Η κινάση εστιακής προσκόλλησης (FAK) είναι μια πρωτεϊνική κινάση τυροσίνης (nRPTK) με κυτταροπλασματική κατανομή στην περιφέρεια του κυττάρου. Εντοπίζεται κυρίως σε περιοχές του κυττάρου γνωστές ως εστίες προσκόλλησης, με τις οποίες το κύτταρο προσκολλάται στην εξωκυτταρική ύλη. Συμμετέχει σε σημαντικές κυτταρικές διεργασίες όπως στην κυτταρική κίνηση και μετανάστευση, στον κυτταρικό πολλαπλασιασμό, στην κυτταρική επιβίωση και απόπτωση. Η FAK ενεργοποιείται από πολλά ερεθίσματα που μπορούν να επάγουν φωσφορυλίωση της σε αμινοξέα τυροσίνης (Υ397, Υ576, Υ577, Υ861, Υ925) όπως για παράδειγμα αυξητικοί παράγοντες, νευροπεπτίδια, παράγοντες που ενεργοποιούν υποδοχείς συνδεδεμένους με G-πρωτεΐνες και μηχανικά ερεθίσματα αλλά κυρίως μετά από τη διέγερση των ιντεγκρινικών υποδοχέων των κυττάρων. Ο ρόλος της FAK στην μεταγωγή μηνυμάτων, στο πλαίσιο της κυτταρικής επικοινωνίας είναι διττός: δρα ως κινάση φωσφορυλιώνοντας τα υποστρώματα της αλλά και ως πρωτεΐνη συνδετήρας (adaptor protein) δημιουργώντας πολυπρωτεϊνικά σηματοδοτικά σύμπλοκα. Δεδομένης, λοιπόν, της ιδιαίτερης φύσης του μορίου, η FAK θα μπορούσε να χαρακτηριστεί ως ενεργοποιούμενη πρωτεΐνη ικρίωμα (activable scaffold protein). Στην παρούσα εργασία μελετήσαμε την ενεργοποίηση της FAK κατά την κυτταροφαγία του βακτηρίου E. coli. Έτσι, χρησιμοποιήθηκαν φαρμακολογικοί αναστολείς για να ανιχνευθεί η συμμετοχή τελεστών της μεταγωγής σημάτων στην ενεργοποίηση της FAK. Επιπλέον, διερευνήθηκε με ανοσοκατακρημνίσεις η συμπλοκοποίηση της FAK με πρωτεΐνες-κλειδιά σημαντικών κυτταρικών μονοπατιών σε κύτταρα που βρίσκονταν σε εναιώρημα. / Cells respond to extracellular stimuli with the recruitment of multiple cytoskeletal and regulatory proteins, which form specialized complexes. These complexes are usually involved in the promotion of integrin-mediated signals to the nucleus. In this study, we investigated whether any signal transduction complexes are constitutively present, in Medfly hemocyte suspension, in the absence of external stimuli. Hemocytes from the 3rd instar larvae were isolated and protein crude extracts were prepared. In hemocyte lysates, the presence of FAK (focal adhesion kinase), Integrin β1 and the adaptor protein Paxillin was identified with immunoprecipitation and immunoblot analysis. The presence of these proteins was also confirmed with immunofluorescence microscopy, in attached hemocytes on glass slides. Co-immunoprecipitation with FAK and immunoblot analysis with anti-Paxillin, anti-tubulin, anti-actin and anti-ERK revealed the complex formation of FAK with Paxillin, Tubulin, Actin and ERK in hemocyte suspensions. The profiles of this complex and of each one of these proteins, separately, varies, during development and phagocytosis of E.coli. Consequently, it was demonstrated that FAK forms complex with cytoskeletal proteins like paxillin, tubulin, actin and signalling proteins such as ERK, in Medfly hemocytes, without the influence of any extracellular stimuli.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 167-195).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references.

