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Functional Characterization of TRIM24 and TRIM32 Proteins in the Heart Through Their Interaction with Dysbindin

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Functional Characterization of TRIM24 and TRIM32 Proteins in the Heart Through Their Interaction with Dysbindin Aus der Klinik für Innere Medizin III im Universitätsklinikum Schleswig-Holstein, Campus Kiel an der Christian-Albrechts-Universität zu Kiel Direktor: Prof. Dr. Norbert Frey Functional characterization of TRIM24 and TRIM32 proteins in the heart through their interaction with Dysbindin Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel Vorgelegt von Ankush Borlepawar Kiel, 2019 First Examiner: Prof. Dr. Dennis Schade Second Examiner: Prof. Dr. Norbert Frey Date of the oral examination: 05.11.2019 2 Declaration: I hereby declare that, apart from the supervisor’s guidance the content and design of the current thesis is all of my own work. This doctoral thesis has never been submitted either partially or wholly as part of a doctoral degree to another examining body and has never been published nor has been submitted for publication. This thesis has been prepared in accordance to the Rules of Good Scientific Practice of the German Research Foundation. No academic degree has ever been withdrawn from me. Kiel, (Date) Doctoral candidate’s signature 3 Preface The work presented in this doctoral thesis has been performed under supervision of Prof. Dr. Norbert Frey in the Institute of Molecular Cardiology. The experimental work and its dissertation were conducted from August 2015 to November 2019. Various results arising from this doctoral thesis work have been published in renowned scientific journals, which also have been enlisted below. 4 1 Index 2 Summary .................................................................................................................................. 12 3 Zusammenfassung ................................................................................................................... 13 4 Introduction ............................................................................................................................. 15 4.1 Prelude .............................................................................................................................. 15 4.2 Cardiomyopathies ............................................................................................................. 16 4.3 Cardiac hypertrophy ......................................................................................................... 17 4.3.1 Dysbindin is a potent inducer cardiac of hypertrophy ............................................... 18 4.3.2 TRIM24 and TRIM32 are potential cardiac interaction partners of Dysbindin .......... 19 4.4 Proteostasis is essential for normal heart functioning ..................................................... 20 4.5 Components of the cellular proteostasis machinery ........................................................ 22 4.5.1 Component 1: Heat shock proteins............................................................................ 22 4.5.2 HSPs in cardioprotection ............................................................................................ 23 4.5.3 Component 2: Autophagy .......................................................................................... 25 4.5.4 Autophagy in the heart .............................................................................................. 26 4.5.5 Component 3: Ubiquitin-Proteasome system (UPS) .................................................. 28 4.5.6 E3 ligases provide substrate recognition specificity .................................................. 30 4.5.7 E3 ubiquitin ligases in the heart ................................................................................. 30 4.6 TRIM E3 ubiquitin ligases .................................................................................................. 31 4.6.1 N-terminal invariable domains in TRIM24 and TRIM32 ............................................. 32 4.6.2 C-terminal variable domains occurring in TRIM24/32 ............................................... 34 4.6.3 Roles of TRIM family in cardiac proteostasis ............................................................. 35 5 Aims of the study ..................................................................................................................... 39 6 Materials .................................................................................................................................. 40 6.1 Hardware and consumables ............................................................................................. 40 6.2 Chemicals .......................................................................................................................... 42 6.3 Enzymes ............................................................................................................................ 44 6.4 Antibodies ......................................................................................................................... 45 6.5 Vectors .............................................................................................................................. 46 5 6.6 Kits ..................................................................................................................................... 47 6.7 Buffers and solutions ........................................................................................................ 48 6.8 Media ................................................................................................................................ 50 6.9 Bacteria ............................................................................................................................. 50 7 Methods ................................................................................................................................... 51 7.1 Microbiological methods .................................................................................................. 51 7.1.1 Generation of electro-competent bacteria ................................................................ 51 7.1.2 Electroporation ........................................................................................................... 51 7.1.3 Agar plate preparation ............................................................................................... 51 7.1.4 Spreading of bacterial cultures .................................................................................. 52 7.1.5 The growth of the bacteria in liquid-culture .............................................................. 52 7.1.6 Storage of positive clones as glycerol stocks ............................................................. 52 7.1.7 Plasmid DNA extraction: Mini preparation ................................................................ 52 7.1.8 Plasmid DNA extraction: Midi preparation ................................................................ 53 7.1.9 Agarose gel electrophoresis ....................................................................................... 53 7.1.10 DNA extraction from agarose gels ........................................................................... 54 7.1.11 DNA and RNA concentration measurement ............................................................ 54 7.2 Cell culture methods ......................................................................................................... 54 7.2.1 Culturing of immortalized HEK293A cell line ............................................................. 54 7.2.2 Cell Transfection in HEK293A cells ............................................................................. 55 7.2.3 Cryo-conservation of the HEK293A cell line ............................................................... 55 7.2.4 Isolation of neonatal rat ventricular cardiomyocytes ................................................ 56 7.3 Generation of mammalian vectors ................................................................................... 58 7.3.1 Open reading frame (ORF) PCRs using target specific and attB site-specific primers ................................................................................................................................................. 58 7.3.2 Recombination using BP and LR clonase from Gateway® technology ....................... 60 7.3.3 Generation of artificial micro-RNA constructs using the Gateway®-compatible Block- iT system ................................................................................................................................. 60 7.3.4 Generation of adenoviruses for recombinant protein or miRNA expression ............ 61 7.3.5 Virus particle titration ................................................................................................ 62 6 7.4 Interaction studies ............................................................................................................ 63 7.4.1 Co-immunoprecipitation in HEK293A cells ................................................................ 63 7.4.2 Co-immunoprecipitation in NRVCMs ......................................................................... 64 7.4.3 Co-localization in NRVCMs: Confocal immunofluorescence microscopy .................. 65 7.4.4 Co-localization in NRVCMs: Analysis .......................................................................... 65 7.5 Animal experiments .......................................................................................................... 66 7.5.1 Transverse Aortic Constriction (TAC) ......................................................................... 66 7.5.2
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