GRASP, Groupe de Recherche Axé sur la Structure des Protéines, 3649 Promenade Sir William Osler, McGill University, Montréal, QC, H3G 0B1, Canada.

Correlation between conformational dynamics and enzyme function has been well established for discrete enzyme systems; however, approaches for characterizing dynamical properties across diverse sequence homologs and their correlation with enzyme activity remain challenging. Members of the pancreatic-type ribonuclease (RNase) superfamily share similarities in structure and fold, but display large variations in catalytic efficiencies and dynamics, making them ideal model systems to probe the relationship between conformational motions and function. Using a combination of bioinformatics, molecular dynamics simulations and NMR approaches, we characterized the dynamical properties of over 20 diverse RNase homologs, whose three-dimensional structures have been determined using X-ray crystallography or NMR approaches, over a wide range of time-scales.Our results show that while the different RNase homologs used for the analysis share a common structural fold, the dynamical properties of these enzymes are significantly different. Clustering these RNase sequences into evolutionarily distinct sub-families showed similar dynamical properties within sub-family members and significant differences between distinct sub-families. Interestingly, sequences sharing the same biological function also display similar dynamical patterns, suggesting that biological function, among other factors, may potentially impact dynamical properties influencing sequence, structure and function.

Crystallization and Structure Determination of PieE: A Flavin-binding Hydroxylase


Mahder Manenda1,2,3,4, Marie-Ève Picard1,2,3,4, Changsheng Zhang5, Rong Shi1,2,3,4
1Département de biochimie, microbiologie et bio-informatique 2Université Laval 3PROTEO 4Institut de biologie intégrative et des systèmes 5south China Sea Institute of Oceanology, Chinise Academy of Sciences

Flavin-binding protein PieE is involved in post-PKS hydroxylation of certain members of a family of natural products known as piericidins. Due to their structural resemblance to ubiquinone, piericidins are potent inhibitors of bacterial NADH–ubiquinone oxidoreductase and the mitochondrion. In this project we have successfully crystallized and determined the structure of PieE by X-ray crystallography at 1.98Å resolution. The globular structure of PieE exhibits three well-known domains in flavoenzyme monooxygenases: an N-terminal FAD-binding domain, a central domain and a C-terminal thioredoxin-like fold. In the active site of the enzyme, residues such as W296 and P323 stack against the isoalloxazine ring of FAD placing the C4α at 6.5Å from the NE2 of the imidazole ring of a putative catalytic residue H54.  The determination of structure of the protein-ligand-substrate complex could shade more light in the mechanism of catalysis and are underway.

Deep functional proteomic identification of alternative proteins gives insight into their functions


Vivian Delcourt1,2, Jean-François Lucier1, Sondos Samandi1, Jean-François Jacques1, Julien Franck2, Isabelle Fournier2, Xavier Roucou1
1Université de Sherbrooke 2INSERM U-1192 PRISM - Université Lille 1 - France

With recent advances in Mass spectrometry and bottom-up proteomics, it is now feasible to identify several thousands of proteins in a single sample. However, even if an important number of MS/MS spectra match known proteins or reference proteins (RefProt), several thousands of MS/MS spectra remain unidentified. Many of them can be attributed to the “hidden proteome”. The hidden proteome contains alternative proteins (AltProts) that are translated from alternative open reading frames (AltORFs). AltORFs are present within 5’ and 3’ UTR regions,   overlap the coding sequence of the RefProt but in a frameshifted reading frame or in putative non-coding-RNA. The identification software PeptideShaker was adapted to CalculQC’s computer cluster Mammoth which allowed the analysis of about 6000 raw MS data out of four reference publications in an outstanding time effectiveness. Interrogation of these large scale proteomics datasets with a custom database containing RefProts and in-silico translated AltORFs led us to identify several thousands of AltProts. More interestingly, analysis of functional proteomics datasets such as deep phosphoproteomics studies or large scale pull-down experiments on RefProts gave us clues for potential biological functions. We observed that AltProts could be specifically phosphorylated during key events of cell life such as mitosis or after an epidermal growth factor stimulation. We also noticed that certain AltProts were enriched in many known protein complexes involved in important biological processes such as mRNA degradation or cell cycle. These findings suggest that the proteome has been largely underestimated and that key biological functions remain to be discovered.

 

Deep transcriptome annotation provides functional insights for thousands of novel proteins


Sondos Samandi1, Jean-François Lucier1, Jules Gagnon1, Alan A Cohen1, Christian Landry2, Michelle Scott1, Xavier Roucou1
1Université de Sherbrooke 2Université Laval

INTRODUCTION - In eukaryotes, current protein annotations rely on the principle that mature mRNAs contain a single protein coding sequences and that lncRNAs do not harbour protein coding sequences. In recent years, functional, proteomic and ribosome profiling approaches have concurrently demonstrated the translation of alternative open reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). AltORFs are defined as ORFs embedded either within the purportedly “untranslated” regions (UTRs), or ORFs overlapping the CDS in non-canonical +2 and +3 reading frames. Here, proteins translated from currently annotated CDSs are termed reference proteins, and proteins translated from altORFs are termed alternative proteins. Our goal was to provide functional annotations of alternative proteins translated from altORFs.

METHODS - We generated databases containing the sequences of all predicted alternative proteins translated from cellular RNAs, with a minimum length of at least 30 amino acids, from different organisms (Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Bos taurus, Mus musculus, Pan troglodytes, Homo sapiens).  An InParanoid-like approach was used for detecting putative orthologous/paralogous alternative and annotated proteins between pairs of species. This is based on pairwise similarity scores, calculated using NCBI-Blast (Blastp), between two complete proteomes. For conservation of altORFs overlapping CDSs, the Basewise conservation scores for the third nucleotide of each codon within the reference ORF for the alignment of 100 vertebrate genomes was extracted from UCSC genome browser. The average conservation score within the alternative ORF region was compared to the average score for the complete CDS. Analysis of human alternative protein signatures (families, domains, motifs, sites) was performed using a local version of InterproScan. Gene ontology (GO terms) and pathway annotations were also reported if available.

RESULTS - We show that the majority of small eukaryotic proteins are coded by altORFs. Conservation analyses show that in mammals, the number of homologous alternative proteins exceeds the number of currently known homologous proteins and comparative patterns of altORFs and their CDSs indicate that many are paired evolutionary units. The most abundant class of alternative proteins with at least one InterPro entry are C2H2 zinc finger proteins with 110 alternative proteins containing 187 C2H2-type/integrase DNA-binding domains, 91 C2H2 domains and 23 C2H2-like domains. The correspondence between InterPro domains of alternative proteins and their corresponding reference proteins coded by the same transcripts suggests functionnal coupling.

DISCUSSION - In light of the increasing evidence that the one mRNA-one canonical CDS assumption is being challenged, our findings provide the first clear functional insight into novel protein-coding sequences. While many observed altORFs are likely evolutionary accidents with no functional role, several independent lines of evidence support a functional role for thousands of alternative proteins: (1) overrepresentation of altORFs relative to shuffled sequences; (2) overrepresentation of altORF kozak sequences; (3) correlated altORF-CDS conservation, but with overrepresentation of highly conserved and fast evolving altORFs; (4) underrepresentation of altORFs in repeat sequences; (5) overrepresentation of identical InterPro signatures between alternative and reference proteins encoded in the mRNA. Our deep annotation of the transcriptome reveals that the vast majority of small eukaryotic proteins are still officially unannotated and that many small and large proteins coded by the same mRNA probably cooperate.

Délivrance quantitative de médicament par effet de tampon


Arnaud Desrosiers1
1Université de Montréal

Le développement de mécanismes ou stratégies permettant de délivrer des médicaments directement à des sites affectés à l’intérieur du corps humain et de manière contrôlée permettrait d’augmenter grandement la performance des médicaments tout en diminuant leurs effets secondaires. À cet égard, depuis des millions d’années, la nature a développé une multitude de nano-transporteurs à base de protéines qui permettent de transporter des molécules à des endroits spécifiques dans le corps. La protéine hémoglobine, par exemple, a évolué de manière à relâcher l’oxygène plus efficacement dans les régions du corps qui démontrent une plus grande activité (ex. en présence d’acide lactique). Le développement de nanomachines semblables à l’hémoglobine, mais qui permettraient de transporter et de libérer un médicament à des sites d’intérêt, comme des tumeurs, offrirait de nouvelles pistes de traitements innovantes. Pour ce faire, nous tirerons avantage des récentes avancées en nanotechnologie à base d’ADN, un polymère beaucoup programmable que les protéines. Dans un premier temps, nous construirons une nanomachine d’ADN simplifiée dans le but de bien caractériser sa mécanique. À partir de ces connaissances, nous développerons une seconde génération de nanomachines permettant de relâcher de manière contrôlée le médicament doxorubicine utilisé dans le traitement de plusieurs cancers.

Dengue fusion peptide: determination of its binding parameters upon insertion into Langmuir monolayers mimicking cell membranes


Thaís F. Schmidt1,2,3,4, Karin A. Riske1, Christian Salesse2,3,4
1Department of Biophysics, Federal University of São Paulo, UNIFESP, São Paulo, SP, Brazil 2CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec 3Département d’ophtalmologie, Faculté de médecine, Université Laval 4Regroupement stratégique PROTEO, Université Laval

The mechanisms of action of fusion peptides in virus systems, particularly in Dengue fever, are still unclear at a level to improve protocols for the treatment and achievement of new drugs. In this context, studies to control the dissemination of the virus involved in this neglected disease are increasingly necessary. The Langmuir technique is very useful to study peptide-lipid interactions. Indeed, the utilization of lipid monolayers allows controlling several parameters, such as the lipid composition, peptide concentration, pH, surface pressure and many others of interest. Here we present a study of the interaction of the Dengue fusion peptide (FLAg) with Langmuir monolayers composed of different types of lipids regarding the number of unsaturation and also the charge at the polar headgroup, including single unsaturated lipids (POPC, POPG, POPS, POPE, POPI) double unsaturated lipids (DOPC, DOPG, DOPS, DOPE) as well as saturated lipids (DPPC, DPPG, DPPS, DPPE). In order to assess peptide maximum insertion pressure (MIP) upon binding the Langmuir model membrane, kinetic measurements were performed. The kinetics of FLAg binding onto the phospholipid monolayers was monitored until the equilibrium surface pressure (Πe) was reached. The MIP was determined by injecting FLAg at different initial surface pressures (Πi) of the lipid monolayers. Results showed high surface activity of the peptide in the absence of lipid monolayers. For the binding parameters, an increase in surface pressure during adsorption kinetics experiments with monolayers revealed in general a higher affinity for unsaturated lipids and anionic lipids that evidences the role of charged membranes in the fusion process. The results showed that the FLAg peptide has strong interactions with different types of lipids monolayers. Altogether, these data clearly demonstrate that useful information on the binding FLAg to membranes can be obtained by performing measurements of their monolayer binding parameters.

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


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

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

Development of a SPR immunosensor for monitoring silent hypersensitivity to L-asparaginase during chemotherapy for leukemia


David M. Charbonneau1,2,3, Alexandra Aubé1, Julien Breault-Turcot1, Natalie M. Rachel1,2,3, Vanessa Guerrero1, Kevin Delorme1, Jean-François Masson1, Joelle N. Pelletier1,2,3
1Université de Montréal 2PROTEO Network 3Center for Green Chemistry and Catalysis (CCVC)

The Escherichia coli type II L-asparaginase (EcAII) has been an essential component of chemotherapy for acute lymphoblastic leukemia (ALL) for many decades. Clinical hypersensitivity to EcAII occurs in around 30% of patients and may lead to severe complications such as anaphylaxis. However, antibodies produced against EcAII do not always lead to clinical hypersensitivity as silent hypersensitivity and/or silent inactivation occur in 5-45% of the patients. It is therefore crucial to monitor early production of silent anti-EcAII antibodies in patients in order to change the treatment in a timely manner. We developed a portable surface plasmon resonance (SPR) device and specific bioreceptors for monitoring anti-EcAII antibodies in serum. We investigated two immobilization strategies and several modes of presentation of the EcAII antigenic bioreceptor on SPR sensor chips coated with functionalized antifouling peptides: random immobilization using covalent binding, and oriented immobilization by metal coordination of N- or C-terminal His-tags. We characterized the antigenicity of the bound EcAII, and considered whether it binds in its native tetrameric form or as a monomer. Using the best surface-immobilization mode, our device allowed for detecting antibodies in the low nanomolar range directly in serum. Analysis of serum samples from children undergoing ALL chemotherapy at CHU Sainte-Justine revealed clear correlation between results obtained with our immunosensor and by the gold standard ELISA.

Development of a whole cell high-throughput screening of P450 BM3 for the generation of efficient and reliable computational training sets in a mutation prediction algorithm for desired substrate conversion


Olivier Rousseau1, Maximilian Ebert1, Josh Pottel2, Nicolas Moitessier2, Joelle Pelletier1
1Université de Montréal 2McGill University

Cytochrome P450 enzymes are able to catalyze the oxidation of non-activated carbon atoms and can be engineered towards a variety of different substrates. In collaboration with Mcgill University, an algorithm is build to predict mutational hotspots for novel substrates. To train the algorithm a library of point mutations in P450 BM3 is created using site-directed saturation mutagenesis. The positions A82, F87, V78, A264 and T268 were primarily selected based on the literature and rational design. To be able to screen rapidly and efficiently the library an automated high-throughput method is developed using the fluorescence of the consumed NADPH co-factor.

In a first round of screening, the library is tested using the substrate indole, which upon hydroxylation can be converted to indigo. Already, three new mutations A82E, A82Q and A82K were found to have a similar indigo conversion rate than the previously characterized mutations A82W and A82F. The formed indigo was extracted and quantified by LC-MS in order to rank the mutations for the computational training set. This method was further optimized to work in whole cells to screen the mutations without the necessity of purified protein or lysate. In a second round of screening, the mutations predicted by the trained algorithm will be used to discover new substrate conversions.

Development of new β-lactamases inhibitors using a sulfahydantoin scaffold


Pierre-Alexandre Paquet-Côté1,2, 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 impairs greatly 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.

 

For this study, we have synthesised 21 new compounds with a sulfahydantoin core using a versatile approach. This approach uses amino acids as starting materials and allows an easy substituent addition on the two nitrogens. These first generation compounds were tested in an enzymatic assay on TEM-1 using the CENTA chromogenic substrate. The results confirmed the activity of sulfahydantoin compounds as β-lactamase inhibitors. We are further developing compounds using this heterocycle scaffold towards improved β-lactamase inhibitors.

Développement d’un procédé en deux étapes pour la purification de vésicules pseudotypées avec le VSV-G


Juliette Champeil1,2,3, Mathias Mangion1,2,3, Rénald Gilbert3,4, Alain Garnier1,2,3, Bruno Gaillet1,2,3
1Université Laval 2PROTEO 3ThéCell 4Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, Canada

Différents types cellulaires peuvent être efficacement transfectées par de l’ADN étranger, lorsque celui-ci est couplé à un cargo composé de vésicules dont la membrane comprend la protéine d’enveloppe G du virus de la stomatite vésiculaire (VSV). Ces vésicules (V-VSV-G) sont produites par transfection transitoire de cellules HEK293 avec un plasmide codant la protéine VSV-G. Un des obstacles à l’utilisation de ces vésicules en thérapeutique est le manque de méthodes efficaces permettant de les purifier. Nous proposons de développer un procédé de purification en deux étapes des V-VSV-G, composé d’une ultracentrifugation sur coussin de sucrose suivie d’une chromatographie échangeuses d’ions. Actuellement cette méthode de purification nous permet de récupérer 27.8 % des protéines totales présentes dans le concentré obtenu après ultracentrifugation du milieu de culture. La pureté des lots purifiés par chromatographie a été étudiée par SDS-PAGE coloré au coomasie orange et révèle que l’Albumine de Sérum Bovin (BSA), provenant du sérum utilisé lors de la production des vésicules, est encore présente après purification. Nous avons également étudié l’efficacité de transfection de cellules HEK293 à l’aide des V-VSV-G obtenues après purification en les complexant avec un plasmide codant pour la GFP. Les résultats obtenus démontrent que les V-VSV-G sont actives après purification : près de 45 % des cellules sont transfectées lorsqu’on les met en contact avec 2,5 µg de protéines totales contenues dans l’extrait purifié. Les techniques de purification par chromatographies étant régulièrement utilisées à l’échelle industrielle, notre procédé sera par conséquent facilement transposable. Par la suite, nous chercherons à améliorer la récupération des particules en développant une méthode de purification par chromatographie d’affinité, grâce à l’intégration d’une étiquette histidine à la surface des VSV-G. Une méthode de quantification spécifique des V-VSV-G sera également mise au point.

Discovery and Structure-Activity Relationship of a Bioactive Fragment of ELABELA that Modulates Vascular and Cardiac Functions


Alexandre Murza1, Xavier Sainsily1, David Coquerel1, Jérôme côté1, Patricia Marx1, Elie Besserer-Offroy1, Jean-Michel Longpré1, Olivier Lesur1, Mannix Auger-Messier1, Philippe Sarret1, Eric Marsault1
1Université de Sherbrooke

ELABELA (ELA) was recently discovered in 2013 as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis.1 The structure-activity relationship (SAR) of ELA and its cardiovascular effects remain to be elucidated. Moreover, to explore the full therapeutic potential of ELA, it is important to decipher the key pharmacophores of this 32-mer peptide, with the additional goal to reduce its size. To discover a bioactive fragment of ELA, we examined the susceptibility of ELA to proteolytic cleavage in human plasma. Then, we performed an alanine scan on the active fragment to identify the key pharmacophores. ELA and shorter analogues were assessed in binding, signaling and receptor internalization assay. Left ventricular developed pressure using isolated-perfused rat hearts and in vivo hemodynamic and echocardiographic measurements were carried out on ELA and its potent bioactive fragment. We discovered a significantly smaller bioactive fragment consisting of the last 14 amino acids of native ELA which binds APJ, activates Gαi1 and β-arrestin-2 signaling pathways, and induces receptor internalization similarly to the parent peptide. The alanine scan revealed that the C-terminal moiety is the most critical for binding and signaling. This SAR sharply contrasts with apelin-13, in which key pharmacophores are mainly located at the N-terminal portion of the peptide. ELA and its fragment caused hypotensive effects and enhanced left ventricular contractility.2 This study is the first to characterize the cardiovascular effects of ELA and its  potent fragment. This represents a promising avenue for further exploitation of the therapeutic potential of ELA and the apelinergic system in cardiovascular diseases. 

