Le but de ce projet était d'identifier de manière méthodique et standardisée les partenaires interagissant avec la protéine dystrophine dans les cellules musculaires squelettiques humaines différenciées et découvrir de nouveaux rôles de la dystrophine. Des cellules immortalisées ont été obtenue en sur-exprimant de manière stable hTERT / CDK4. Nous avons réalisé une analyse transcriptomique comparant des lignées immortalisées avec leurs populations primaires correspondantes, à l’état de prolifération et de différentiation. Nous avons constaté que l'immortalisation n'a pas d'effet mesurable sur le programme myogénique ou sur tout autre processus cellulaires, et qu'elle avait un effet protecteur contre le processus de sénescence. Les lignées de cellules musculaires humaines constituent donc de bon model in vitro pour l’étude de l’interactome de la dystrophine. Nous avons déterminé l’interactome de la dystrophine en utilisant l’approche proteomique ‘QUICK’. Nous avons identifié 18 nouveaux partenaires directs de la dystrophine, partenaires étant impliqués dans le transport vésiculaire ou étant des protéines d'adhésion. Ces résultats renforcent les données précédemment publiées suggérant un lien entre la dystrophine et le trafic vésiculaire, ainsi que dystrophine et adhesion cellulaire. Ces nouveaux partenaires ont été ajoutés à l’interactome de la dystrophine, interactome accessible sur le Web: sys-myo.rhcloud.com/dystrophin-interactome. Ce site web est dédié à être un outil facile d’utilisation permettant d’explorer et de visualiser l’interactome de la dystrophine du muscle squelettique. / The aim of this project was to systematically identify new interaction partners of the dystrophin protein within differentiated human skeletal muscle cells in order to uncover new roles in which dystrophin is involved, and to better understand how the global interactome is affected by the absence of dystrophin. hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research however, disruption of the cell cycle has the potential to affect many other cellular processes to which it also linked. A transcriptome-wide analysis of healthy and diseased lines comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states testing their myogenic character by comparison with non-myogenic cells found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the senescence. In this context the human muscle cell lines are a good in vitro model to study the dystrophin interactome. We investigated dystrophin’s interactors using the high-sensitivity proteomics ‘QUICK’ approach. We identified 18 new physical interactors of dystrophin which displayed a high proportion of vesicle transport related proteins and adhesion proteins, strengthening the link between dystrophin and these roles. The proteins determined through previously published data together with the newly identified interactors were incorporated into a web-based data exploration tool: sys-myo.rhcloud.com/dystrophin-interactome, intended to provide an easily accessible and informative view of dystrophins interactions in skeletal muscle.

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The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24533号 / 医科博第147号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 篠原 隆司, 教授 松田 道行, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer.

Ein Schlüsselprozess in der Immunantwort ist die durch das proinflammatorische Zytokin Interferon-gamma (IFNg) induzierte transiente Akkumulation von neu synthetisierten defekten Proteinen, die durch Anknüpfen von Polymeren des Proteins Ubiquitin (Ub) post-translational modifiziert werden. Die Ubiquitinierung ist das Schlüsselsignal für den Abbau dieser Proteine. Die Abbauprodukte dienen unter anderem als Quelle für die Prozessierung von Antigenen. Um die frühe Immunantwort besser zu verstehen, wurden im Rahmen dieser Arbeit die Identität und Charakteristika dieser neu synthetisierten Proteine, sowie die Topologie ihrer post-translationalen Modifizierung durch Ub untersucht. Dazu wurde die massenspektrometrischen Analyse ubiquitinierter Proteine weiterentwickelt, indem die experimentellen Konditionen auf deren Analyse optimiert wurden. Insbesondere konnte gezeigt werden, dass die kombinierte Verdauung der Ub-Konjugate mittels zweier Peptidasen die Identifizierung der Proteinpeptide und der Indikatorpeptide für Ubiquitinierungsstellen entscheidend verbessert. Es wurde demonstriert, dass eine selektive Isotopenmarkierung der neu synthetisierten Proteine möglich ist. Mit Hilfe dieser Methode gelang es, Veränderungen der Ub Modifikationen bezüglich der Topologie sowie quantitative Unterschiede der ubiquitinierten Proteine aus humanen HeLa-Zellen in An- und Abwesenheit von IFNg zu identifizieren. Nach Induktion durch IFNg wurden drei polyubiquitinierte, drei mono- oder polyubiquitinierte und 111 potentiell ubiquitinierte Proteine identifiziert. Diese Proteine zeigten, dass keine generelle Ubiquitinierungspräferenz für die durch IFNg verstärkt transkribierten Gene besteht. Die Ergebnisse dieser Arbeit erweitern das Verständnis der frühen zellulären Immunantwort und tragen zum Verständnis von Krebs- und Autoimmunerkrankungen sowie chronischen Entzündungsprozessen bei. Möglicherweise bilden sie die Grundlage für die Weiterentwicklung entsprechender Therapien. / A key process within the immune response of organisms is the transient accumulation of newly