Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 105-106). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38818

The organization of two or more biological macromolecules into a functioning assembly is critical for many biological functions to occur. This phenomenon is the result of subtle interplay between complimentary structural and electrostatic factors. While a growing protein data bank of solved protein structures provides experimental evidence for studying the structural factors that stabilize protein-protein interface, there has been little advance in experimental determination of the electrostatic contributions. This lack of experimental investigation into protein electrostatics results in an inability to describe or predict how protein-protein complexes are arranged and stabilized. This problem is addressed in this dissertation by use of vibrational Stark effect (VSE) spectroscopy in which the spectral transitions of a vibrational probe are directly related to the strength and direction of the electric fields in the vicinity of the probe. The work presented here details an approach using VSE spectroscopy coupled with molecular dynamics simulation (MD) to interpret the role that electrostatics play in organizing the signaling protein Ras' interactions with its downstream effectors Raf and Ral guanosine dissociation simulator (RalGDS). Each chapter describes a specific set of experiments and MD simulations designed to understand the nature of protein-protein interactions. In Chapter 3, changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface was found. The areas of conserved Stark shifts are used to help describe electrostatic factors that stabilize the Ras-Ral interface. In Chapter 4, VSE is used to describe an electrostatic origin to the binding tilt between complexes formed between Ras and its two effectors Raf and Ral. There are three regions of conserved Stark effect shifts upon docking with WT Ras between the two effectors, indicating that the docked complexes conserve electrostatic fields, resulting in different binding orientation of otherwise structurally similar proteins. Chapter 5 details the use of MD simulation in correlation with VSE data for 18 mutants of the Ras at the oncogenic position 61 site. The combination of experimental and simulations support the hypothesis that position 61 on Ras is used to coordinate an active site water molecule during native guanosine triphosphate (GTP) hydrolysis. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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RAS is one of the most commonly mutated genes in human cancer; its aberrant activation drives tumor cell proliferation and survival. However, RAS mutations are rare in some cancers, including breast cancer, even though the Ras pathway is hyperactivated, suggesting that alternative mechanisms deregulate Ras signaling in these settings. The RasGAPs are negative regulators of Ras and, as such, are poised to function as tumor suppressors whose loss might contribute to Ras pathway hyperactivation in cancer. However, the RasGAPs remain an understudied family of genes whose role in cancer has not been fully explored. In this Dissertation I identify a previously uncharacterized RasGAP, RASAL2, as the newest tumor suppressor in this gene family.

Τα αρνητικά και θετικά κατά Gram βακτήρια E. coli και S. αureus αντίστοιχα αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων της C. capitata. Η πρόσδεση των βακτηρίων ενεργοποιεί τόσο τις β1 ιντεγκρίνες, όσο και σηματοδοτικά μονοπάτια που περιλαμβάνουν τα μόρια μεταγωγής σήματος Ras, FAK, Src και MAP κινάσες. Οι παραπάνω ενεργοποιήσεις, σε συνδυασμό με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης, καταλήγουν στην επαγωγή της έκκρισης μορίων απαραίτητων για την κυτταροφαγία των βακτηρίων που είναι και το τελικό αποτέλεσμα των παραπάνω διαδικασιών. Τα σφαιρίδια λάτεξ -αλλά και πιθανόν και άλλοι αβιοτικοί παράγοντες- παρότι δεν έχουν καμία προηγούμενη εξελικτική σχέση με τα αιμοκύτταρα, ως σύγχρονο προϊόν της ανθρώπινης γνώσης, αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων από άγνωστους μέχρις στιγμής υποδοχείς. Η κυτταροφαγία τους προωθείται μέσω ενεργοποίησης σηματοδοτικών μονοπατιών που περιλαμβάνουν την ενεργοποίηση των μορίων FAK, Src και MAP κινασών καθώς και με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης. Ο LPS αναγνωρίζεται και δεσμεύεται στην επιφάνεια των αιμοκυττάρων, ενεργοποιεί άγνωστους μέχρις στιγμής υποδοχείς και διαμέσου σηματοδοτικών μονοπατιών που περιλαμβάνουν τις Ras, ενεργοποιεί τις MAP κινάσες και το σύστημα της έκκρισης. Αν και ενεργοποιεί και τις τρεις MAP κινάσες, μόνο η ERK και η p38 απαιτούνται τόσο στη διαδικασία της έκκρισης, όσο και στη διαδικασία της ενδοκυττάρωσής του. Η FAK, αν και ενεργοποιείται από τον LPS, δεν εμπλέκεται στην διαδικασία της ενδοκυττάρωσής του. Τα παραπάνω δείχνουν ότι τα αιμοκύτταρα έχουν αναπτύξει διακριτούς μηχανισμούς για την κυτταροφαγία των παθογόνων, των μικρομορίων και των αβιοτικών παραγόντων, γεγονός που δείχνει την ικανότητα εξέλιξης των εντόμων έτσι ώστε να καλύπτουν τις ανάγκες της επιβίωσή τους. / Gram negative and positive bacteria E. coli and S. αureus respectively, are recognized and binded on C. capitata hemocyte surface. After binding, they activate β1 integrins and intracellular signalling pathways involving the kinases Ras, FAK, Src and MAP. This signal transduction, with the participation of the cytoskeleton of actin and tuboulin, leads to a regulated secretion that is a prerecuisite for phagocytosis. Latex beads and probably other abiotic factors, despite having no previous evolutionary relation to the hemocytes being a new product of human knowledge, are recognized and binded on hemocyte surface by so far unknown receptors. They activate intracellular signalling pathways that involves FAK, Src and MAP kinases and promote -with the participation of actin and tuboulin cytoskeleton- their phagocytosis. LPS is recognized and binded on hemocyte surface and activates so far unknown receptors and through unknown intracellular signalling pathways involving Ras, activates the MAP kinases and the regulated secretion. Although it activates all three MAPKs, only the ΕΡΚ and p38 are required not only for the secretion, but also for its internalization. Although FAK is activated by LPS, does not get involved in the process of its internalization. All the above mentioned results indicate that the hemocytes have developed distinct mechanisms for phagocytosis of pathogens, micromolecules and abiotic factors, a fact that underlines insects evolutionary adaptations, so that they can survive.