 

 

 

1. Chng et al, Dev. Cell 2013, 27, 672-680

2. Murza et al, J Med Chem 2016, in press

Dissecting the functional role of the non-conserved N-terminal region of Drosophila melanogaster Small Heat Shock Protein DmHSP27.


Mohamed Taha MOUTAOUFIK1, Finet Stéphanieb2, Morrow Genevièvea1, Tanguay M Roberta1
1IBIS, Université Laval 2Université pierre et marie curie Paris 6

Background: Small heat-shock proteins (sHsps) are a family of molecular chaperone originally discovered because of their heat inducibility. These proteins are potential therapeutic targets in many disorders involving protein folding (neurodegenerative diseases, cancer, etc.). Their activity is related to their dynamic structure and their polydispersity.

Objective: Study regions important for DmHSP27 structure and function, a small nuclear stress protein of Drosophila melanogaster.

Methods: DmHSP27 mutants have been constructed based on sequence alignement and bioinformatic. The resulting mutants were then analyzed by size exclusion chromatography (SEC) and native electrophoresis to unveil their oligomeric structure and their ability to prevent protein aggregation was tested by spectroscopy.

Results: Unlike other sHSPs, wild type of DmHsp27 forms two populations of oligomers. Together or separately these two populations are able to prevent the aggregation of citrate synthase. We show that N-terminal region is implicated in both chaperone activity and the formation of higher-order oligomers. Complete removal of the N-terminal region, showed full loss of chaperone like activity. Conserved glycine 30, acts as a regulator of oligomerisation, as its mutation greatly enhanced formation of higher-order oligomers. Intriguingly, the oligomeric structure of DmHSP27 was dependent on temperature. DmHSP27 wild type and mutants have the same oligomeric sate after heating at 45°C and have the same chaperon like activity.

Conclusion: This project allowed us to characterize the oligomeric structure of DmHSP27 and identify N-terminal residues important for the quaternary structure. A better understanding of the mechanisms of aggregation would open the way for development of therapy against diseases involving protein folding.

Diversity among five newly isolated phages infecting Citrobacter freundii


Sana Hamdi1,2, Geneviève M. Rousseau1, Simon J. Labrie1, Rim Saied Kourda2, Denise M. Tremblay1, Sylvain Moineau1, Karim Ben Slama2
1Université Laval 2Université Tunis El Manar

Citrobacter freundii is the cause of opportunistic infections in humans and animals, which are increasingly difficult to treat due to increased antibiotic resistance. In fact, Citrobacter infections are now a public health problem. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. Here, we report on the isolation and characterization of five new virulent phages that infect various strains of C. freundii. Morphological and genomic analyses revealed that phages SH1, SH2, SH3, SH4, and SH5 belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double stranded DNA genomes are ranging from 39,158 pb to 39,832 pb and are terminally redundant with direct repeats between 183 pb and 242 pb. Based on genomic comparisons and on DNA polymerase phylogeny, we assigned the five phages to the T7likevirus genus but separated them into two different groups. Phages SH1 and SH2 are similar to phages phiSG-JL2 and phiYe03-12 infecting respectively Salmonella enterica and Yersinia enterocolitica. They also share more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are similar to phages K1F and Dev2 infecting respectively Escherichia coli and Cronobacter turicensis. The five phages were stable from pH 5 to 10. No genes coding for known virulence factor were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships of T7likeviruses.

Échange conformationnel dans les doigts de Zinc de Miz-1 : Implication de résidus His non-conservés


Jean-Michel Moreau1, Mikaël Bédard1, Martin Montagne1, Cynthia Tremblay1, Pierre Lavigne1
1Université de Sherbrooke

La protéine Miz-1 est un facteur de transcription de type BTB/POZ et possède 13 doigts de zinc (ZF) de type C2H2. Miz-1 active la transcription de gènes impliqués dans plusieurs aspects de la biologie de la cellule tel que des gènes cytostatiques (e.g.  p15INK4b) et autres impliqués dans l’inflammation et la réparation de l’ADN. Miz-1 active la transcription de ces gènes en recrutant différents co-activateurs comme l’histone acétyltransférase p300 et la nucléophosmine. L’oncogène c-Myc peut se lier à la protéine Miz-1 pour réprimer la transcription des gènes cytostatiques, ce qui contribue à son caractère oncogénique.

 

Les ZF C2H2 sont des domaines d’environ 30 acides aminés, ayant un repliement ββα stabilisé par un atome de Zn2+ chélaté par 2 C et H. La séquence consensus est (F/Y)-X-C-X2-5-C-X3-(F/Y)-X5-L-X2-H-X3-4-H, où X peut être n’importe quel résidu. Contrairement à ce consensus, des ZF de Miz-1 (e.g. ZF1 et 6) possèdent des H à la place des F/Y avant la première C. À l’opposé des ZFs qui répondent au consensus, les ZF 1 et 6 subissent un échange conformationnel substantiel sur l’échelle de la µs-ms. Afin de vérifier l’implication des H dans ce phénomène, nous avons remplacé l’H du ZF1 par une Y. Cette mutation a complètement aboli l’échange conformationnel démontrant ainsi l’implication de l’H dans l’induction de ce phénomène. Nous discuterons du mécanisme et du rôle potentiel de cet échange conformationnel dans l’affinité de liaison à l’ADN par Miz-1.

Élucidation du mécanisme d’activité enzymatique de la lécithine rétinol acyltransférase par des études structurales en résonance magnétique nucléaire


Sarah Roy1,2,3,4,5, Line Cantin2,3,5, Stéphane Gagné1,4,5, Christian Salesse2,3,5
1Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie, Université Laval 2Département d’ophtalmologie, Faculté de médecine, Université Laval 3CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec 4Institut de biologie intégrative et des systèmes de l'Université Laval 5Regroupement stratégique PROTEO, Université Laval

Le pigment visuel des photorécepteurs de la rétine est composé d’une protéine et d’un chromophore, le 11-cis rétinal, un rétinoïde dérivé de la vitamine A. L’absorption de la lumière par ce pigment visuel mène à l’isomérisation de son chromophore en tout-trans rétinal. Ce rétinoïde est ensuite transporté à l’épithélium pigmentaire rétinien, où il subit diverses modifications enzymatiques au cours du cycle visuel des rétinoïdes pour régénérer le 11-cis rétinal. La lécithine rétinol acyltransférase (LRAT) est une enzyme du cycle visuel qui est responsable de catalyser l'estérification du tout-trans rétinol en tout-trans rétinyl esters. Des mutations dans sa séquence mènent à la dégénérescence des photorécepteurs, ce qui engendre une perte de la vision. Nous avons déterminé auparavant que les mutations de la LRAT tronquée, sans ses segments hydrophobes en N-terminal et en C-terminal (tLRAT), mènent à une perte presque complète de son activité. L'objectif de ces travaux consiste donc à élucider le mécanisme d’activité enzymatique de la tLRAT et à déterminer l’impact de ses mutations sur son activité par des études structurales en résonance magnétique nucléaire (RMN). La tLRAT a été surexprimée et ensuite purifiée par chromatographie d'affinité. L'information sur sa structure tertiaire est obtenue par RMN en utilisant un échantillon de tLRAT marqué avec du 13C et du 15N. Des résultats obtenus auparavant par RMN ont démontré que les mutations n’ont pas d’effet sur le repliement global de la tLRAT. Nous avons utilisé le double mutant C161S/C168S-tLRAT pour éliminer l’acylation hétérogène et avons attribué 100 % des amides du squelette peptidique de cette protéine, ce qui a permis de déterminer le contenu suivant en structure secondaire : 57 % hélice α, 43 % random coil/coudes et 0 % feuillet β. Nous procédons présentement à l’analyse des spectres de la protéine native pour obtenir de l'information détaillée sur sa structure tertiaire et celle de ses mutants afin de mieux comprendre son mécanisme d'activité enzymatique ainsi que l'effet de ces mutations délétères sur cette activité. On remarque qu'il y a seulement des changements mineurs dans les déplacements chimiques comparativement à ceux du double mutant. L'information obtenue de la structure tertiaire de la tLRAT permettra de mieux comprendre son mécanisme d'activité enzymatique et l'effet de ses mutations sur son activité.

Enzyme-Operated DNA-Based Nanodevices


Erica Del Grosso1,2, Anne-Marie Dallaire2, Alexis Vallée-Bélisle2, Francesco Ricci1
1University of Rome Tor Vergata 2University of Montreal

DNA-based nanodevices and nanomachines have attracted a growing interest for their potential use in life science and nanomedicine, in particular, due to their versatility and modularity they appear extremely promising. A limitation of such devices is represented by the limited number of molecular stimuli that can be used to control and regulate their function. Here we propose the possibility to expand the available toolbox of molecular stimuli used to control and regulate this DNA-based nanomachines. In particular we use enzymes and enzymatic substrates as molecular cues to rationally control and regulate DNA-based reactions and nanodevices. To demonstrate the versatility of our approach we have employed three model DNA-based systems: a conformational-changing nanoswitch, a ligand-releasing nanomachine and a DNA-based strand-displacement reaction. For each of these model systems we show that we can finely control and regulate their activity by using different enzymes (belonging to several classes, i.e. transferases and hydrolases) and specific substrates. The possibility to use enzymes and enzymatic substrates as possible cues in DNA-based processes and reactions appears particularly promising to expand the available toolbox of molecular stimuli to be used in the field of DNA nanotechnology and could open the door to many applications including enzyme-induced drug delivery and enzyme-triggered nanostructures assembly.

 

 

Del Grosso, E.; Dallaire, A. M.; Vallée-Bélisle, A.; Ricci, F. Nano Lett. 2015, 15, 8407-8411.

Étude des réseaux signalétiques de GRB2 dans la signalisation des récepteurs de la famille HER


Alice Beigbeder1,2,4, Nicolas Bisson1,2,3,4
1Centre de Recherche sur le Cancer 2PROTEO 3Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Université Laval 4Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec

GRB2 est une protéine adaptatrice régulant la transduction du signal en aval de récepteurs tyrosine kinase (RTK). En interagissant avec les RTK et différentes combinaisons d’effecteurs, GRB2 permet d’augmenter la spécificité et la diversité des réponses cellulaires. La famille de récepteurs HER comprend 4 membres : HER1 ou EGFR, HER2, HER3 et HER4. L’activation de ces récepteurs et des voies de signalisation en aval de ces derniers contrôle des processus cellulaires fondamentaux tels que la prolifération ou la migration. Le récepteur HER2 joue un rôle très particulier dans la signalisation de la famille HER. En effet, ce dernier ne possède aucun ligand connu, son activation peut donc être due à sa dimérisation avec d’autres membres HER activés par leur ligand, ou bien à une homo-dimérisation lorsque le niveau d’expression est élevé. HER2 joue ainsi un rôle de tampon dans la signalisation par les récepteurs HER, en ayant la capacité d’augmenter le niveau de dimères de récepteurs activés par une quantité de ligand donnée. La protéine GRB2 peut interagir avec tous les membres de la famille HER, et joue un rôle clé dans le recrutement de leurs effecteurs cytoplasmiques. Nous avons donc analysé les réseaux de signalisation de GRB2 dans un contexte de surexpression de HER2. Nous avons pour cela utilisé une méthode de protéomique, l’AP-MS (Purification d’Affinité suivie d’analyses de Spectrométrie de Masse) pour identifier les partenaires de GRB2 dans une lignée humaine de cancer du sein surexprimant HER2. Nous avons caractérisé des complexes connus, ainsi que de nouvelles interactions non répertoriées dans la littérature comme GRB2-MPZL1. Pour évaluer le rôle de cette interaction  dans la signalisation HER-dépendante, nous avons validé cette interaction par des expériences d’AP suivies d’analyses des interactions par western blot. En utilisant différents mutants de GRB2, nous avons pu déterminer que cette interaction dépend du domaine SH2 de GRB2. Comme la protéine MPZL1 ne présente aucun site consensus de liaison à ce domaine de GRB2, nous avons cherché à évaluer la nécessité pour l’interaction GRB2-MPZL1 de la protéine PTPN11, connue pour interagir avec ces deux dernières, et retrouvée dans nos expériences initiales. Des résultats préliminaires suggèrent que PTPN11 est impliquée dans l’interaction MPZL1-GRB2. Nous cherchons maintenant à identifier la fonction de cette interaction dans les processus cellulaires régulés par la signalisation des récepteurs HER comme la prolifération et la migration.

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


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

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

 

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

 

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

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

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

Functionalized Low Generation Dendrimers Modulate Islet Amyloid Polypeptide Self-Assembly and Cytotoxicity


Phuong Trang Nguyen1, Rishi Sharma2, Rabindra Rej2, Carole Anne De Carufel1, René Roy2, Steve Bourgault1
1UQAM 2Pharmaqam

Besides insulin resistance, type II diabetes mellitus is characterized by a loss of β-cell mass as a result of the deposition of misfolded Islet Amyloid Polypeptide (IAPP) in the pancreatic islets. Recent studies have revealed that the amyloidogenic process mediates cell death whereas mature amyloid fibrils are poorly cytotoxic. Therefore, therapeutic strategies to prevent b-cell degeneration associated with amyloid deposition should either sequester prefibrillar proteospecies and/or modulate the early steps of amyloid formation. Besides, a few studies have reported that dendrimers can modulate amyloid formation from the amyloid-β peptide derivatives and inhibit its neurotoxicity. In this context, we evaluated the effects of low-generation dendrimers on IAPP assembly and cytotoxicity. Densely functionalized dendritic scaffolds harboring 4 to 16 hydroxyl, amine, carboxylate or sulfonate units were designed. Whereas polyhydroxyl and polyamine dendrimers did not affect the kinetics of amyloid assembly, carboxylated dendrimers accelerated IAPP fibrillization proportionally to surface group density. Interestingly, as revealed by thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy, the G0 sulfonated dendrimers inhibited amyloid formation whereas G1 sulfonated dendritic scaffolds dramatically hastened IAPP self-assembly into long amyloid fibrils. G1 dendrimers decorated with carboxylate or sulfonate groups attenuated IAPP induced toxicity on pancreatic b-cells and the protective effect was proportional to the number of functional units attached. This study indicates that IAPP amyloidogenesis and cytotoxicity can be controlled with low generation dendrimers and offer novel mechanistic insights for the design of molecular identities to manipulate peptide self-assembly.

Générer des mutants par sauvetage évolutif afin de mieux comprendre les bases moléculaires de la réponse au stress


Clara Bleuven1,2,3, Isabelle Gagnon-Arsenault1,2,3, Alexandre K. Dubé1,2,3, Christian Landry1,2,3
1Université Laval 2Institut de Biologie Intégrative et des Systèmes (IBIS) 3Regroupement PROTÉO

La plupart des organismes subissent des variations environnementales au cours de leur existence, à différentes échelles : spatiale, temporelle, de manière prédictible ou aléatoire. Lorsque l’environnement se détériore jusqu’à un niveau létal, les organismes doivent évoluer pour s’adapter aux nouvelles conditions et éviter l’extinction. Le sauvetage évolutif est le processus correspondant au changement adaptatif assurant la survie de la population déclinante. Des études aussi bien théoriques qu’expérimentales ont pu démontrer que la rapidité du changement environnemental, la taille initiale de la population, la diversité génétique disponible influencent la probabilité de sauvetage évolutif. Cependant les bases moléculaires et leur universalité sont encore peu connues. En utilisant des techniques d’évolution expérimentale chez la levure Saccharomyces cerevisiae, nous avons créé un protocole automatisé afin de provoquer des sauvetages évolutifs dans un environnement quasi-létal. Nous pouvons comparer les capacités de sauvetage et les cibles de la sélection en utilisant différents contextes génétiques de S. cerevisiae et de nombreuses populations indépendantes. Le séquençage génomique permettra l’identification des gènes clés dans le sauvetage et celle des réseaux fonctionnels associés. Nous déterminerons ainsi si les bases moléculaires du sauvetage évolutif sont dépendantes des contextes génétiques ou bien universelles. Par la suite, en utilisant des approches transcriptomiques et protéomiques, nous étudierons l’effet des mutations à différents niveaux d’organisation cellulaire : au niveau des éléments traduits, des différences d’expression protéique et leurs conséquences sur les phénotypes des mutants. Cette étude dans son ensemble apportera des éléments de compréhension sur la capacité de persistance des populations et sur les mécanismes adaptatifs associés dans un contexte d’hétérogénéité spatiotemporelle complexe.

