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Decoding the Structure and Function of WWP2 in the Tgfβ Signalling Pathway

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Decoding the Structure and Function of WWP2 in the Tgfβ Signalling Pathway Decoding the structure and function of WWP2 in the TGFβ signalling pathway Lloyd Christian Wahl A thesis submitted for the degree of Doctor of Philosophy at the School of Biological Science, University of East Anglia, March 2016 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract The secret to the specificity of the ubiquitin-proteasome system lies in the protein-protein interaction domains of the diverse group of E3 enzymes. WWP2 is one such E3 enzyme, and the relevant protein-protein interaction domain is the WW domain. WWP2 has four WW domains which are used to interact with proline-rich motifs found in the sequences of the Smad signalling proteins that propagate or inhibit the TGFβ pathway, and in so doing, allows WWP2 to regulate its Smad targets. WWP2 has three isoforms that are known to participate in regulation of TGFβ signalling, but, even amongst isoforms of the same E3, they exhibit different specificities for components of the pathway. The reason for this is unknown, but it is likely to be due to the different domain composition of WWP2, since each of the isoforms has a different combination of WW domains. The aim of this thesis is to investigate the structure of the domains of WWP2, and to explore how this relates to the selectivity of different isoforms in the TGFβ pathway. Overexpression of recombinant WWP2 domains in a bacterial host, and affinity and size- exclusion chromatography have been used to produce pure, high concentration protein samples. Both NMR spectroscopy and crystallography have been used in an attempt to elucidate the structure of WWP2 domains. NMR spectroscopy, the more successful of the two approaches, has allowed the elucidation of the structure of the fourth WW domain of WWP2. By observing ligand interaction using NMR, the binding site of WW4 is revealed and the substrate preference of WW4 and WW3 domains is observed on a molecular level. Evidence of phospho-regulation of substrate selectivity is presented, and a structural basis for this selectivity is proposed. In addition, a further layer of complexity is added to the WWP2 isoform-mediated regulation of the TGFβ signalling pathway, as a new isoform is discovered. i Contents Abstract ............................................................................................ i List of Tables ................................................................................................... viii List of Figures .................................................................................. xi List of Abbreviations ................................................................... xviii Acknowledgements ..................................................................... xxii 1. Introduction ......................................................................... 1 1.1 Protein Degradation ................................................................................... 3 1.2 The Ubiquitin System ................................................................................. 4 1.2.1 The Proteasome .......................................................................................... 7 1.2.2 Ubiquitin ..................................................................................................... 8 1.3 E1 Ubiquitin Activator .............................................................................. 10 1.4 E2 Ubiquitin Conjugators .......................................................................... 12 1.5 E3 Ubiquitin Ligases .................................................................................. 14 1.5.1 RING Domain E3 Ligases ........................................................................... 15 1.5.2 HECT Domain E3 Ligases ........................................................................... 17 1.6 TGFβ signalling .......................................................................................... 22 1.6.1 Latent TGFβ ............................................................................................... 22 1.6.2 TGFβ receptors ......................................................................................... 23 1.6.3 Smads 2/3 ................................................................................................. 23 1.6.4 TGFβ gene programs and Cancer .............................................................. 25 1.6.5 Smad7 ....................................................................................................... 26 1.7 NEDD4 Ubiquitin E3 Ligases ..................................................................... 27 ii 1.7.1 WW domains ............................................................................................ 28 1.7.2 NEDD4 family WW domains ...................................................................... 31 1.7.3 WWP2 HECT E3 ligase ............................................................................... 33 1.8 Aims of the thesis ..................................................................................... 35 2. Materials and Methods ...................................................... 37 2.1 Recipes ...................................................................................................... 38 2.1.1 Lysogeny Broth (LB) .................................................................................. 38 2.1.2 LB Agar ...................................................................................................... 38 2.1.3 Minimal Essential Medium (MEM) ............................................................ 38 2.1.4 10x M9 salts .............................................................................................. 38 2.1.5 1000x Micronutrient mix .......................................................................... 39 2.1.6 Immobilised metal ion affinity (IMAC) buffers .......................................... 39 2.1.7 Gel filtration buffer ................................................................................... 39 2.1.8 PBS ............................................................................................................ 39 2.1.9 NMR sample buffer ................................................................................... 40 2.1.10 Polyacrylamide gels ................................................................................. 40 2.1.11 SDS-PAGE buffer ..................................................................................... 40 2.1.12 Tricine gels .............................................................................................. 40 2.1.13 Tricine gel buffers ................................................................................... 41 2.1.14 Silver stain solutions ............................................................................... 41 2.1.15 Silver stain developing solution............................................................... 41 2.1.16 2x Laemmli buffer ................................................................................... 41 2.1.17 1% Agarose-TAE gel ................................................................................ 41 2.1.18 50x TAE ................................................................................................... 42 2.2 In-Fusion cloning ....................................................................................... 43 2.3 Heat shock transformation ....................................................................... 46 2.4 Overexpression of recombinant proteins - LB.......................................... 46 iii 2.5 Immobilised metal ion affinity chromatography...................................... 47 2.6 Size-exclusion chromatography................................................................ 48 2.6.1 Column calibration .................................................................................... 48 2.6.2 Recombinant protein size-exclusion chromatography .............................. 48 2.7 3C protease digest .................................................................................... 49 2.8 Crystallisation trials .................................................................................. 49 2.8 Overexpression of isotopically enriched recombinant proteins .............. 50 2.9 Thrombin digest ........................................................................................ 50 2.10 NMR sample preparation ....................................................................... 51 2.11 NMR spectroscopy .................................................................................. 51 2.12 Spectral processing ................................................................................. 52 2.13 Resonance assignment ........................................................................... 52 2.14 Structure calculation .............................................................................. 52 2.15 Bacterially expressed Smad7 peptide .................................................... 53 2.15.1 Cloning .................................................................................................... 54 2.15.2 Overexpression and purification ............................................................. 55 2.15.3 ULP-1 protease digest ............................................................................
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