Identification de nouveaux substrats des kinases Erk1/2 par une approche bio-informatique, pharmacologique et phosphoprotéomique

La phosphorylation est une modification post-traductionnelle omniprésente des protéines Cette modification est ajoutée et enlevée par l’activité enzymatique respective des protéines kinases et phosphatases. Les kinases Erk1/2 sont au cœur d’une voie de signalisation importante qui régule l’activité de protéines impliquées dans la traduction, le cycle cellulaire, le réarrangement du cytosquelette et la transcription. Ces kinases sont aussi impliquées dans le développement de l’organisme, le métabolisme du glucose, la réponse immunitaire et la mémoire. Différentes pathologies humaines comme le diabète, les maladies cardiovasculaires et principalement le cancer, sont associées à une perturbation de la phosphorylation sur les différents acteurs de cette voie. Considérant l’importance biologique et clinique de ces deux kinases, connaître l’étendue de leur activité enzymatique pourrait mener au développement de nouvelles thérapies pharmacologiques. Dans ce contexte, l’objectif principal de cette thèse était de mesurer l’influence de cette voie sur le phosphoprotéome et de découvrir de nouveaux substrats des kinases Erk1/2. Une étude phosphoprotéomique de cinétique d’inhibition pharmacologique de la voie de signalisation Erk1/2 a alors été entreprise. Le succès de cette étude était basé sur trois technologies clés, soit l’enrichissement des phosphopeptides avec le dioxyde de titane, la spectrométrie de masse haut débit et haute résolution, et le développement d’une plateforme bio-informatique nommée ProteoConnections. Cette plateforme permet d’organiser les données de protéomique, évaluer leur qualité, indiquer les changements d’abondance et accélérer l’interprétation des données. Une fonctionnalité distinctive de ProteoConnections est l’annotation des sites phosphorylés identifiés (kinases, domaines, structures, conservation, interactions protéiques phospho-dépendantes). Ces informations ont été essentielles à l’analyse des 9615 sites phosphorylés sur les 2108 protéines identifiées dans cette étude, soit le plus large ensemble rapporté chez le rat jusqu’à ce jour. L’analyse des domaines protéiques a révélé que les domaines impliqués dans les interactions avec les protéines, les acides nucléiques et les autres molécules sont les plus fréquemment phosphorylés et que les sites sont stratégiquement localisés pour affecter les interactions. Un algorithme a été implémenté pour trouver les substrats potentiels des kinases Erk1/2 à partir des sites identifiés selon leur motif de phosphorylation, leur cinétique de stimulation au sérum et l’inhibition pharmacologique de Mek1/2. Une liste de 157 substrats potentiels des kinases Erk1/2 a ainsi été obtenue. Parmi les substrats identifiés, douze ont déjà été rapportés et plusieurs autres ont des fonctions associées aux substrats déjà connus. Six substrats (Ddx47, Hmg20a, Junb, Map2k2, Numa1, Rras2) ont été confirmés par un essai kinase in vitro avec Erk1. Nos expériences d’immunofluorescence ont démontré que la phosphorylation de Hmg20a sur la sérine 105 par Erk1/2 affecte la localisation nucléocytoplasmique de cette protéine. Finalement, les phosphopeptides isomériques positionnels, soit des peptides avec la même séquence d’acides aminés mais phosphorylés à différentes positions, ont été étudiés avec deux nouveaux algorithmes. Cette étude a permis de déterminer leur fréquence dans un extrait enrichi en phosphopeptides et d’évaluer leur séparation par chromatographie liquide en phase inverse. Une stratégie analytique employant un des algorithmes a été développée pour réaliser une analyse de spectrométrie de masse ciblée afin de découvrir les isomères ayant été manqués par la méthode d’analyse conventionnelle.Phosphorylation is an omnipresent post-translational modification of proteins that regulates numerous cellular processes. This modification is controlled by the enzymatic activity of protein kinases and phosphatases. Erk1/2 kinases are central to an important signaling pathway that modulates translation, cell cycle, cytoskeleton rearrangement and transcription. They are also implicated in organism development, glucose metabolism, immune response and memory. Different human pathologies such as diabetes, cardiovascular diseases, and most importantly cancer, are associated with misregulation or mutations in members of this pathway. Considering the biological and clinical importance of those two kinases, discovering the extent of their enzymatic activity could favor the development of new pharmacological therapies. In this context, the principal objective of this thesis was to measure the influence of this pathway on the phosphoproteome and to discover new substrates of the Erk1/2 kinases. A phosphoproteomics study on the pharmacological inhibition kinetics of the Erk1/2 signaling pathway was initiated. The success of this study was based on three key technologies such as phosphopeptides enrichment with titanium dioxide, high-throughput and high-resolution mass spectrometry, and the development of ProteoConnections, a bioinformatics analysis platform. This platform is dedicated to organize proteomics data, evaluate data quality, report changes of abundance and accelerate data interpretation. A distinctive functionality of ProteoConnections is the annotation of phosphorylated sites (kinases, domains, structures, conservation, phospho-dependant protein interactions, etc.). This information was essential for the dataset analysis of 9615 phosphorylated sites identified on 2108 proteins during the study, which is, until now, the largest one reported for rat. Protein domain analysis revealed that domains implicated in proteins, nucleic acids and other molecules binding were the most frequently phosphorylated and that these sites are strategically located to affect the interactions. An algorithm was implemented to find Erk1/2 kinases potential substrates of identified sites using their phosphorylation motif, serum stimulation and Mek1/2 inhibition kinetic profile. A list of 157 potential Erk1/2 substrates was obtained. Twelve of them were previously reported and many more have functions associated to known substrates. Six substrates (Ddx47, Hmg20a, Junb, Map2k2, Numa1, and Rras2) were confirmed by in vitro kinase assays with Erk1. Our immunofluorescence experiments demonstrated that the phosphorylation of Hmg20a on serine 105 by Erk1/2 affects the nucleocytoplasmic localization of this protein. Finally, phosphopeptides positional isomers, peptides with the same amino acids sequence but phosphorylated at different positions, were studied with two new algorithms. This study allowed us to determine their frequency in an enriched phosphopeptide extract and to evaluate their separation by reverse-phase liquid chromatography. An analytical strategy that uses one of the algorithms was developed to do a targeted mass spectrometry analysis to discover the isomers that had been missed by the conventional method