Glycosaminoglycan-mediated amyloid assembly of the neuropeptide hormone PACAP


Mathew Sebastiao1, Phuong Trang Nguyen1, Noé Quittot1, Isabelle Marcotte1, Steve Bourgault1
1Université du Québec à Montréal

The amyloid quaternary structure has been historically associated with various pathologies, although there are several cases where functional amyloids have been identified in various organisms, including humans. This supramolecular structure rich in cross-β-sheets is a generic characteristic of the peptidic backbone and the vast majority of endogenous peptides and proteins can adopt this structure. It has been suggested that the neurohormone pituitary adenylate cyclase-activating peptide (PACAP), a 27-residue peptide, can be stored as amyloids in the pituitary secretory granule. This idea is intriguing since the peptide alone shows no propensity to assemble in vitro, and the molecular basis of amyloid assembly and hormone liberation remains elusive. Nonetheless, the unique physicochemical properties and stability of the amyloid nanostructure offer novel perspectives for the development of unique applications in peptide drug delivery and formulation. In this context, we investigated the fibrillization of PACAP in vitro by a combination of biophysical approaches. We probed the assembly of this otherwise non-aggregating peptide in the presence of two different sulfated glycosaminoglycans (GAGs): standard molecular weight heparin and a low-molecular weight fraction of heparin, the effect of seeding and the effect of pH on the rate of fibril formation. In order to accomplish this, PACAP was incubated with a low-molecular weight fraction of heparin (LMWH) and aliquots were analyzed by thioflavin T (ThT) fluorescence, circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). Amyloid fibrils were initially absent, whereas after 3 days incubation, the presence of amyloid fibrils could be detected. There were no changes in CD spectra, ThT fluorescence or AFM morphology after 3 days. After 7 days, the peptide alone had shown no changes in secondary structure, confirming that it was not prone to amyloid formation in the absence of GAGs. ­­The amyloid formation of PACAP27 with the LMWH was compared with that of the peptide with a common mixed-weight heparin, and the lag-phase, i.e. the time required before amyloids could be detected, was somewhat shorter with the LMWH. Finally, the amyloid fibrils were tested for the ability to dissociate and release monomeric peptide from the fibrils. This was accomplished with a dialysis system, in which pre-formed amyloid fibrils were dialysed against a different pH buffer and were monitored for changes in ThT, CD and tyrosine fluorescence. Overall, this study demonstrates that a non-amyloidogenic peptide hormone readily forms well-defined amyloid fibrils when the peptide is incubated in presence of a highly sulfated biopolymer.

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


Jacynthe Toulouse1,2, Dominic Bastien1, Delphine Forge3, Brahm Yachnin4, Daniel Deon5, Marc Gagnon5, Edward Ruediger5, Kévin Saint-Jacques6, Thaddeus J. Edens7, Genbin Shi8, Xinhua Ji8, Amee R. Manges9, Anne Marinier5, Albert Berghuis2,4, Jean Jacques Vanden Eynde3, Joelle Pelletier1,2
1Université de Montréal 2PROTEO, the Québec Network for Research on Protein Structure, Function and Engineering 3Laboratoire de chimie organique, Université de Mons, Belgium 4McGill University 5Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Qc, Canada 6Département de chimie, Université de Sherbrooke, Qc, Canada 7Devil’s Staircase Consulting, BC, Canada 8Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, USA 9School of Population and Public Health, University of British Columbia, BC, Canada

The widespread use of the antibiotic trimethoprim (TMP) in clinical and veterinary applications against infections and for preventive measures provides positive pressure for the development of antibiotic resistance. TMP inhibits the bacterial chromosomal dihydrofolate reductase (DHFR). The selective pressure produced by TMP has resulted in the emergence of an alternative family, the TMP-resistant plasmid-borne type II DHFRs. To date, seven type II DHFR genes are known, among which dfrB1 (R67 DHFR) is the best studied. There is little knowledge of the prevalence of type II DHFR genes: those genes have occasionally been observed by tracking integron-related elements but have not been systematically tracked. A library of hundreds of TMP-resistant blood samples from clinical E. coli urinary tract infections was screened in silico for those type II DHFR genes. The relatively new dfrB4 gene was found in two samples as a mobile genetic element in a plasmid with multiple resistance genes. The expression and the purification of DfrB4 are underway.

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

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

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

Identification de nouveaux acteurs de la signalisation dépendante du récepteur aux androgènes par approches combinées de protéomique


Lauriane Vélot1,2,3,4, Dominique Levesque5, Francois-Michel Boisvert5, Frédéric Pouliot1,3,6, Nicolas Bisson1,2,3,4
1Centre de Recherche sur le Cancer 2PROTEO 32Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec 4Département de Biologie Moléculaire, Biochimie Médicale et Pathologie, Université Laval 5Faculté de Médecine et des sciences de la santé, département d’anatomie et de biologie cellulaire, Université de Sherbrooke 6Département de chirurgie, Université Laval

Le récepteur aux androgènes (AR) est un récepteur nucléaire qui appartient à la superfamille des récepteurs stéroïdiens. Il joue un rôle essentiel dans le développement normal et pathologique de la prostate. En absence de ligand, le AR est maintenu sous forme inactive dans le cytoplasme par différentes protéines chaperonnes. La liaison de la Dihydrotestosterone (DHT) induit l’homodimérisation du AR, conduisant à un changement de sa conformation et induisant ainsi sa translocation au noyau. La forme phosphorylée du AR va alors se lier aux ARE (Androgen Response Element) présents sur l’ADN, afin d’induire la transcription de gènes cibles impliqués dans la croissance, la prolifération et la survie cellulaire. Le AR activé peut recruter des co-régulateurs pour faciliter la transcription. De plus, le AR peut être activé par phosphorylation en absence de son ligand par des voies alternatives, notamment celles des MAPK ou  la voie PI3K/Akt. Ainsi, toute modification des voies régulant l’activation du AR, sa translocation au noyau, ou l’activation de ses gènes cibles modifiera l’expression de gènes directement impliqués dans le développement tumoral du cancer de la prostate (CaP). Par une combinaison de deux approches de protéomique, nous souhaitons donc identifier de nouveaux acteurs impliqués dans la régulation de la signalisation AR-dépendante via un modèle cellulaire de CaP. Nous avons tout d’abord utilisé une méthode de biotinylation de proximité appelée BioID pour déterminer les réseaux AR-dépendants modifiés après stimulation à la DHT. Nous avons caractérisé 202 partenaires du AR, dont 177 n’avaient pas été préalablement identifiés. Ensuite, nous avons étudié par phosphoprotéomique combiné à un marquage SILAC (Stable Isotope Labelling by Amino-acids in Cell culture) la régulation des voies de signalisation après différents traitements. Nous observons des augmentations de phosphorylation de nombreuses protéines par comparaison entre des lignées sensibles et résistantes à l’Enzalutamide, un anti-androgène utilisé dans le traitement du CaP. Nous retrouvons notamment des protéines impliquées dans la prolifération et la survie cellulaire. Ceci suggère l’importance de la régulation du phosphoprotéome dans l’activité fonctionnelle du AR. Nous souhaitons maintenant caractériser le rôle de ces protéines dans la régulation de la signalisation AR-dépendante.

Identification de nouvelles protéines effectrices dans la signalisation des récepteurs Eph


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

Les récepteurs Eph (EphR), la plus vaste famille de récepteurs tyrosine kinase, régulent une large gamme de processus cellulaires comme l’adhésion et la migration. Cependant, les mécanismes moléculaires permettant d’assurer les fonctions biologiques de ces récepteurs sont loin d’être compris. Dans cette étude, nous cherchons à identifier de nouveaux acteurs de la signalisation dépendante des EphR pour déterminer leurs fonctions dans la signalisation via ces récepteurs. Afin d'explorer les interactions protéiques dans la signalisation des EphR, nous avons utilisé deux approches complémentaires de protéomique: (i) des purifications d’affinité suivies de spectrométrie de masse (AP-MS) et (ii) du marquage de proximité in vivo (BioID). Nous avons généré des lignées de cellules HEK293 exprimant pour trois EphR: EphA4, -B2 et -B4, fusionnés à un épitope Flag et à une biotine ligase appelée BirA*. Nous avons validé la localisation membranaire des constructions chimériques et confirmé que ces dernières possèdent toujours leur activité kinase. Par nos criblages protéomiques, nous avons pu identifier certains interacteurs connus des EphR ainsi que plusieurs nouveaux candidats intéressants. Pour relier nos résultats de protéomique à la signalisation EphR-dépendante, nous avons entrepris des expériences pour étudier l’effet de la perte de fonction de certains candidats (dsiRNA) dans des essais d'arrondissement et de séparation des cellules. En prenant avantage d’approches protéomiques, ces travaux vont conduire à l'élucidation des réseaux d'interactions protéiques qui sont fonctionnellement pertinents et qui régulent la signalisation par les EphRs. Cela fournira des informations importantes sur les mécanismes qui affectent le comportement cellulaire.

Impact de la charge des lipides et de la fluidité membranaire, ainsi que du calcium sur la recoverine, une neuroprotéine des photorécepteurs sensible au calcium


Kim Potvin-Fournier1,2,3,4,5,7, Thierry Lefèvre1,2,3,4,5, Audrey Picard-Lafond1,2,3,4,5, Catherine Marcotte1,2,3,4,5, Caroline Dufresne1,2,3,4,5, Geneviève Valois-Paillard1,2,3,4,5,7, Line Cantin1,2,6,7, Christian Salesse1,2,6,7, Michèle Auger1,2,3,4,5
1Université Laval 2PROTEO 3CERMA 4CQMF 5Département de chimie, Faculté des sciences et de génie 6Département d'ophtalmologie et d'ORL-CCF, Faculté de médecine 7CUO–Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec

La recoverine est une protéine périphérique présente dans les photorécepteurs. C’est un membre de la famille des neuroprotéines sensibles au calcium (NCS). La conformation de plusieurs membres de cette famille varie en fonction de la concentration en calcium. À faible concentration en calcium, elles sont cytosoliques avec leur groupement myristoyle séquestré dans une poche hydrophobe. Cependant, la liaison de 1 à 4 ions calcium résulte en l’extrusion du groupement myristoyle. Ce phénomène s’appelle "calcium myristoyl switch". L’étude de la liaison membranaire de la recoverine en absence et en présence de calcium est importante en considérant la composition particulière des membranes des photorécepteurs. En effet, les photorécepteurs contiennent plus de 60% de lipides polyinsaturés, ainsi que ~20% de lipides chargés négativement, ce qui pourrait être responsable de moduler l’interaction membranaire, la liaison et l’agrégation de la recoverine. La conformation de la recoverine en présence de lipides a été étudiée par spectroscopie infrarouge. L’interaction entre la recoverine et les lipides avec différentes têtes polaires et longueurs de chaînes acyles a été étudiée par la combinaison de la spectroscopie infrarouge et résonance magnétique nucléaire à l’état solide. Les lipides chargés lient les ions calcium et ainsi réduisent la stabilité thermique de la recoverine. La recoverine modifie la température de transition de phase des lipides chargés négativement et réduit la formation des complexes entre ces lipides et le calcium. L’hydratation et les chaînes acyles sont influencées différemment selon la charge de la tête polaire des lipides. L’immobilisation du groupement myristoyl de la recoverine est influencée par la fluidité de la membrane. La liaison d’un maximum d’ions calcium par la recoverine permet de maintenir sa stabilité thermique et de favoriser son interaction avec les lipides chargés négativement. Des lipides dans une phase fluide sont nécessaires pour immobiliser de façon optimale le groupement myristoyle de la recoverine.

Incorporation of Crown Ether Peptides in Model Membranes : A Study Using Synchrotron Radiation-Based Oriented Circular Dichroism Spectroscopy


Jean-Daniel Savoie1, François Otis1, Jochen Bürck2, Anne S. Ulrich2, Normand Voyer1
1Université Laval 2Karlsruhe Institute of Technology

Ion channels are transmembrane proteins that regulate the flow of ions through cell membranes and are required for the proper functioning of the cell. However, some ion channels such as non-gated nanopores may act as toxins by enabling the uncontrolled passage of ions, destroying the usual ionic gradients of a cell and leading to its death. Targeting non-gated nanopores towards cancer cells would be very promising for the development of new nanochemotherapeutic agents to treat resistant cancer cells. In this intent, a new family of synthetic ion channels was developed in our laboratory using a transmembrane helical peptide as framework bearing six crown ethers to create a transmembrane channel. Even though biophysical studies have shed light on several aspects of these channels, the mechanism of action by which they incorporate into membranes remains unclear. Therefore, in an attempt to assess what drives the incorporation of crown ether-modified peptides into membranes, we have used synchrotron radiation-based oriented circular dichroism (SR-OCD) spectroscopy. Specifically, we will report our results on the orientation of crown-ether peptides with lipid membranes using a wide range of peptide:lipid ratios going from 1:20 to 1:1000, different N- and C-terminal functionalities and different bilayer thickness.

Influence de la composition chimique de la membrane plasmique du spermatozoïde sur l'action de la protéine BSPA1-A2 chez les bovins


Dominic Lauzon1, Puttaswamy Manjunath2, Michel Lafleur1
1Département de chimie, Université de Montréal 2Département de biochimie et médecine moléculaire, Université de Montréal

La capacitation est un phénomène biologique qui rend les spermatozoïdes aptes  à la fécondation de l'ovule. Elle se résume par une maturation des spermatozoïdes dans le tractus génital féminin qui se traduit par une suite de changements intracellulaires et membranaires facilitant la fixation à la zone pellucide. L'un de ces changements est un efflux lipidique de la membrane plasmique du spermatozoïde. Un des principaux vecteurs du liquide séminal qui cause ces perturbations est la BSPA1-A2, une protéine de la famille des BSP (« Binder of SPerm »). Cette protéine induit un efflux de lipide dans un ratio d’environ 20 lipides par protéine en se liant spécifiquement aux phosphocholines des lipides composant la membrane plasmique. Toutefois, l’origine de cette spécificité lipidique n’est pas clairement établie. Par l'utilisation de membranes lipidiques modèles à base de phosphoglycérolipides et de cholestérols, cette étude vise à démontrer l’influence des lipides qui composent la membrane lipidique sur les actions de la BSPA1-A2 extraite du liquide séminal bovin. De plus, la capacité d'inhiber les perturbations induites par la BSPA1-A2 sera aussi examinée par l'utilisation d'un extendeur commercial (BIOXcell) utilisé dans le but de préserver les semences bovines. Divers mélanges protéiques du liquide séminal bovin seront étudiés pour mieux en comprendre l'efficacité.  

Investigating the kinetics of hypoxia-inducible factors heterodimerization by surface plasmon resonance


Billel Djerir1, Noé Quittot1, Marc P. Lussier1, Steve Bourgault1
1Université du Québec à Montréal

It is known that cancer cells establish multiple stress adaptation mechanisms in order to maintain their survival and their growth. For example, hypoxia is a condition that deprives the cellular environment of adequate oxygen supply, in a case of poorly vascularized solid tumor. Several studies have recently demonstrated that the regulation of hypoxia-inducible factors (HIFs) coordinate cellular response to oxygen deprivation. Particularly, the modulation of HIFs activity, including the heterodimerization of HIF-2α with the nuclear aryl hydrocarbon translocator receptor (ARNT), mediates the adaptive transcriptional response to hypoxia in both normal and tumor cells. Our study aims at implementing biophysical assays to characterize the kinetics and thermodynamics that drive HIF-2α heterodimerization for potentially supporting the development of small molecules that inhibits HIF-2α. To reach this goal, we expressed the PAS-B domains of HIF-2α and ARNT in Escherichia coli, followed by affinity chromatography purification, GST-tag cleavage and size exclusion chromatography. Afterwards, the kinetics of HIF-2α / ARNT heterodimerization was studied by means of surface plasmon resonance to implement a high-throughput assay for the identification of inhibitors. Overall, these data dissecting this molecular interaction will support the development of a new generation of potent and selective inhibitors of HIF-2α activation and could ultimately lead to novel cancer treatments.
 

Investigation of the mechanism of action of synthetic peptides displaying antimicrobial potential


Matthieu Fillion1, Burkhard Bechinger2, Normand Voyer1, Michèle Auger1
1Université Laval 2Université Strasbourg

A wide variety of organisms produce antimicrobial peptides as part of their first line of defense. Among them, the naturally occurring cationic antimicrobial peptides represent a promising alternative to fight against multiresistant bacteria which are an important clinical problem. Despite their diversity, cationic antimicrobial peptides generally share common characteristics such as a short length of amino acids, a positive charge and an amphiphilic character. Contrary to conventional antibiotics which alter a specific target, the main target of cationic antimicrobial peptides is the membrane(s) of pathogens. We have previously shown that a non-natural peptide composed of 14 residues (10 leucines and 4 phenylalanines modified with a crown ether) has a helical secondary structure, and is able to disrupt lipid bilayers but is not selective towards bacterial membranes. To gain specificity against negatively charged membranes, several leucines of this 14-mer have been substituted by positively charged residues (lysine, arginine, and histidine). Solid-state NMR experiments performed on model membranes and lipids oriented between glass plates were used to better characterize the mode of action of the cationic analogs. An important feature of the mechanism of action is the membrane topology, i.e. the location of the peptide in the membrane. Membrane topology of the α-helical peptides has been assessed by performing experiments with nitrogen-15 labeled peptides in oriented samples. Furthermore, we have measured internuclear distances between the peptides and the phospholipids by using the REDOR technique. Fluorescence spectroscopy has been useful in determining the membrane activity of these peptides and the results show a strong correlation between the release activity and the peptide conformation. We have also demonstrated that the cationic peptides impose a positive curvature to the membrane.

IsoMif - Detection of binding site molecular interaction field similarities


Matthieu Chartier1, Rafael Najmanovich1
1Université de Sherbrooke

Methods that find similarities between two protein binding sites of proteins can be used for the prediction of molecular function, prevention of cross-reactivity, drug repurposing and identification of polypharmacological targets. Such methods can use sequence, structure and a few use molecular interaction fields (MIFs). We developped IsoMIF, a computational algorithm that detects MIF similarities between binding sites, regardless of fold or sequence homology.

The MIF used by IsoMIF is composed of 6 probes that measure potential hydrophobic, aromatic, hydrogen-bonding and charged interactions in binding sites. Two binding sites with a high level of MIF similarities, that is a similar geometrical disposition of probes of the same chemical type, are likely to bind similar ligands as they exhibit similar physico-chemical environments.