For nearly five decades, the simple eukaryote Saccharomyces cerevisiae has been used as a model for understanding the eukaryotic cell cycle. One vein of this research has focused on understanding how chromosome structure is regulated in relation to the cell cycle. This work characterizes a new mechanism that modulates the chromatin organizing condensin complex, in hopes of furthering the understanding of chromosome structure regulation in eukaryotes. During mitosis, chromosomes are condensed to facilitate their segregation through a process mediated by the condensin complex. Upon interphase onset, condensation is reversed, allowing for efficient transcription and replication of chromosomes. This work demonstrates that Ycg1, the Cap-G subunit of budding yeast condensin, is cell-cycle regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. The cyclical expression of Ycg1 is unique amongst condensin subunits, and is established by a combination of cell cycle-regulated transcription and constitutive proteasomal degradation. Interestingly, when cyclical expression of Ycg1 is disrupted, condensin formation and chromosome association increases, and cells exhibit a delay in cell-cycle entry. These results demonstrate that Ycg1 levels limit condensin function, and suggest that regulating the expression of an individual condensin subunit helps to coordinate chromosome conformation with the cell cycle. These data, along with recent corroborating results in Drosophila melanogaster suggest that condensin regulation through limiting the expression of a single condensin subunit may be broadly conserved amongst eukaryotes.

Aperçu du contrôle de la dégradation des ARNm par les protéines YTHpendant la transition de la méiose à la mitose chez les levures.Le cycle cellulaire est contrôlé par des processus complexes et interconnectés. Un gène est transcrit en ARNm qui est traduit en protéines mais de nombreux processus de régulation travaillent pour contrôler chaque étape de ce processus apparemment simple. Parmi ces points de contrôle, la régulation post-transcriptionnelle est importante, et la formation d'un complexe protéine-ARN peut diriger le destin cellulaire. Parmi ces protéines de liaison à l'ARN, les protéines contenant des domaines YTH n’ont été découvertes qu’à la fin des années 90. Les protéines contenant des domaines YTH sont abondantes chez les eucaryotes et absentes chez les procaryotes. Elles constituent la majorité des protéines « readers » capables de reconnaître spécifiquement la modification m6A. L’Homme possède cinq protéines YTH, YTHDF1-3, YTHDC1,2 (Hazra, D., C. Chapat, et Graille, M. (2019). Destin de l'ARNm de m6A : enchaînés au rythme par les protéines contenant de la YTH. , 10 (1), 49.). Bien qu'il soit évident que ces protéines contrôlent le destin cellulaire, la fonction de chaque protéine et son réseau d’interaction restent à élucider. Chez les levures, une seule protéine YTH est présente: Pho92 chez Saccharomyces cerevisiae et Mmi1 chez Schizosaccharomyces pombe. Hormis le domaine YTH, il n'y a pas d'homologie de séquence entre ces deux protéines mais leur fonction cellulaire est similaire.Il est bien établi que Mmi1 est responsable de la dégradation des transcrits spécifiques de la méiose au cours de la croissance végétative des cellules chez la levure S. pombe. Mmi1 forme un complexe stable avec une petite protéine, Erh1 (complexe Erh1-Mmi1 ou EMC). Le complexe EMC peut physiquement interagir avec la sous-unité Not1 du complexe CCR4-Not et la recruter pour la dégradation des ARNm contenant des motifs DSR (déterminant de l'élimination sélective). L'action de Mmi1 est à son tour régulée par une protéine possédant un domaine RRM, Mei2. Au cours de la méiose, Mei2, avec l’aide d’un lncRNA meiRNA, séquestre Mmi1 dans un point nucléaire, le rendant inactif et assurant la continuité de la méiose. Ces trois protéines, Mmi1-Erh1-Mei2, jouent un rôle clé dans la transition de la mitose vers la méiose.Chez S. cerevisiae, Pho92 est impliquée dans la dégradation des transcrits de PHO4, contribuant à la voie du métabolisme du phosphate, pendant la privation en phosphate et participe également à la dégradation des ARNm contenant les marques épitranscriptomiques de N6-méthyladénosine (m6A). Comme pour S. pombe Mmi1, Pho92 recrute le complexe CCR4-Not via une interaction physique avec Not1.Au cours de ma thèse, j'ai tenté d'élucider le rôle de ces deux protéines du domaine YTH de deux organismes modèles, S. cerevisiae et S. pombe, dans la dégradation de l'ARNm et la régulation du cycle cellulaire par des approches biochimiques et structurales.Pho92 de S. cerevisiae interagit physiquement avec Not1 du complexe CCR4-Not, nous avons pu déterminer les limites des domaines impliqués dans cette interaction. L’interaction entre ces deux protéines a été étudiée par anisotropie de fluorescence. Le complexe protéique a été purifié avec succès et des essais de cristallisation sont en cours.Chez S. pombe, la structure de Mei2-RRM3 a été résolue avec et sans ARN. Les propriétés de liaison à l'ARN de Mei2-RRM3 ont été étudiées par ITC. La