The myelin basic protein (MBP) family arises from different transcription start sites of the Golli (gene of oligodendrocyte lineage) gene, with further variety generated by differential splicing. The “classic” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath, but also have multiple protein interactions. As an intrinsically disordered protein, MBP has proven to have complex structural and functional relationships with proteins in vitro including actin, tubulin, Ca2+-calmodulin, and multiple protein kinases. The investigations reported in this thesis were to further examine the multifunctionality, and protein:protein interactions of MBP with cytoskeletal and SRC homology 3 domain (SH3) proteins in cells using an oligodendrocyte (OLG) model system to better understand MBP’s contributions to membrane structure, formation, and elaboration in the developing OLG. A new function of MBP has been described showing that classic MBPs can modulate voltage operated calcium channels (VOCCs) by direct or indirect protein-protein interactions at the OLG cytoplasmic leaflet. These interactions contribute to global calcium homeostasis, and may play a complex developmental and spatiotemporal role in the regulation of oligodendrocyte precursor cell (OPC) migration and OLG differentiation. The importance of MBPs SH3 ligand binding domain within its central amino acid region was investigated with the protein-tyrosine kinase Fyn. Co-expression of MBP with a constitutively-active form of Fyn in OLGs resulted in membrane process elaboration, a phenomenon that was abolished by amino acid substitutions within MBP’s SH3-ligand domain. These results suggest that MBP’s SH3-ligand domain plays a key role, and may be required for proper membrane elaboration of developing OLGs. Lastly, interactions of MBP with the OLG cytoskeleton were investigated in OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it had increased co-localization with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. The results presented here advance our understanding regarding protein:protein interactions of MBP, and its multifunctionality in OLGs with regards to membrane formation and elaboration. / This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, G.H., RG121541). G.S.T.S. was a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18138号 / 医博第3858号 / 新制||医||1002(附属図書館) / 30996 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 坂田 隆造, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

SH3-Domänen sind Proteinmodule, die über die Erkennung prolinreicher Peptidsequenzen Protein-Protein-Interaktionen in einer Vielzahl von zellulären Prozessen vermitteln. In der vorliegenden Arbeit wurde die Erkennung von Peptidliganden durch ausgewählte SH3–Domänen hinsichtlich Selektivität versus Promiskuität untersucht. Diese Arbeiten dienten als Vorversuche für einen kompletten proteomischen Ansatz aller SH3-Domänen aus S. cerevisiae. Für die vier ausgewählten SH3-Domänen, nämlich Myo5, Abp1, Yhr016 und Rvs167, wurden die Ligandenpräferenzen durch Interaktionsanalysen mit einem vollständigen Satz prolinreichen Peptidliganden, abgeleitet aus dem Hefeproteom ermittelt. Hierzu wurde ein Peptid–Array Ansatz gewählt. Mit der SPOT-Technologie wurden 2953 15mere Peptide als Array auf einem Celluloseträger Schritt für Schritt synthetisiert. Hierfür wurde zuerst die Art des Celluloseträgers festgelegt und anschließend überprüft, ob sich ein Array mehrmals in Interaktionsexperimenten verwenden lässt. Es zeigte sich, dass eine Regeneration der Arrays nicht möglich war, so dass für jedes Interaktionsexperiment ein neues Peptidarray synthetisiert werden musste. Die Interaktionsstudien bestätigten die Bindung prolinreicher Sequenzmotive als eine universelle Eigenschaft der vier SH3-Domänen, wobei die einzelnen SH3–Domänen-Peptid-Interaktionen von zusätzlichen, spezifitätsdetermierenden Resten im Liganden abhängig waren. Darüber hinaus zeigten die Experimente deutliche Unterschiede hinsichtlich Selektivität und Promiskuität im Erkennungsverhalten der vier Domänen. Die Domänen konnten nach steigender Selektivität an Hand der Bestimmung von Grenzwerten und Integralen geordnet werden, nämlich Myo5 < Abp1 < Yhr016 < Rvs167. Ein gemeinsamer Ligand für alle vier Domänen konnte nicht identifiziert werden. Dagegen konnten gemeinsame Liganden für die Domänenpaare Myo5/Abp1 und Yhr016/Rvs167 bestimmt werden. Die Bindungspräferenzen zweier ausgewählter Liganden, dem Myo5/Abp1 Liganden ERPKRRAPPPAPKKP und dem Yhr016/Rvs167 Liganden VQQDSLPKLPFRSWG, wurden mit Hilfe von Substitutions- und Längenanalysen detailliert charakterisiert. Es zeigte sich deutlich, dass die selektiveren SH3-Domänen Yhr016 und Rvs167 einen klar definierten und kurzen Konsensus im Liganden binden, hingegen die mehr promiskuitiven Domänen Myo5 und Abp1 ein stärker variables und längeres Motiv im Liganden erkennen. Die Ergebnisse dieser Arbeit zeigten, dass die hier gewählte Methode geeignet ist, die proteomische Charakterisierung aller dreißig Hefe SH3-Domänen in Angriff zu nehmen. / SH3-Domains are protein modules, which recognize polyproline sequences. The Domains are involved in a variety of cellular processes. The recognition rules between peptide ligands and selected SH3-Domains were analysed in respect of selectivity versus promiscuity. This dissertation is a preliminary test for subsequent scanning the whole number of yeast SH3-Domains. The SH3-Domains Myo5, Abp1, Yhr016 and Rvs167 were selected and chosen for interaction with all yeast polyproline ligands. A peptide array was chosen and built up with SPOT-Synthesis. 2953 15-mere peptides were spotted as an array on a cellulose membrane and inspected for regeneration method. The regeneration failed and therefore four arrays were synthesised. The scanning experiments revealed an interaction between Ligand and Domain with a common polyproline core. However, these experiments demonstrated as well that specific residues were needed for operative ligand domain interaction. In addition, these four SH3-Domains exhibited clear differences in selectivity and promiscuity of recognition profiles. The integrals and the results after the setting of the threshold showed a distinct increase in selectivity. The arrangement was based on the increasing selectivity: 1. Myo5 2. Abp1 3. but cases of consistency were found between Abp1- and Myo5-SH3, as well as ligands between Yhr16- and Rvs167-SH3. The Myo5-/Abp1-Ligand ERPKRRAPPPAPKKP and the Yhr016-/Rvs167 ligand VQQDSLPKLPFRSWG were selected for characterisation of the peptide domain interaction through the analysis of substitution and length. The data showed that more selective Domains like Yhr016 and Rvs167 had a well-defined and short consensus, whereas more promiscuous Domains like Yhr016 and Rvs167 showed a mutable and longer consensus. In conclusion, the data showed that the SPOT-Synthesis is a suitable technique for a proteomic characterisation of the whole 30 yeast SH3-Domains.

Wie auch andere bakterielle Pathogene überträgt C. trachomatis Effektorproteine in die Wirtszelle, um deren Funktionen zu manipulieren. Das während der Invasion sekretierte Effektorprotein Tarp besitzt N-terminale SH2-Bindungsstellen und eine C-terminale SH3-Bindungsstelle für die Interaktion mit Wirtszellproteinen. Zur Bestimmung dieser Interaktionen wurden Protein-Microarrays mit nahezu alle humanen SH2- und SH3-Domänen verwendet. Zahlreiche neue Interaktionen wurden detektiert, wobei das Adaptorprotein SHC1 eine der stärksten SH2-abhängigen Interaktionen mit Tarp zeigte. Mittels Transkriptionsanalyse SHC1-abhängiger Genregulation während der Infektion konnten Gene identifiziert werden, welche an der Apoptose- und Zellwachstumskontrolle beteiligt sind. Infizierte Wirtszellen mit SHC1-Knockdown wiesen eine erhöhte Apoptoserate nach Stimulation mit TNF-alpha auf. Diese Ergebnisse offenbaren eine wichtige Rolle von SHC1 im Kontext des frühen, Chlamydien-induzierten Wirtszellüberlebens und deuten darauf hin, dass Tarp als vielseitige Signaltransduktionsplattform dient. Um Wirtszelllipide aufzunehmen, nutzt C. trachomatis Transportrouten der Wirtszelle und modifiziert Lipide bei der Aufnahme. Zur Bestimmung der Lipidzusammensetzung der Wirtszelle wurde diese mittels MALDI-TOF-Massenspektrometrie analysiert. Dabei hatte die Infektion den stärksten Einfluss auf Phosphatidylinoslitol (PI)- und Cardiolipin (CL)-Spezies. Des Weiteren