Fakultät für Medizin III. Medizinische Klinik und Poliklinik The role of the deubiquitinase USP24 in multiple myeloma Laura Häusler Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Medizin (Dr. med.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Jürgen Schlegel Prüfer der Dissertation: 1. Prof. Dr. Florian Bassermann 2. Prof. Dr. Christian Peschel Die Dissertation wurde am 10.02.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 28.03.2018 angenommen. Table of Contents Abstract ....................................................................................................................... 5 List of Figures ............................................................................................................ 6 List of Tables .............................................................................................................. 7 Abbreviations ............................................................................................................. 8 1 Introduction ........................................................................................................ 11 1.1 Multiple myeloma ........................................................................................ 11 1.1.1 Genetic aberrations and molecular pathogenesis .................................. 11 1.1.2 Clinical manifestation and diagnosis ....................................................... 14 1.1.3 Current treatment .................................................................................... 16 1.2 The ubiquitin proteasome system in health and disease ....................... 19 1.2.1 Ubiquitin and the proteasome ................................................................. 19 1.2.2 Ubiquitination .......................................................................................... 20 1.2.2.1 The ubiquitin cascade ....................................................................... 20 1.2.2.2 Ubiquitin-ligases ................................................................................ 21 1.2.3 Deubiquitination ...................................................................................... 24 1.2.4 The Usp24 gene locus in MM ................................................................. 26 1.3 The TGF-beta signaling pathway ............................................................... 28 1.3.1 The TGF-beta superfamily ...................................................................... 28 1.3.2 SMAD family-dependent TGF-beta signaling ......................................... 29 1.3.3 Regulation of the TGF-beta signaling pathway ....................................... 32 1.3.4 The TGF-beta system in MM .................................................................. 35 1.4 Aim of the study .......................................................................................... 37 2 Materials and Methods ...................................................................................... 38 2.1 Materials ....................................................................................................... 38 2.1.1 Chemicals ............................................................................................... 38 2.1.2 Media and supplements for cell culture .................................................. 40 2.1.3 Antibiotics ............................................................................................... 40 2.1.4 Transfection reagents ............................................................................. 40 2.1.5 Enzymes ................................................................................................. 40 2.1.6 Inhibitors ................................................................................................. 40 2.1.7 Molecular weight standards .................................................................... 41 2.1.8 Molecular biological Kits ......................................................................... 41 THE ROLE OF USP24 IN MM 2 2.1.9 Buffers .................................................................................................... 41 2.1.10 Antibodies ............................................................................................. 43 2.1.11 Plasmids ............................................................................................... 44 2.1.12 Oligonucleotides ................................................................................... 45 2.1.13 Bacteria ................................................................................................. 47 2.1.14 Cell lines ............................................................................................... 47 2.1.15 Cell culture dishes ................................................................................ 47 2.1.16 Membranes ........................................................................................... 47 2.1.17 Machinery and equipment .................................................................... 48 2.1.18 Software and databases ....................................................................... 48 2.2 Methods ....................................................................................................... 49 2.2.1 Molecular biology .................................................................................... 49 2.2.1.1 Agarose gel electrophoresis .............................................................. 49 2.2.1.2 DNA restriction .................................................................................. 49 2.2.1.3 DNA ligation ...................................................................................... 49 2.2.1.4 Molecular cloning and DNA extraction .............................................. 50 2.2.1.5 Gene silencing .................................................................................. 50 2.2.1.6 PCR ................................................................................................... 51 2.2.1.7 Reverse transcription ........................................................................ 51 2.2.1.8 qPCR ................................................................................................. 52 2.2.2 Protein biochemistry ............................................................................... 52 2.2.2.1 SDS-gel electrophoresis ................................................................... 52 2.2.2.2 Western blot analysis ........................................................................ 53 2.2.2.3 Cell lysis and fractionation ................................................................ 54 2.2.2.4 Immunoprecipitation .......................................................................... 54 2.2.2.5 Mass spectrometry ............................................................................ 55 2.2.2.6 SILAC ................................................................................................ 56 2.2.2.7 Ubiquitination assay .......................................................................... 57 2.2.3 Eukaryotic cell culture ............................................................................. 57 2.2.3.1 Plasmid and siRNA transfections ...................................................... 58 2.2.3.2 Lentivirus production ......................................................................... 59 2.2.3.3 Viral infection ..................................................................................... 59 2.2.3.4 Proteasome inhibition ........................................................................ 60 2.2.3.5 Ribosome inhibition ........................................................................... 60 2.2.3.6 TGF-beta signaling ............................................................................ 60 THE ROLE OF USP24 IN MM 3 2.2.3.7 Flow cytometry .................................................................................. 61 2.2.4 Bacterial cell culture and transformation ................................................. 62 2.2.5 Data mining and processing ................................................................... 62 3 Results ................................................................................................................ 64 3.1 Loss of the Usp24 gene locus in MM ........................................................ 64 3.1.1 FISH analysis of MM patient samples .................................................... 64 3.1.2 Usp24 mRNA and protein levels in different MM cell lines ..................... 65 3.2 USP24 inhibits proliferation in U2OS cells ............................................... 67 3.3 WWP2 as important binding partner of USP24 ........................................ 69 3.3.1 Specific binding of USP24 to WWP2 ...................................................... 71 3.3.2 WWP2 is not a substrate of USP24 ........................................................ 73 3.4 USP24 and proteins of the SMAD family .................................................. 76 3.4.1 Binding of USP24 to activating SMAD2 and 3 ........................................ 76 3.4.2 USP24 depletion and phosphorylation of SMAD3 .................................. 77 3.5 Proteins of the SMAD family as possible substrates of USP24 ............. 79 3.5.1 USP24 has no influence on SMAD stability ............................................ 79 3.5.2 SMAD2 is not a substrate