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In Vitro Studies of Protein-Lipid Interactions Modulating Autophagosome Elongation

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In Vitro Studies of Protein-Lipid Interactions Modulating Autophagosome Elongation Tesis doctoral Autophagy-related proteins: in vitro studies of protein-lipid interactions modulating autophagosome elongation Unidad de Biofísica (CSIC, UPV/EHU) Departamento de Bioquímica y Biología Molecular Facultad de Ciencia y Tecnología Universidad del País Vasco (UPV/EHU) Memoria presentada por D. Javier Hervás Hidalgo para optar al grado de Doctor por la Universidad del País Vasco (UPV/EHU) Directoras: Prof. Alicia Alonso Izquierdo y Dra. Lidia Ruth Montes Burgos Doctoral Thesis Autophagy-related proteins: in vitro studies of protein-lipid interactions modulating autophagosome elongation Candidate: Javier Hervás Hidalgo Supervisors: Prof. Alicia Alonso Izquierdo Dr. Lidia Ruth Montes Burgos (c)2016 JAVIER HERVAS HIDALGO A Aita, Ama y Marta Contents Contents Abbreviations v Index of Experimental Protocols ix Chapter 1: Introduction and Aims 3 1.1 Cell Membranes 3 1.1.1 Common Properties and Functions 3 1.1.2 Membrane Lipids 6 1.1.2.1 Classification of Membrane Lipids 6 1.1.2.2 Lipid Distribution and Asymmetry 8 1.1.2.3 Lipid Polymorphism 10 1.1.2.4 Lipid Geometry 12 1.1.3 Membrane Fusion 13 1.1.4 Protein-lipid Interactions 16 1.2 Autophagy 17 1.2.1 Origin 17 1.2.2 Types of Autophagy 18 1.2.3 Molecular Machinery of Macroautophagy 19 1.2.4 Autophagy Signalling Cascade 22 1.2.5 Autophagy is a Lipid-modulated Process 24 1.2.6 Autophagy in Health and Disease 25 1.2.7 Atg8 Conjugation System 26 1.2.8 ATG3, the E2-like Enzyme in the Atg8 Conjugation System 30 1.2.9 ATG9: the Only Transmembrane Atg Protein 31 1.3 Aims 35 i Contents Chapter 2: Experimental Techniques 2.1 Molecular Biology Methods 39 2.1.1 DNA Amplification and Cloning 39 2.1.2 Site-directed Mutagenesis 41 2.2 Recombinant Protein Expression and Purification 43 2.2.1 Protein Expression 43 2.2.2 Purification of GST-tagged Proteins (LC3, GATE-16, GABARAP and ATG3) 44 2.3 Baculovirus Expression Vector System 45 2.3.1 Baculovirus as Expression Vector 46 2.3.2 Insect Cell Lines 47 2.3.3 Recombinant Protein Expression Using the Baculovirus- Insect Cell System 47 2.3.4 hATG7 Protein Cloning 49 2.3.5 hATG9 Protein Cloning 50 2.3.6 Transfection with Recombinant Bacmid 51 2.3.7 Purification of 6xHis-tagged Human ATG7 and ATG9 52 2.4 Membrane Lipid Model Systems 53 2.4.1 Lipid Vesicles (Liposomes) 53 2.4.2 Multilamellar Vesicles (MLVs) 54 2.4.3 Large Unilamellar Vesicles (LUVs) 55 2.4.4 Small Unilamellar Vesicles (SUVs) 56 2.4.5 Giant Unilamellar Vesicles (GUVs) 57 2.4.5.1 GUV in Solution 58 2.4.5.2 GUV for Direct Microscopy 60 2.4.6 Lipid Monolayers 61 2.5 Phospholipid Assay (FISKE Assay) 62 2.6 Vesicle Size Measurement by Dynamic Light Scattering (DLS) 64 2.7 Langmuir Balance 65 2.7.1 Surface Pressure Measurements 65 2.8 Equilibrium Sucrose Gradient Centrifugation of Liposomes 68 ii Contents 2.9 Vesicle Aggregation Measurements 69 2.10 Protein Analysis 70 2.10.1 Protein Concentration Measurements 70 2.10.1.1 Biscinchoninic Acid Assay (BCA) 70 2.10.1.2 Absorbance at 280 nm 71 2.10.2 Protein Electrophoresis 71 2.10.3 Western Blotting 