"Une grande union pour tous les travailleurs" : la One Big Union au Québec (1919-1929)

La One Big Union (OBU) a marqué l’histoire du mouvement ouvrier au Canada. Associée au courant du syndicalisme industriel révolutionnaire, l’OBU s’est développée au Québec dès 1919, tout particulièrement à Montréal. Ce mémoire nous permettra mieux comprendre les stratégies d’implantation du syndicat dans la province jusqu’en 1929, en nous attardant sur ses objectifs et ses moyens d’action. Notre recherche mettra en lumière la culture politique de l’organisation et de ses militants, ses périodes d’avancées et de reculs, de même que ses rapports parfois conflictuels avec le reste du mouvement ouvrier

Head-to-tail cyclization of side chain-protected linear peptides to recapitulate genetically-encoded cyclized peptides

Genetically‐encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution‐phase macrocyclization of side chain‐protected linear peptides obtained from standard solid‐phase peptide synthesis. Cyclic peptide targets, including cyclo‐[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically‐encoded counterparts. Synthetic products were characterized by tandem high‐resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation‐induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost‐effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens

GPR61 anchoring of PKA consolidates GPCR and cAMP signaling

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions

The SysteMHC Atlas project.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts

"Neither Rome nor Moscow" : the itinerary of French-speaking libertarian communists activists in Montreal during the interwar period