structure de Erh1 a également été résolue révélant une organisation en homodimere. Nous avons montré que la formation de cet homodimere est important pour la fonction biologique de Mmi1. Des essais de co-cristallisation ont été réalisés avec de l'ARN et les protéines Mmi1 et Mei2, mais sans succès et nous avons obtenu des cristaux de Mmi1. / Insights into the control of mRNA decay by YTH proteinsduring the transition from meiosis to mitosis in yeasts.Keywords: Epitranscriptomics, mRNA decay, meiosis, multi-protein complexes, YTH domainCell cycle is controlled by multi-layered processes. A gene is transcribed in mRNA which is translated in proteins but innumerable regulation processes are working to control every step of this apparently simple process. Among these regulatory check points, post-transcriptional regulation is an important one, where formation of a protein-RNA complex may direct the cellular fate. Among these RNA binding proteins, YTH domain proteins are most novel, discovered in late 90s. YTH domain proteins are abundant in eukaryotes and absent in prokaryotes. YTH domain proteins constitute the majority of reader proteins that can specifically identify m6A modification. Human beings have five YTH domain proteins YTHDF1-3, YTHDC1-2 (Hazra, D., Chapat, C., & Graille, M. (2019). m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins. Genes, 10(1), 49.). Although it is evident that these proteins are controlling cellular fate, the function of each protein and their network is yet to be elucidated. In yeast, there is only one YTH domain protein present: Pho92 in Saccharomyces cerevisiae and Mmi1 in Schizosaccharomyces pombe. Apart from the YTH domain there is no sequence homology between these two proteins but their cellular function is similar.It is well established that Mmi1 is responsible for degradation of meiosis specific transcripts during vegetative growth of the cell. Mmi1 forms a tight complex with a small protein, Erh1 (Erh1-Mmi1 complex or EMC). EMC can physically interact with Not1 of CCR4-Not complex and recruit it for degradation of DSR (determinant of selective removal) containing RNAs. The action of Mmi1 is in turn regulated by an RRM domain protein, Mei2. During meiosis, Mei2, along with a lncRNA meiRNA sequesters Mmi1 in a nuclear dot, rendering it inactive and ensuring smooth continuance of meiosis. These three proteins, Mmi1-Erh1-Mei2 play a key role in mitosis to meiosis switch.In S. cerevisiae, Pho92 is involved in the degradation of PHO4 transcripts contributing to phosphate metabolism pathway, during phosphate starvation and also participates in the degradation of mRNAs containing the N6-methyladenosine (m6A) epitranscriptomics marks. Similarly, to S. pombe Mmi1, Pho92 recruits CCR4-Not complex by physical interaction with Not1.During my PhD, I have tried to elucidate the role of these two YTH domain proteins from two model organisms, S. cerevisiae and S. pombe, in mRNA degradation and cell cycle regulation using biochemical and structural approaches.Pho92 of S. cerevisiae physically interacts with Not1 of CCR4-Not complex, we were able to determine the boundaries of this interaction. The interaction between these two proteins was studied by Fluorescence anisotropy. The protein complex was successfully purified and crystallization trials are ongoing.From S. pombe, structure of Mei2-RRM3 was solved with and without an RNA. RNA binding properties of Mei2-RRM3 was studied by ITC. The structure of Erh1 was also solved and we tried to elucidate its importance for biological function of Mmi1. A co-crystallization trial was performed with Mmi1-Mei2-RNA but it was unsuccessful and we ended up with Mmi1 crystals.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Recently, linear ubiquitin assembly complex (LUBAC)-mediated linear ubiquitination has come into focus due to its emerging role in activation of NF-κB in different biological contexts. However, the role of LUBAC in LMP1 signaling leading to NF-κB and interferon regulatory factor 7 (IRF7) activation has not been investigated. We show here that RNF31, the key component of LUBAC, interacts with LMP1 and IRF7 in Epstein-Barr virus (EBV)-transformed cells and that LUBAC stimulates linear ubiquitination of NEMO and IRF7. Consequently, LUBAC is required for LMP1 signaling to full activation of NF-κB but inhibits LMP1-stimulated IRF7 transcriptional activity. The protein levels of RNF31 and LMP1 are correlated in EBV-transformed cells. Knockdown of RNF31 in EBV-transformed IB4 cells by RNA interference negatively regulates the expression of the genes downstream of LMP1 signaling and results in a decrease of cell proliferation. These lines of evidence indicate that LUBAC-mediated linear ubiquitination plays crucial roles in regulating LMP1 signaling and functions. IMPORTANCE We show here that LUBAC-mediated linear ubiquitination is required for LMP1 activation of NF-κB but inhibits LMP1-mediated IRF7 activation. Our findings provide novel mechanisms underlying EBV-mediated oncogenesis and may have a broad impact on IRF7-mediated immune responses.

