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A Proteomic Screen Reveals Novel Fas Ligand Interacting Proteins Within Nervous System Schwann Cells

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A Proteomic Screen Reveals Novel Fas Ligand Interacting Proteins Within Nervous System Schwann Cells A Proteomic Screen Reveals Novel Fas Ligand Interacting Proteins within Nervous System Schwann cells Peter Thornhill Department of Physiology McGill University June, 2007 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Master of Science (MSc.). © Peter Thornhill, 2007 Libraryand Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 978-0-494-38437-4 Our file Notre référence ISBN: 978-0-494-38437-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell theses le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ACKNOWLEDGEMENTS: Firstly, 1 would like to greatly thank my supervisor, Dr. Julie Desbarats, for guiding me well, supporting part of my tenure in her lab, and giving such a young graduate student like myself such a long leash to try many of my own experimental ideas, sorne of which worked, and unfortunately many of which didn't. 1 would like to thank Dr. Yaël Mamane of the Sonenberg lab for advice in co­ immunoprecipitation. 1 would like to thank Dr. Kurt Dejgaard for his expertise and sage advice during the interpretation and preparation part of my mass spectrometry experiments. 1 would like to thank Dr. Veen a Sangwan of the Park lab for advice and furnishing me with several antibodies. 1 would like to thank Gillian Drury for many valuable discussions both lab related and otherwise and for helping to guide me through problems with the molecular biology experiments. Thanks to Manuelle Rongy for originaIly purifying the primary Schwann cells 1 used to create the stable FasL expressing celllines. 1 would like to thank Ken McDonald for his expertise in ceIl sorting/flow cytometry and for being an all around ni ce guy. Thanks to Dr. Peter McPherson for generously providing antibodies to alpha and gamma adaptins Thanks to the National Science and Engineering Research Council of Canada for supporting my masters degree. 1 would like to thank the proteomic platform at the Institute for Research in Immunology and Cancer, led by Dr. Pierre Thibault, for performing the mass spectrometry. This work was supported by grant 53337 from the Canadian Institute of Health Research (CIHR). My collaborator, Jason B. Cohn, was supported by a CIHR Doctoral Fellowship and my supervisor, Dr. Julie Desbarats, by a CIHR New Investigator award. Thornhill, pg. 2 CONTRIBUTIONS OF AUTHORS: This rnanuscript based thesis contains two rnanuscripts. One is in revision after subrnission to FEBS Letters and the second will be subrnitted shortly, also to FEBS Letters. I, Peter Thornhill, attest that I performed aIl of the experiments presented within this thesis myself, except for the following contributions by other authors: Gillian Drury - Helped to perform RT-PCR experirnents for Fig. IC of Chapter 2 to compare FasL to enhanced green fluorescent protein expression at the mRNA level. Jason B. Cohn - Developed and provided the author with antiserurn against Sorting Nexin 18/Sorting Nexin Associated Golgi protein 1 (SNXI8/SNAGI), as weIl as advice for its use. Dr. William L. Stanford - Supervised Jason B. Cohn. Dr. Alan Bernstein - Aiso supervised Jason B. Cohn. Dr. Julie Desbarats- Supervised the author, as weIl as performed sorne of the bench work found in Fig 3A, B of Chapter 3. y oon Kow Young - Counted a large number of FasL staining puncta within Schwann cells to generate the statistics found in Fig. 3E of Chapter 3. Others- Other non-authors have also helped in ways that do not merit authorship and are outlined in the acknowledgements section. I, Dr. Julie Desbarats, attest to the accuracy of these statements: I, Peter Thornhill, attest to the accuracy of these statements: Thornhill, pg. 3 ABSTRACT: ~ 1 Fas Ligand (FasL) binds to the Fas receptor to induce apoptosis or activate other signaling pathways. FasL can also transduce "reverse signais" and thus participate in bidirectional signaling. The FasL intracellular domain contains consensus sequences for phosphorylation and a proline rich protein interaction domain. This latter region of FasL has previously been implicated in FasL reverse signaling and regulation of FasL surface expression. In this report, we sought to identify novel FasL interacting proteins to help understand signaling through and trafficking of this death factor Using mass spectrometry, we identified sorting nexin 18, adaptin ~, Grh2, PACSIN2 and PACSIN3 as FasL interacting proteins. RNAi mediated knockdown of Grh2 significantly reduced the surface expression of FasL and increased its expression intracellularly. Our data show that Grb2 con troIs the suhcellular localization of FasL. AlI other proteins identified in our screen could be classified as trafficking-associated proteins, highlighting the complex regulation of the surface expression of this death factor. Thornhill, pg. 4 RÉSUMÉ: La protéine Fas Ligand (FasL) se lie au récepteur Fas afin d'induire l'apoptose ou activer d'autres voies de signalisation. La protéine FasL est aussi impliquée dans la signalisation rétrograde et participe à une signalisation bidirectionnelle. Le domaine intracellulaire de FasL contient une séquence consensus de phosphorylation ainsi qu'un domaine d'interaction protéique riche en proline. Ce domaine a précédemment été impliqué dans la signalisation rétrograde de FasL et dans la régulation de son expression à la surface cellulaire. Dans cette thèse, nous désirions identifier de nouvelles interactions protéiques avec FasL afin de contribuer à la compréhension du trafic cellulaire et de la signalisation de ce facteur apoptotique. Grâce à la spectrométrie de masse, nous avons identifié Sorting Nexin 18, Adaptin ~,PACSIN2, Grb2 et PACSIN3 comme étant des protéines interagissant avec FasL. En utilisant la méthode de l'ARN par interférence, nous avons diminué l'expression de Grb2 et observé que l'expression de FasL à la surface cellulaire a été significativement réduite alors que son expression intracellulaire a été augmentée. Nos résultats démontrent que Grb2 est impliquée dans le contrôle de la localisation subcellulaire de FasL. Les autres protéines identifiées dans notre criblage pourraient être classifiées comme protéines associées au trafic cellulaire, suggérant ainsi la complexité de la régulation de ce facteur apoptotique à la surface cellulaire. Thornhill, pg. 5 TABLE OF CONTENTS: Acknowledgements ........................................................................................................ 2 Contributions of Authors ................................................................................................. .3 Abstract. .................................................................................................................... .4 Résumé ................................................................................................................................ 5 Abbreviations ........................................................................................................................7 Chapter 1: Introduction .......................................................................................................... 10 Fas: the Molecule ...................................................................................................... 11 Fas Death Signaling .................................................................................................. .12 Non Apoptotic Fas signaling ........................................................................................ 12 FasL: the Molecule .................................................................................................... 14 FasL Expression and Function ....................................................................................... 15 Regulation of FasL .................................................................................................... 19 FasL Reverse
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