How well can IsoMIF identify binding sites that bind similar molecules from proteins that share no sequence or structural homology? Compared to other methods with performance reported in the litterature, IsoMIF performed best in average and more consistently across 4 datasets.
We built a web interface freely available at bcb.med.usherbrooke.ca/imfi that allows a query protein to be compared to pre-calculated MIFs or to user defined target protein structures.

Using IsoMif, many cases of drug repurposing and side-effect mechanism prediction have been identified.

La caspase-7 a un faible pour PARP-1


Alexandre Desroches1, Dave Boucher1, Éric Marsault2, Jean-Bernard Denault1
1Université de Sherbrooke 2IPS - Université de Sherbrooke

Les caspases sont des peptidases à cystéine qui, par le clivage de nombreux substrats, sont impliquées dans l’apoptose et l’inflammation. Un de ces substrats est PARP-1, une enzyme impliquée entre autres dans la réparation de l’ADN. Nous avons découvert que la caspase-7 possède un exosite qui interagit avec PARP-1. Un exosite est un domaine d'interaction avec le substrat qui se trouve en dehors de la pochette catalytique et qui a pour effet d'augmenter l'efficacité d’une enzyme. Malgré cette découverte, les déterminants de cette interaction exosite-substrat sont encore méconnus.

 

Afin de caractériser l’interaction entre la caspase-7 et PARP-1, des mutants de délétion du domaine N-terminal de la caspase-7 liés à la GST ont été générés. L’affinité des mutants pour PARP-1 a été évaluée grâce à des essais de précipitation à la GST, ce qui nous a permis de déterminer que le module minimal de l’exosite se trouve entre les résidus 34 à 47 de la caspase-7. Par la suite, des mutants de délétion de PARP-1 ont été générés. Les mutants ont été testés dans des essais de clivage, ce qui nous a permis d’identifier les domaines Zn3 et BRCT comme étant ceux qui interagissent avec l’exosite. L’efficacité de clivage de la caspase-7 sur les deux formes de PARP-1 (automodifiée ou non) a aussi été testée et c'est la forme non modifiée qui est préférentiellement clivée.

 

En somme, nos travaux permettent de mieux comprendre comment les caspases reconnaissent et interagissent avec leurs substrats.

La fonction des protéines S100A10 et annexine A2 dans la réparation membranaire


Xiaolin YAN1,2,3,4, Elodie Boisselier1,2,4
1Université Laval 2Département d'ophtalmologie et d'ORL-CCF, Faculté de médecine 3Département de biochimie microbiologie et bio-informatique, Faculté de sciences et génie 4CUO-Recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, CHU de Québec

Les protéines appartenant à la famille S100 et les annexines interviennent lors de différents mécanismes membranaires vitaux. En effet, le complexe protéique S100A10/annexine A2 permettrait le recrutement de la protéine AHNAK à la membrane avant de former une plateforme qui initierait la réparation membranaire. Cependant, aucune donnée moléculaire n’est à ce jour disponible sur la liaison membranaire des différentes protéines de ce complexe. L’objectif général de ces travaux est donc de caractériser l’interaction membranaire des protéines S100A10 et annexine A2 et de leur complexe. Dans un premier temps, les protéines S100A10 et annexine A2 sont surexprimées et purifiées. Le modèle des monocouches de Langmuir sera utilisé pour mimer les membranes cellulaires et caractériser l’interaction des protéines avec différents phospholipides spécifiques. L’influence de la concentration calcique sur leurs interactions membranaires sera étudiée car l’annexine A2 subit un changement conformationnel en présence de calcium. L’influence de la liaison des protéines S100A10 et annexine A2 sur l’état physique des phospholipides sera suivie par microscopie à fluorescence. En effet, en utilisant des phospholipides et ces protéines marquées avec des sondes fluorescentes différentes, il est possible d’observer l’état physique des phospholipides et leur évolution en présence de protéines. Les phospholipides dans l’état physique solide-condensé miment les microdomaines retrouvés dans les cellules. Nous pourrons alors confirmer la capacité de l’annexine A2 à lier les microdomaines, tel que suggéré dans la littérature. La surexpression des protéines S100A10 et annexine A2 est actuellement en cours. L’annexine A2 est surexprimée dans la souche BL21(DE3) d’E. coli et purifiée selon un protocole qui utilise différentes étapes de dilution, centrifugation, filtration et dialyse. La stratégie proposée pour la purification de la protéine S100A10 consiste à la surexprimer en fusion avec la Glutathion S-transférase (GST). Ce projet de recherche permet donc de développer les connaissances actuelles sur les protéines S100 et les annexines ainsi que sur la complexité des processus membranaires auxquels ces protéines participent.

La modification du métabolisme des adipocytes humain pour la prévention du diabète de type 2.


Thierry Chénard1, André Carpentier1, André Tchernof2, Rafael Najmanovich1
1Université de Sherbrooke 2Université Laval

La résistance à l’insuline et la déficience de sécrétion de l’insuline en réponse au glucose sont les deux principales caractéristiques physiopathologiques du diabète de type 2 (DT2). La résistance à l’insuline précède le développement du diabète de type 2 et la déficience de sécrétion de l’insuline en réponse au glucose est considérée comme une condition sine qua non à la présence du DT2. En effet, une réponse normale des cellules β du pancréas permet de compenser pour la résistance à l’insuline et de maintenir un niveau de glucose sanguin normal. Les données expérimentales dans les modèles animaux et chez l’humain supportent que la surexposition des tissues maigres aux acides gras (AG) joue un rôle important dans le développement de la résistance à l’insuline et de la dysfonction pancréatique. Les tissus adipeux jouent un rôle central dans la régulation des flux d’AG sanguin. Un problème dans le stockage d’AG dans les tissus adipeux a été associé à une augmentation des flux circulant d’AG se produisant dans les stages précoce du développement du DT2. Deux mécanismes généraux gouvernent l’expansion des tissus adipeux nécessaire au stockage des AG: 1) l'hyperplasie cellulaire (augmentation du nombre d'adipocytes, qui sont alors plus nombreux et plus petits); et 2) l'hypertrophie cellulaire (augmentation de la taille des adipocytes, qui sont alors moins nombreux et plus gros). Un default d’hyperplasie, un recrutement de pré-adipocytes et une différenciation en adipocyte mature diminués mène à une expansion pathologique de type hypertrophique qui contribue au développement du DT2.

Pour analyser les mécanismes pouvant influencer l’expansion des adipocytes et le développement du DT2, nous avons développé un réseau métabolique d’adipocyte humain en corrigeant les erreurs présentes dans un réseau publié puis avons réalisé une analyse sur ce réseau pour identifier des gènes ayant le potentiel d’influencer les mécanismes d’expansion des tissus adipeux (hypertrophie et/ou hyperplasie).

Nous avons réalisé une délétion de gènes in-silico sur notre réseau métabolique d’adipocytes humain, iTC1390adip, utilisant l’analyse de balance de flux. Cette analyse a permis de prédire l’effet de la délétion génique sur la production optimal de biomasse et de gouttelette lipidique utilisé comme représentation de l’hyperplasie et de l’hypertrophie des adipocytes respectivement. Nous avons comparé l’effet des délétion sur la production de biomasse à des résultats expérimentaux de délétion génique dans différentes lignées cancéreuses pour évaluer la capacité prédictive de notre réseau et établir un seuil pour la production de biomasse donnant la meilleure capacité prédictive de l’essentialité des gènes pour une croissance optimale. Suivant l’analyse des délétion génique, 26 gènes ont été prédits comme ayant le potentiel de réduire la production de gouttelette lipidique avec peu d’effet sur la production de biomasse donc qui pourrais avoir un effet positif sur l’hypertrophie des adipocytes. Certains des gènes qui ont été identifié, tels que LCAT et DGAT1, possèdent des résultats expérimentaux qui supportent nos prédictions alors que d’autres, tels que FAR2 et HSD17B12, pourrais servir de nouvelles cibles potentiel dans le remodelage des tissus adipeux et dans le traitement du DT2 si leur rôle est validé expérimentalement. Nous avons aussi utilisé les données d’expressions géniques d’adipocytes viscéraux et sous-cutanées pour déterminé les voies métaboliques ayant des différences d’expression significative pouvant être relié à la différence d’importance des différents dépôts dans le développement du DT2. Ces résultats guideront de nouvelles études in-vivo et in-cellulo pour valider de nouvelles cibles thérapeutiques pouvant affecter le développement du DT2.

Les liaisons dangereuses de la caspase-7 et de p23.


Cyrielle Martini1, Mikaël Bédard1, Pierre Lavigne1, Jean-Bernard Denault1
1Université de Sherbrooke

Les caspases sont des peptidases à cystéine impliquées dans l’apoptose et l’inflammation. Deux d’entre elles, les caspases 3 et 7, partagent la même séquence préférée de reconnaissance de substrat, soit DEVD*(G/A/S). Bien que la caspase-3 possède une meilleure spécificité catalytique (kcat/KM), la caspase-7 est plus efficace à cliver certains substrats, dont la protéine cochaperone p23. Nous avons précédemment montré que la caspase-7 possède un exosite constitué de quatre résidus lysine (K38KKK) présent dans le domaine N-terminal (DNT) de la caspase-7 qui est critique pour une protéolyse efficace du substrat apoptotique PARP-1. L’implication de cet exosite dans le clivage d’autres substrats de la caspase-7 reste à démontrer.

Nous avons montré que la mutation des 4 résidus lysine constituant l’exosite de la caspase-7 induisait une perte de son efficacité de clivage de la protéine p23. Cependant, on observe une perte plus importante lorsque la caspase-7 ne possède pas de DNT. On a donc émis l’hypothèse qu’il existait d’autres déterminants du DNT impliqués dans la reconnaissance de p23. En réalisant des délétions successives du DNT, nous avons déterminé que les résidus 40 à 45 sont critiques pour une protéolyse efficace de p23. Cependant, un balayage alanine de cette région nous a permis de montrer qu’il n’y avait pas de résidus spécifiques impliqués dans la reconnaissance de p23. Donc, seuls l’environnement et la structure du DNT semblent être importants pour une protéolyse efficace de p23 par la caspase-7.

Afin d’identifier les résidus de p23 interagissant avec l’exosite de la caspase-7, nous avons réalisé des expériences de résonance magnétique nucléaire (RMN) en solution avec la protéine p23 et des concentrations successives de peptide 34-47 de la caspase-7, correspondant à la région du DNT contenant l’exosite précédemment identifié. L’analyse des spectres [1H][15N]-HSQC obtenus met en évidence le déplacement chimique de plusieurs résidus localisés sur une région précise de p23. Or la mutation de 3 résidus présents dans cette région ne permet pas d’observer une diminution de l’efficacité de clivage par la caspase-7.

Afin de confirmer la localisation de leur site d’interaction, nous avons ensuite séparé le corps de p23 de sa queue C-terminale non structurée. Nous avons pour cela remplacé le corps de p23 par la protéine GST et nous avons réalisé des tests de clivage avec la caspase-7. Nous avons ainsi pu mettre en évidence que l’exosite de la caspase-7 ne se fixe pas au corps de p23 puisque son remplacement par la GST n’induit pas de perte d’efficacité de clivage. Des dissections de la queue C-terminale sont actuellement en cours afin de déterminer les résidus impliqués dans cette liaison.

Ces travaux nous permettront de mieux comprendre les mécanismes moléculaires impliqués dans la reconnaissance du substrat p23 par la caspase-7 et de réaliser le premier modèle d’interaction entre une caspase et un de ses substrats.

Ligand Binding Induces Enhanced Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans


Donald Gagné1, Nhung Nguyen1, Chitra Narayanan1, Louise Roux1, Joseph S. Brunzelle2, Jean-Francois Couture 3, Pratul Argawal4, Nicolas Doucet1
1INRS-Université du Québec 2Feinberg School of Medicine 3Universite d'Ottawa 4Oak Ridge National Laboratory

Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited due to relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on timescales orders of magnitude faster than the catalytic time frame. Here, we used NMR titration, chemical shift projection analysis (CHESPA) and relaxation dispersion experiments (15N-CPMG) in combination with computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft while the catalytically impaired E87A variant only displays millisecond dynamics in the fingers, demonstrating the long-range effect of mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 xylanases, providing essential dynamic information to help improve protein engineering and design applications.

Macrocycle mimics active conformation of apelin-13


Kien Tran1, Alexandre Murza1, Xavier Sainsily1, Jérôme Côté1, Philippe Sarret1, Éric Marsault1,2
1Université de Sherbrooke 2IPS - Université de Sherbrooke

Objectives:

The apelin receptor (APJ) is a member of the G-protein coupled receptor family. Apelin involves in the regulation of blood pressor (vasodilation/ inotropic effect) and has a closed link with insulin sensitivity and vasopressin hormone. The shortest ligand, apelin-13, has been an interesting starting point for the pharmacological study and drug development process over the past decade. However, its stability versus proteolytic degradation still present an unsolved problem limiting the scope of many researches. Here, we describe a potential solution for this issue using mimetic macrocycles.

 

Method:

In the first step, we performed a “rolling loop scan”1 to explore the inner space of the receptor and identified macrocycle’s positions where a benefit effect was obtained. All synthesized compounds were evaluated in binding affinity, and in signaling (Gi/o, β-arrestin recruitment using BRET assays), and the capability of modulating blood pressor in vivo in Sprague-Dawley rats.

 

Result:

The C-terminus and N-terminus of apelin-13 has been determined as favored regions for cyclization. These results suggest a very large hydrophobic pocket in the bottom of binding site. The central amino acids are supposed to bind with a flexible surface of APJ where rigid macrocycles were not tolerated. In this study, compound KT01_11 showed an identical signaling profiles compared to that of apelin-13 (mimic active conformation) and a potential stability by blocking hydrophilic sites in N-terminus.

 

Conclusion:

Mimetic macrocycles represent a useful tool to examine the space inside the receptor and mimic active conformation of the endogenous ligand. KT01_11 was identified as an interesting lead to develop a new stable, selective generation of ligand and validate apelinergic as promising drug target.

 

(1)       Reichwein et al. Angew. Chem. Int. Ed. 1999, 38 (24), 3684–3687.

Measuring protein-protein assemblies with a molecular ruler in living cells


Andrée-Ève Chrétien1,2,3,4, Isabelle Gagnon-Arsenault1,2,3,4, Claudine Lamothe1,2,3,4, Alexandre K Dubé1,2,3,4, Anne-Marie Dion-Côté1,2,3,4, Christian R Landry1,2,3,4
1Institut de biologie intégrative des systèmes (IBIS) 2PROTEO 3Département de biologie 4Université Laval

Protein quaternary assembly is central to many cellular processes. Several methods have been developed to detect and measure protein-protein interactions in model organisms. Some methods allow the identification of members of complexes but do not provide information on their spatial relationships. Other methods allow the detection of binary and direct interactions between physically associated proteins but do not provide direct insights into the larger quaternary architecture. In order to dissect the organisation of protein complexes both in terms of composition and spatial relationship among the subunits, it would be optimal to have a method that combines the two approaches described above. Here, we show that Protein-fragment Complementation Assay (PCA) can meet these needs. PCA is based on the association of complementary reporter fragments that reveals protein-protein interactions upon complementation. The reporter fragments are fused to the proteins of interest by a short peptide linker that determines the maximal physical distance at which fragments can complement. We found that we can modulate the length of the linkers to detect novel protein-protein interactions on a proteome-wide scale and more distant protein-protein interactions within large protein complexes.  Overall, we show that this modified PCA can act as a molecular ruler for measuring protein-protein interactions and proximity in living cells.

Measuring the temperature around an enzyme using DNA thermometers


Scott Harroun1, Arnaud Desrosiers1, David Gareau1, Alexis Vallée-Bélisle1
1Université de Montréal

There has been much research into the evolution of enzymes, but many fundamental questions remain unanswered. For example, how does heat produced at the active site diffuse into the surrounding medium?1 Can the heat released by an enzyme destabilize its structure; in other words, can the enzyme “overheat”? In this project, we propose to use programmable DNA-based nanothermometers (DNA-NTs) recently reported by our group2 to measure local temperature rise in the vicinity of an enzyme. ­The stability, and thus melting temperature, of these DNA-NTs can be readily tuned by varying the ratio of G-C to A-T base pairs in the stem. Structures with more G-C base pairing in the stem are found to have higher stability, and thus unfold at higher temperatures.2 By attaching a fluorophore and quencher pair at both extremities of the stem loop, one can thus obtain a library of fluorescent nanothermometers with a specific linear dynamic range spanning 12°C. The 0GC/5AT and 1GC/4AT stem sequences have melting temperatures near 37 °C, and thus have linear changes in fluorescence in this range. Accordingly, DNA-NTs are anchored onto an enzyme to measure local rise in temperature due to heat released during the enzymatic reaction by monitoring fluorescence variation. Initial results have found that the local change of temperature in the vicinity of the enzyme contrasts with the global temperature of the sample, which remains relatively constant before, during and after the reaction. As a proof-of-concept measurement, our first design involved coupling of biotinylated DNA-NTs to streptavidin alkaline phosphatase (AP). The AP enzymatic reaction for conversion of para-nitrophenylphosphate (pNPP) to para-nitrophenol (pNP) is highly exothermic (ΔH = -43.5 kJ/mol),3 and we have measured local temperature rise during this reaction. More generally, our project proposes to measure local change in temperature to experimentally validate the rate of temperature diffusion, and to determine if enzymes “overheat” when functioning at high rates. In addition to improving fundamental understanding of enzymes, by using these thermosensitive nanoswitches attached to enzymes, it may be possible to trigger drug release via local temperature rise around an enzyme.