konnten im Infektionsverlauf PI- und CL-Spezies mit einem Massenunterschied von 14 Da detektiert werden, was auf verzweigtkettige Fettsäurereste chlamydialen Ursprungs und eine Beteiligung der cytosolischen Phospholipase A2 (cPLA2) hindeutet. Entsprechend zeigten infizierte Zellen mit einem Knockdown von cPLA2 oder der Cardiolipinsynthase 1 eine signifikant reduzierte Bildung infektiöser Bakterien. Dies unterstreicht die Bedeutung von CL und einer funktionellen Nährstoffversorgung für die erfolgreiche Vermehrung von C. trachomatis. / Like many bacterial pathogens, C. trachomatis translocates effector proteins into the host cell to manipulate host cell functions. The early phase effector protein Tarp harbors N-terminal SH2 binding sites and a C-terminal SH3 binding site for the interaction with host cell proteins. To assess these interactions, protein microarrays comprising virtually all human SH2 and SH3 domains were used. Numerous novel interactions were discovered, while the adaptor protein SHC1 was among Tarp’s strongest SH2-dependent interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to TNF-alpha-induced apoptosis. These findings reveal a critical role for SHC1 in early Chlamydia-induced cell survival and suggest that Tarp functions as an important multivalent signaling hub. To acquire host-derived lipids, C. trachomatis hijacks cellular trafficking pathways and modifies lipids during the acquisition. To assess infection-dependent changes of the host cell lipid composition, cells were analyzed by MALDI-TOF mass spectrometry. Phosphatidylinositol (PI) and cardiolipin (CL) levels were most prominently influenced by C. trachomatis infection. Furthermore, PI and CL species with a 14 Da mass difference were detected during the course of infection, indicating the presence of Chlamydia-derived branched chain fatty acids and a role of cytosolic phospholipase A2 (cPLA2) in this process. Accordingly, infection of cPLA2 or cardiolipin synthase 1 knockdown cells resulted in a significantly reduced formation of infectious particles. These data demonstrate the importance of cardiolipin and a functional nutrient supply for the successful propagation of C. trachomatis.

No description available.

CD2AP is a member of the CD2AP/CIN85 family of adaptors and involved in several cellular processes, such as kidney podocyte development, actin mediated membrane trafficking and T cell activation. It contains three SH3 domains whose binding properties and interaction partners remain largely unexplored. The CD2AP SH3 interaction with the novel partner Rab5-activating GEF RIN3 was studied extensively by isothermal titration calorimetry (ITC), peptide scanning arrays, mutagenesis and X-ray crystallography. Mapping of the interaction regions showed that human RIN3 contains two binding sites for the CD2AP SH3 domains. From these studies, the CD2AP SH3 recognition motif P-x-P/A-x-x-R emerged. Two crystal structures (1.65 &Aring; and 1.2 &Aring;) of the SH3 1 and SH3-2 domains in complex with RIN3 epitopes 1 and 2 respectively revealed that these residues serve as anchoring points. With the aid of bioinformatics tools, this motif was used to conduct a peptide array-based screen for additional signalling partner candidates. One of the hits was the Arf-GAP ARAP1. ITC data indicate that the three SH3 domains differentially recognise three ARAP1 epitopes, with the first ARAP1 epitope binding to SH3-2 in the nanomolar range. A crystal structure (1.6 &Aring;) of the SH3-2 domain in complex with the first ARAP1 epitope implicates two additional anchoring residues that extend beyond the PPII helical region of the canonical motif. The CD2AP/ARAP1 interaction was confirmed in podocytes and cancer cells at the endogenous protein level. Even though RIN3 and ARAP1 are involved in membrane trafficking, a direct link to CD2AP had not been reported before. Other candidates from the peptide array analyses were also investigated by ITC. In conclusion, this study led to the elucidation of the CD2AP SH3-1 and SH3-2 domain binding signatures and the identification of putative novel binding partners for all three SH3 domains. Lastly, insight was gained into the binding preferences of CD2AP SH3-3.