Normal tissue structure and function are maintained by a dynamic interaction between epithelial cells and the stroma consisting of fibroblasts, adipose, vasculature and resident immune cells, and a multitude of cytokines and growth factors. Stroma was usually studied in the background of the malignant lesion, only in recent years researchers have started considering its role before carcinogenic lesions appear. Recent studies have shown that stromal cells and their products can cause the transformation of adjacent cells through transient signaling during phenomena like adipogenesis and inflammation by secreting various cytokines and chemokines into the matrix which can lead to apoptosis resistance, proliferation, mutations etc. Research in the last few years has demonstrated a functional role for stroma in the initiation and progression of breast, colon and prostate carcinomas. In this study effect of adipogenesis and/or inflammation on prostaglandin biosynthesis is investigated and the effects that these prostaglandins can have on epithelial cells is highlighted. This work demonstrates that normal colonic fibroblasts CCD18Co can produce anti-tumorigenic and pro-tumorigenic prostaglandins during adipogenesis and that this signaling is mediated via COX-2 activation. Although deoxycholic acid (DCA), a secondary bile acid that is responsible for inflammation in the gastro-intestinal tract, induces COX-2 signaling in the fibroblasts the downstream signaling of prostaglandin synthases is suppressed. Adipogenesis also leads to an increased polyamine catabolism. Effects of the prostaglandins were studied on various epithelial colon cancer cell lines. It was seen that 15d-PGJ2 causes growth inhibition and apoptosis in all cell lines tested and it was demonstrated that an activated K-RAS suppressed this phenomena. It was also seen that 15d-PGJ2 treatment could induce MAPK signaling and that an activated K-RAS suppressed JNK activation via AKT and MKK4. In conclusion this work reports that colonic fibroblasts can produce anti-tumorigenic factors like 15d-PGJ2 which may then induce apoptosis in epithelial cancer cells. This would be suppressed by an activated K-RAS and at the same time 15d-PGJ2 mediated MAPK signaling could confer a growth advantage for these cells and thus aid in tumor progression.

Telomerase is a ribonucleoprotein that maintains telomere. It is activated in greater than 85% of human neoplasms. Traditionally, reactivation of telomerase during tumorigenesis was thought to be required solely to impart an indefinite lifespan. Recently, however, several studies have suggested that telomerase may contribute to tumorigenesis via an additional mechanism that is independent of its role in telomere lengthening. We sought to identify the region(s) of hTERT that contribute to this non-classical role of telomerase. We proposed to identify such regions by their ability to impart a tumorigenic phenotype in ALT cells transduced with activated Ras. Also, we attempted to develop methods to demonstrate that this role is not dependant of telomerase localizing to the telomere. The strategies employed and the progress gained toward each goal is presented in this thesis.