References

[1] A. J. Wand. Nature. 2015, 517, 149-150

[2] D. Gareau, A. Desrosiers, A. Vallée-Bélisle. Nano Letters. Accepted Manuscript. http://doi.org/10.1021/acs.nanolett.6b00156

[3] C. Riedel, R. Gabizon, C. A. M. Wilson, K. Hamadani, K. Tsekouras, S. Marqusee, S, Pressé, C. Bustamante. Nature. 2015, 517, 227-230.

Modeling the GAPDH – SIAH1 Death Complex


Vinod Parmar1,2, Kalle Gehring 1,3, Gilles H Peslherbe 2, Ann M English 1,2
1PROTEO 2Department of Chemistry and Biochemistry and Centre for Research in Molecular Modeling, Concordia University 3GRASP and Department of Biochemistry, McGill University

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-known glycolytic enzyme that exhibits a multitude of moonlighting functions depending on its oligomeric state. For example, the GAPDH tetramer possesses dehydrogenase activity in the cytosol whereas the GAPDH monomer is implicated in DNA repair in the nucleus. Intriguingly, S-nitrosation of GAPDH triggers its binding to SIAH1 (seven in absentia homolog 1), an E3 ubiquitin ligase. SIAH1’s nuclear localization signal mediates nuclear translocation of the GAPDH-SIAH1 complex, and the enhanced stability of SIAH1 within this complex facilitates ubiquitination resulting in apoptotic cell death. The SIAH1 dimer has been previously docked to dimeric GAPDH using PatchDock to give a 2:1 SIAH1-dimer / GAPDH-tetramer complex but we hypothesize that this death complex may actually be composed of a 1:1 complex of the SIAH1 dimer with the GAPDH monomer since chemical modification of its active-site Cys152 frequently leads to GAPDH deoligomerization. We performed molecular dynamics (MD) simulations of the GAPDH monomer and subjected the data to principal component analysis (PCA) to extract functionally important motions. Principal components 1, 2 and 3 of the monomer are dominated by high fluctuations of the S-loop and we speculate that conversion of this loop into an intrinsically disordered domain in the monomer promotes the binding of GAPDH to its multitude of protein partners in the cell including SIAH1. Thus, we compared the interaction of the SIAH1 dimer and monomer with the GAPDH tetramer and monomer using protein-protein docking. Our preliminary results provide new insight into the GAPDH-SIAH1 binding interaction and highlight the possible importance of GAPDH deoligomerization as an apoptosis trigger.

Modelling conformational entropy in the scoring function of the molecular docking software FlexAID


Louis-Philippe Morency1, Rafael Najmanovich1
1Université de Sherbrooke

A major goal of molecular docking is to predict the experimentally observed binding mode between a biomolecule, i.e. a polymer of amino acids, DNA or RNA, and a ligand, e.g. small-molecules, peptides or nucleic acids. However, there is no current molecular docking methods that properly account for the configurational entropy of the predicted complexes.  It has been previously shown that the estimation of configurational entropy a posteriori significantly enhances the precision of popular scoring functions and may favour the prediction of binding poses of synthetic drugs. Our research group develops FlexAID, a competitive ligand and biomolecule docking method that includes ligand flexibility, biomolecule side chains and large scale backbone flexibility. Here we introduce the methods used to implement FlexAID’s newest feature that allows its scoring function to estimate the configurational entropy from the occupancy of the observed binding modes. FlexAID redefines the static binding mode usually predicted in molecular docking as a dynamic collection of poses scored altogether. The higher accuracy of FlexAID on dynamic target, the addition of novel features, i.e. the configurational entropy, its accessibility and its easy-to-use graphical user interface place FlexAID in an interesting position to tackle biologically and pharmacologically relevant situations currently ignored by other methods.

 

FlexAID is available as source code, pre-compiled executables or through the NRGsuite, a PyMOL integrated user interface allowing the user to use FlexAID in an interactive, intuitive and visual manner   —   http://bcb.med.usherbrooke.ca/flexaid.

Molecular insights into the interplay between metal coordination and DNA binding by the Campylobacter jejuni Ferric Uptake Regulator (CjFur)


Sabina Sarvan1, Karim Francois Charih1, Momen Askoura1, James Butcher1, Alain Stintzi1, Jean-Francois Couture 1
1University of Ottawa

Transition metals are crucial components of several metabolic pathways and are critical for DNA, RNA and protein synthesis. However, when found in excess, these metal ions are toxic. The Ferric uptake regulator (Fur) protein is an important regulator of iron homeostasis, however its functions extend beyond iron metabolism. Fur protein regulates a wide variety of functionally diverse pathways including flagellar and capsule biogenesis, energy metabolism, oxidative stress defense, uptake of other metal ions such as molybdate, tungsten, zinc and nickel as well as the regulation of non-coding RNAs via four general mechanisms namely apo-Fur activation/repression and holo-Fur activation/repression. Given that the Fur protein employs diverse regulatory mechanisms, we hypothesized that the ability of Fur to adopt different structural conformations underlies these peculiar functional differences. To address this important question, we solved the crystal structure of the Fur protein from Campylobacter jejuni. Structural analysis revealed that protein adopts a V-shaped conformation harboring an evolutionary conserved cluster of positively charged residues on the surface. Using an extensive library of mutants and electrophoretic mobility shift analysis, we found that substituting residues forming the positively charged surface is detrimental for Fur interaction with DNA. Furthermore, our in vivo studies suggest that these positively charged residues are important for the regulation of CjFur target genes and that different mechanisms modulate the activity of Fur family of metalloregulators depending on the number of occupied metal binding sites. Finally, we showed that the disruption of metal binding sites of CjFur significantly reduces DNA binding in vitro and is deleterious for the repression of Fur target genes in C. jejuni and the colonization of animal’s gut. Overall, our structural studies suggest that Fur protein employs a common surface and requires intact metal binding sites to bind DNA that regulate gene expression and contribute to bacterial pathogenicity.

New insight on experimental data using a streamlined, naive protocol for molecular dynamics analysis


Laurent Bruneau Cossette1,2, Jérôme Cabana1,2, Philippe Sarret2, Éric Marsault2, Pierre Lavigne1
1Université de Sherbrooke 2IPS - Université de Sherbrooke

Hidden Markov Models (HMMs) and time-lagged Independent Component Analysis (tICA) are simple, informative and accurate tools for the analysis or Molecular Dynamics (MD) data. They provide accurate information on both the thermodynamics (populations of states) and kinetics (timescales of state transitions) of proteins. However, they are relatively new: HMMs lack the analysis tools and validation that Markov State Models already have and tICAs, like PCAs, are hard to interpret.

The use of both techniques in a single set of tools, complemented with naive model selection criteria and analysis and display tools, provides new insight on experimental results on the structure and dynamics of STARD6, a globular protein, and AT1-R, a GPCR. It has been found that STARD6 exhibits movements in the μs timescales at the expected testosterone binding site, and that these movements may serve as initiators to ms-scales movements, as shown by CPMG NMR. It has also been found that AT1-R conformations, a few μs after ligand binding, are distributed in three macrostates, and that these macrostates could correspond to different signaling behaviors. The transitions between macrostates show major contributions from highly conserved residues, most likely involved in ligand binding and signaling. The information provided by this set of tools could be used to explore further the dynamics of STARD6 and AT1-R, as well as other proteins, in targeted structural and dynamics research (e.g. mutants, docking).

Nouveau protocole de recherche d’antibiotiques appliqué à Clostridium difficile.


Mathieu Larocque1
1Université de Sherbrooke

INTRODUCTION
Clostridium difficile est un pathogène nosocomial du tractus intestinal. Cette bactérie infecte son hôte suite à un déséquilibre de la flore normale, souvent relié à la prise d’antibiotiques. Elle produit des toxines menant à des diarrhées, des colites pseudomembraneuses et même la mort dans 10-15% des cas. Il s’agit du principal agent causal de diarrhées associées à ce type de médication. Les traitements couramment utilisés pour contrer la bactérie maintiennent la dysbiose ce qui favorise les réinfections (24% pour la vancomycine et de 12% pour la fidaxomicine). Considérant que chaque rechute augmente les risques de complication, la découverte d’agents thérapeutiques mieux adaptés au pathogène est une urgente nécessité. 


HYPOTHÈSE
Pour ce faire, nous avons élaboré un nouveau protocole de recherche d’antibiotique alliant science expérimentale et bio-informatique. Cette approche intégrative utilise les réseaux métaboliques pour identifier des cibles thérapeutiques potentielles du pathogène d’intérêt. Le potentiel pharmaceutique de chaque gène est par la suite évalué afin d’identifier les cibles d’intérêt supérieur qui seront validées expérimentalement. Des ligands pour ces protéines sont ensuite identifiés par arrimage moléculaire en prenant en considération les besoins de spécificité et d’innocuité qui devront être retrouvés dans la molécule finale dont l’affinité et l’efficacité seront validée expérimentalement.


RÉSULTATS
Un réseau métabolique de C. difficile, nommé iMLTC806cdf, contenant 911 réactions et 806 gènes, a été créé et a permis l’identification de 163 cibles thérapeutiques potentielles de la bactérie sous différentes conditions. Une précision de 89% pour la prédiction de l’essentialité des gènes en milieu riche fut atteinte. Parmi ces cibles, nadA et ribD ont été identifiés comme gènes d’intérêt supérieur. Ces cibles sont essentielles uniquement lorsque le produit finale de leur voie métabolique respective est absent du milieu de croissance, une condition qui, selon nous, se rapproche de la réalité in-vivo. Cette caractéristique facilite leur validation expérimentale puisque des souches mutantes pour chaque gène peuvent être maintenues en culture en milieu riche.


Des mutants pour chacun des gènes ont été générés et l’essentialité en milieu minimum du gène nadA a été démontrée expérimentalement. Une validation similaire est en cours pour ribD alors que des essaies d’infection dans un modèle murin, permettant de valider l’importance du gène nadA in-vivo, sont aussi en cours. La spécificité d’un futur antibiotique contre C. difficile étant une nécessité pour ne pas maintenir la dysbiose à l’origine de l’infection, la recherche de ligands qui s’effectue présentement compare l’affinité prédite des molécules pour des modèles des protéines de C. difficile à celle de proches homologues commensaux présents dans l’intestin. Les molécules avec une bonne spécificité pour C. difficile seront testées expérimentalement.


MÉTHODES
Les bases de données KEGG, Biocyc et Transport DB furent utilisées comme point de départ du réseau métabolique iMLTC806cdf. Le réseau fut manuellement amélioré et validé en se basant sur des données de la littérature. L’analyse de balance de flux (FBA) et l’accessibilité synthétique (SA) furent utilisées pour prédire l’essentialité des gènes, considérant les deux méthodes comme complémentaires. Les souches mutantes furent générées via le protocole d’insertion d’un intron de groupe II (ClosTron). Les modèles par homologie des protéines NadA et RibD furent crées en utilisant la suite I-Tasser et les essaies d’arrimage moléculaire furent réalisés grâce à FlexAid ; un logiciel développé au sein du laboratoire. 


CONCLUSION
Grâce à cette approche intégrative nous espérons identifier les bases de nouveaux antibiotiques plus spécifiques et mieux adaptés à C. difficile ce qui réduirait les risques de rechute en ne perturbant pas autant la flore intestinale normale qui a un effet protecteur contre ce pathogène.

Optimisation d’un milieu de culture défini pour la culture de myoblastes humains


Alexandre Côté1, Bruno Gaillet1, Juneau PM1, Maltais C1, Gravel WE1, Tremblay JP1, Garnier A1
1Université Laval

La Dystrophie musculaire de Duchenne (DMD) est  une maladie rare qui touche un garçon sur 3 500. Elle est causée par la mutation du gène de la dystrophine qui ne peut alors plus prévenir la dégradation progressive et irréversible des muscles. La thérapie cellulaire basée sur l'injection de myoblastes est un traitement potentiel prometteur pour la DMD. Cette approche en est présentement au stade des essais cliniques de phase 2. Pour ce faire, on utilise les cellules d’un donneur sain et compatible ou encore la greffe autologue de cellules corrigées génétiquement. Peu importe la source cellulaire, celles-ci sont d'abord multipliées in vitro afin d'en produire un nombre suffisant pour la greffe. Le choix du milieu de culture nécessaire pour cette expansion est important, car il doit être performant, sécuritaire et peu coûteux.

Le premier milieu sans sérum sanguin développé dans notre laboratoire, le LOBSFM, a connu un certain succès et a d'ailleurs été breveté. Cependant, son coût est élevé (1500$/L) et il ne semble pas pouvoir produire des cellules d'aussi grande qualité que le milieu contenant du FBS. En effet, il a été remarqué que la morphologie cellulaire ainsi que l'efficacité de greffe étaient différentes pour des cellules cultivées dans ces deux milieux.  Pour optimiser cet environnement de manière systématique et rapide, nous avons fait l'hypothèse que le changement morphologique des myoblastes était lié à l'efficacité de leur greffe. Nous avons ainsi pu tester un grand nombre de combinaisons de composants du milieu de culture sur la croissance ainsi que la morphologie des myoblastes humains, dans l'espoir d'obtenir des cellules fonctionnelles en grand nombre.

 Les plans statistiques d’expérience et l’analyse automatique d’images ont été utilisés pour réaliser cette optimisation. Le milieu finalement conçu permet de réduire de 55% les coûts par rapport au milieu de culture sans sérum original tout en obtenant un temps de dédoublement, une morphologie cellulaire et une efficacité de greffe équivalents ou supérieurs aux cellules multipliées en milieu de culture contenant du FBS.

 

Optimization and characterization of synthetic antimicrobial peptides


Nicolas Poulin1,2, Rachelle Séguin1,2, Pierre-Alexandre Paquet-Côté1,2, Michèle Auger1,2, Normand Voyer1,2
1Université Laval 2PROTEO

We have designed a membrane active 14 amino acid long peptide consisting of 10 leucines and 4 crown ether modified phenylalanines. This peptide is known for mimicking the membrane activity of natural antimicrobial peptides, but with a poor selectivity.  In this study, the effect of the global charge of analogous 14-mer peptides on the structure and the antimicrobial activity is investigated by replacing neutral residues (leucines) by cationic residues (arginine). The increase of the net positive charge by one or two has already shown to improve the antimicrobial activity by influencing the secondary structure of the peptidic chain. In an attempt to determine the link between the secondary structure and the antimicrobial selectivity, we will report our recent results on analogs having an increased number of positive charges by incorporating more arginines. The antimicrobial selectivity is evaluated by comparing the hemolytic activity with the antimicrobial activity (towards Escherichia coli and Streptococus epidermidis). The secondary structure the 14-mers adopted to permeabilize cell membranes is determined mainly by circular dichroism of the peptide using POPC and POPG vesicles and by other biophysical techniques.

Peptides macrocycliques pour l'exploration conformationnelle de l'interaction Neurotensine-NTS1


Marc Sousbie1,2, Élie Besserer-Offroy1,2, Jean-Michel Longpré1,2, Philippe Sarret1,2, Richard Leduc1,2, Éric Marsault1,2
1Université de Sherbrooke 2IPS - Université de Sherbrooke

Les RCPG (récepteurs couplés aux protéines G) constituent la plus grande famille de récepteurs retrouvés à la membrane plasmique. NTS1 est un RCPG d'intérêt dans le traitement de la douleur chronique. Les études actuelles lui accordent un effet antinociceptif(1) comparable aux opiacés couramment utilisés en clinique, mais avec potentiellement beaucoup moins d'effets secondaires tels que l'addiction ou l'arrêt cardiaque.

Pour qu'un ligand active un RCPG , il doit non seulement s'y lier, mais aussi stabiliser une ou un ensemble de conformations actives. Restreindre la conformation d'un ligand peptidique peut avoir un effet bénéfique sur la liaison au récepteur car la différence d'entropie est diminuée. Ceci est valable seulement si la conformation restreinte est proche de celle que le ligand adopte au sein de la poche de liaison.

Nous avons par conséquent décidé d'étudier les effets de la macrocyclisation sur le peptide neurotensine 8-13 (NT 8-13), qui active le récepteur NTS1 et a une affinité de l'ordre du bas nanomolaire.

 

Les peptides sont synthétisés sur résine, en stratégie Fmoc, et cyclisés par métathèse (RCM, ring-closing metathesis). Cela implique la présence de résidus comportant une double liaison C=C, qui est apportée soit par intégration de l'acide aminé allylglycine ou bien d'acides aminés modifiées à partir de la sérine, de la proline ou de la tyrosine, et ayant divers effets sur la conformation du peptide.

La caractérisation des analogues consiste à mesurer leur affinité pour le récepteur NTS1 (capacité à déplacer le ligand endogène radiomarqué), et à évaluer leur efficacité à activer les protéines G par BRET (bioluminescence resonance energy transfer).


 

Les analogues testés ont une affinité pour le récepteur NTS1 de l'ordre du bas µM (1,1 µM pour le meilleur) et activent la voie Gq, qui est considérée comme la voie canonique de ce récepteur.

Les essais de liaison au récepteur, couplés à de la modélisation, nous permettent de nous rapprocher de la conformation endogène.

Phytochemical investigation of northern Quebec lichens


Claudia Carpentier1, Emerson Ferreira-Queiroz2, Muriel Cuendet2, Jean-Luc Wolfender2, Normand Voyer1
1Université Laval 2University of Geneva

 

Lichens are a symbiotic association of a combination of fungi and algae or cyanobacteria.1 Due to the defense response to ecological and climatic growth conditions, lichens produce a vast diversity of bioactive natural products (NPs) to protect the symbiotic partners.1,2 Therefore, lichens of Northen Quebec remain an underexplored source of potential bioactive secondary metabolites. We have started recently an investigation on such lichens.The dichloromethane extract of Cladonia stellaris demonstrated significant in vitro inhibitory activity against nuclear factor kappa B. Targeted isolation of the active compounds by MPLC led to the isolation and characterization of perlatolic acid (1), usnic acid (2), olivetolic acid (3) and its derivative (4). Inhibitory activity against three major pro-inflammatory targets (mPGES-1, 5-LO and NF-KB) of perlatolic acid have been reported in the literature.1 Inhibitory activity of compounds 3 and 4 were also previously reported.1 To investigate their potency as pharmacological agents, we have undertarken the synthesis of compounds 3, 4 and 1. We will report on the isolation, the synthesis and the bioactivity of NPs from Cladonia stellaris, which represents a potential source of anti-inflammatory compounds.
1: Oettl, S. K.; Gerstmeier, J. ; Khan, S. Y.; et al. PloS one, 2013, 8, e76929.
2: Vasconsuelo, A.; Boland, R. Plant Science, 2007, 172, 861–875.

