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The Search for Deconjugating Enzymes of FAT10

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The Search for Deconjugating Enzymes of FAT10 The Search for Deconjugating Enzymes of FAT10 Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Faiz Ahmad an der Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie Day of oral examination: 22 April 2016 Referee: Prof. Dr. Marcus Groettrup Referee: Prof. Dr. Martin Scheffner Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-334261 Table of contents I Table of Contents Abbreviations ............................................................................................................. III Tables ...................................................................................................................... VIII Figures ....................................................................................................................... IX Summary .................................................................................................................... X Zusammenfassung .................................................................................................... XI 1. Introduction ........................................................................................................... 1 1.1 Ubiquitin proteasome system ...................................................................... 1 1.1.1 Proteasome ................................................................................ 1 1.1.2 Ubiquitination.............................................................................. 4 1.1.3 E3 ligases ................................................................................... 6 1.1.4 Deubiquitination.......................................................................... 7 1.2 Ubiquitin-like modifiers ................................................................................ 9 1.2.1 ISG15 ....................................................................................... 10 1.2.2 SUMO....................................................................................... 11 1.2.3 NEDD8 ..................................................................................... 12 1.3 Ubiquitin-like modifier (FAT10) .................................................................. 13 1.4 Deconjugating enzymes (DUBs) ............................................................... 16 1.4.1 USP Domain DUBs .................................................................. 17 1.4.2 OTU Domain DUBs .................................................................. 17 1.4.3 JAMM/MPN+ Domain DUBs .................................................... 17 1.4.4 UCH Domain DUBs .................................................................. 18 1.4.5 Josephin Domain DUBs ........................................................... 18 1.5 Catalytic mechanism ................................................................................. 19 1.6 DUBomics ................................................................................................. 20 1.7 Deubiquitinase specificity .......................................................................... 20 1.8 Regulation of DUB activity ......................................................................... 24 1.9 DUBs as a component of protein degradation machineries ...................... 25 1.9.1 Proteasome associated DUBs ................................................. 25 1.9.2 ESCRT DUBs ........................................................................... 25 1.9.3 Cdc48/p97/VCP associated DUB ............................................. 26 1.10 DUBs as a player in cellular processes ................................................... 27 1.11 Tissue and subcellular distribution of DUBs ............................................ 27 1.12 Immunoregulatory DUBs ......................................................................... 27 1.13 Cytokine inducible DUBs ......................................................................... 30 1.14 DUBs in human health and disease ........................................................ 31 1.14.1 Ubiquitin specific protease 7 (USP7) ...................................... 31 1.14.2 DUB3 (USP17L2) ................................................................... 32 1.14.3 USP11 .................................................................................... 32 1.14.4 USP6 and USP16................................................................... 32 1.14.5 CYLD ...................................................................................... 33 1.14.6 VCIP135 ................................................................................. 33 1.14.7 A20 and BAP1 ........................................................................ 33 1.14.8 ATXN3 .................................................................................... 34 1.15 SENPs ..................................................................................................... 34 Table of contents II 1.16 DUBs and cancer .................................................................................... 34 1.17 DUBs as a druggable targets .................................................................. 35 1.18 DUB inhibitors ......................................................................................... 36 1.19 DUBs in human pathogens ..................................................................... 37 2. Aim of the Thesis ................................................................................................ 39 3. Results ................................................................................................................. 40 3.1 Search for FAT10 Deconjugating Enzymes .............................................. 40 3.2 The Impact of p53 overexpression on FAT10 ........................................... 64 3.3 Investigation of FAT10 and Foxp3 interaction ........................................... 69 3.4 Investigation of FAT10 (diubiquitin) or FAT10 conjugates stimulate 26S proteasome activity ......................................................... 71 4. Discussion ........................................................................................................... 79 5. Material and Methods ......................................................................................... 91 5.1.1 E. coli competent cell preparation ......................................... 91 5.1.2 Transformation of chemically competent bacteria ................. 92 5.1.3 Plasmid DNA purification ....................................................... 92 5.1.4 Agarose gel electrophoresis .................................................. 93 5.2 Cell lines .................................................................................................... 94 5.2.1 Transfection, FAT10 induction .............................................. 95 5.2.2 Immunoprecipitation (IP) ....................................................... 95 5.2.3 SDS-PAGE ............................................................................ 96 5.2.4 SDS-PAGE staining .............................................................. 97 5.2.5 Immunoblotting ...................................................................... 98 5.3. Quantification of gene expression ............................................................. 99 5.3.1 siRNA mediated knockdown ................................................. 99 5.3.2 Total RNA extraction ........................................................... 100 5.3.3 Complementary DNA synthesis .......................................... 100 5.3.4 Quantitative reat time PCR (qRT-PCR) ............................... 101 5.4 GST Pull-down assay .............................................................................. 102 5.5 Generation of stable cells ........................................................................ 103 5.5.1 Antibiotic killing curve ........................................................... 104 5.6 DUB activity assay.. ................................................................................ 105 5.6.1 Elution by Flag-peptide or HA-peptide ................................ 105 5.6.2 In-vitro conjugation assay .................................................... 106 5.6.3 In-vitro transcription and translation of p53 (IVTT) .............. 106 5.6.4 In-vitro p53 ubiquitination assay .......................................... 107 5.6.5 In-vitro p53 de-ubiquitination assay ..................................... 107 5.7 Peptidase activities of the 26S proteasome ............................................ 108 5.8 Softwares ................................................................................................ 109 5.9 Statistical analysis ................................................................................... 109 6. References ........................................................................................................ 110 7.9 Record of contributions ........................................................................... 128 5.9 Acknowledgements ................................................................................. 129 Abbreviations III Abbreviations Abx antibiotic AIPL1 aryl hydrocarbon receptor interacting protein-like 1 AMC 7-amino-4-methylcoumarin AMSH associated molecule with the SH3 domain of STAM AMV avian myeloblastosis virus APC anaphase promoting complex ATG15 autophagy related protein 15 ATP adenosine triphosphate BAP1 BRCA1 associated protein-1;
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