UNIVERSITE PARIS SUD ÉCOLE DOCTORALE 418 DE CANCÉROLOGIE Laboratoire : UMR1138 Apoptosis, Cancer & Immunity THESE Présentée et soutenue publiquement par FEDERICO PIETROCOLA Le 2 septembre 2015 REGULATION OF AUTOPHAGY BY ACETYL COENZIME A: FROM THE MECHANISMS TO A REVISED DEFINITION OF CALORIC RESTRICTION MIMETICS Composition du jury Directeur de thèse Pr. Guido KROEMER Directeur de Recherche UMR1138 (CRC Paris) Rapporteur Pr. Pascal FERRE Directeur CRC (CRC Paris) Dr. Patricia BOYA Directeur de Laboratoire (CIB-CSIC- Madrid) Examinateur Pr. Jose M. FUENTES Directeur de Laboratoire (UNEX RODRIGUEZ Caceres) Pr. Laurence ZITVOGEL Directeur de Recherche UMR1015 (IGR Villejuif) Dr. Maria Chiara MAIURI Chargé de Recherche UMR1138 (CRC Paris) INDEX ABBREVIATIONS ........................................................................................................................................ 2 I. SUMMARY ........................................................................................................................................... 11 II. INTRODUCTION .............................................................................................................................. 14 A. GENERAL PRINCIPLES OF THE AUTOPHAGIC PATHWAYS ........................................................... 14 B. MECHANISMS AND REGULATION OF AUTOPHAGOSOME FORMATION ................................. 16 C. HOUSEKEEPING FUNCTIONS OF AUTOPHAGY ................................................................................... 26 D. AUTOPHAGIC RESPONSE TO METABOLIC STRESS: THE CROSS-TALK BETWEEN AUTOPHAGY AND METABOLISM ....................................................................................................................... 29 1. Energy Charge/ Glucose deprivation ........................................................................................................ 30 2. Hypoxia and ROS .............................................................................................................................................. 32 3. NADH/NAD Ratio ............................................................................................................................................. 33 4. Response to amino acids starvation ........................................................................................................... 35 5. Lipids ...................................................................................................................................................................... 38 6. Sensing of micronutrients: depletion of iron .......................................................................................... 40 7. Metabolic byproducts: Ammonia ................................................................................................................ 40 8. Transcription Factors, Metabolism and Autophagy ............................................................................ 40 9. Metabolic Regulation of Autophagy in vivo ............................................................................................ 42 E. ROLE OF AUTOPHAGY IN THE PATHOGENESIS OF DISEASE ........................................................ 45 1. Autophagy and Cancer .................................................................................................................................... 46 2. Autophagy in metabolic disease .................................................................................................................. 52 3. AUTOPHAGY AND AGING ......................................................................................................................... 55 III. AIM OF THE WORK ..................................................................................................................... 57 IV. RESULTS ........................................................................................................................................... 70 V. MATERIALS AND METHODS ..................................................................................................... 72 VI. DISCUSSION .................................................................................................................................... 90 VII. REFERENCES .............................................................................................................................. 