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Dissertation Submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University O Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Kai Richter, M.Sc. born in: Bremen, Germany Oral examination: 26th October 2017 Identification and characterization of novel regulators of the tumor suppressor and ubiquitin ligase SCF-FBXW7 Referees: Prof. Dr. Ingrid Hoffmann Prof. Dr. Frauke Melchior Hiermit erkläre ich an Eides statt, dass ich die vorliegende Dissertation selbstständig und ohne unerlaubte Hilfsmittel durchgeführt habe. Heidelberg, den 27. Juli 2017 …………………………………………. (Kai Richter) Table of contents Table of contents I. Summary ................................................................................................................. 1 II. Zusammenfassung ................................................................................................ 3 1. Introduction ............................................................................................................ 5 1.1. The ubiquitin system ........................................................................................ 5 1.1.1. The ubiquitylation reaction ........................................................................ 5 1.1.2. Types of ubiquitin ligases .......................................................................... 7 1.1.3. The ubiquitin code ..................................................................................... 8 1.1.4. Effects of substrate ubiquitylation ............................................................. 8 1.1.5. Ubiquitin-like modifiers .............................................................................. 9 1.2. The SCF complex is a Cullin-RING ubiquitin ligase ......................................... 9 1.2.1. Cullin-RING ubiquitin ligases (CRLs) ........................................................ 9 1.2.2. Regulation of CRL activity ....................................................................... 11 1.2.3. The SCF complex uses F-box proteins to target its substrates .............. 11 1.3. FBXW7 is a tumor suppressor protein ........................................................... 13 1.3.1. FBXW7 is conserved from yeast to human ............................................. 13 1.3.2. Domain organization of human FBXW7 .................................................. 14 1.3.3. Human FBXW7 isoforms ........................................................................ 14 1.3.4. The CDC4 phosphodegron ..................................................................... 15 1.3.5. FBXW7 substrates .................................................................................. 16 1.3.5.1. FBXW7 mediates the degradation of important oncoproteins ......... 16 1.3.5.2. Regulation of transcription by FBXW7 ............................................. 17 1.3.5.3. FBXW7 is a regulator of cell survival ............................................... 18 1.3.5.4. Other substrates of FBXW7 ............................................................. 18 1.3.6. FBXW7 is frequently mutated in human cancers .................................... 18 1.3.7. Regulation of FBXW7 ............................................................................. 19 1.3.7.1. Regulation of FBXW7 expression by transcription factors ............... 19 1.3.7.2. Regulation of FBXW7 expression by microRNAs ............................ 19 1.3.7.3. Regulation of FBXW7 protein stability ............................................. 19 1.3.7.4. Regulation of FBXW7 protein localization ....................................... 20 1.3.7.5. Regulation of SCF-FBXW7 ubiquitin ligase activity ......................... 21 1.4. MYCBP2 and FBXO45 form a ubiquitin ligase complex ................................ 21 1.5. RAE1 is a mitotic regulator that interacts with MYCBP2 ................................ 22 Table of contents 1.6. XIAP is an anti-apoptotic ubiquitin ligase ....................................................... 23 1.7. Objectives ....................................................................................................... 23 2. Materials and methods ........................................................................................ 25 2.1. Materials ......................................................................................................... 25 2.1.1. Chemicals and reagents ......................................................................... 25 2.1.2. Laboratory equipment ............................................................................. 27 2.1.3. Buffers and media ................................................................................... 29 2.1.4. Antibodies ............................................................................................... 31 2.1.4.1. Primary antibodies ........................................................................... 31 2.1.4.2. Secondary antibodies ...................................................................... 32 2.1.5. Small interfering RNAs (siRNAs) ............................................................ 32 2.1.6. Primers .................................................................................................... 33 2.1.7. Plasmids ................................................................................................. 36 2.1.8. Bacterial strains ...................................................................................... 38 2.1.9. Cell lines ................................................................................................. 39 2.1.10. Kits ........................................................................................................ 39 2.1.11. Antibiotics .............................................................................................. 39 2.2. Methods .......................................................................................................... 40 2.2.1. Methods in molecular biology ................................................................. 40 2.2.1.1. Polymerase chain reaction (PCR) ................................................... 40 2.2.1.2. Agarose gel electrophoresis ............................................................ 41 2.2.1.3. Restriction digest of DNA ................................................................. 41 2.2.1.4. Extraction of DNA fragments from agarose gels ............................. 41 2.2.1.5. Ligation of DNA fragments ............................................................... 42 2.2.1.6. Transformation of chemically competent E. coli .............................. 42 2.2.1.7. Isolation of plasmid DNA from E. coli .............................................. 42 2.2.1.8. Determination of DNA concentration ............................................... 43 2.2.2. Methods in cell biology ............................................................................ 43 2.2.2.1. Cell culture ....................................................................................... 43 2.2.2.2. Harvesting and freezing of cells ....................................................... 43 2.2.2.3. Transient transfection of mammalian cells ...................................... 44 2.2.2.3.1. Transfection of cells with plasmid DNA using polyethylenimine (PEI) .............................................................................. 44 2.2.2.3.2. Transfection of cells with siRNA using Lipofectamine 2000 ..... 44 Table of contents 2.2.2.4. Cell cycle synchronization ............................................................... 45 2.2.2.5. MG132 and cycloheximide treatment .............................................. 45 2.2.2.6. Live-cell imaging .............................................................................. 45 2.2.3. Methods in protein biochemistry ............................................................. 46 2.2.3.1. Preparation of protein extracts from mammalian cells ..................... 46 2.2.3.2. Determination of protein concentration ............................................ 46 2.2.3.3. SDS polyacrylamide gel electrophoresis (SDS-PAGE) ................... 47 2.2.3.4. Electrotransfer and Western blot ..................................................... 47 2.2.3.5. Immunoprecipitation assays ............................................................ 48 2.2.3.5.1. Immunoprecipitation of Flag-tagged proteins using Flag M2 affinity beads ............................................................................................ 48 2.2.3.5.2. Immunoprecipitation of endogenous proteins using NHS- activated sepharose ................................................................................. 48 2.2.3.5.3. Sequential immunoprecipitation for the analysis of protein complexes .................................................................................... 49 2.2.3.6. Colloidal Coomassie staining ........................................................... 49 2.2.3.7. Mass spectrometry analysis ............................................................ 50 2.2.3.8. In vivo ubiquitylation assays ............................................................ 50 2.2.3.9. Expression and purification of recombinant proteins ....................... 51 2.2.3.9.1. Expression and purification of MBP-tagged proteins ................ 51 2.2.3.9.2. Expression and purification of His-tagged proteins .................. 52 2.2.3.10. In vitro transcription and translation and in vitro
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