Itch est une ligase de l’ubiquitine impliquée dans la reconnaissance et la dégradation des protéines par le protéasome. Itch contient trois sites phosphorylés par JNK et il a été démontré que la phosphorylation de ces résidus est nécessaire pour que Itch puisse reconnaître et ubiquityler les protéines c-Jun et JunB. Ces sites de phosphorylation se retrouvent dans le domaine PRD responsable des interactions de Itch avec les protéines à domaine SH3. Si la phosphorylation de Itch par JNK est importante pour réguler son activité avec c-Jun et JunB, on connaît peu de choses sur les interactions de Itch avec les protéines à domaine SH3 ainsi que l’implication de la phosphorylation dans leur régulation. Nous avons donc créé des mutants de Itch par mutagenèse dirigée où les sites de phosphorylation étaient remplacés par des alanines (mutant non phosphorylable) et où l’un des trois sites était remplacé par un acide aspartique (mutant constitutivement phosphorylé). Ces mutants sont utilisés dans des tests d’interaction et d’ubiquitylation, dans le but de déterminer l’impact de la phosphorylation de Itch dans la reconnaissance et l’ubiquitylation des protéines SH3. Nos résultats montrent que, contrairement au modèle proposé, la phosphorylation de Itch n’est pas essentielle à l’interaction de Itch avec l’endophiline, mais la phosphorylation de Itch module l’ubiquitylation ainsi que la dégradation de l’endophiline. La régulation de l’interaction de Itch avec ses substrats est donc différente selon le substrat. / Itch is an ubiquitin ligase involved in protein recognition and degradation by the proteasome. Itch has three phosphorylation sites targeted by JNK. These sites overlap a small proline rich domain responsible for Itch binding to SH3 domain proteins. Phosphorylation of Itch is important for Itch interaction with c-Jun and JunB. However, little is known about Itch interaction with SH3 proteins and the impact of phosphorylation on Itch ability to recognize and ubiquitinate SH3 proteins. We created a phosphomimic mutant of Itch and a mutant of Itch that cannot be phosphorylated by JNK. We tested these mutants in interaction and ubiquitination assays to determine their effect on Itch ability to bind and ubiquitinate Endophilin, an SH3 domain protein. Phosphorylation is not a prerequisite for Itch binding to Endophilin but phosphorylation of Itch modulates Itch ability to ubiquitinate Endophilin. Phosphorylation of Itch also modulates Endophilin fate, as phosphomimic Itch induces degradation of Endophilin compared to non-phosphorylated Itch. These results show that phosphorylation regulates Itch activity towards differents substrates in different ways.

Grâce aux récents progrès technologiques et à l'arrivée des séquenceurs de nouvelle génération, la quantité de données génomiques croît exponentiellement, alors que l'écart avec le nombre de structures résolues se creuse. Dans l'idéal, on aimerait pouvoir prédire par informatique la structure 3D de n'importe quelle protéine à partir de l'information de séquence seule, même en l'absence d'homologie. En effet, en dessous de 30% d'identité de séquence, les mesures de similarité de séquences ne sont plus suffisantes pour détecter l'homologie. Il faut donc mettre en place d'autres méthodes afin de venir à bout de cette zone d'ombre. Pour une structure donnée (et donc une fonction biologique), on ne dispose souvent que d'une petite quantité de séquences natives y correspondant, et parfois assez peu identiques. Il est alors difficile de construire un profil de recherche d'homologues pour retrouver ces séquences dont on ne connaîtrait pas la structure. Alors comment disposer de bases de données de séquences plus conséquentes pour chaque structure ? Ainsi, le design computationnel de protéine (CPD) tente de répondre à cette problématique : si l'on connaît un repliement, est-il possible de retrouver l'ensemble des séquences qui lui correspondent ? Le principe du CPD consiste à identifier parmi toutes les séquences compatibles avec le repliement d'intérêt, celles qui vont conférer à la protéine, la fonction désirée. La procédure générale est réalisée en deux étapes. La première consiste à calculer une matrice d'énergie contenant les énergies d'interactions entre toutes les paires de résidus de la protéine en autorisant successivement tous les types d'acides aminés dans toutes leurs conformations possibles. La seconde étape, ou "phase d'optimisation", consiste à explorer simultanément l'espace des séquences et des conformations afin de déterminer la combinaison optimale d'acides aminés étant donné le repliement de départ. Une première phase d'analyse de covariances de positions d'alignements de séquences théoriques a été menée. Nous avons ainsi pu mettre au point une méthode statistique pour repérer des ensembles de positions qui muteraient ensemble pour une structure donnée. La construction d'un profil avec toutes ces séquences théoriques moyennant trop l'information en acides aminés, nous avons pu améliorer la recherche d'homologues en construisant plusieurs profils à partir de groupes de séquences classées grâce à des motifs sur ces positions considérées comme covariantes. Pour mieux appréhender la qualité de ces prédictions de séquences théoriques, il fallait mettre en place un protocole de sélection des meilleurs protéines mutantes afin de les tester in vivo. Mais comment déterminer qu'une séquence théorique est meilleure qu'une autre? Sur quels critères se baser pour les caractériser? Aussi, un ensemble de descripteurs a été choisi, permettant de trier sur plusieurs critères les séquences théoriques pour n'en choisir qu'une vingtaine. Ensuite, ces protéines mutantes ont été soumises à des simulations de dynamique moléculaire afin d'évaluer leur stabilité théorique. Pour quelques protéines mutantes plus prometteuses, nous avons réalisé des expériences de sur-expression, de purification et de détermination structurale, tentant d'obtenir une validation biologique du modèle de CPD. Ces protocoles d'analyse et de validation semblent être de bons moyens permettront à notre équipe de tester d'autres protéines mutantes dans l'avenir. Ils pourront ainsi modifier des paramètres lors de la génération par CPD et s'appuyer sur des résultats expérimentaux pour les ajuster.

