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Proquest Dissertations THE ROLE OF CASP AND SORTING NEXIN 27 IN IMMUNITY by Adam J. MacNeil Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Dalhousie University Halifax, Nova Scotia December 2008 © Copyright by Adam J. MacNeil, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-50156-6 Our file Notre reference ISBN: 978-0-494-50156-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque 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 telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. 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 DALHOUSIE UNIVERSITY To comply with the Canadian Privacy Act the National Library of Canada has requested that the following pages be removed from this copy of the thesis: Preliminary Pages Examiners Signature Page (pii) Dalhousie Library Copyright Agreement (piii) Appendices Copyright Releases (if applicable) DEDICATION This thesis is dedicated to my loving mother and father, Charlene and Harold MacNeil. iv TABLE OF CONTENTS LIST OF TABLES x LIST OF FIGURES xi ABSTRACT xiii LIST OF ABBREVIATIONS xiv ACKNOWLEDGMENTS xvii CHAPTER 1 INTRODUCTION 1 CHAPTER 2 SORTING NEXIN 27 INTERACTS WITH THE CYTOHESIN-ASSOCIATED SCAFFOLDING PROTEIN (CASP) IN LYMPHOCYTES 6 2.1 ABSTRACT 7 2.2 INTRODUCTION 7 2.3 MATERIALS AND METHODS 9 2.3.1 CELLS AND LYSATES 9 2.3.2 DNA AND RECOMBINANT PROTEINS 10 2.3.3 GST PULLDOWNS, WESTERN ANALYSIS AND ESI-TANDEM MASS SPECTROMETRY 11 2.3.4 CO-IMMUNOPRECIPITATION 11 2.3.5 IMMUNOCYTOCHEMISTRY AND TRANSFECTIONS 12 2.4 RESULTS 13 2.4.1 SNX27 INTERACTS WITH THE CARBOXY-TERMINAL PDZ-BINDING MOTIF OF CASP IN LYMPHOCYTES 13 2.4.2 PHYSICAL INTERACTION WITH CASP is MEDIATED BY THE PDZ DOMAIN OF SNX27 14 2.4.3 SNX27 CO-IMMUNOPRECIPITATES WITH CASP AND CYTOHESIN-1 IN LYMPHOCYTES 14 v 2.4.4 ENDOGENOUS SNX27 CO-LOCALIZES WITH CASP AT EARLY ENDOSOMES IN YT CELLS AND RECRUITS TRANSFECTED CASP TO ENDOSOMES IN HEK-293 CELLS 15 2.5 DISCUSSION 22 2.6 ACKNOWLEDGEMENT 24 2.7 REFERENCES 25 2.8 LINKING PARAGRAPH 28 CHAPTER 3 POLARIZATION OF ENDOSOMAL SNX27 IN MIGRATING AND TUMOR-ENGAGED NATURAL KILLER CELLS 29 3.1 ABSTRACT 30 3.2 INTRODUCTION 30 3.3 MATERIALS AND METHODS 32 3.3.1 CELLS AND ANTIBODIES 32 3.3.2 IMMUNOCYTOCHEMISTRY 33 3.3.3 CONJUGATION ASSAYS 33 3.4 RESULTS 33 3.4.1 ENDOGENOUS SNX27 LOCALIZES TO THE EARLY ENDOSOMES IN NK-92 CELLS 33 3.4.2 SNX27 POLARIZES TO THE IMMUNOLOGICAL SYNAPSE IN VITRO AND IS DISTINCT FROM THE CYTOTOXIC PERFORIN- CONTAINING GRANULES 34 3.4.3 SNX27 POLARIZES TO THE LEADING EDGE OF MIGRATING NK-92 CELLS 35 3.5 DISCUSSION 41 3.6 ACKNOWLEDGEMENT.. 44 3.7 REFERENCES 45 3.8 LINKING PARAGRAPH 48 vi CHAPTER 4 GENE DUPLICATION IN EARLY VERTEBRATES RESULTS IN TISSUE- SPECIFIC SUBFUNCTIONALIZED ADAPTOR PROTEINS: CASP AND GRASP 49 4.1 ABSTRACT 50 4.2 INTRODUCTION 51 4.3 MATERIALS AND METHODS 53 4.3.1 IDENTIFICATION AND RETRIEVAL OF CASP AND GRASP HOMOLOGUES 54 4.3.2 ANALYSIS OF CASP AND GRASP GENE STRUCTURES AND PROTEIN ALIGNMENTS 54 4.3.3 ALIGNMENTS AND PHYLOGENETIC ANALYSIS OF GRASP AND CASP HOMOLOGUES 55 4.3.4 ANALYSIS OF AMPHIOXUS, ZEBRAFISH, AND HUMAN GENOMIC LOCI 56 4.4 RESULTS 57 4.4.1 CASP AND GRASP SHARE HIGHLY SIMILAR FUNCTIONAL DOMAINS, MOTIFS, AND GENOMIC STRUCTURE ..57 4.4.2 MAMMAL, BIRD, AND REPTILE CASP SHARE A UNIQUE AND RECENTLY ACQUIRED AMINO ACID IN THE COILED-COIL MOTIF 58 4.4.3 THE PDZBM OF VERTEBRATE CASPS AND GRASPS SHOW CONSIDERABLE CONSERVATION 59 4.4.4 PHYLOGENETIC ANALYSIS IDENTIFIES A CASP/GRASP GENE DUPLICATION PREVIOUS TO THE VERTEBRATE RADIATION 61 4.4.5 EXAMINATION OF GENETIC LOCI IN VARIOUS CHORDATES SHOWS THAT GRASP is THE MOST LIKELY ANCESTRAL GENE AND THAT THE ANCESTRAL NR4A GENE WAS CO-DUPLICATED 62 4.5 DISCUSSION 74 vii 4.6 ACKNOWLEDGEMENTS 79 4.7 REFERENCES 80 4.8 LINKING PARAGRAPH 84 CHAPTER 5 GETTING A GRASP ON CASP: PROPERTIES AND ROLE OF THE CYTOHESIN-ASSOCIATED SCAFFOLDING PROTEIN IN IMMUNITY....85 5.1 ABSTRACT 86 5.2 INTRODUCTION 87 5.2.1 DISCOVERY AND EARLY YEARS 87 5.3 TRANSCRIPTIONAL ACTIVATION AND TRANSCRIPT PROPERTIES 88 5.4 PROTEIN PROPERTIES AND INTERACTIONS 91 5.4.1 THE CYTOHESIN/ARNO FAMILY 92 5.4.2 SNX27 AND THE SORTING NEXIN FAMILY 94 5.5 INTRACELLULAR LOCALIZATIONS AND FUNCTIONAL ACTIVITIES 99 5.6 FUNCTIONAL ASSESSMENT IN THE CONTEXT OF IMMUNITY: KNOCKOUTS AND RNAI 107 5.6.1 ROLE IN IMMUNE CELL POLARIZATION 108 5.6.2 ROLE IN CELL MIGRATION 110 5.7 ADAPTOR PROTEIN FAMILY: CASP AND GRASP 115 5.8 PROGRESS TO DATE AND OUTLOOK 116 5.9 ACKNOWLEDGEMENT.. 