Prédiction d'un site d'inhibition allostérique spécifique à une ectonucléotide pyrophosphatase/phosphodiestérase 1 humaine (ENPP1) par modélisation moléculaire


Xavier Barbeau1,3, Elnur Elyar Shayhidin1,4, Elsa Forcellini1,3, Marie-Chloé Boulanger1,4, Jean-François Paquin1,3, Patrick Mathieu1,4, Patrick Lagüe1,2
1Université Laval 2Département de biochimie, de microbiologie et de bio-informatique 3Département de chimie 4Laboratoire d’Études Moléculaires des Valvulopathies (LEMV), Groupe de Recherche en Valvulopathies (GRV), Institu Universitaire de Cardiologie et de Pneumologie de Québec (IUCPQ)

La calcification de la valve aortique est la troisième maladie cardiovasculaire en importance, touchant principalement la population de 65 ans et plus1. Le seul traitement disponible est le remplacement chirurgicale de la valve aortique. Ce traitement est offert seulement aux patients sévèrement atteints. Ainsi, la mise au point d’un composé pharmacologique pour le traitement de la calcification de la valve aortique serait une avancée scientifique majeure en permettant de d’alléger significativement la lourdeur du traitement pour les patients tout en diminuant les coûts.

Le groupe du Pr Patrick Mathieu (IUCPQ) a mis en évidence le rôle de l’ectonucléotide pyrophosphatase/phosphodiestérase 1 (ENPP1)2 dans le processus de la calcification de la valve aortique. En effet, cette protéine,  est surexprimée dans les tissus calcifiés3. Lorsque surexprimée, elle agit sur le processus de calcification en dérégulant la concentration de pyrophosphate inorganique extracellulaire (PPi), un inhibiteur de la calcification. Des tests en lignées cellulaires ont démontré que l’inhibition de ENPP1 empêchait la calcification3. ENPP1 est donc une cible pharmaceutique potentielle contre la calcification de la valve aortique. 

L’ARL 67156 est un analogue de l’ATP et présente une constate d’inhibition (ki) de 12 uM contre ENPP1, mais possède aussi une activité inhibitrice sur les autres membres de la famille de ENPP1. Le QPS1 est de type quinazoline et inhibe ENPP1 avec un ki de 0.059 uM4,5. En plus de sont haut niveau d’inhibition, le QPS1 est spécifique à ENPP1. Toutefois, le QPS1 présente un profil d’inhibition de type non compétitif, son site de liaison sur ENPP1 n’est présentement pas connu, limitant le processus de design rationnel.

Mon projet de doctorat a consisté en l’étude de ENPP1 par modélisation moléculaire. La modélisation par homologie a été utilisée pour générée la structure de ENPP1 humaine à partir de celle de souris6. Cette structure a ensuite été étudiée par dynamique moléculaire selon deux approches. La première approche a consisté à étudier la dynamique de ENPP1 en incluant une molécule d’ATP dans le site catalytique. Cela a permis l’étude de la structure de l’ATP au site actif. La deuxième approche a consisté en l’utilisation du protocol SILCS7, ce qui a permis de prédire un site de liaison pour le QPS1.

 

Références:
1- R.V. Freeman, C.M. Otto 2005, Circulation, 111 p 3316
2- Côté N. et al. 2012, J. Mol. Cell. Cardiol. , 52, 1191-1202
3- Côté N. et al. 2012, European Journal of Pharmacology, 689 p 139-146
4- Patel S. D. 2009, Bioorganic & Medicinal Chemistry Letters, 19 p 3339-3343
5- Shayhidin E. E., Forcecllini E. et al., Br. J. Pharmacol. 2015 
6- Jansen S. et al. 2012, Structure, 20 p 1948-1959
7- Guvench O. MacKerell A. D. Jr 2009, Plos Computational Biology

Prédiction de structure et de fonction de toutes les protéines du phage Lactococcus lactis P680


Wiem Kristou1,2,3, Sylvain Moineau1,3, Stéphane Gagné1,2,3
1PROTEO 2IBIS 3Département de biochimie, de microbiologie et de bioinformatique, Université Laval

Les Lactococcus lactis sont les principales bactéries utilisées par l'industrie laitière dans la transformation du lait. Ce processus peut cependant être altéré par la présence de phage virulents qui infectent ces bactéries et dont les phages de type 936 sont fortement retrouvés dans l'industrie du fromage. Afin de mieux comprendre le processus d'infection, l'utilisation de plusieurs outils de bio-informatique structurale nous a permis d'effectuer des prédictions de structure et de fonction des 49 protéines du phage P680. Un criblage des banques par le programme BLAST a permis d'identifier les protéines pouvant être modélisées par homologie (par MODELLER). L'utilisation d'un logiciel de « meta-threading » (LOMETS) a permis de classer les protéines par niveau de difficulté de prédiction. La modélisation de la structure tridimensionnelle des protéines caractérisées comme moyennement faciles ou faciles à prédire a pu être entamée grâce à la version stand-alone de I-TASSER. Les protéines catégorisées comme difficiles à modéliser, ont fait l'objet d'une prédiction par la méthode ab initio moyennant le logiciel QUARK. Après avoir prédit les structures tridimensionnelles des protéines, l'utilisation du logiciel COACH a permis l'obtention de prédiction de fonction pour les meilleures prédictions de structure tridimensionnelles. Des logiciels supplémentaires ont été utilisés pour la prédiction de désordre, de protéines constituées de plusieurs domaines et d'hélices transmembranaires.

Preparation of Various 2,3,3-triarylacrylic Acid Esters, a Particular Class of 1,2,2-triarylethene Compounds, using Suzuki-Miyaura Coupling Reactions


Sébastien Cardinal1, Normand Voyer1
1Université Laval

We recently developed a new strategy to access 2,3,3-triarylacrylic acid esters, a class of 1,2,2-triarylethene compounds bearing an a,b-unsaturated ester functionality.1 Our approach implies the preparation of a gem-dibromoalkene precursor from a a-ketoester compound, followed by the installation of two aryl groups by Suzuki-Miyaura coupling reactions on the two C-Br bonds. Many 2,3,3-triarylacrylic acid esters with various arrangements regarding the aryl rings diversity on the 1,2,2-triarylethe scaffolds could be obtained from a single gem-dibromoalkene. Efficient conditions for double coupling and for stereoselective mono-coupling were found, in a way to give access to all those classes of compounds. This presentation will report the preparation of compounds with one, two or three types of aryl groups, in a way to demonstrate the versatility of our synthetic strategy. The exploitation of this methodology for the total synthesis of quebecol, a promising polyphenolic compound found in maple syrup, will also be discussed.2

1. Cardinal, S.; Voyer, N. Synthesis 2016, 48, 1202.

2. Cardinal, S.; Voyer, N. Tetrahedron Letters 2013, 54, 5178.

Protein engineering of the CalB lipase to synthesize methyl salicylate


Ying Chew Fajardo1, Yossef Lopez de los Santos1, Guillaume Brault1, Nicolas Doucet1
1INRS-Institut Armand-Frappier

A computationally-guided semi-rational protein design approach will be used to improve the enzymatic selectivity and catalytic efficiency of the lipase B from Pseudozyma antarctica (CalB) to synthesize methyl salicylate. This fatty acid ester is a flavoring and fragrance compound with significant relevance in the biotechnological industry. CalB is one the most widely used lipases for the enzymatic hydrolysis and synthesis of esters [1,2,3,4,5], offering potential for the biological production of flavoring agents. However, the relatively confined organization of its active site precludes the recognition of more complex substrates. To overcome this limitation, in silico docking analyses of the best clones obtained from a previous mutant library generated in the Doucet lab will be undertaken. This will allow identification of the most significant amino acid residues involved in methyl salicylate precursor binding and recognition. These “hot spots” will be subjected to combinatorial mutagenesis to synthesize a ‘second generation’ library of CalB variants, which will further be screened for the desired activity. Finally, up scaling production of the most efficient variants will be tested to help develop a biocatalyst for the proper industrial enzymatic synthesis of this flavor.

 

References:

[1] Faber K. 2011. Biotransformations in Organic Chemistry. DOI 10.1007/978-3-642-17393-6_2, # Springer-Verlag Berlin Heidelberg.

[2] Jaeger, K. E. & T. Eggert. (2002). Lipases for biotechnology. Current Opinion in Biotechnology. 3:390-397.

[3] Reetz, M. T. (2002). Lipases as practical biocatalysts. Current Opinion in Biotechnology 6:145-150.

[4] Lee, M. Y. & Dordick, J. S. (2002). Enzyme activation for nonaqueous media. Current Opinion in Biotechnology 13:376-384.

[5] Gotor-Fernandez, V., Busto, E. & Gotor, V. (2006). Candida antarctica lipase B; an ideal biocatalyst for the preparation of nitrogenated organic compounds. Advanced Synthesis & Catalysis 348:797-812.

Protein-protein interactions of a thermosensitive allele in different environments


Véronique Hamel1,2,3,4, Isabelle Gagnon-Arsenault1,2,3,4, Marie Filteau1,2,3,4, Christian R. Landry1,2,3,4
1Institut de Biologie Intégrative et des Systèmes (IBIS) 2Département de Biologie 3PROTEO 4Université Laval

Deleterious mutations segregate and often fix in small populations during the course of evolution. These deleterious mutations can be compensated for by secondary mutations known as compensatory mutations. We examined if the trajectory of compensatory evolution to a strongly deleterious mutation is dependent on the environment and the genetic background in which compensatory evolution takes place. We performed an evolutionary rescue experiment in a yeast model for the Wiskott-Aldrich syndrome (mutation in LAS17) in two genetic backgrounds and two carbon sources.  We found that compensatory mutations tend to be overrepresented in the physical interaction network surrounding Las17p, showing that protein interaction partners are prime targets for compensatory mutations. In addition, we found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment and their combination. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target and the associated fitness cost varied across contexts. Overall, our results show that the course of evolution following the fixation of a deleterious allele is highly contingent on the initial conditions in which a deleterious mutation occurs. 

Proteins and oligopeptides as green catalysts for chiral epoxidations


Christopher Bérubé1,2, Corinne Bouchard1,2, Normand Voyer1,2
1Université Laval 2PROTEO

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

 

Inspired by those works, our objective is to developed a novel ecofriendly way for the chiral epoxidation of α,β-unsaturated ketones in water without any co-solvent. Our hypothesis is based on the use of oligopeptides or proteins as solubilizing agents and chiral catalysts for the substrates. Indeed, performing efficiently the reactions in water in absence of co-solvent is highly favourable for economic and environmental reasons, reducing chemical waste in large scale processes. Towards such greener synthetic epoxidation methods, our group has been investigating the use of different proteins and oligopeptides as efficient asymmetric epoxidation catalyst in pure water.

 

We will report the results of our initial investigation on the enantioselective epoxidation of enones using bovine serum albumin, sheep serum albumin and poly-L-leucine in pure water. Results demonstrated that no organic co-solvent is necessary for the Juliá-Colonna epoxidation of a wide variety of electron deficient enones with high enantioselectivities. 

Recréer et comprendre les mécanismes de reconnaissance moléculaire à l'aide d'interrupteurs d'ADN


Carl Prévost-Tremblay1, Alexis Vallée-Bélisle1
1Université de Montréal

Avec l'avènement des biotechnologies, les principes fondamentaux qui gouvernent le fonctionnement des biomolécules naturelles font l'objet d'un intérêt grandissant.  Une meilleure compréhension des mécanismes de liaison, tels que l'ajustement induit et la sélection conformationnelle, responsables du couplage entre la liaison et le changement conformationnel, fournirait sans aucun doute des outils efficaces pour la conception de biomolécules finement régulées.  À cette fin, nous proposons de recréer ces deux mécanismes de liaison classiques à l'aide d'interrupteurs d'ADN fluorescents nous permettant de varier indépendamment les paramètres thermodynamiques et structurels de l'interrupteur.  Nos résultats démontrent que l'affinité de l'état inactif de l'interrupteur pour le ligand est un paramètre déterminant pour la sélection du mécanisme.  Par exemple, un site de liaison partiellement accessible dans la conformation inactive rend possible une activation par ajustement induit (liaison avant changement conformationnel).  Nous avons montré que ce mécanisme permet d'accélérer l'activation de l'interrupteur d'au moins 10 000 fois comparativement au mécanisme de sélection conformationnelle.  Contrairement au mécanisme d'ajustement induit, la cinétique d'activation par sélection conformationnelle est en grande partie  contrôlée par l'équilibre conformationnel de l'interrupteur.  Nous avons montré que cette cinétique peut-être modulée d'au moins 200 fois simplement en variant cet équilibre conformationnel. Toutefois l'optimisation de ces deux mécanismes implique un compromis. Dans le cas du mécanisme par déplacement de population, une accélération du mécanisme via une déstabilisation de l'état inactif entrainera également un augmentation du bruit de fond de l'interrupteur (une fraction d'interrupteur sera active en absence de ligand). Dans le cas du mécanisme par ajustement induit, une accélération du mécanisme via une augmentation de l'affinité de l'état inactif diminuera la capacité du ligand a payer pour le changement conformationnel  (une fraction de l'interrupteur lié restera sous forme inactive). En conclusion, notre étude met en valeur les principes qui permettent de concevoir et d'optimiser les mécanismes de régulation par liaison des biomolécules.  En plus d'améliorer notre compréhension des mécanismes de reconnaissance moléculaire naturels et de leur rôle dans l'évolution, ces principes permettront la conception de biomolécules plus performantes et mieux adaptées.

Régulation par phosphorylation de la fonction des domaines SH3 de NCK1/2


Ugo Dionne1,3, François Chartier1, David N. Bernard3,4, Michel Tremblay1, Gerald Gish5, Patrick Laprise1,2, Nicolas Doucet3,4, Nicolas Bisson1,2,3
1Centre de recherche du CHU de Québec, ULaval, axe oncologie 2Département de biologie moléculaire, biochimie médicale et pathologie 3PROTEO 4INRS-Université du Québec 5Lunenfeld-Tanenbaum Research Institute

Les signaux extracellulaires relayés par certains récepteurs tyrosine kinase (RTK) impliquent des protéines adaptatrices comme NCK1/2. Leur fonction est de coupler les signaux entre des RTK activés, via leur domaine SH2 (Src-Homology 2), et des effecteurs cytoplasmiques via leurs trois domaines SH3. L’objectif de ce projet est de déterminer si les protéines NCK1/2 peuvent être phosphorylées, et de caractériser la fonction de cette modification. Un résidu tyrosine conservé dans l’évolution des domaines SH3 (de la levure à la souris) a été retrouvé phosphorylé dans 5/6 des domaines SH3 de NCK1/2. Le RTK EphA4, un partenaire direct SH2-dépendant de NCK1/2, a été confirmé comme étant la kinase responsable de cette phosphorylation, in vitro avec des protéines purifiées et in vivo dans des cellules en culture. Il a été montré in vitro que la phosphorylation du premier domaine SH3 de NCK1/2 par EphA4 inhibe sa liaison avec son partenaire CD3ε. De plus, il a été observé qu’un triple mutant de NCK2 mimant la phosphorylation (Y/E) ne peut plus lier ses partenaires SH3 directs comme WASP et PAK dans des cellules en culture. Il a aussi été montré que EGFR et FGFR pourraient phosphoryler la tyrosine des 3 domaines SH3 de NCK2. De plus, chez la drosophile, le mutant Y/E est incapable de transmettre le signal via ses SH3, résultant en un défaut de guidance axonale des photorécepteurs. Ces travaux suggèrent que les RTK peuvent non seulement recruter des partenaires suite à leur activation, mais aussi les phosphoryler pour terminer le signal en aval.

Selective binding of functionalized porphyrin compounds at the homodimeric interface of human galectin-7


Philippe Egesborg1, David Bernard1, Agathe Urvoas2, Donald Gagné1, Jean-Pierre Mahy2, Rémy Ricoux2, Nicolas Doucet1,3,4
1INRS-Université du Québec 2Université Paris-Sud XI 3PROTEO 4GRASP

Galectins are small soluble lectins that bind beta-galactosides via their carbohydrate recognition domain (CRD). Their ability to dimerize is critical for the crosslinking of glycoprotein receptors and subsequent cellular signaling. This is particularly important for their immunomodulatory role via the induction of T-cell apoptosis. Because galectins play a central role in many pathologies, including cancer, they represent valuable therapeutic targets for drugs or as biomarkers. At present, most inhibitors have been directed towards the CRD, a challenging task in terms of specificity given the high structural homology of the CRD among galectins. However, while the CRD β-galactoside binding site remains highly similar throughout galectin homologues, they display little sequence identity. This observation raises the possibility of targeting various galectins through the use of unusual ligands that would specifically bind galectins in a carbohyrate-independent manner. Here, we report non-carbohydrate ligands, porphyrin compounds functionalized with zinc ions, that specifically bind human galectin-7 (hGal-7). The medical appeal and relevance of porphyrins as photosensitizers in cancer treatment has been amply demonstrated, especially in tumor imaging and photodynamic therapy, potentially providing a means to use these binding affinities and intrinsic physicochemical imaging properties as hGal-7 markers in cancerous tissue progression. We used a combination of fluorescence and NMR titration experiments to specifically define and map the low-micromolar, non-carbohydrate binding sites of porphyrins on the surface of hGal-7. We found that these porphyrin ligands offer limited selectivity with respect to charge and metal, and that their binding affinity to hGal-7(~20µM) is stronger than the previously characterized interactions mediated by glycan-binding residues in the CRD pocket, suggesting that the distinctively high porphyrin affinity to hGal-7 may be biologically significant. To our knowledge, these results highlight the first distinct and structurally characterized non-carbohydrate binding site on the surface of hGal-7, in addition to portraying the only structural characterization of porphyrin binding to human galectins to date.