Src homology 3 (SH3) domains are one family of the peptide recognition modules (PRMs), which bind peptides rich in proline or positively charged residues in the target proteins, and play important assembly or regulatory functions in dynamic eukaryotic cellular processes, especially in signal transduction and endocytosis. SH3 domains are conserved from yeast to human, and improper SH3 domain mediated protein-protein interaction (PPI) leads to defects in cellular function and may even result in disease states. Since commonly used large-scale PPI mapping strategies employed full-length proteins or random protein fragments as screening probes and did not identify the particular PPIs mediated by the SH3 domains, I employed a combined experimental and computational strategy to address this problem. I used yeast two-hybrid (Y2H) as my major experimental tool, as well as individual SH3 domains as baits, to map SH3 domain mediated PPI networks, “SH3 domain interactomes”. One of my important contributions has been the improvement for Y2H technology. First, I generated a pair of Y2H host strains that improved the efficiency of high-throughput Y2H screening and validated their usage. These strains were employed in my own research and also were adopted by other researchers in their large-scale PPI network mapping projects. Second, in collaboration with Nicolas Thierry-Mieg, I developed a novel smart-pooling method, Shifted Transversal Design (STD) pooling, and validated its application in large-scale Y2H. STD pooling was proven to be superior among currently available methods for obtaining large-scale PPI maps with higher coverage, high sensitivity and high specificity. I mapped the SH3 domain interactomes for both budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans, which contain 27 and 84 SH3 domains, respectively. Comparison of these two SH3 interactomes revealed that the role of the SH3 domain is conserved at a functional but not a structural level, playing a major role in the assembly of an endocytosis network from yeast to worm. Moreover, the worm SH3 domains are additionally involved in metazoan-specific functions such as neurogenesis and vulval development. These results provide valuable insights for our understanding of two important evolutionary processes from single cellular eukaryotes to animals: the functional expansion of the SH3 domains into new cellular modules, as well as the conservation and evolution of some cellular modules at the molecular level, particularly the endocytosis module.

Src homology 3 (SH3) domains are one family of the peptide recognition modules (PRMs), which bind peptides rich in proline or positively charged residues in the target proteins, and play important assembly or regulatory functions in dynamic eukaryotic cellular processes, especially in signal transduction and endocytosis. SH3 domains are conserved from yeast to human, and improper SH3 domain mediated protein-protein interaction (PPI) leads to defects in cellular function and may even result in disease states. Since commonly used large-scale PPI mapping strategies employed full-length proteins or random protein fragments as screening probes and did not identify the particular PPIs mediated by the SH3 domains, I employed a combined experimental and computational strategy to address this problem. I used yeast two-hybrid (Y2H) as my major experimental tool, as well as individual SH3 domains as baits, to map SH3 domain mediated PPI networks, “SH3 domain interactomes”. One of my important contributions has been the improvement for Y2H technology. First, I generated a pair of Y2H host strains that improved the efficiency of high-throughput Y2H screening and validated their usage. These strains were employed in my own research and also were adopted by other researchers in their large-scale PPI network mapping projects. Second, in collaboration with Nicolas Thierry-Mieg, I developed a novel smart-pooling method, Shifted Transversal Design (STD) pooling, and validated its application in large-scale Y2H. STD pooling was proven to be superior among currently available methods for obtaining large-scale PPI maps with higher coverage, high sensitivity and high specificity. I mapped the SH3 domain interactomes for both budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans, which contain 27 and 84 SH3 domains, respectively. Comparison of these two SH3 interactomes revealed that the role of the SH3 domain is conserved at a functional but not a structural level, playing a major role in the assembly of an endocytosis network from yeast to worm. Moreover, the worm SH3 domains are additionally involved in metazoan-specific functions such as neurogenesis and vulval development. These results provide valuable insights for our understanding of two important evolutionary processes from single cellular eukaryotes to animals: the functional expansion of the SH3 domains into new cellular modules, as well as the conservation and evolution of some cellular modules at the molecular level, particularly the endocytosis module.