Departement de Biochimie Characterization of Tollip in the Interleulkin-1 Receptor/Toll Like Receptors signaling pathways Thèse de doctorat ès sciences de la vie (PhD) Présentée à la Faculté de Biologie et de Médecine de l’Université de Lausanne Par Brian Brissoni Diplômé en Biochimie, Université de Lausanne Jury de thèse: Jürg Tschopp, Directeur de thèse Nicolas Mermod, Président de jury et rapporteur Kimberly Burns-DiMarco, Expert Jean-Claude Sirard, Expert Frederic Levy, Expert Lausanne, 2005 Brian Brissoni Thesis Report SUMMARY ......................................................................................................................................................5 RESUME .........................................................................................................................................................6 GENERAL INTRODUCTION: 1. IMMUNITY AND INFLAMMATION .........................................................................................................11 1.1 THE IMMUNE RESPONSE ........................................................................................................................ 11 1.1.1 The Innate immunity............................................................................................................................13 1.1.1.1 Pathogens/danger recognition ...............................................................................................................13 1.1.1.2 Innate immunity induced response.........................................................................................................14 1.1.2 The adaptive response.........................................................................................................................16 1.2 THE INFLAMMATORY RESPONSE ............................................................................................................. 18 2. THE TIR RECEPTORS SUPERFAMILY ..................................................................................................19 2.0. INTRODUCTION .................................................................................................................................... 19 2.1 THE TOLL RECEPTORS .......................................................................................................................... 21 2.2 MAMMALIAN TOLL-LIKE RECEPTORS AND THEIR LIGANDS......................................................................... 21 2.3 THE IL-1R-LIKE FAMILY AND LIGANDS .................................................................................................... 24 2.3.1 Interleukin-1.........................................................................................................................................24 2.3.2 The interleukin-1 Receptors................................................................................................................25 2.4 TIR RECEPTOR SUPERFAMILY SIGNALING................................................................................................ 27 2.4.1 MyD88-depdendent and independent signaling ................................................................................27 2.4.2 IL-1R/TLR Signaling pathways ..........................................................................................................28 CHAPTER 1: Tollip deficient mice characterization 1. INTRODUCTION: TOLLIP (TOLL INTERACTING PROTEIN)........................................................32 1.1 TOLLIP STRUCTURE AND FEATURES ........................................................................................................ 32 1.2 TOLLIP AND TLR/IL-1R SIGNALING ........................................................................................................ 32 2. MATERIAL AND METHODS ......................................................................................................................34 3. RESULTS AND DISCUSSION ......................................................................................................................37 3.0 OBJECTIVES ......................................................................................................................................... 37 Rational .........................................................................................................................................................37 Overall objective: .........................................................................................................................................37 3.1 GENERATION OF TOLLIP DEFICIENT MICE ................................................................................................. 38 3.2 ANALYSIS OF IκBα DEGRADATION AND MAPK PHOSPHORYLATION IN TOLLIP-/- CELLS ............................... 40 3.3 ACTIVATION OF IMMUNE EFFECTORS CELLS IN TOLLIP-/- MICE .................................................................... 43 3.3.1 Normal TLR-induced activation and proliferation in Tollip-/- cells .................................................43 3.3.2 IL-1 and IL-18 induced activation and proliferation in Tollip-/- cells..............................................45 3.4 TOLLIP-/- MICE PRODUCE LESS CYTOKINE FOLLOWING IL-1β AND LPS STIMULATION .................................. 47 4. CONCLUSIONS...............................................................................................................................................50 5. DISCUSSION AND PERSPECTIVE............................................................................................................50 CHAPTER 2: Regulation of IL-1R endocytosis by Tollip and its CUE domain 1. INTRODUCTION ............................................................................................................................................57 1.1 UBIQUITINATION AND UBIQUITIN FUNCTIONS ..................................................................................... 57 1.1.1 Ubiquitin ..............................................................................................................................................57 1.1.2 The ubiquitination mechanism............................................................................................................57 1.1.2 Different forms of ubiquitination and their functions .......................................................................58 1.2 UBIQUITIN SIGNAL IN THE ENDOCYTIC PATHWAY....................................................................................... 60 1.2.1 The endocytosis and Endosome structure..........................................................................................60 1.2.2 Ubiquitin-dependent internalization and trafficking in the endocytic pathway..............................63 1.2.2.1 Ubiquitination of transmembrane receptors in yeast and mammals ...................................................63 1 Brian Brissoni Thesis Report 1.2.2.2 Ubiquitin as internalization and sorting signal for cargo proteins into the endosome......................65 1.2.3 Ubiquitin-receptors as endocytic adaptors........................................................................................66 1.3 THE CUE DOMAIN, AN UBIQUITIN BINDING DOMAIN ................................................................................. 67 2. MATERIAL AND METHODS ......................................................................................................................70 3. RESULTS AND DISCUSSIONS....................................................................................................................74 3.0 OBJECTIVES ......................................................................................................................................... 74 Rational: ........................................................................................................................................................74 Overall objectives .........................................................................................................................................74 3.1 TOLLIP CUE DOMAIN BINDS UBIQUITIN SPECIES ...................................................................................... 75 3.2 TOLLIP IS UBIQUITINATED ....................................................................................................................... 76 3.3 TOLLIP CUE IN NECESSARY FOR TOLLIP UBIQUITINATION ......................................................................... 80 3.4 GENERATION OF UBIQUITIN DEFICIENT MUTANT CUE................................................................................ 82 3.5 TOLLIP’S CUE-UBIQUITIN DEPENDENT IL-1R INTERACTION ..................................................................... 84 3.5 IL-1R CHAINS UBIQUITINATION............................................................................................................... 86 3.4 TOLLIP AND IL-1R INTERNALIZATION ...................................................................................................... 88 3.5 TOLLIP AND IL-1RS BINDING PROTEINS FOR SIGNALING AND ENDOCYTOSIS ............................................... 95 3.5.1 Tom1 as endocytic adaptor interacting with Tollip ..........................................................................95 3.5.2 Tom1 binds IL-1Rs with Tollip ...........................................................................................................96 3.5.3 TOLLIP INTERACTS WITH TRAF6, AND FORMS A COMPLEX WITH RAB PROTEINS: .................................... 99 3.5.3.1 Tollip interact