Ce travail de thèse repose sur le développement de méthodes en spectrométrie de masse native et mobilité ionique pour la caractérisation structurale de protéines d’intérêt thérapeutique et de complexes multiprotéiques. L’optimisation fine et conséquente de la préparation d’échantillon et des conditions analytiques ont permis la caractérisation de protéines membranaires solubilisées en milieu détergent, protéines hydrophobes habituellement réfractaires à l’analyse par MS. D’autre part, une nouvelle approche de mobilité ionique appelée Collision Induced Unfolding a été évaluée et mise en place au laboratoire. Elle a permis une caractérisation conformationnelle approfondie et originale de plusieurs formats d’anticorps monoclonaux thérapeutiques. Enfin, les techniques de MS native et de mobilité ionique ont été utilisées pour caractériser des complexes multiprotéiques d’hétérogénéité variable mettant ainsi en lumière leurs avantages et les progrès réalisés dans le domaine de la MS structurale. / This PhD work focuses on developments in native mass spectrometry and ion mobility methods for the structural characterization of therapeutic proteins and multiprotein complexes. First, careful optimizations of sample preparation and analytical conditions allowed the characterization of membrane proteins, which are hydrophobic proteins difficult to analyze by MS approaches in detergent environment. Then, a new ion mobility-based activation approach called Collision Induced Unfolding has been set up and evaluated. CIU allowed extensive and original conformational characterization of several therapeutic monoclonal antibody formats. Finally, native MS and ion mobility techniques were used for the characterization of heterogeneous multiprotein complexes depicting their benefit when combined to other biophysical techniques for the structural characterization of multiprotein complexes.

Recent studies suggest adipose tissue plays a critical role in regulating whole body energy homeostasis in both animals and humans. In particular, activating brown adipose tissue (BAT) activity is now appreciated as a potential therapeutic strategy against obesity and metabolic disease. However, the signaling circuits that coordinate nutrient uptake and BAT function are poorly understood. Here, I investigated the role of the nutrient-sensing mTOR signaling pathway in BAT by conditionally deleting Rictor, which encodes an essential component of mTOR Complex 2 (mTORC2) either in brown adipocyte precursors or mature brown adipocytes. In general, inhibiting BAT mTORC2 reduces glucose uptake and de novo lipogenesis pathways while increases lipid uptake and oxidation pathways indicating a switch in fuel utilization. Moreover, several key thermogenic factors (Ucp1, Pgc1α, and Irf4) are elevated in Rictor-deficient BAT, resulting in enhanced thermogenesis. Accordingly, mice with mTORC2 loss in BAT are protected from HFD-induced obesity and metabolic disease at thermoneutrality. In vitro culture experiments further suggest that mTORC2 cell-autonomously regulates the BAT thermogenic program, especially Ucp1 expression, which depends on FoxO1 activity. Mechanistically, mTORC2 appears to inhibit FoxO1 by facilitating its lysine-acetylation but not through the canonical AKT-mediated phosphorylation pathway. Finally, I also provide evidence that β-adrenergic signaling which normally triggers thermogenesis also induces FoxO1 deacetylation in BAT. Based on these data, I propose a model in which mTORC2 functions in BAT as a critical signaling hub for coordinating nutrient uptake, fuel utilization, and thermogenic gene expression. These data provide a foundation for future studies into the mTORC2-FoxO1 signaling axis in different metabolic tissues and physiological conditions.

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.