RasGRP1 is an intracellular signaling protein expressed in lymphocytes that is responsible for activating Ras GTPases. Positive regulation of RasGRP 1 requires translocation to cellular membranes where lipid-anchored Ras can be accessed. Plasma membrane localization of RasGRP 1 in response to antigen receptors requires both the Cl domain and the plasma-membrane targeting (PT) domain. The Cl domain binds to diacylglycerol (DAG) at membranes. The PT domain binds its putative ligand at the plasma membrane and is negatively regulated by an adjacent suppressor of PT (SuPT) domain. RasGRP1 also contains a pair of EF-hands, with Ca²⁺-binding capability, but with no known regulatory role. In DT4O cells, RasGRP1 translocates to the plasma membrane and activates the Ras ERK pathway in response to B cell receptor (BCR) signaling. By introducing point mutations in the Ca²⁺-binding loops of each of the EF-hands, I found that a potential Ca²⁺- interaction loop in the first EF-hand is required for RasGRP1 translocation and the consequential activation of the Ras-ERK pathway in response to BCR signaling. However, RasGRP1 translocation is not regulated by BCR-generated Ca²⁺ flux. EF-hands were not required for Cl domain-mediated membrane localization, but were needed for PT-mediated plasma membrane targeting. EF-hands enhanced PT-domain mediated plasma membrane localization by repressing the SuPT domain. The REM and GEF domains, which co ordinately bind to and catalyze guanine nucleotide exchange on Ras GTPases, needed to be present and Ras-bound for this EF-hand mechanism to be effective. When not bound to Ras, the REM-GEF domain complex suppressed both plasma membrane and endomembrane targeting of RasGRP 1 by an EF-hand independent mechanism. Finally, membrane localization and activation of a naturally occurring splice variant of RasGRP 1, found overexpressed in systemic lupus erythematosus (SEE) patients, was examined. This splice variant lacks exon 11, which encodes the segment of RasGRP1 between the GEF domain and the first EF-hand. Removal of exon 11 resulted in a defect in plasma membrane localization that was partially overridden by deletion of SuPT, while membrane localization control via the REM-GEF complex was not affected. Therefore, exon 11 deletion via alternative splicing appears to functionally disable the first EF-hand of RasGRP1.

Baltymų tarpusavio sąveika užtikrina įvairių ekstraląstelinių ir viduląstelinių signalų perdavimą. Sąveikas sąlygoja specializuoti baltymų domenai, vieni pagrindinių jų yra Src homologijos (SH) domenai, kurie aptinkami daugelyje baltymų. Nck šeimos baltymai, Nck-α ir Nck-β, yra sudaryti iš trijų SH3 ir vieno SH2 domenų ir neturi jokių katalitinių domenų. Nck veikia kaip tarpininkai per SH2 ir SH3 domenus sujungdami įvairius viduląstelinius procesus reguliuojančius baltymus. Šiame darbe nustatėme, kad Nck šeimos nariai sąveikauja su Ras GTPazę aktyvuojančiu baltymu (RasGAP) pastoviai in vitro ir in vivo, bet po PDGFR-β stimuliacijos su Nck asocijuojančio RasGAP kiekis padidėja. Pastovi sąveika priklauso nuo Nck-α SH2 ir vieno arba kelių SH3 domenų. Po PDGFR-β aktyvacijos, už papildomo RasGAP kiekio surišimą yra atsakingas SH2 domenas. Taip pat nustatėme, kad Nck baltymai gali sąveikauti su RasGAP tiesiogiai. Vienas arba keli Nck-α SH3 domenai yra atsakingi už Nck-a tiesioginę sąveiką su RasGAP. Imunofluorescencijos tyrimais parodėme, kad Nck-a ir RasGAP baltymų lokalizacijos profilis yra panašūs. / Extracellular and intracellular signals are mediated by protein-protein interactions. These interactions are determined by specialized protein domains, for example, Src homology (SH) domains found in many proteins. Nck family proteins, Nck-α and Nck-β, are composed of three SH3 and one SH2 domains and have no any putative catalytic domain. Nck proteins act as adaptors by linking via SH2 and SH3 domains proteins that regulate various intracellular processes. Here we show that Nck family proteins associate with RasGAP constantly in vivo and in vitro, but after PDGFR-β stimulation quantity of Nck-bound RasGAP increases. Constant interaction depends on Nck-α SH2 and one or several SH3 domains. SH2 domain is responsible for binding of additional RasGAP after PDGFR-β stimulation. We have also determined that Nck proteins are able to interact with RasGAP directly. One or several SH3 domains are responsible for Nck-a direct association with RasGAP. Immunofluorescence assay show that localization patterns of Nck-a and RasGAP are similar.