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Characterization of MAGED1 As a Component of E3 Ubiquitin Ligase Complexes

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Characterization of MAGED1 As a Component of E3 Ubiquitin Ligase Complexes Characterization of MAGED1 as a component of E3 ubiquitin ligase complexes Nora Riems Student number: 01206813 Promoter: Prof. Dr. Mathieu Bertrand Scientific supervisor: Dario Priem Master’s dissertation submitted to Ghent University to obtain the degree of Master of Science in Biochemistry and Biotechnology. Major Biomedical Biotechnology. Academic year: 2016 - 2017 Ghent University – Department of Biomedical Molecular Biology VIB – Center for Inflammation Research Research Group: Molecular Signalling and Cell Death Acknowledgments Many people have contributed to the realization of this master dissertation and I would like to give a well-deserved thank you to everybody. First of all, I would like to thank my promotor Mathieu Bertrand and scientific supervisor Dario Priem for giving me the opportunity to work on this project. I would like to express my sincere appreciation for your constant guidance and the immense amount of feedback. Without it, this project would have remained uncompleted. Thank you. I would also like to thank Ria Roelandt and Inge Bruggeman for the help and guidance throughout my project. You have not only helped met with practical work but you were also the persons I could turn to with all my questions. Next, I would like to warmly thank all the people of the research group for the fun moments in the lab. In particular Wannes, you have supported me in an emotional way and never failed to make me smile. Finally, I would like to thank my parents. Although the last couple of years have not always been the easiest, you never failed to continuously encourage and support me. You have not only helped me to accomplish my years of study but you also have set the best example I could wish for on the person I want to become. Nora Table of contents List of abbreviations ........................................................................................................................ 1 Resume............................................................................................................................................ 2 Abstract ........................................................................................................................................... 3 Introduction .................................................................................................................................... 4 1. Ubiquitination ................................................................................................................................... 5 1.1. Ubiquitin activating enzyme (E1) .............................................................................................. 7 1.2. Ubiquitin conjugating enzyme (E2) ........................................................................................... 7 1.3. Ubiquitin ligase (E3) .................................................................................................................. 8 1.3.1. HECT E3 ligases.................................................................................................................. 8 1.3.2. RING E3 ligases .................................................................................................................. 8 1.3.3. RING-IBR-RING E3 ligases .................................................................................................. 9 1.3.4. Multi-subunit RING ubiquitin ligase complexes .............................................................. 10 1.3.4.1. The cullin-RING ligase complexes ............................................................................... 10 1.3.4.2. The MAGE-RING ligase complexes .............................................................................. 11 1.4. DUBs ........................................................................................................................................ 13 2. Ubiquitination in the regulation of immune signaling pathways ................................................... 13 2.1. Pattern recognition receptor-mediated activation of the MAPKs and NF-κB pathways ........ 13 2.2. RING E3 ligases in the regulation of immune signaling pathways .......................................... 15 Aim ................................................................................................................................................ 17 Contributions of third parties ....................................................................................................... 18 Results ........................................................................................................................................... 19 1. Cloning of MAGE and RING-containing proteins into a bacterial expressing vector ...................... 19 1.1. Cloning of PELI3 ....................................................................................................................... 20 1.2. Cloning of the other proteins .................................................................................................. 22 2. Protein expression and purification ................................................................................................ 23 2.1. Determining optimal expression conditions ........................................................................... 23 2.1.1. Expression conditions for MAGED1 ................................................................................ 24 2.1.2. Expression conditions for XIAP ....................................................................................... 25 2.1.3. Expression conditions for the other GST-fusion proteins ............................................... 26 2.2. Production and purification of the GST-fusion proteins ......................................................... 26 2.2.1. Production and purification of MAGED1 and MAGEC2 .................................................. 27 2.2.2. Cleavage of MAGED1 and MAGEC2 ................................................................................ 29 2.3. Small scale production of recombinant E3 ligases .................................................................. 33 3. In vitro ubiquitination assays .......................................................................................................... 34 3.1. Effect of MAGEC2 on the enzymatic activity of TRIM28 ......................................................... 34 3.1.1. Set up of the TRIM28 ubiquitination assay ..................................................................... 34 3.1.2. Effect of MAGEC2 on TRIM28 activity............................................................................. 35 3.2. Effect of MAGEC2 on the enzymatic activity of XIAP, cIAP1 and cIAP2 .................................. 36 3.2.1. Set up of the XIAP, cIAP1 and cIAP2 ubiquitination assays ............................................ 36 3.2.2. Effect of MAGEC2 on XIAP, cIAP1 and cIAP2 activities ................................................... 38 Discussion and conclusion ............................................................................................................ 40 1. General discussion .......................................................................................................................... 40 2. Conclusion ....................................................................................................................................... 44 Materials and methods ................................................................................................................. 45 Cloning .................................................................................................................................................... 45 Screen for best expression conditions .................................................................................................... 45 Recombinant protein production + purification ..................................................................................... 46 Quickchange mutagenesis ...................................................................................................................... 46 GST fusion-protein removal .................................................................................................................... 46 In vitro ubiquitination assay .................................................................................................................... 47 References .................................................................................................................................... 48 Addendum 1: Protocols ................................................................................................................ 55 Addendum 2: List of primers ........................................................................................................ 72 Addendum 3: Construction of expression vectors ....................................................................... 73 Addendum 4: Determining the best expression conditions ......................................................... 80 Addendum 5: Determining the concentration of recombinant E3 ligase .................................... 86 List of abbreviations List of abbreviations AIM2 Absent in melanoma-2 NIK NF-κB inducing kinase BIR Baculovirus IAP protein repeat NLR NOD-like receptor BSA Bovine serum albumin NOD Nucleotide-binding and oligomerization domain CHD Cullin homology domain NS Non-soluble cIAP Cellular inhibitor of apoptosis ORF Open reading frame CLR C-type lectin receptor OTU Ovarian tumor CRL Cullin-RING
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