TECHNISCHE UNIVERSITÄT MÜNCHEN Fakultät für Medizin III. Medizinische Klinik und Poliklinik Klinikum rechts der Isar Dissecting aberrant proteolytic ubiquitin networks in cancer Carmen Gloria Richter (M.Sc.) Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Dr. Andreas Pichlmair Prüfer der Dissertation: 1. Prof. Dr. Florian Bassermann 2. Prof. Dr. Bernhard Küster 3. Prof. Dr. Sebastian Theurich Die Dissertation wurde am 01.08.2019 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 07.04.2020 angenommen. Content 1 Summary ................................................................................................................... 1 2 Introduction .............................................................................................................. 3 2.1 Multiple myeloma ............................................................................................................3 2.1.1 Pathophysiology .........................................................................................................3 2.1.2 Clinical manifestation and diagnosis ...........................................................................5 2.1.3 Treatment ...................................................................................................................6 2.1.4 The role of MYC in multiple myeloma .........................................................................7 2.2 The ubiquitin code ...........................................................................................................7 2.2.1 Ubiquitin .....................................................................................................................8 2.2.2 Ubiquitylation cascade ...............................................................................................9 2.2.3 SCF family of ubiquitin E3 ligases .............................................................................10 2.2.4 The human F-box protein FBXL13 ............................................................................12 2.2.5 Deubiquitinases ........................................................................................................12 2.2.6 The OTU family DUB OTUD6B ..................................................................................14 2.2.7 The proteasome ........................................................................................................15 2.3 LIN28 ..............................................................................................................................16 2.3.1 Physiological role of LIN28 .......................................................................................16 2.3.2 The role of LIN28B in cancer.....................................................................................18 2.4 The cell cycle .................................................................................................................20 2.4.1 Overview of the cell cycle .........................................................................................20 2.4.2 G1/S transition ..........................................................................................................22 2.5 Centrosomes .................................................................................................................22 2.5.1 Centrosome functions and structure .........................................................................22 2.5.2 The centrosome cycle ...............................................................................................23 2.6 Aim of the study ............................................................................................................25 3 Material ................................................................................................................... 27 3.1 Devices and instruments ..............................................................................................27 3.2 Consumables .................................................................................................................28 3.3 Chemicals and reagents ...............................................................................................29 3.4 Commercial kits ............................................................................................................32 3.5 Enzymes .........................................................................................................................32 3.6 Oligonucleotides ...........................................................................................................33 3.6.1 Cloning oligonucleotides ...........................................................................................33 I 3.6.2 Oligonucleotides for amplicon deep-sequencing ...................................................... 34 3.6.3 qPCR oligonucleotides ............................................................................................. 34 3.6.4 Sequencing oligonucleotides .................................................................................... 35 3.7 Sequences of shRNAs and siRNAs .............................................................................. 35 3.7.1 Sequences of shRNAs .............................................................................................. 35 3.7.2 Sequences of siRNAs ............................................................................................... 36 3.8 Plasmids ........................................................................................................................ 36 3.9 Bacteria .......................................................................................................................... 38 3.10 Standards for DNA and proteins electrophoresis ....................................................... 38 3.11 Antibodies ...................................................................................................................... 38 3.11.1 Primary antibodies .................................................................................................... 38 3.11.2 Custom made primary antibodies ............................................................................. 39 3.11.3 Conjugated primary antibodies ................................................................................. 39 3.11.4 Conjugated secondary antibodies ............................................................................ 39 3.12 Cell lines ........................................................................................................................ 40 3.13 Cell culture media and supplements ........................................................................... 40 3.14 Patient samples ............................................................................................................. 41 3.15 Solutions and buffers .................................................................................................... 41 3.16 Software and databases ............................................................................................... 44 4 Methods................................................................................................................... 45 4.1 Molecular cloning .......................................................................................................... 45 4.1.1 Polymerase chain reaction (PCR) .............................................................................. 45 4.1.2 Agarose gel electrophoresis and gel purification ...................................................... 45 4.1.3 DNA restriction digest and ligation............................................................................ 45 4.1.4 DNA mutagenesis ..................................................................................................... 46 4.1.5 Bacterial transformation ............................................................................................ 46 4.1.6 DNA extraction from bacteria ................................................................................... 47 4.1.7 Annealing of shRNA oligonucleotides ....................................................................... 47 4.2 Generation of CRISPR/Cas9 sgRNA libraries ............................................................. 47 4.2.1 Designing CRISPR/Cas9 libraries ............................................................................. 47 4.2.2 Annealing and dilution of libraries ............................................................................. 48 4.2.3 Vector digest and ligation of libraries ........................................................................ 48 4.2.4 Bacterial transformation and harvesting of libraries .................................................. 48 4.2.5 CRISPR/Cas9 screen ................................................................................................ 49 4.2.6 Sample preparation for Illumina next-generation sequencing ................................... 49 II 4.2.7 Illumina MiSeq sequencing .......................................................................................50 4.3 Gene expression analysis .............................................................................................51 4.3.1 RNA extraction from eukaryotic cells ........................................................................51