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


Carlos Eduardo DulceyJordan1, Eric Déziel1, Nicolas Doucet1
1INRS-Institut Armand-Frappier, Université du Québec, Laval, QC, Canada

Rhamnolipids are non-toxic and biodegradable surfactants mainly produced by Pseudomonas aeruginosa. They demonstrate an excellent potential as substitutes for synthetic surfactants and are currently found in formulations of household cleaning products. Numerous other applications for these biosurfactants are currently evaluated in cosmetics, detergents and in the bioremediation of soils. The bacterial strains that produce rhamnolipids generate a mixture of congeners with different lipophilic chain lengths. In Pseudomonas aeruginosa, the predominant chain length is 10 carbons. Since the physicochemical properties of rhamnolipids are directly influenced by their molecular structure, modification or improvement of their surfactant properties can be acquired by controlling the length of the carbon chains. In Pseudomonas aeruginosa, the enzyme RhlA is responsible for diverting the 10-carbon hydroxy fatty acids to the β-oxidation pathway, linking them to form dimer precursors of rhamnolipids. We have shown that it is possible to control the length of the fatty acid chain by changing the recognition pattern of the RhlA substrate through semi-rational mutagenesis. The main objective of this project is the functional and structural characterization of RhlA, improving its catalytic efficiency and changing its affinity for hydroxy fatty acids with different chain lengths to produce rhamnolipids with distinct surfactant properties. Preliminary mutagenesis results of RhlA will be presented.

Similarities Between Conformational Exchange Patterns in Members of the Ribonuclease 3 Subfamily


David N. Bernard1, Donald Gagné1, Nicolas Doucet1
1INRS-Université du Québec

Enzymes are increasingly being used in pharmaceutical and industrial environments, particularly as greener and more efficient alternatives to chemical catalysts. However, engineering new enzyme reactions is an arduous and inefficient process, mainly because the predictable outcome of protein engineering on 3D structure, function and dynamics remains elusive. Recent experimental evidence suggests that conformational exchange may be involved in promoting catalysis in many enzyme systems, but the mechanisms underlying this atomic flexibility remain unclear. It is still unknown whether sequence and/or structure are evolutionarily conserved to promote flexibility events linked to biological function among protein homologs. Understanding phenomena underlying protein dynamics is thus an important step in facilitating protein engineering. In order to tackle these interrogations, we have used NMR to characterize the millisecond timescale conformational exchange in various members of the ribonuclease A superfamily. While these enzymes display very similar structure, their evolutionary distance and diversified biological activities complicate flexibility-function analyses. To solve this issue, we have investigated mammalian homologs of human ribonuclease 3 (Eosinophil Cationic Protein, ECP), comparing the human enzyme with its close ECP homologs from Pongo pygmaeus and Macaca fascicularis. Our findings show that conformational exchange in the monkey enzymes strongly resembles that of their human counterpart, providing insights into the effects of sequence and phylogenetic diversity on protein dynamics. Further experiments are required to determine the exact biological roles of these enzymes and their dependence on atomic flexibility.

Smart mutational exploration of the CalB lipase active site using a combination of virtual docking and iterative saturation mutagenesis


Yossef Lopez de los Santos1, Guillaume Brault1, Nicolas Doucet1,2,3
1INRS-Université du Québec 2PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications 3GRASP, Groupe de Recherche Axé sur la Structure des Protéines

One of the key elements for proper directed evolution of proteins is the cyclic use of mutagenesis and selection processes, giving rise to libraries containing millions of mutants.  However, analyzing such an important number of mutants is not a trivial task, as the identification of active variants among millions of possibilities quickly becomes exhaustive and inefficient. Here we describe a semi-rational combinatorial approach supported by virtual docking to generate smaller and smarter libraries. Because of its ability to perform the synthesis of esters in organic media, lipase B from Pseudozyma antarctica (CalB) was used as an industrially-relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was aimed at the development of CalB variants with enhanced synthetic activity towards bulky substrates. Substrate-imprinted docking was used to uncover target positions involved with the stabilization of the enzyme-substrate complex, identifying “hot spots” that are most likely to yield active improvements for desired ligands. The Iterative Saturation Mutagenesis strategy was employed to sequentially incorporate favorable mutations, further increasing our chances of selecting improved variants with a concomitant reduction in screening effort. We tested a limited number of 164 mutants that explored 6 residue positions in the active-site cavity of CalB. For a single round of mutagenesis and selection against 2 different substrates, a number of variants showed up to 5-fold increase in activity relative to WT CalB. These results represent the first stage in the development of additional CalB variants with improved activity towards bulky esters.

Spatial distribution of an anti-cancer drug in mouse skin studied by infrared microspectroscopy.


Quoc Chon Le1, Thierry Lefèvre1, René C.-Gaudreault2, Gaétan Laroche3, Michèle Auger1
1Département de chimie, PROTEO, CERMA, CQMF, Université Laval 2Départementde médecine moléculaire, Université Laval 3Départementde génie des mines, métallurgie et matériaux, CERMA, CQMF, Université Laval

Infrared microspectroscopy has been proven to be very efficient for skin tissues1 and percutaneous absorption characterization2,3,4. This technique explores the molecular structure information and the spatial distribution of a molecule through the skin without staining the tissues. Our group applies mid-infrared microspectroscopy to investigate the in vitro diffusion of a derivative of arylchloroethylurea (CEU), an anti-cancer drug5, through mouse skin. This study aims at understanding the diffusion and distribution of CEU within skin tissues. We use deuterated CEU molecules to easily distinguish them from the complex composition of the skin matrix. Mouse skin samples were freshly obtained, conserved at -80 °C and subcutaneous fat removed for diffusion experiments. Finally, the skin samples was sectioned by cryo-microtome for IR microspectroscopy characterization. Our results indicate that CEU penetrates across the skin and accumulates in several regions. The distribution of CEU within the skin is not spatially homogeneous and does not clearly show a correlation with the distribution of neither proteins nor lipids.

 

References

1. Leroy, M., 2014. La microscopie vibrationnelle comme outil de caractérisation de la peau normale humaine et reconstruite. Ph.D. dissertation, Université Laval.

2. Caspers P. J., Lucassen G. W., Puppels G., J. Biophys. J. 2003, 85, 572-580.

3. Mendelsohn R., Flach, C.R., Moore, D.J., Biochim. Biophys. Acta. 2006, 1758, 923-933.

4. Zhang, Q., 2014. Vibrational spectroscopy and microscopic imaging of skin. Ph.D. dissertation, Rutgers University.

5. Saint-Laurent A., Boudreau, N., Larivière, D., Legault, J., C.-Gaudreault, R., Auger, M., Chem. Phys. Lipids. 2001, 111, 163-175.

Spectroscopic Investigation of α-Synuclein 71-82, a Peptide Derived from a Protein Involved in Parkinson’s Disease


Benjamin Martial1, Laurie Bédard1, Thierry Lefèvre1, Michèle Auger1
1Université Laval

Our research project focuses on a specific peptide of α-synuclein which is an amyloid protein known to be involved in the neurodegenerative Parkinson’s disease. Aggregates of this protein are found in Parkinson’s disease patients’ brain, and more specifically, in the nerve tissues. This protein can be found in several forms, comprising monomers, oligomers and protofibrils. The secondary structure of these compounds has been described as a parallel β-sheet structure. All along this 140 amino acids protein, a specific portion plays an important role in the aggregation process, namely the non-amyloid-β component (NAC, sequence 61-95). In the core of this NAC, the amino acid sequence 71-82 appears to be crucial in the fibril formation process.

In the present study, we have investigated the secondary structure and thermal stability of the peptide fragment 71−82, α-syn 71−82, as a function of concentration and temperature, as well as its interactions with phospholipid model membranes using various spectroscopic techniques. The data show that α-syn 71−82 is mainly disordered in solution with the presence of a few β-sheet structure elements. The peptide reversibly forms intermolecular β-sheets with increasing concentration and decreasing temperature, suggesting that it is subjected to a thermodynamic equilibrium between a monomeric and an oligomeric form. This equilibrium seems to be affected by the presence of zwitterionic membranes. Conversely, the influence of the peptide on zwitterionic lipid bilayers is small and concentration-dependent. By contrast, α-syn 71−82 is strongly affected by anionic vesicles. The peptide indeed exhibits a dramatic conformational change, reflecting an extensive and irreversible self-aggregation, the majority of the amino acids being involved in a parallel β-sheet conformation. The aggregates appear to be located near the membrane surface but do not perturb significantly the membrane order. Comparing these results with the literature, it appears that α-syn 71−82 shares several general properties and structural similarities with its parent protein. These common points suggest that the sequence 71−82 may overall contribute to the behavior and properties of α-syn.

Further studies on this peptide will be conducted to investigate its behavior at the molecular level via:

- Calcein release experiments to probe the effect of the peptide on membrane permeability

- Deuterium solid-state NMR to identify the effect of the peptide on the order of lipid acyl chains

- PISEMA experiments to determine the membrane topology of the peptide

- REDOR internuclear distance measurements to determine the membrane insertion of the peptide

Altogether, this information on the peptide conformation and membrane interactions will broaden the knowledge on the mode of action of a-syn 71−82, and thereby of a-syn.

Structural insights into the assembly of the Ash2L/DPY-30 heterotrimer


John Haddad1,2, Véronique Tremblay1,2, Jean-Francois Couture 1,2
1University of Ottawa 2Ottawa Institute of Systems Biology

In eukaryotes, the SET1 family of methyltransferases carries out the methylation of Lysine 4 on Histone H3 (H3K4). Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the molecular underpinnings controlling the formation of the Ash2L/DPY-30 complex have remained largely unexplored. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. The structure shows that Ash2L C-terminus folds in two distinct domains that include a b-sandwich composed of 12 b-strands and a long a-helix located at the C-terminal of the protein. This amphipathic a-helix makes several hydrophobic interactions with DPY-30 homodimer. Disruption of these interactions is deleterious for the Ash2L/DPY-30 complex formation in vitro and in erythroid cells. Interestingly, close inspection of the Ash2L/DPY-30 heterotrimer reveals a large positive fourier map located on the surface of the complex structurally analogous to a lipid. Binding assays show that the Ash2L/DPY-30 complex binds to anionic lipids in vitro with a preference for cardiolipin, a phospholipid found in mitochondrial membranes. Altogether, these results show that hydrophobic interactions are pitoval for the formation of the Ash2L/DPY-30 complex and that lipids may play a role in epigenetic signaling in modulating histone H3K4 methylation.

 

Structural Investigations of Supercontracted Spider Dragline Silk


Justine Dionne1, Thierry Lefèvre1, Michèle Auger1
1Université Laval

Spider dragline silk is a semicrystalline polymer of great interest in materials science due to its unique mechanical properties and biocompatibility. Spun from the major ampullate glands, this type of silk possesses an exceptional combination of elasticity, strength and toughness. At the molecular level, spider silk is composed of fibrous proteins organized into crystalline nanodomains embedded in an amorphous matrix.

 

In the presence of liquid water or high humidity, the amorphous phase is plasticized and its hydrogen bonding network disrupted, which results in the shrinking of the fiber up to 50% of its initial length. This phenomenon, known as supercontraction, is triggered by the entropic folding of the polypeptide chains resulting from the water-induced increase in chain mobility.

 

However, disagreements exist in the literature as to whether a change in the secondary structure of the protein occurs or not during supercontraction. Furthermore, although it has already been established that supercontraction induces a disorientation of the molecular units, this effect has not been quantified yet. Therefore, we will investigate and quantify these variations by Raman spectromicroscopy, a particularly useful technique to probe silk molecular structure and orientation. Furthermore, the magnitude of supercontraction will be examined for the golden orb-web spider Nephila clavipes.

 

Structure prediction of a conserved membrane peptide from Lactococcus lactis phages from molecular dynamics simulations


Christine J. LeBlanc1,2,3, Sylvain Moineau1,3, Patrick Lagüe1,2,3, Stéphane Gagné1,2,3
1PROTEO 2IBIS 3Département de biochimie, de microbiologie et de bioinformatique, Université Laval

Lactococcus lactis phages 936-type are the most virulent phages in the dairy industry. This study is focused on a small conserved peptide that might play an important role in this process.
53 of the 56 genomes of Lactococcus lactis 936-type phages currently in NCBI have a highly conserved peptide of an average length of 39 amino acids.
It was predicted with high confidence to be an α-helical transmembrane (TM) protein by TM-predictors such as TOPCONS and TMHMM 2..
It is annotated as an uncharacterized protein (no function reported yet).
There is no known 3D structure for this peptide or its homologs in the Protein Data Bank (PDB).
The consensus sequence corresponds to orf33 of the phi7 genome.
The objective is to carry a structural characterization for orf33 phi7 from molecular dynamics simulations, and to gain insights into the potential function of this conserved peptide.

Structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes


Sophie Gobeil1, Maximilian Ebert1, Jaeok Park2, Donald Gagné3, Christopher Clouthier1, Jürgen Pleiss4, Nicolas Doucet3, Albert Berghuis2, Joelle Pelletier1
1Université de Montréal 2McGill University 3INRS-Institut Armand-Frappier 4Université de Stuttgart

Our understanding of the contribution of protein dynamics to function is still emergent. In a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? Using high resolution crystal structures, NMR relaxation dispersion and µs molecular dynamics simulations, we compare two naturally evolved homologous class A β-lactamases, TEM-1 and PSE-4 which share a high degree of structural and functional conservation. We observed a conservation of dynamics on a catalytically relevant timescale. This is consistent with dynamics being an evolutionarily conserved feature. However, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. The laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. This tolerance of β-lactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins.

Study of the interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact gram+ and gram - bacteria by solid-state NMR


Marwa Laadhari1, Alexandre Arnold1, Isabelle Marcotte1
1UQAM

Solid-state nuclear magnetic resonance (SS-NMR) is commonly used to probe the effect of antimicrobial agents on bacterial membranes, generally with model membranes. Ideally, the interaction of molecules with membranes should be studied in vivo considering the membrane complexity. The objective of this work was to investigate the interaction of two antimicrobial peptides (AMPs) with intact Escherichia coli and Bacillus subtilis using deuterium SS-NMR. More specifically, we studied caerin 1.1 and aurein 1.2 isolated from the skin of Australian tree frogs. First, we have determined the minimal inhibitory concentration (MIC) values of both peptides for the E. coli and B. subtilis, which was about 100 µg/mL and 30 µg/mL, respectively. Then, we have successfully established a protocol to deuterate the membrane phospholipids of non-mutated E. coli and B. subtilis using deuterated palmitic acid. 2H NMR spectra combined to spectral moment analysis support the interaction of the two AMPs with the hydrophobic core of the bacterial membranes. Indeed, the presence of peptides decreases the order of the lipid acyl chains of both bacteria but at different concentration of peptides. The Lipoteichoic acid (LTA) present in the peptidoglycan seems to interact with peptides and decrease their concentration on the membrane surface. Our results reveals the strong dependence of action mechanism to the cellular environment and the importance of studying the intact bacteria. These results are consistent with previous studies on model membranes, and are promising for the study of interactions between the antimicrobial peptides and all the membrane components by two-dimensional 13C-13C NMR.

STUDY OF THE STRUCTURE AND FUNCTION OF THREE DROSOPHILA MELANOGASTER HSP22 ARGININE MUTANTS


Afrooz Dabbaghizadeh1, Geneviève Morrow1,2, Robert Tanguay3
1Laval university 2Laval university 3Laval University

Introduction: The small heat shock protein (sHSP) family is ubiquitous and is composed of proteins sharing the alpha-crystallin domain (ACD) and having molecular masses in the range of 12 to 43 kDa. As molecular chaperones, they protect cells from different kinds of stress. A R120G mutation of HspB5 located in the ACD is genetically linked to desmin-related myopathy and results in high molecular weight oligomers and lost of chaperone-like function. Similar observations have been made for the corresponding mutation in HspB4 (R116C), which is generally linked to congenital cataracts. These arginines lies in the most highly conserved region of sHSPs.

Objective: In order to obtain inactive mutants of Drosophila melanogaster DmHsp22, three different arginine mutants located in the ACD were obtained and characterized.

Method: Blue native electrophoresis (BN-PAGE) and size exclusion chromatography (SEC) were used to determine the structure of DmHsp22 and mutants. Chaperone like activity was done to analyze their ability to prevent heat-induced aggregation of substrates in vitro.

Results: The double mutant (R109/110G) displayed a different behavior comparatively to the wild-type DmHsp22 as revealed by SEC and BN-PAGE. Using single mutations revealed that the R110G mutation was responsible of the double mutant phenotype. In contrast to the R120G mutation of HspB5 and R116C mutation of HspB4, the R110G mutation of DmHsp22 resulted in smaller oligomers than the wild-type protein. Moreover, in vitro chaperone like activity using citrate synthase and malate dehydrogenase demonstrated that all three mutants are as efficient chaperones as wild-type DmHsp22 to prevent heat-induced aggregation of different substrates.

Conclusion: The R110G mutation decreases the size of DmHsp22 oligomers and does not affect its chaperone-like activity, which suggest that it is not the ortholog of the HspB5 R120G mutation as expected. While the mutation did not result in an inactive mutant of DmHsp22, it may prove useful in future experiments aimed at understanding the mechanism by which DmHsp22 interacts with its substrates.