Cette thèse retrace l’itinéraire collectif d’un groupe de militants communistes libertaires de langue française pendant l’entre-deux-guerres à Montréal rassemblés autour d’Albert Saint-Martin (1865-1947). Figure importante du mouvement ouvrier au Québec, l’itinéraire politique de Saint-Martin est multiforme : on le retrouve associé au Parti socialiste du Canada, à la One Big Union, au Parti socialiste (communiste), à la Ligue des sans travail, à l’Association révolutionnaire Spartakus, à l’Université ouvrière, à l’Association humanitaire, à la Ligue du Réveil féminin et à de nombreuses coopératives de consommation et de production. Saint-Martin est entouré de camarades provenant de divers horizons politiques. Notre thèse nous a permis d’identifier plus de 300 individus ayant pris part à des activités militantes à ses côtés. À travers l’analyse croisée de leurs parcours individuels, nous cherchons à mieux comprendre les modalités de leur engagement collectif avant, pendant et après la Première Guerre mondiale, leur représentation de la société idéale et les moyens d’y parvenir, la nature et la diversité de leurs liens de sociabilité, les territoires où se déploient leurs réseaux, la fréquence et les thèmes de leurs réunions de même que les symboles et les rituels qui y sont rattachés. Nous faisons l’hypothèse que celles et ceux qui participent aux activités de ce milieu partagent une même culture révolutionnaire articulée autour des notions de communisme, d’anticapitalisme, d’anticléricalisme et d’internationalisme, débouchant sur une critique des institutions autoritaires : l'État, l’Église catholique, la propriété privée, l’armée, le mariage, etc. Les stratégies d’émancipation individuelle et collective mises de l’avant par ces militants reposent sur l’éducation et l'action directe. C’est cet ensemble de principes théoriques, stratégiques et tactiques que nous regroupons sous le terme de communisme libertaire.This thesis retraces the collective itinerary of a group of French-speaking libertarian communist militants during the inter-war period in Montreal gathered around Albert Saint-Martin (1865-1947). An important figure in Quebec’s labour movement, Saint-Martin’s political itinerary is multifaceted: he is associated with the Socialist Party of Canada, the One Big Union, the Socialist Party (Communist), the Workers' University, the Spartakus Revolutionary Association, the Humanitarian Association, the Women's Awakening League, the Montreal Unemployed League and various consumers and production cooperatives. Saint-Martin is surrounded by comrades from various political backgrounds. Our thesis allowed us to identify more than 300 individuals who took part in militant activities at his side. Through the cross-analysis of their individual journeys, we seek to better understand the terms of their collective commitment, their representation of the ideal society and the means to achieve it, the nature and diversity of their sociability links, the territories in which their networks are deployed, the frequency and themes of their meetings, as well as the symbols and rituals attached to them. We hypothesize that those who participate in the activities of this milieu share a same revolutionary culture articulated around the notions of communism, anti-capitalism, anticlericalism and internationalism, leading to a critique of authoritarian institutions: the State, the Catholic Church, private property, army, marriage, etc. The strategies of individual and collective emancipation put forward by these activists are based on education and direct action. It is this set of theoretical, strategic and tactical principles that we group together under the term libertarian communism

"Ni Rome, ni Moscou" : l'itinéraire des militants communistes libertaires de langue française à Montréal pendant l'entre-deux-guerres

