MuRF3 binds to the retromer subunit SNX5 inhibiting its MuRF2-mediated degradation and leading to its stabilization Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von Diplom Biologin Jida Hamati Präsident der Humboldt-Universität zu Berlin: Prof. Dr. Jan-Hendrik Olbertz Dekan der Lebenswissenschaftlichen Fakultät: Prof. Dr. Richard Lucius Gutachter: 1. Prof. Dr. Thomas Sommer 2. PD Dr. med. Jens Fielitz 3. Prof. Dr. Oliver Daumke Tag der mündlichen Prüfung: 01.12.2015 Index Table of Contents List of Figures ......................................................................................................................................... 4 List of Tables ........................................................................................................................................... 5 Abstract ................................................................................................................................................... 6 Zusammenfassung ................................................................................................................................... 7 1 Introduction ......................................................................................................................................... 8 1.1. The skeletal muscle .................................................................................................................... 8 1.1.1. Structure and composition ................................................................................................ 8 1.1.2. Skeletal muscle physiology - the sarcomere .................................................................. 10 1.2. Skeletal muscle atrophy ............................................................................................................ 11 1.3. Ubiquitin proteasome system ................................................................................................... 12 1.4. Muscle specific ubiquitin E3 ligases ........................................................................................ 15 1.4.1. Muscle specific RING finger protein 1 (MuRF1) .......................................................... 15 1.4.2. Muscle specific RING finger protein 2 (MuRF2) .......................................................... 16 1.4.3. Muscle specific RING finger protein 3 (MuRF3) .......................................................... 17 1.4.4. Association of MuRF2 and MuRF3 with microtubules ................................................. 18 1.5. Membrane trafficking pathways in skeletal muscle ................................................................. 19 1.5.1. Endosomal trafficking pathway ..................................................................................... 20 1.5.2. Endosome maturation ..................................................................................................... 22 1.5.3. Retrograde and recycling pathways and the Retromer - complex .................................. 23 1.5.4. Endosome motility along microtubules .......................................................................... 26 1.6. Sorting nexin 5 (SNX5) ............................................................................................................ 27 1.7. SNX5 and the GLUT4 trafficking pathway.............................................................................. 28 1.8. Aim of the study ....................................................................................................................... 30 2 Materials and Methods ...................................................................................................................... 31 2.1. Materials and instruments ......................................................................................................... 31 2.1.1. Equipment and instruments ............................................................................................ 31 2.1.2. Reagents and chemicals ................................................................................................. 33 2.1.3. Antibodies ...................................................................................................................... 36 2.1.4. Primers ........................................................................................................................... 37 2.1.5. Expression plasmids ....................................................................................................... 38 2.2. Molecular biological methods .................................................................................................. 38 2.2.1. Polymerase chain reaction (PCR) .................................................................................. 38 2.2.2. Site - directed mutagenesis PCR .................................................................................... 39 2.2.3. Transformation of competent bacteria with DNA .......................................................... 41 2.2.4. Preparation of plasmid DNA by alkaline lysis with SDS............................................... 41 1 Index 2.2.5. Cleavage of DNA using restriction endonucleases ........................................................ 42 2.2.6. Ligation of DNA fragments ........................................................................................... 42 2.2.7. DNA sequence analysis .................................................................................................. 43 2.2.8. DNA gel electrophoresis ................................................................................................ 43 2.2.9. Extraction of DNA fragments from agarose-gels ........................................................... 43 2.2.10. RNA isolation from cells and tissues ............................................................................. 44 2.2.11. cDNA synthesis .............................................................................................................. 44 2.2.12. Quantitative real - time polymerase chain reaction (qRT-PCR) .................................... 44 2.3. Protein biochemical methods.................................................................................................... 45 2.3.1. Immunocytochemistry (ICC) of mammalian cells ......................................................... 45 2.3.2. Isolation of proteins from cells and tissues .................................................................... 46 2.3.3. Affinity purification- and co-immunoprecipitation-assays ............................................ 47 2.3.4. Sodium Dodecyle Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).............. 48 2.3.5. Western blot analysis ..................................................................................................... 49 2.3.6. Ubiquitination assays ..................................................................................................... 49 2.3.7. Establishing MBP-MuRF recombinant proteins ............................................................ 50 2.3.8. Mass spectrometric analysis ........................................................................................... 51 2.4. Cell culture methods ................................................................................................................. 52 2.4.1. Cell lines and culturing of cells ...................................................................................... 52 2.4.2. Differentiation and atrophy of mouse skeletal muscle cells ........................................... 52 2.4.3. Cryoconservation and thawing of cells .......................................................................... 53 2.4.4. Transfection of mammalian cells ................................................................................... 53 2.4.5. Establishing stably transfected cell lines ........................................................................ 54 2.4.6. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) ................................. 55 2.4.7. Cycloheximide chase assays .......................................................................................... 55 2.5. Animal experiments .................................................................................................................. 55 2.5.1. Denervation-induced skeletal muscle atrophy ............................................................... 56 2.5.2. Starvation-induced skeletal muscle atrophy ................................................................... 56 2.6. Fluorescence and confocal microscopy .................................................................................... 56 3 Results ............................................................................................................................................... 57 3.1. Detection of novel interaction partners for MuRF1, 2 and 3 using SILAC-AP-MS ................ 57 3.1.1. Cloning and over-expression of MuRF1, MuRF2, and MuRF3 and SILAC .................... labeling of cells.............................................................................................................. 57 3.1.2. SILAC- labeling of mouse skeletal muscle cells ............................................................ 58 3.1.3. Detection of MuRF1, 2, or 3- interaction partners in C2C12 myoblasts ....................... 59 3.1.4. Detection