synthesized defective proteins affected by the proinflammatory cytokine interferon-gamma(IFNg). These proteins are post-translationally modified by attachment of polymers of the protein ubiquitin (Ub), which represents the key signal for targeting them to degradation. The resulting peptides can serve as sources for antigen processing. In order to get an insight into the details of the cellular early immune response, the identity and characteristics of the newly synthesized proteins and the topology of their post-translational modification with Ub were investigated. An essential progress of the mass spectrometric approach was achieved by optimizing the experimental conditions with regard to the requirements of the analysis of the targeted proteins. In particular, it could be shown that a combined digestion of Ub-conjugates with two peptidases leads to an improved detection of protein peptides and indicator peptides for ubiquitination sites. It was shown that a selective isotopic labeling of the newly synthesized proteins was possible. With this method, a decisive step forward was made in the understanding of changes of the Ub modifications with respect to the topology and in clarifying the quantitative differences between Ub-conjugates from IFNg treated and untreated human HeLa cells. Three IFNg induced polyubiquitinated proteins, three mono- or polyubiquitinated as well as 111 potential Ub-substrates could be identified. These proteins did not show any general ubiquitination preference for genes whose transcription is enhanced in presence of IFNg. The results obtained in this work help to broaden and refine the general picture of the early cellular immune response. They contribute to the knowledge on molecular processes of cancer, autoimmune diseases or chronic inflammation and, potentially, can give hints for the continued development of corresponding therapies.

Le génôme des cellules eucaryotes est condensé au sein d'une structure complexe hiérarchiquement organisée : la chromatine. La chromatine est composée d'ADN, de protéines histone et non-histone. Cette thèse a pour but d'étudier le rôle de la chromatine dans la réponse cellulaire aux dommages de l'ADN (DDR) par les méthodologies de génomique fonctionnelle et de protéomique. Nous avons tout d'abord analysé les modifications post-traductionnelles (PTM) des histones dans le cadre des pontages inter-brins (ou "Interstrand Crosslinks", ICL), type particulier de lésions de l'ADN, en choisissant le modèle de l'Anémie de Fanconi (FA). Ceci a été réalisé grâce aux techniques de protéomique quantitative SILAC (Stable Isotope Labeling of Amino acid during Cell culture) et de spectrométrie de masse (MS). Nous avons ainsi réussi à identifier et à quantifier de nombreuses PTMs dans les histones H3 et H4, et à démontrer que certaines de ces PTM sont dépendantes d'une voie fonctionnelle de la signalisation de FA. Nous avons également approfondi l'étude des DDR dans les cellules de FA par une approche de génomique fonctionnelle. Pour cela, nous avons analysé le profil l'expression d'enzymes associées à l'acétylation et à la méthylation des histones. Nos résultats suggèrent l'existence de corrélations entre le profil d'expression de ces enzymes et les PTMs des histones. Des études complémentaires sont nécessaires en vue de confirmer ces corrélations. Nous avons également comparé le transcriptome de deux lignées cellulaires de FA (mutée en FANCC et corrigée en FANCC) après induction de dommages à l'ADN. Afin de différencier les changements spécifiquement associés à la voie de signalisation de FA en réponse aux ICL de l'ADN des réponses plus générales aux dommages de l'ADN, nous avons inclus des cellules traitées par rayonnement ionisant. En réalisant une analyse d'interactions factorielles, nous avons pu identifier une réponse transcriptionnelle aux dommages de l'ADN nécessitant une voie fonctionnelle de la signalisation de FA. Nous avons également tenté de pallier aux limitations rencontrées dans l'analyse des PTMs des histones. En effet, les PTMs des histones que nous avons identifiées représentent l'ensemble des modifications, c'est-à-dire les PTMs concernant les histones se trouvant immédiatement à proximité du site du dommage et en relation directe avec celui-ci, et les PTMs se trouvant à distance du dommage et pouvant ne pas être en relation directe avec celui-ci. Les approches courantes pour identifier les PTMs se trouvant à des loci particuliers sont basées sur l'immunoprécipitation classique de la chromatine où l'utilisation de formaldéhyde altère les protéines, ce qui en rend impossible l'analyse par MS. Nous avons proposé une nouvelle méthodologie basée sur la biotinylation expérimentale d'histones situées à proximité d'une protéine particulière, suivie de la purification des nucléosomes contenant ces histones biotinylées. Contrairement àl'immunoprécipiatation classique de la chromatine, cette méthode n'induit pas d'altération des protéines, permettant ainsi de purifier les histones à partir d'un locus spécifique et d'analyser à grande échelle leurs PTMs par MS. Cette approche permet aussi de suivre dans le temps les PTMs d'une fraction des histones juste après leur biotinylation. Enfin, elle présente l'avantage de pouvoir étudier le profil des PTMs de différents états fonctionnels de la chromatine grâce à l'utilisation de variants d'histones. / In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.