72 2.11 Circular Dichroism 72 2.12 Fluorescence Spectroscopy Techniques 73 2.12.1 Lipid Mixing Assay 73 2.12.2 Aqueous Contents Mixing Assay 76 2.12.3 Vesicle Contents Efflux Measurement (Leakage) Assay 77 2.13 Cryo-electron Microscopy 80 2.14 Fluorescence Confocal Microscopy 81 Chapter 3: Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-mediated Model Autophagosomal Elongation 3.1 Introduction 85 3.2 Materials and Methods 87 3.3 Results 92 3.4 Discussion 109 Chapter 4: Human ATG3 Membrane Association Induces Tethering of Negatively-charged Membranes 4.1 Introduction 115 4.2 Materials and Methods 117 4.3 Results 122 4.4 Discussion 138 iii Contents Chapter 5: Preliminary Results on hATG9 Purification in E.coli and Insect Cells, and its Implication in Lipid Delivery 5.1 Introduction 145 5.2 Materials and Methods 147 5.3 Results 151 5.4 Discussion 159 Chapter 6: Overview and Conclusions 163 Capítulo 6: Resumen y Conclusiones 173 References 185 Publications 203 Acknowlegments 207 iv Abbreviations Abbreviations AC: Alternating Current BV: Baculovirus AcMNPV: Autographa californica CBB: Coomassie Brilliant blue nucleopolyhedrovirus CD: Circular Dichroism AD: Adenylation Domain Cer: Ceramide Akt: RAC-alpha serine/threonine-protein Chol: Cholesterol kinase CL: Cardiolipin ALPS: Amphipathic Lipid Packing Cryo-EM: Cryo-Electron Microscopy Sensor CTD: C-terminal Domain Amp: Ampicillin DAG: Diacylglycerol ANTS: 8-aminonapthalene-1,3,6 trisulfonic acid DAPI: 4',6-diamidino-2- phenylindole AP: Autophagosome Deptor: DEP domain containing ArfGAP1: ADP-ribosylation factor MTOR-interacting protein GTPase-activating protein 1 DFCP1: Double FYVE-containing ATG: AuTophaGy-related protein 1 ATP: Adenosine Triphosphate DLS: Dynamic Light Scattering BAR: Bin–Amphiphysin–Rvs DOPE: 1,2-dioleoyl-sn-glycero-3- domain phosphoethanolamine Barkor: Beclin 1-associated DPPC: 1,2-dipalmitoyl-sn-glycero- autophagy-related key 3-phosphocholine regulator DPX: p-xylene-bis-pyridinium BCA: Bicinchoninic acid assay bromide Beclin-1: Coiled-coil myosin-like DTT: DL-Dithiothreitol BCL2-interacting protein ECTD: Extreme C-terminal domain BEVS: Baculovirus Expression Vector System EDTA: Ethylenediaminetetraacetic acid bI-PI: bovine-liver PhosphatidylInositol ER: Endoplasmic Reticulum BmNPV: Bombyx mori FCS: Fetal Calf serum nucleopolyhedrovirus FIP200: FAK family kinase- BSA: Bovine Serum Albumin interacting protein of 200 kDa v Abbreviations FR: Flexible Region Lα: Fluid, Liquid-disordered or Liquid-crystalline lamellar FRET: Förster Resonance Energy lipid phase Transfer Lβ: Gel or Solid-ordered FT: Flow-Through lamellar lipid phase GABARAP: GABA(A) Receptor- Lβ´: Oblique lamellar lipid phase Associated Protein LβI: Interdigitated lamellar lipid GATE-16: Golgi-associated ATPase phase enhancer of 16 kDa MCF7: Michigan Cancer GPI: Glycosylphosphatidyl- Foundation-7 cells Inositol MLV: Multilamellar vesicle GSL: Glycolsphingolipid MOI: Multiplicity of Infection GST: Glutathione S-Transferase mTORC1: mammalian target of GβL: G protein beta subunit-like rapamycin complex 1 HEK293: Human Embryonic Kidney NBD: 7-nitrobenz-2-oxa-1,3- 293 cells diazol-4-yl HEPES: 2-[4-(2- NTD: N-terminal Domain hydroxyethyl)piperazin-1- yl]ethanesulfonic acid OD: Optical Density HOPS: Homotypic Fusion and OG: