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Biophysical and Biochemical Investigations of CARD-CARD Interactions

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Biophysical and Biochemical Investigations of CARD-CARD Interactions Biophysical and biochemical investigations of CARD-CARD interactions Manuela Hess The Francis Crick Institute Mill Hill Laboratory The Ridgway London NW7 1AA, UK Department of Structural and Molecular Biology University College London Thesis submitted to University College London for the degree of Doctor of Philosophy December 2015 1 Declaration of authenticity The work presented in this thesis has been completed in the laboratory of Dr Katrin Rittinger at the Francis Crick Institute - Mill Hill Laboratory. I, Manuela Hess, confirm that this work is my own. Where information has been derived from other sources, this has been indicated in the thesis. 2 [Diä Doktorarbeit isch mine Elterä und Grosselterä gwidmet, wo mich und mini Karriere immer unterstützt händ.] 3 Abstract Innate and adaptive immunity are regulated by complex signalling pathways that require the assembly and activation of apoptotic and inflammatory signalling complexes, which ultimately induce gene expression and alter cellular behaviour. Key players in regulating the assembly of many of these signalling complexes are CARD-containing proteins that are members of the death domain superfamily. However, the molecular mechanisms that underline the function and regulation of CARDs remain unclear. This project was aimed to investigate the biochemical and biophysical characteristics of CARD-CARD interactions to gain insights into the binding specificity and/or stoichiometry of CARD-mediated protein complexes and specifically, to elucidate if there is a common mode of interaction within this protein subfamily. It was known that CARDs have a tendency to aggregate when purified individually, therefore all human CARDs were prepared for small scale expression and solubility screening with the aim to perform co-expression and pull-down studies. These screens identified the highly soluble, yet structurally and biophysically uncharacterised CARMA1-CARD. Consequently, one focus was set on the signalling complexes involving CARMA1 and CARD9, both of which bind BCL10 via CARD-CARD interactions. Preliminary work had been performed in the group focusing on NOD2 and RIP2, therefore, these two proteins were taken as a second system for further investigations with the aim to describe the intramolecular interaction between the tandem CARDs of NOD2 and its intermolecular interaction with the CARD of RIP2 in molecular detail. Further insights into the binding surfaces involved in these interactions were gained using mutagenesis combined with biophysical assays. To follow up on the newest development in the field concerning CARDs forming filamentous assemblies, this work tested both, RIP2- and BCL10-CARD for their ability to polymerise using fluorescence polymerisation assays. However, no polymerisation could be detected in this work, questioning the relevance of filament formation as a general behaviour of CARDs. 4 Table of contents Declaration of authenticity ....................................................................................... 2 Abstract .................................................................................................................... 4 Table of contents ...................................................................................................... 5 List of figures and tables ......................................................................................... 10 Abbreviations ......................................................................................................... 14 Acknowledgments .................................................................................................. 17 Chapter 1 - Introduction.......................................................................................... 18 1.1 The immune system - Innate and adaptive immunity .......................................... 19 1.2 Transmembrane pattern recognition receptors ................................................... 20 1.2.1 Toll-like receptors ........................................................................................... 20 1.2.1.1 TLR signalling pathways .......................................................................... 22 1.2.2 C-type lectin receptors ................................................................................... 24 1.2.2.1 Dectin-1 signalling pathway .................................................................... 25 1.3 Intracellular pattern recognition receptors .......................................................... 28 1.3.1 RIG-I like receptor signalling pathway ............................................................ 28 1.3.2 NOD-like receptor signalling pathways .......................................................... 31 1.3.2.1 The apoptosome ..................................................................................... 33 1.3.2.2 The NLRP3 inflammasome ...................................................................... 34 1.3.2.3 The NOD2-RIP2 signalling pathway......................................................... 35 1.4 Activation of transcription factors NF-κB and IRF3/IRF7 ...................................... 39 1.4.1 Nuclear factor-kappaB (NF-κB) - the canonical pathway ............................... 39 1.4.2 Interferon regulatory factors .......................................................................... 40 1.5 Antigen receptors (AgR) ........................................................................................ 42 1.5.1 B-cell receptors (BCR) ..................................................................................... 42 1.5.2 T-cell receptors (TCR) ...................................................................................... 43 1.5.3 Antigen receptor mediated activation and regulation of NF-κB .................... 43 1.5.3.1 CARMA1 activation ................................................................................. 44 1.5.3.2 CBM-L complex formation and downstream signalling .......................... 46 1.6 The death domain superfamily ............................................................................. 48 1.6.1 Complex formation within death domain subfamilies ................................... 50 1.6.2 The death domain (DD) subfamily .................................................................. 52 1.6.2.1 Intermolecular interaction ...................................................................... 52 1.6.3 The death effector domain (DED) subfamily .................................................. 54 5 1.6.3.1 MC159 ..................................................................................................... 55 1.6.3.2 Caspase-8 ................................................................................................ 56 1.6.4 The pyrin domain (PYD) subfamily ................................................................. 58 1.6.5 The caspase recruitment domain (CARD) subfamily ...................................... 58 1.6.5.1 Tandem CARDs and their intramolecular interactions ........................... 61 1.6.5.2 Intermolecular interactions .................................................................... 67 1.7 Project aims ........................................................................................................... 71 Chapter 2 - Materials and methods ......................................................................... 72 2.1 Materials ................................................................................................................ 73 2.2 Molecular biology .................................................................................................. 73 2.2.1 Bioinformatics ................................................................................................. 73 2.2.2 Construction of expression plasmids .............................................................. 74 2.2.3 Polymerase chain reaction (PCR). ................................................................... 75 2.2.4 Agarose gel electrophoresis .......................................................................... 76 2.2.5 Determination of DNA concentration ........................................................... 76 2.2.6 Restriction enzyme digestion ......................................................................... 77 2.2.7 Ligation independent cloning ......................................................................... 77 2.2.8 Infusion cloning ............................................................................................... 79 2.2.9 Transformation of cloning products ............................................................... 79 2.2.10 Identification of positive plasmids and purification ..................................... 80 2.2.11 Plasmid DNA purification, storage and sequencing ..................................... 80 2.2.12 Site-directed mutagenesis ............................................................................ 81 2.3 Protein expression and purification ...................................................................... 82 2.3.1 Bacterial expression strains ............................................................................ 82 2.3.2 Overexpression ............................................................................................... 83 2.3.2.1 Small-scale expression ............................................................................ 83 2.3.2.2 Large-scale expression ............................................................................ 83 2.3.3 Buffers
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