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Purification of Full-Length Bcl-2 Family Proteins and Molecular Analysis of Bim-DLC1 Interaction

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Purification of Full-Length Bcl-2 Family Proteins and Molecular Analysis of Bim-DLC1 Interaction Purification of full-length Bcl-2 family proteins and molecular analysis of Bim-DLC1 interaction Inaugural-Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Aristomenis Roukounakis geboren in Heraklion, Griechenland Freiburg im Breisgau Februar 2018 Die Untersuchungen zur vorliegenden Arbeit wurden von Oktober 2013 bis Februar 2018 am Institut für Medizinische Mikrobiologie und Hygiene des Universitätsklinikums der Albert- Ludwigs-Universität Freiburg unter der Leitung von Prof. Dr. Georg Häcker durchgeführt. Dekan der Fakultät für Biologie: Prof. Dr. Bettina Warscheid Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuer der Arbeit: Prof. Dr. Georg Häcker Referent: Prof. Dr. Georg Häcker Koreferent: Prof. Dr. Tilman Brummer Drittprüfer: Prof. Dr. Christof Meisinger Datum der mündlichen Prüfung: 07.05.2018 Publication Major part of this thesis has been published in Genes & Development journal (doi: 10.1101/gad.302497.117). Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis Prafull Kumar Singh* Aristomenis Roukounakis* Daniel O. Frank, Susanne Kirschnek, Kushal Kumar Das, Simon Neumann, Josef Madl, Winfried Römer, Carina Zorzin, Christoph Borner, Ana Garcia-Saez, Arnim Weber and Georg Häcker* *equal contribution Table of contents I. Summary .............................................................................................................................. 1 II. Introduction ......................................................................................................................... 3 1. Programmed cell death ..................................................................................................... 3 1.1 History of programmed cell death ................................................................................ 3 1.2 Morphology of programmed cell death ........................................................................ 3 2. Apoptosis .......................................................................................................................... 4 2.1 How important is apoptosis? ........................................................................................ 4 2.2 Molecular pathways of apoptosis ................................................................................. 5 2.2.1 Extrinsic apoptosis pathway .................................................................................. 8 2.2.2 The mitochondrial or intrinsic apoptotic pathway ................................................ 8 2.3 Necroptosis and other forms of programmed cell death .............................................. 9 3. The Bcl-2 protein family: gatekeepers of mitochondrial apoptosis ............................... 10 3.1 Interaction models among members of the Bcl-2 protein family ............................... 12 3.1.1 Direct activation model ....................................................................................... 12 3.1.2 Indirect activation model ..................................................................................... 13 3.1.3 Unified model ...................................................................................................... 13 3.2 Executioner proteins Bax, Bak and their ‘cousin’ Bok .............................................. 14 3.3 Anti-apoptotic Bcl-2 proteins ..................................................................................... 16 3.4 Pro-apoptotic BH3-only proteins ............................................................................... 17 3.5 The Bcl-2 family in disease and therapy .................................................................... 19 4. The pro-apoptotic BH3-only protein Bim ...................................................................... 21 4.1 The structure of Bim .................................................................................................. 21 4.2 Regulation of Bim expression and activity ................................................................ 24 4.3 Bim, Bmf and their (non-)common regulators ........................................................... 25 4.4 The dynein adaptor protein: DLC .............................................................................. 26 III. Objectives .......................................................................................................................... 29 IV. Results .............................................................................................................................. 30 1. Purification of several key Bcl-2 protein family-members .............................................. 30 2. Recombinant BimL exhibits similar cytochrome c releasing activity to tBid .................. 38 3. Recombinant Mcl-1 is able to inhibit the cytochrome c releasing activity of BimL ........ 39 4. Recombinant Bak shows no reliable activity ................................................................... 39 5. DLC1 promotes Bim-Bim interaction, leading to the formation of large MW complexes .............................................................................................................................................. 41 6. Evidence of Bim-containing larger structures on mitochondria of healthy cells ............. 43 7. Higher order Bim-DLC1 complexes on liposomes require an excess of DLC1 .............. 46 8. DLC1 and DLC2 interact equally with Bim and induce a similar shift in the MW of Bim- containing complexes on isolated mitochondria .................................................................. 47 9. DLC1 is recruited by Bim to artificial membranes .......................................................... 49 10. Interaction of Bim with DLC1 mildly compromises its Bax-activating capacity .......... 51 11. DLC1 promotes the interplay between Bim and anti-apoptotic members of the Bcl-2 family ................................................................................................................................... 53 12. DLC1 induces formation of high order Bmf-complexes on LUVs ................................ 55 13. Bmf is a sensitizer that cannot directly activate Bax...................................................... 56 14. In vitro Bim-Bmf interaction is facilitated by DLC1 and DLC2 ................................... 58 15. Attempts at BimL crystallization .................................................................................... 59 16. DLC1 induces conformational changes in BimL ............................................................ 60 V. Discussion ........................................................................................................................ 63 1. Importance of cell-free systems for the analysis of mitochondrial apoptosis .................. 63 2. Establishing an efficient purification strategy for Bcl-2 family members ....................... 64 3. Evaluating the activity of purified components ............................................................... 67 4. Regulating Bim in non-apoptotic cells: role of DLC1 ..................................................... 68 5. Characterizing full-length recombinant Bmf and evaluating its interaction with DLC ... 71 6. Structural aspects of the Bim-DLC1 interaction .............................................................. 72 VI. Conclusions ...................................................................................................................... 74 VII. Materials and Methods .................................................................................................... 76 1. Materials ........................................................................................................................... 76 1.1 Cell lines ................................................................................................................ 76 1.2 Cell culture reagents .............................................................................................. 76 1.3 Immunoprecipitation (IP) materials ....................................................................... 77 1.4 Molecular biology kits ........................................................................................... 77 1.5 Protein biology kits ................................................................................................ 78 1.6 Recombinant proteins ............................................................................................ 78 1.7 Antibodies .............................................................................................................. 78 a. Primary antibodies .................................................................................................... 78 b. Secondary antibodies ................................................................................................ 79 1.8 Western blot reagents ............................................................................................. 79 1.9 Common buffers and solutions .............................................................................. 80 1.10 Buffers for BN-PAGE ........................................................................................ 81 1.11 Cloning and mutagenesis primers ...................................................................... 81 2. Methods ...........................................................................................................................
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