Molecular Characterisation of a Novel Vipar Interacting Protein in Health and Disease
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MOLECULAR CHARACTERISATION OF A NOVEL VIPAR INTERACTING PROTEIN IN HEALTH AND DISEASE ! BY BLERIDA BANUSHI ! A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY ! !! ! ! College of Medicine & Dentistry The University of Birmingham August 2013 I University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Arthrogryposis, Renal dysfunction, and Cholestasis (ARC) syndrome is a multisystem disorder caused by mutations in genes encoding two proteins VPS33B or VIPAR, which appear to be critical regulators of cell polarity. VPS33B and VIPAR may function as part of a multi-protein complex that interacts with an active form of RAB11A and is involved in the transcriptional regulation of E-cadherin. VPS33B and VIPAR are shown to interact at a protein level forming a stable binary complex. A novel interacting partner of VIPAR was identified, PLOD3, a post- translational modification enzyme with lysyl hydroxylase (LH), collagen galactosyltransferase (GT), and glucosyltransferase (GGT) activities (Wang et al., 2012). In mIMCD-3 polarized cell lines VPS33B and VIPAR are involved in PLOD3 trafficking from the TGN compartment via RAB11A positive vesicles. Study of the topology of this ternary protein complex evidenced that VIPAR is a transmembrane protein with its luminal N-terminal interacting with PLOD3 and its cytosolic C-terminal being involved in the interaction with VPS33B. VPS33B/VIPAR mediates the trafficking of PLOD3 from the TGN to collagen carrier structures where the binding of PLOD3 with collagen takes place. The PLOD3-collagen binding is required for collagen trafficking and an abnormal accumulation of intracellular collagen, associated with failure in PLOD3 delivery to collagen, is observed in Vipar knockdown mIMCD-3 cells. Abnormal collagen modifications and trafficking in Vps33b/Vipar deficiency can explain the down-regulation of E-cadherin that characterises some polarized cell types in ARC and the cell model for this syndrome. These findings establish a role for VPS33B/VIPAR in the intracellular trafficking of collagen. II Acknowledgments I am very thankful to all the members of the Medical and Molecular Genetics Department at the University of Birmingham and all members of the MRC-LMCB University College of London for their help and support through the time I spent in each department during my PhD. I would like to sincerely thank my supervisor Prof. Paul Gissen for the continuous support during all the time of my PhD research and writing of this thesis, for his friendly guidance and patience, for his motivation and great enthusiasm. I would also like also to thank Federico Forneris for his important help through the project, the discussions, the suggestions and his collaborative and enthusiastic support. Special thanks go to Ania Straatman-Iwanowska and Holly Smith for their precious help throughout the project and for allowing me to work everyday in a very supportive and friendly environment. I would like to thank Holly also for her help in improving my English (a part from the ‘biscUit’ word ☺) for her firiendship and all the sushi and icecreams I had thanks to her (and all the spinning to fix things back)! I would like to thank Ania also for her friendship and support, her patientce (and sometimes impatience ☺) that definitely helped me in becoming a better lab worker. Now hopefully nothing is between us and our plan to go for walks! I would like to thank Chris Bruce and Danai Bem for their help during my permanence at the University of Birmingham. I would like to thank Holly Smith, Clare Rogerson and Anne-Marie Lyne for corrections and help with the thesis. Special thank also to the Biology of Liver and Pancreatic Development and Disease (BOLD) network for finding this project. Finally I would like to thank my family and friends for their continued support and encouragement throughout this project. III Table of contents List of figures VII List of tables X List of abbreviations XI CHAPTER ONE: INTRODUCTION 1 1.1 - ARC syndrome 2 1.2 - VPS33B and ARC 4 1.3 - VIPAR and ARC 8 1.4 - Insights into vesicular transport 12 1.4.1 - Endocytic and biosynthetic pathway: the central role of TGN 12 1.4.2 - Polarized cells and vesicular trafficking 15 1.4.3 - Steps in vesicular transport: from budding to fusion 16 1.4.4 - Vesicular trafficking machinery 18 1.4.4.1 - SNARE proteins 18 1.4.4.2 - SM proteins 20 1.4.4.3 - Rab GTPases 21 1.4.4.3.1 - The RAB11 GTPase subfamily 25 1.4.4.4 - Tethering factors 27 1.4.4.4.1 - HOPS complex 28 1.4.4.4.2 - The CORVET complex 30 1.5 - Extracellular matrix 31 1.5.1 - Extracellular Matrix Components 32 1.5.1.1 - Adhesion molecules 32 1.5.2 - Adherens Junctions 34 1.5.2.1 - Regulation of adherens junctions: EMT 