118 5.10 REFERENCES 119 5.11 LINKING PARAGRAPH 129 CHAPTER 6 SUPPORTING AND PRELIMINARY RESULTS AND DISCUSSION 130 6.1 SNX27 RECRUITMENT OF CASP TO ENDOSOMES 130 viii 6.1.1 CASPAPDZBM HAS ALTERED INTRACELLULAR DISTRIBUTION 130 6.1.2 MATERIALS AND METHODS 130 6.2 ADDITIONAL INTERACTIONS: CASP IS CLEAVED BY GRANZYME B IN VITRO 132 6.2.1 CASP AS A TARGET FOR GRB AND FURTHER INTERACTIONS 132 6.2.1 MATERIALS AND METHODS 133 6.3 GRASP INTERACTS WITH SNX27 IN VITRO 137 6.4 LINKING PARAGRAPH 139 CHAPTER 7 CONCLUSION 140 REFERENCES 145 APPENDIX A DEVELOPMENT OF SNX27 ANTISERUM 159 APPENDIX B COPYRIGHT PERMISSIONS 162 ix LIST OF TABLES Table 2.1 ESI-tandem mass spectrometry identification of SNX27 interacting with the carboxy terminus of CASP. 17 Table 5.1 Summary of CASP binding partners, related localizations and functions. 98 x LIST OF FIGURES Figure 2.1 Identification of proteins interacting with the carboxy terminus of CASP 18 Figure 2.2 SNX27 interaction with the carboxy terminus of CASP is mediated via its PDZ domain in vitro 19 Figure 2.3 Co-immunoprecipitation of endogenous SNX27 with CASP and cytohesin-1 and detection of SNX27 in lymphocytes 20 Figure 2.4 Imrnunocytochemistry shows partial co-localization of endogenous CASP with SNX27 in YT cells 21 Figure 3.1 Endogenous SNX27 co-localizes with early endosomes and not with lysosomal or Golgi markers in NK-92 cells 37 Figure 3.2 Imrnunocytochemistry of killer :target conjugates shows SNX27 polarization to the IS in vitro 38 Figure 3.3 SNX27 polarization to the IS in conjugates is distinct from the similarly polarized perforin-containing cytotoxic granules in NK-92 cells 39 Figure 3.4 SNX27 polarizes to the leading edge, opposite the CD 18 enriched uropod of migrating NK-92 cells 40 Figure 4.1 A comparison of the domain distribution of human CASP and GRASP proteins 65 Figure 4.2 Comparison of CASP and GRASP gene structures 66 Figure 4.3 Alignment of vertebrate CASP and GRASP protein sequences in the PDZ-binding motif region 6 8 Figure 4.4 Phylogenetic analysis of vertebrate CASP and GRASP proteins including invertebrate homologues 69 Figure 4.5 Comparison of gene loci from amphioxus, zebrafish, and human genomes 71 Figure 4.6 A simplified model depicting the sequence of events and relative evolutionary time line (left) for duplication of the primordial GRASP and NR4A locus 73 xi Figure 5.1 Domain and motif architecture in the CASP interactome. Protein domain distributions of CASP, the cytohesin/ARNO family, and SNX27 97 Figure 5.2 A model implicating CASP in putative novel polarized hematopoietic endosomal sorting events 106 Figure 5.3 A model depicting the role of CASP expression in antigen presentation by DCs 114 Figure 6.1 SNX27 recruits transfected CASP to the endosomal compartment, dependent on the PDZ-PDZbm interaction 131 Figure 6.2 Recombinant CASP is cleaved by granzyme B in vitro 135 Figure 6.3 Predicted CASP proteolytic cleavage site by granzyme B 136 Figure A.l New antibodies for SNX27 immunoprecipitate endogenous protein 160 Figure A.2 SNX27a antiserum detects endogenous, endosomal SNX27 in YT cells 161 xn ABSTRACT Cytohesin-associated scaffolding protein (CASP) is a novel human adaptor protein, expressed exclusively in immune cells. CASP participates in the assembly and recruitment of protein complexes associated with intracellular trafficking and signaling.
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