n-octyl--D- Protein Sorting glucopyranoside hpi: hours post-infection OV: Occluded Virus HR: Handle Region PA: Phosphatidic Acid HRP: Horse-Radish Peroxidase PAS: Preautophagosomal Structure H5: High Five cells PBS: Phosphate Buffered Saline IPTG: Isopropil-β-D-1- tiogalactopiranósido PC: PhosphatidylCholine LAMP2: Lysosomal Associated PDI: Polydispersity Index Membrane Protein-2 PE: Phosphatidylethanolamine LB: Luria Broth PEmal: 1,2-dioleoyl-sn-glycero-3- LC3B: Microtubule associated phosphatidylethanolamine- protein 1 light chain 3 B p-maleimidomethyl- cyclohexanecarboxamide LPC: Lysophosphatidylcholine PG: Phosphatidylglycerol LUV: Large unilamellar vesicle PI3P: Phosphatidylinositol 3- Lo: Liquid-ordered lamellar lipid phosphate phase vi Abbreviations PIK3C1: class I phosphatidylinositol- ROS: Reactive Oxigen Species 3-kinase SDS-PAGE: Sodium dodecyl sulfate PITPα: Phosphatidylinositol polyacrylamide gel Transfer Protein alpha electrophoresis PLC: Phospholipase C SM: Sphingomyelin PLD1: Phospholipase D1 SUV: Small unilamellar vesicle polh: Polyhedrin TCEP: tris(2-carboxyethyl) phosphine PRAS40: Proline-rich AKT substrate 40 kDa TEV: Tobacco Etch Virus nuclear-inclusion-a PS: Phosphatidylserine endopeptidase PTEN: Phosphatidylinositol 3,4,5- Tm: Melting temperature trisphosphate 3- phosphatase and dual- TSC1-2: Tuberous sclerosis 1-2 specificity protein protein phosphatase UBL: Ubiquitin-Like Pβ´: Rippled lamellar gel phase ULK1-ULK2: Uncoordinated-51 (unc-51)- QELS: quasi-elastic light scattering like kinase 1 or 2 Rab: Ras-related GTP-binding UVRAG: UV-radiation resistance protein associated gene Raptor: regulatory associated WIPI1-2: WD repeat domain protein of mTOR phosphoinositide interacting 1 and 2 rBV: recombinant Baculovirus Πc: Critical surface pressure Rho: Rhodamine Rho-PE: 1,2-dioleoyl-sn-glycero-3- phosphatidylethanolamine- N-(lissamine rhodamine B sulfonyl) vii Index of Experimental Protocols Index of Experimental Protocols Protocol 1. Site-directed Mutagenesis 42 Protocol 2. Multilamellar Vesicles (MLVs) 55 Protocol 3. Large Unilamellar Vesicles (LUVs) 56 Protocol 4. Giant Unilamellar Vesicles in Solution 59 Protocol 5. GUVs Attached to a Platinum Wire for Direct Microscopy 60 Protocol 6. Phospholipid Assay (FISKE Assay) 63 Protocol 7. Vesicle Aggretation Assay 69 Protocol 8. Total Lipid Mixing Assay 74 Protocol 9. Inner Lipid Mixing Assay 74 Protocol 10. Vesicle Contents Efflux Measurement (Leakage) Assay 79 ix 1. Introduction and Aims Introduction CHAPTER 1: Introduction and Aims 1.1 Cell Membranes 1.1.1 Common Properties and Functions Living cells need to be connected with and protected from the outer environment. Membranes confer the main protection against the different environmental conditions for survival. Plasma membrane surrounds the cytoplasm of living cells and physically separates the intracellular components from the extracellular ones. Membranes constitute a selective barrier that regulates the exchange of molecules between defined compartments. Unlike prokaryotes, eukaryotic cells have not only a plasma membrane but also intracellular membranes surrounding various organelles. All of these membranes are part of an extensive endomembrane system. The plasma membrane
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