36 1.5.3 - Collagens 37 1.5.3.1 - Collagen type IV - the collagen of basement 40 membranes 1.5.3.2 - The Biosynthesis of Collagen 42 1.5.4 - Lysyl hydroxylases: localisation and activities 47 1.5.4.1 - Lysyl hydroxylase 3 deficiency 52 CHAPTER TWO: MATERIALS AND METHODS 53 2.1 - Suppliers 54 2.2 - Cloning system 54 2.2.1 - Proofreading Polymerase Chain Reaction 55 2.2.2 - Agarose Gel Electrophoresis 56 2.2.3 - PCR purification 56 2.2.4 - Restriction Digestion 57 2.2.5 - Agarose Gel Extraction 58 2.2.6 - Ligation 58 2.2.7 - Transformation 59 IV 2.2.8 - Colony Screening 59 2.2.9 - Plasmid DNA Purification, Miniprep 60 2.2.10 - Plasmid Sequencing 61 2.2.10.1 - Sequencing Reaction: precipitation and analysis 61 2.3 - Protein harvesting and quantification 62 2.3.1 - Mammalian protein extraction 62 2.3.2 - Protein detection and quantification 63 2.3.2.1 - Sodium dodecyl sulphate polyacrylamide gel 63 electrophoresis 2.3.2.2 - Page Blue protein staining and Drying of SDS-PAGE 64 Gels 2.3.3 - Western Blotting 65 2.3.3.1 - Electrophoretic Transfer to PVDF Membranes 65 2.3.3.2 - Western Blot detection 65 2.4 - Immunoprecipitation 67 2.5 - Cell biology 68 2.5.1 - Cell lines culture conditions and maintenance 68 2.5.1.1 - Culture of fully polarised mIMCD-3 cells 69 2.5.2 - Transfection of HEK293, HeLa and mIMCD-3s using 70 Lipofectamine™ 2000 2.5.3 - Immunofluorescence microscopy 71 2.5.4 - mIMCD-3 stable knockdowns 73 CHAPTER THREE: VPS33B and VIPAR interact forming a stable binary 74 complex 3.1 - INTRODUCTION AND OVERVIEW 75 3.2 - AIMS 86 3.3 - METHODS 86 3.3.1 - Mammalian expression system 86 3.3.2 - Mammalian protein extraction 89 3.3.3 - Mammalian protein purification 89 3.3.4 - Dialysis of Recombinant Proteins 90 3.3.5 - Size exclusion chromatography 90 3.3.6 - Multi-Angle Laser Light Scattering (MALLS) 91 3.4 - RESULTS 92 3.4.1 - VPS33B interacts directly with VIPAR forming a binary 92 complex 3.4.2 - VPS33B and VIPAR form a stable complex 95 3.4.3 - VPS33B and VIPAR interact in the cell with Fluorescence 97 Resonance Energy Transfer (FRET) 3.4.4 - VPS33B interacts with the globular C-terminal domain of 100 VIPAR 3.5 - DISCUSSION 102 3.6 - Supplementary Bioinformatics analysis of VPS33B and VIPAR 104 proteins V CHAPTER FOUR: PLOD3 is a novel interactor for VPS33B/VIPAR 110 4.1 – INTRODUCTION AND OVERVIEW 111 4.2 - AIMS 115 4.3 - METHODS 116 4.3.1 - Strep-tactin pull-down assay 116 4.3.2 - Liquid chromatography electrospray ionisation tandem mass spectrometry ESI-LC-MS/MS 118 4.4 - RESULTS 121 4.4.1 - Mass spectrometry analysis of VPS33B-VIPAR binding proteins 121 4.4.2 - PLOD1 does not co-localise with VPS33B-VIPAR complex 122 4.4.3 - Confirmation of the PLOD3-VIPAR interaction 124 4.4.3.1 - Reciprocal co-immunoprecipitation of PLOD3 and 124 VIPAR 4.4.3.2 - PLOD interacts with VPS16 in D. melanogaster 125 4.4.3.3 - Significant co-localisation between VPS33B/VIPAR 126 complex and PLOD3 4.4.3.4 - Further evidence for VIPAR-PLOD3 intracellular 129 interaction from FRET experiments 4.4.3.5 - PLOD3 is likely to interact with the N-terminal domain 133 of VIPAR 4.5 - DISCUSSION 135 CHAPTER FIVE: VPS33B/VIPAR is involved in PLOD3 intracellular 138 delivery: a novel mechanism for Collagen trafficking 5.1 - INTRODUCTION AND OVERVIEW 139 5.2 - AIMS 149 5.3 - METHODS 149 5.3.1 - Separating cytosol from crude membrane fractions in mIMCD-3 149 and HeLa cells 5.3.2 - Cell fractionation 150 5.4 - RESULTS 151 5.4.1 - Endogenous PLOD3 does not co-localise with PDI: PLOD3 151 intracellular localisation is different from PLOD1 5.4.2 - Endogenous PLOD3 and endogenous VIPAR co-localise with 154 TGN markers and RAB11A in HeLa cells 5.4.3 - PLOD3 is abnormally trafficked in Vps33b/Vipar knockdown 159 mIMCD-3 cells 5.4.4 - VPS33B/VIPAR and PLOD3 express co-ordinately in mIMCD-3 164 cells 5.4.5 - The topology of VPS33B/VIPAR/PLOD3 interaction: VIPAR is 168 a transmembrane protein 5.4.6 - Collagen type IV and E-cadherin are abnormally expressed in 175 Vps33b/Vipar and Plod3 knockdown cell lines 5.4.7 - VPS33B-VIPAR complex is involved in PLOD3 delivery to 183 collagen 5.5 - DISCUSSION 187 VI CHAPTER SIX: DISCUSSION 192 6.1 - VPS33B interacts with VIPAR at a protein level forming a stable 193 binary complex 6.2 - PLOD3 is a novel interactor for VIPAR 194 6.3 - VIPAR is a transmembrane protein with its luminal N-terminal 195 interacting with PLOD3 and its cytosolic C-terminal interacting with VPS33B 6.4 - VPS33B/VIPAR complex is involved in PLOD3 delivery to collagen 197 6.5 - Abnormal collagen in Vps33b/Vipar/Plod3 deficiency and its relevance 201 to ARC 6.6 - Future experiments 203 CHAPTER SEVEN: APPENDICES 206 CHAPTER EIGHT: REFERENCES 211 List of figures Figure 1.1 - Typical features of a patient with ARC syndrome 3 Figure 1.2 - Immunostaining of kidney biopsies from ARC patients and age 4 matched controls.