107 1 ABBREVIATIONS 2-DG = 2-DeoxyGlucose 4EBP1 = Eukaryotic translation initiation factor 4E Binding Protein 1 ACAC = Acetyl CoA Carboxylase AcCoA = AcetylCoA ACLY = ATP Citrate Lyase ACO1 = Aconitase 1 ACSS = Acyl-CoA Synthetase Short-chain family Akt = v-Akt murine thymoma viral oncogene homolog 1 ALDH = Aldehyde Dehydrogenase AMBRA1 = Activating Molecule in BECN-1 Regulated Autophagy protein 1 AMP = Adenosine MonoPhosphate AMPK = Adenosine MonoPhosphate-activated Protein Kinase APP = Aβ-extracellular amyloid plaques derived from Amyloid Precursor Protein ATF4 = Activating Transcription Factor 4 ATG = Autophagy related 2 ATP = Adenosine TriPhosphate Bak = BCL2-antagonist/killer Bax = BCL2-associated X protein BCAA = Branched-Chain Amino Acids BCAT1 = Branched-Chain amino Acid Transaminase 1 BCKD = Branched-Chain α-Ketoacid Dehydrogenase Bcl-2 = member B-cell lymphoma 2 BDH1 = 3-HydroxyButyrate Dehydrogenase, type 1 BECN1 = Beclin-1 BNIP3 = BCL2/adenovirus E1B 19kDa interacting Protein 3-like JNK1 = c-Jun N-terminal Kinase 1 CAMMK2 = Calcium/calmodulin-dependent protein Kinase 2 CLEAR = Coordinate Lysosome Expression And Regulation CMA = Chaperone Mediated Autophagy CNS = Central Nervous System CoA = Coenzyme A 3 CPT1 = Carnitine PalmitoylTransferase 1 CR = Caloric Restriction CRAT = Carnitine O-AcetylTransferase CRM = Caloric Restriction Mimetics CS = Citrate Synthase DAMP = Damage-Associated Molecular Pattern molecules DCA = Dichloroacetate DEPTOR = DEP domanin containing MTOR-interacting protein DFCP1 = Double FYVE Containing Protein 1 DLAT = DihydroLipoamide S-AcetylTransferase DLD = DihydroLipoamide Dehydrogenase DR = Dietary Restriction EGFR = Epithelial Growth Factor Receptor EIF2AK3 = Eukaryotic translation Initiation Factor 2-Alpha Kinase 3 eIF2α = eukaryotic translation Initiation Factor 2 alpha ER = Endoplasmic Reticulum 4 ERGIC = ER-Golgi Intermediate Compartment FIP200 = FAK family kinase activating protein of 200 kDa FOXO1 = Forkhead box O1 GAAC = General Amino Acids Control GAG = Glycosaminoglycans GAP = Guanosine triphosphatase Activating Protein GATOR = GAP activity towards Rag GFP = Green Fluorescent Protein GH = Growth Hormone GLS = Glutaminase GOT2 = Glutamic-Oxaloacetic Transaminase 2 GTP = Guanosine TriPhosphate HSC70 = Heat Shock Cognate 70 HDAC = Histone Deacetylases HFD = High Fat Diet HIF1-α = Hypoxia Inducible Factor 1-alpha 5 HAT = Histone AcetylTransferases HK-II = Hexokinase-II HPV = Human Papilloma Virus ICD = Immunogenic Cell Death IDH1 = Isocitrate Dehydrogenase 1 IGF-1 = Insulin Growth Factor -1 IGFBP = Insulin-like Growth Factor Binding Protein Ikkβ = Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta I IL = Interleukin KAT = Lysine acetyltransferases Keap1 = Kelch-like ECH-associated protein 1 LAMP-2A = Lysosome-Associated Membrane Protein 2 ESCRT = Endosomal Sorting Complex Required for Transport LIR = LC3 Interacting LKB1 = Liver Kinase B1 LRRK2 = Leucine-Rich Repeat Kinase 2 6 MAM = Mitochondria-Associated Membrane MAPK = Mitogen-Activated Protein Kinase MDH1 = Malate Dehydrogenase 1 ME = Malic Enzyme MEF = Mouse Embryo Fibroblast mETC = mitochondrial Electron Transport Chain MPC = Mitochondrial Pyruvate Carrier mTORC1 = mammalian Target Of Rapamycin Complex 1 MVB = Multivescicolar Body NAD = Nicotinamide Dinucleotide NADH = Nicotinamide Adenine Dinucleotide NADP = Nicotinamide Dinucleotide Phosphate NAMPT = Nicotinamide Phosphoribosyltransferase NBR1 = Neighbor of BRCA1 gene 1 Nrf2 = Nuclear Factor, Erythroid 2-Like 2 OIS = Oncogene Induced Senescence 7 OXCT1 = 3-Oxoacid CoA Transferase 1 OXPHOS = Oxidative Phosphorylation PAMP = Pathogen-Associated Molecular Patterns PDH = Pyruvate Dehydrogenase PDHX = Pyruvate Dehydrogenase complex, component X PDK = Pyruvate Dehydrogenase Kinase PDP = Pyruvate Dehydrogenase Phosphatase PE = PhosphatidylEthanolamine PGC-1α = Proliferator-activated receptor Gamma Coactivator 1-alpha PI3KIII = Class III Phosphatidylinositol-3 Kinase PINK1 = PTEN Induced putative Kinase 1 PKD = Protein Kinase D PNPLA5 = Patatin-like phospholipase domain containing 5 PRAS40 = Proline-Rich Akt Substrate of 40 kilodaltons PTEN = Phosphatase and Tensin homolog RAPTOR = Regulatory Associated Protein of mTOR 8 RB1CC1 = RB-1 inducible Coiled-Coil 1 RHEB = Ras Homolog Enriched in Brain ROS = Reactive Oxygen Species RTK = Receptor Tyrosine Kinases SCD1 = Stearoyl-CoA Desaturase SILAC = Stable Isotope Labeling with Aminoacids in cell Culture siRNA = small interfering RNA SIRT1 = Sirtuin 1 SLC = Solute carrier SQSTM1 = Sequestosome 1 SREBP2 = Sterol Regulatory Element Binding Protein 2 TAB1 = Inhibitors TGF-beta activated kinase 1/ MAP3K7 binding protein 1 TAK1 = Transforming growth factor beta-activated kinase 1 TCA = Tricarboxylic Acid TFEB = Transcription Factor EB TSC = Tuberous Sclerosis Complex 9 ULK1 = UNC51-like Ser/Thr kinases UPR = Unfolded Protein Response UVRAG = UV Radiation resistance-Associated Gene VDAC1 = Voltage-Dependent Anion Channel 1 ZKSCAN3 = Zinc finger with KRAB and SCAN domains 3 10 I. SUMMARY Autophagy is a self-digestion process in which cell degrades its own components in order to maintain homeostasis