This thesis retraces the collective itinerary of a group of French-speaking libertarian communist militants during the inter-war period in Montreal gathered around Albert Saint-Martin (1865-1947). An important figure in Quebec’s labour movement, Saint-Martin’s political itinerary is multifaceted: he is associated with the Socialist Party of Canada, the One Big Union, the Socialist Party (Communist), the Workers' University, the Spartakus Revolutionary Association, the Humanitarian Association, the Women's Awakening League, the Montreal Unemployed League and various consumers and production cooperatives. Saint-Martin is surrounded by comrades from various political backgrounds. Our thesis allowed us to identify more than 300 individuals who took part in militant activities at his side. Through the cross-analysis of their individual journeys, we seek to better understand the terms of their collective commitment, their representation of the ideal society and the means to achieve it, the nature and diversity of their sociability links, the territories in which their networks are deployed, the frequency and themes of their meetings, as well as the symbols and rituals attached to them. We hypothesize that those who participate in the activities of this milieu share a same revolutionary culture articulated around the notions of communism, anti-capitalism, anticlericalism and internationalism, leading to a critique of authoritarian institutions: the State, the Catholic Church, private property, army, marriage, etc. The strategies of individual and collective emancipation put forward by these activists are based on education and direct action. It is this set of theoretical, strategic and tactical principles that we group together under the term libertarian communism.Cette thèse retrace l’itinéraire collectif d’un groupe de militants communistes libertaires de langue française pendant l’entre-deux-guerres à Montréal rassemblés autour d’Albert Saint-Martin (1865-1947). Figure importante du mouvement ouvrier au Québec, l’itinéraire politique de Saint-Martin est multiforme : on le retrouve associé au Parti socialiste du Canada, à la One Big Union, au Parti socialiste (communiste), à la Ligue des sans travail, à l’Association révolutionnaire Spartakus, à l’Université ouvrière, à l’Association humanitaire, à la Ligue du Réveil féminin et à de nombreuses coopératives de consommation et de production. Saint-Martin est entouré de camarades provenant de divers horizons politiques. Notre thèse nous a permis d’identifier plus de 300 individus ayant pris part à des activités militantes à ses côtés. À travers l’analyse croisée de leurs parcours individuels, nous cherchons à mieux comprendre les modalités de leur engagement collectif avant, pendant et après la Première Guerre mondiale, leur représentation de la société idéale et les moyens d’y parvenir, la nature et la diversité de leurs liens de sociabilité, les territoires où se déploient leurs réseaux, la fréquence et les thèmes de leurs réunions de même que les symboles et les rituels qui y sont rattachés. Nous faisons l’hypothèse que celles et ceux qui participent aux activités de ce milieu partagent une même culture révolutionnaire articulée autour des notions de communisme, d’anticapitalisme, d’anticléricalisme et d’internationalisme, débouchant sur une critique des institutions autoritaires : l'État, l’Église catholique, la propriété privée, l’armée, le mariage, etc. Les stratégies d’émancipation individuelle et collective mises de l’avant par ces militants reposent sur l’éducation et l'action directe. C’est cet ensemble de principes théoriques, stratégiques et tactiques que nous regroupons sous le terme de communisme libertaire

Occurrence and Detection of Phosphopeptide Isomers in Large-Scale Phosphoproteomics Experiments

The past decade has been marked by the emergence of selective affinity media and sensitive mass spectrometry instrumentation that facilitated large-scale phosphoproteome analyses and expanded the repertoire of protein phosphorylation. Despite these remarkable advances, the precise location of the phosphorylation site still represents a sizable challenge in view of the labile nature of the phosphoester bond and the presence of neighboring phosphorylatable residues within the same peptide. This difficulty is exacerbated by the combinatorial distribution of phosphorylated residues giving rise to different phosphopeptide isomers. These peptides have similar physicochemical properties, and their separation by LC is often problematic. Few studies have described the frequency and distribution of phosphoisomers in large-scale phosphoproteomics experiments, and no convenient informatics tools currently exist to facilitate their detection. To address this analytical challenge, we developed two algorithms to detect separated and co-eluting phosphopeptide isomers and target their subsequent identification using an inclusion list in LC–MS/MS experiments. Using these algorithms, we determined that the proportion of isomers present in phosphoproteomics studies from mouse, rat, and fly cell extracts represents 3–6% of all identified phosphopeptides. While conventional analysis can identify chromatographically separated phosphopeptides, targeted LC–MS/MS analyses using inclusion lists provided complementary identification and expanded the number of phosphopeptide isomers by at least 52%. Interestingly, these analyses revealed that the occurrence of phosphopeptides isomers can also correlate with the presence of extended phosphorylatable amino acids that can act as a “phosphorylation switch” to bind complementary domains such as those present in SR proteins and ribonucleoprotein complexes