Introduction:

 

 

 

Substrate recycling in secondary metabolism: a smart design in microbes


Marie-Ève Picard1,2,3,4, Yiguang Zhu5, Qingbo Zhang5, Julie Barma1,2,3,4, Xavier Murphy Després1,2,3,4, Xiangui Mei5, Liping Zhang5, Jean-Baptiste Duvignaud1,2,3,4, Manon Couture1,2,3,4, Weiming Zhu5, Rong Shi1,2,3,4, Changsheng Zhang5
1Département de biochimie, microbiologie et bio-informatique 2Université Laval 3PROTEO 4Institut de biologie intégrative et des systèmes 5South China Sea Institute of Oceanology, Chinese Academy of Sciences

In today’s world, the importance of recycling has become more prominent. Sustainable urbanism now promotes the use of recycled materials and favors methods that maximize energy savings. Nature itself has elegantly designed ways to maximize its efficiency and its use of resources.
 

Many important substrate salvaging or recycling pathways are commonly found in primary metabolism, such as for nucleosides, uracil and coenzyme B1. However, there are only rare examples of such mechanism in secondary metabolism. Substrate salvage allows the recycling of metabolites derived from regular cellular catabolism back to the general biosynthetic pathway. Herein, we provide the first biochemical and structural evidence for flavoenzyme-mediated substrate recycling in secondary metabolism.  

 

In this study, we report a shunt product recycling pathway in the biosynthesis of caerulomycin A, a 2,2-bipyridine-containing natural product under development as a potent novel immunosuppressive agent. This pathway is mediated by flavine-dependent oxidase CrmK. Biochemical characterizations demonstrate that CrmK plays an unexpectedly important role in caerulomycin A biosynthesis by catalyzing the conversion of an alcohol shunt product to an aldehyde and then to a carboxylate, thus recycling or salvaging the shunt product into the main pathway of caerulomycin A. Moreover, the crystal structures of CrmK in apo form and in complex with the substrate CRM P and site-directed mutagenesis studies provide insights into the catalytic mechanisms and the molecular basis of CrmK in shunt product recycling.

 

Natural product biosynthetic gene clusters (BGCs) are well known to often carry additional genes for resistance, transport, regulation, or carrying out natural protection group chemistry. Now the functional characterization of CrmK in caerulomycin biosynthesis adds a recycling mechanism that may operate in secondary metabolism. The majority of antibiotic BGCs carry genes with unknown or unassigned function, and this work indicates that a salvage/recycling role could be a hypothesis to test for some of such genes. With the exponential growth of characterization of BGCs for secondary metabolites, it was expected that more and more enzymes with a salvaging/recycling function would be found in the future and explored for the yield improvement of secondary metabolites.
 

Sulfur-Aromatic Interactions: Modeling Cysteine and Methionine Binding to Tyrosinate and Histidinium Ions


Esam Orabi1, Ann English1
1Concordia University

Noncovalent sulfur-aromatic interactions are believed to be common in proteins and small molecules. In proteins, the association of cysteine and methionine side chains with those of phenylalanine, tyrosine, tryptophan, and histidine are noted to contribute to protein function and stability. However, little is known about the structural and energetic properties of these interactions, especially those involving charged aromatics. Ab initio quantum mechanical calculations at the MP2(full)/6-311++G(d,p) level are performed in gas phase on complexes of hydrogen sulfide (H2S), methanethiol (MeSH), and dimethyl sulfide (Me2S) with imidazolium and 4-methylimidazolium cations and with phenolate and 4-methylphenolate anions as well as on complexes of H2O with these aromatic and sulfur molecules. The results reveal that imidazole protonation and phenol deprotonation increase the affinity of these aromatics toward the sulfur ligands. From the calculated geometries and binding energies of the stable conformers, we note that the sulfur ligands preferentially bind at the edge of the two aromatic cations and at the face of the anions. Potential energy curves for H2O, MeSH, and Me2S interacting with imidazolium and phenolate ions and for H2O interacting with MeSH and Me2S are used to calibrate non-polarizable force fields for the complexes. The strength of S-imidazolium and S-phenolate interactions in aqueous solution is investigated by calculating the potential of mean force between MeSH and Me2S and the two aromatic ions. The default, non-optimized, force field underestimates the gas-phase affinity of these complexes and predicts week affinity (–0.3 to –0.6 kcal/mol) in water. The optimized models yield binding affinities of –4.3 and –3.1 kcal/mol for the MeSH- and Me2S- imidazolium complexes and –2.9 and –2.1 kcal/mol for the corresponding phenolate complexes. Simulations reveal that the most stable complexes of both ions with the two S-ligands in water are characterized by mainly en face binding conformation. Besides understanding the strength and directionality of the studied sulfur-aromatic complexes, the optimized models are important for reliable molecular dynamics simulations of proteins.

SYNTHÈSE ET ACTIVITÉ IN VITRO D’INHIBITEURS D’UNE ECTONUCLÉOTIDE PYROPHOSPHATASE / PHOSPHODIESTERASE DE TYPE 1 (ENPP1)


Elsa Forcellini1, Elnur Elyar Shayhidin2, Marie-Chloé Boulanger2, Ablajan Mahmut2, Carole-Anne Lefebvre1, Sophie Boutin1, Xavier Barbeau3, Patrick Lagüe3, Patrick Mathieu2, Jean-François Paquin1
1Département de chimie, Faculté des sciences et de génie, Université Laval 2Département de chirurgie, Faculté de médecine, Université Laval 3Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval.

La calcification de la valve aortique (CVA) est l’un des plus importants types de maladies cardiovasculaires. Il s’agit notamment, de la condition la plus fréquente de valvulopathie cardiaque aux États-Unis et en Europe de l’Ouest.

 

Le seul traitement disponible actuellement est le remplacement chirurgical de la valve aortique, et ce, uniquement pour les patients ayant atteint un stade sévère de la maladie.(1) Par conséquent, le développement d'un traitement pharmaceutique qui empêcherait la progression de la CVA, et par le fait même écarterait la nécessité d'une chirurgie, représenterait une avancée majeure.

 

Des études récentes, faites dans le laboratoire du Dr Patrick Mathieu (Faculté de médecine, U. Laval), ont montré qu’une enzyme régulant les niveaux de pyrophosphate inorganique extracellulaire était impliquée dans la maladie. En effet, une augmentation de l'expression et de l'activité d'une ectonucléotide pyrophosphatase/phosphodiestérase-1 (ENPP-1) favorise le processus de minéralisation dans la valve aortique. Enfin, l’inhibition de cette enzyme représente un enjeu majeur, d’où la naissance de mon projet dans le design et la synthèse d’inhibiteurs. Il sera donc présenté la synthèse de potentiels inhibiteurs ainsi que leur activité in vitro.(2)

 

(1) Côté, N.; El Husseini, D.; Pépin, A.; Guauque-Olarte, S.; Ducharme, V.; Bouchard-Cannon, P.; Audet, A.; Fournier, D.; Gaudreault, N.; Derbali, H.; McKee, M. D.; Simard, C.; Després, J.-P.; Pibarot, P.; Bossé, Y.; Mathieu, P. J. Mol. Cell. Cardiol. 2012, 52, 1191. 

(2) Shayhidin, E. E.; Forcellini, E.; Boulanger, M.-C.; Mahmut, A.; Dautrey, S.; Barbeau, X.; Lagüe, P.; Sévigny, J.; Paquin, J.-F.; Mathieu, P. Br. J. Pharmacol. 2015, 172, 4189.

The BDKRB2 Gene Encodes Two Distinct Proteins: The Bradykinin B2 Receptor (B2R) and a Regulator of the B2R Signalling


Maxime Gagnon1, Julie Motard1, Jean-François Jacques1, Sondos Samandi1, Fernand Gobeil1, Xavier Roucou1
1Université de Sherbrooke

Functional, proteomic and ribosome profiling approaches in eukaryotes have concurrently demonstrated the translation of alternative open reading frames (AltORFs) in addition to annotated protein coding sequences (CDSs) present in the same mature mRNAs. AltORFs may be present in UTRs or overlap the CDS in a different reading frame. Hence, translation may produce multiple proteins in addition to the annotated protein. For clarity, proteins translated from AltORFs are termed alternative proteins while proteins translated from annotated CDSs are termed reference proteins.

We are currently studying the multicoding potential of genes from the superfamily of G Protein Coupled Receptors (GPCRs). Using a bioinformatic pipeline developed in our laboratory, we predict 2 897 AltORFs in GPCR genes. The majority are localised in 3'UTRs (48.65%), while a significant portion are localised within the CDSs (38.33%). These proportions are reminiscent of the AltORFs in the overall transcriptome.

In order to assess the function of alternative proteins co-expressed with their reference GPCRs, we are studying the Bradykinin Receptor B2 (B2R), which has an AltORF (AltB2R) embedded within its CDS. Our results show that both proteins are expressed from B2R's coding sequence and they partially co-localise in the endoplasmic reticulum. Moreover, using bi-molecular fluorescence complementation and co-immunoprecipitation, we showed that they interact together. Furthermore, AltB2R is involved in the signaling pathway of B2R. Indeed, AltB2R overexpression accelerates and enhances MAPK signaling after stimulation with either Bradykinin or RMP-7, a B2R selective synthetic agonist.

These evidence indicate that AltB2R is a novel regulator of B2R's function. We will next investigate whether the regulation of GPCRs' activity by alternative proteins co-expressed from the same genes is a general rule.

The binding affinity of StarD6 with different ligands


Patrick Delattre1, Danny Létourneau1, Jean-Guy LeHoux1, Pierre Lavigne2
1Université de Sherbrooke 2Unibersité de Sherbrooke

Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain proteins are proteins responsible for the transfer of lipids between cell membranes. More specifically, StarD6 is responsible for the transport of steroid hormones, such as progesterone and testosterone. The study of these transport molecules could help us understand different mechanism involved in hormone-dependant cancer, such as prostate and breast cancer. In this study, we investigated the affinity of different molecules to determine the preferred ligand of StarD6. We used circular dichroism to follow the thermal denaturation of the protein in the presence of different steroid hormones. We were then able to calculate the binding affinity of these different ligands. Furthermore, we took N15-HSQC of StarD6 with some of the most interesting ligands, testosterone and progesterone. We finally performed docking analyses to elucidate the key residues of the binding site to better understand StarD6 specificity for certain hormones. This kind of study coupled with further analysis of other SART protein will give us insight into the complex signaling mechanism based upon steroid hormone response.

The TagR as a New and Versatile Fusion Protein Tool to Improve Solubility and Purification of Proteins


Line Cantin1,2, Christian Salesse1,2
1Université Laval 2CUO–Recherche, Centre de recherche du CHU de Québec and Département d’ophtalmologie, Faculté de médecine, and Regroupement stratégique PROTEO

It is very difficult to purify proteins using their intrinsic properties. The use of fusion tags is thus widely spread to facilitate protein purification. However, the expression, solubility and purification of recombinant proteins are still challenging in spite of the array of available fusion tags on the market. These problems can often be solved by changing the expression system, the type of host cell or by changing the type of fusion tag. Many fusion tags are available and new tags or new tagging procedures are continously produced. They can be located at either the N- or C-terminus of the protein of interest. The choice of the most appropriate tag depends on the properties of the protein of interest. The most commonly used small affinity tag is the polyhistidine (PolyHis) tag. However, this tag does not allow to improve the solubility of the proteins of interest. The most widely used protein tag is Glutathione S-transferase (GST), which can thus be considered as the « Gold Standard ». In the present poster, data are presented on a new protein tag to demonstrate its usefulness in improving the solubility and purification of proteins of interest. This new tag has been called TagR. The properties of TagR to improve solubility and purification of fusion protein partners have thus been compared with those of the GST tag. This comparaison has been achieved using two proteins of interest which are difficult to solubilize: truncated Lecithin Retinol Acyltransferase (tLRAT) and Retinitis Pigmentosa 2 (RP2). We have obtained caparable levels of expression for TagR and GST in fusion with tLRAT or RP2. However the solubility of tLRAT or RP2 is markedly increased when using TagR as a fusion partner compared to GST. The TagR thus provides a much larger solubility than GST for these particular proteins of interest. Highly purified TagR-RP2 and TagR-tLRAT has been achieved using a single step chromatography with a hydrophobic column. Moreover, quantitative cleavage of TagR has been accomplished by proteolysis with thrombin. RP2 and tLRAT free of their TagR fusion partner have then been purified using the same chromatography column. In addition, the TagR fusion proteins can be purified in the presence of strong detergents, such as sodium dodecyl sulfate (SDS), which is not possible with GST. The TagR thus appears as a new, attractive technology to improve the solubility of proteins of interest. Furthermore, it allows purification of its fusion partner and it can be easily removed by proteolytic cleavage. This technology thus represents an interesting additional tool on the market to purify proteins which are difficult to solubilize.

Une approche de biologie des systèmes appliquée au domaine acéricole mène à l’élaboration d’un nouvel outil pour le contrôle de la qualité de la sève d’érable


Marie Filteau1,2, Christian Landry1,2
1Laval University 2Université Laval

La composition chimique de la sève d’érable est non seulement complexe, mais varie au cours de la période de récolte. Parallèlement, les microorganismes qui se développent dans la sève jouent un rôle crucial sur le développement des saveurs. Nous avons donc utilisé une approche de biologie des systèmes de type “barseq”, traditionnellement utilisée en chimiogénétique et qui utilise la levure comme rapporteur biologique, pour suivre la variation de la sève et du sirop d’érable récolté tout au long de la saison. Cette approche consiste à faire croitre en compétition des milliers de souches de levures comportant chacune la délétion d’un gène et à mesurer individuellement leur aptitude de croissance par séquençage grâce à un code barre intégré dans leur génome. La méthode révèle ainsi quelles sont les fonctions cellulaires importantes pour la croissance dans un environnement tel que la sève d’érable comparativement à un environnement contrôle synthétique. Cette approche a mis en évidence que la voie de dégradation métabolique de l’allantoine est nécessaire pour une croissance optimale dans la sève et le sirop, en particulier les gènes impliqués dans le transport et la dégradation de l’acide allantoique. Une analyse HPLC a confirmé que l’acide allantoique est bel et bien présent dans nos échantillons et constitue vraisemblablement la source majeure d’azote dans la sève et le sirop. Ce résultat comporte une importance écologique pour les microorganismes présents dans cet environnement où l’azote est présent en quantité limitante, mais également pour le procédé de transformation de la sève en sirop. En effet, l’acide allantoique est un uréide pouvant servir de substrat à la réaction de Maillard qui se produit lors de la cuisson de la sève en présence de sucres réducteurs. Nous avons observé une réactivité relativement haute, menant à la production de pigments et de saveurs empyreumatiques qui affectent la qualité du sirop d’érable. Considérant la double importance de cette molécule pour la croissance microbienne et le procédé de transformation, nous avons adapté un test colorimétrique simple permettant de doser qualitativement les uréides directement dans la sève concentrée à l’érablière. Puisque l’acide allantoique constitue une réponse métabolique de l’arbre qui augmente avec la progression de la saison et qui varie au niveau local, l’optimisation de cet outil permettra de mieux contrôler les paramètres d’entreposage de la sève et du concentré, ainsi que les paramètres de cuisson.

VESICULAR STOMATITIS VIRUS-BASED VESICLES USED FOR NUCLEIC ACIDS DELIVERY.


Mathias Mangion1,2,3, Alexandre Audy1,2,3, Igor Slivac1,2,3, Rénald Gilbert3,4, Jacques-P. Tremblay3,5, Bruno Gaillet1,2,3
1université Laval 2PROTEO 3ThéCell 4Human Health Therapeutics Portfolio, National Research Council 5Laboratory of Human Genetics Unit CHUL-CHUQ

Gene delivery methods are essential to understand fundamental cellular mechanisms and to develop new therapies. Recombinant viruses are efficient to transfer nucleic acids but their safety is a concern. In addition, some commercial transfection reagents (including lipids and cationic polymers) and electroporation methods are cytotoxic. Interestingly, the sole expression of G envelope protein of the vesicular stomatitis virus (VSV) in mammalian cells can lead to the formation of VSV-G pseudotyped vesicles (V-VSV-G). In presence of polybrene, these vesicles are able to transfer plasmids in animal cells. Unfortunately, the production of V-VSV-G and their use for nucleic acids delivery is poorly documented. Here we propose to improve this promising method of transfection. At first we developed a V-VSV-G production process by transient transfection of HEK-293 cells using polyethylenimine (PEI). Two modes of production were compared: cells cultivated in adherence in T-flask and in suspension attached on micro-carriers. Also we demonstrated that the quantity of vesicles produced depends on the VSV-G sequence used. Then, several parameters potentially involved in the formation and the transfer efficiency of V-VSV-G/DNA complex were studied: polybrene concentration, order of addition of mix transfection components, incubation time of the complexes, medium of transfection, etc. Stability studies also demonstrated that V-VSV-G are robust particles: DNA transfer capacity of V-VSV-G is efficient after 10 freeze/thaw cycles and V-VSV-G can be stored for long term at +4 °C, -20 °C and -80 °C. Finally, V-VSV-G/DNA ratio was optimized for three different cell types. Transfection efficiency of 70 % and 55 % were obtained for HEK-293 and HeLa cells respectively, with 1 µg of V-VSV-G and 0.4 µg of DNA. Transfection of refractory cells such as human myoblasts, reached 25 % with 5 µg of V-VSV-G and 0.8 µg of DNA. V-VSV-G can also deliver large plasmids (18 kb). Furthermore, no cytotoxicity was observed in cells transfected with these complexes. Presently, the potential of V-VSV-G to transfer siRNA is investigated. In conclusion, V-VSV-G is a powerful tool for nucleic acids delivery which could be useful for several applications oriented toward cell and gene therapies.