Die Muscle-RING-finger (MuRF) Proteine sind E3-Ubiquitin-Ligasen, die im Muskelgewebe den Ubiquitin-Proteasom-System abhängigen Abbau von Proteinen vermitteln. MuRF1 wird in der Muskelatrophie verstärkt synthetisiert, was zu einem gesteigerten Proteinabbau und damit zum Verlust von Muskelmasse führt. Zudem sind Mäuse, denen MuRF1 fehlt vor Muskelatrophie geschützt. E3-Ubiquitin-Ligasen fungieren oftmals in Multiproteinkomplexen. Dies wurde für MuRF-Proteine bisher nicht gezeigt. Aufgrund dessen sollten neue MuRF-Multiproteinkomplex assoziierte Faktoren mittels Hefe-Zwei-Hybrid-System und SILAC AP-MS identifiziert und deren Einfluss auf die MuRF-Funktion charakterisiert werden. Es wurden sowohl neue als auch publizierte MuRF-Interaktionspartner (Iap) gefunden. Von den neu entdeckten MuRF-Iap wurde der Fokus auf WDR42A gelegt, da das Protein mit beiden Methoden identifiziert wurde und zudem funktionell hoch interessant ist. WDR42A homologe Proteine bilden zirkuläre β-Propeller Strukturen die Multiproteinkomplexe koordinieren. Die Interaktion zwischen MuRF-Proteinen und WDR42A wurde mittels Ko-IP Experimenten und Kolokalisationsstudien bestätigt. Cycloheximid-Abbau-Experimente deuten darauf hin, dass WDR42A kein MuRF1 Substrat-Protein ist. Da die MuRF-Proteine spezifisch im Muskel hergestellt werden, sollte überprüft werden ob WDR42A ebenfalls im Muskelgewebe synthetisiert wird. Es wurde gezeigt, dass WDR42A ubiquitär sowie im Muskelgewebe und in immortalisierten Muskelzellen hergestellt wird. Analog zu MuRF1 wird WDR42A in der Denervations-induzierten Skelettmuskelatrophie und der Muskelentwicklung verstärkt synthetisiert. Die Herunterregulation von WDR42A mittels siRNA in C2C12 Myotuben schützte diese Zellen vor dem Auftreten von Atrophie. Diese Ergebnisse zeigen, dass WDR42A wie MuRF1 an der Entstehung von Muskelatrophie beteiligt ist. Aufgrund der WDR42A Domänenstruktur wird vermutet, dass WDR42A als Scaffolding-Protein MuRF1-Multiproteinkomplexe reguliert. / The muscle-RING-finger (MuRF) proteins are E3 ubiquitin ligases which coordinate the ubiquitin-proteasome system dependent protein degradation in muscle tissue. MuRF1 is up-regulated under muscle atrophy conditions. This leads to enhanced proteolysis and thereby to loss of muscle mass and strength. Furthermore are MuRF1 knockout mice resistant to muscle atrophy. E3 ubiquitin ligases often operate in multi-protein complexes. This has not been shown for MuRF proteins. Therefore we used yeast-two-hybrid and SILAC-AP-MS to identify and subsequently characterize new MuRF multi-protein complex associated proteins. We found new and also published MuRF interaction partners (Iap) with both methods. Amongst the new Iap, we focused on WDR42A, because it was found with both techniques and his interesting functional potential. WDR42A exhibits seven consecutive arranged WD40-repeat domains. This domain arrangement leads in homologues proteins to the formation of seven-bladed β-propeller structures, which act as protein interaction platforms that coordinate multi-protein complexes. The protein interaction between the MuRFs and WDR42A was confirmed with Co-IP and co-localization experiments. Cycloheximide decay experiments indicated that WDR42A is not a MuRF1 substrate protein. The MuRF proteins are muscle specific, therefore we tested if WDR42A is also synthetized in muscle tissue. We could show that WDR42A is ubiquitously, but also in muscle tissue as well as in immortalized muscle cells produced. WDR42A is similar to MuRF1 up-regulated under denervation-induced skeletal muscle atrophy as well as in muscle development. Furthermore are C2C12 myotubes resistant to muscle atrophy after siRNA down-regulation of WDR42A. These results demonstrate that WDR42A is like MuRF1 important for the development of muscle atrophy. Due to the domain structure of WDR42A, we hypothesize that WDR42A regulates MuRF1 multi protein complexes as scaffolding protein.