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An Investigation Into the Localisation and Possible Protein Partners of the Base Excision Repair Protein Ogg1

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An Investigation Into the Localisation and Possible Protein Partners of the Base Excision Repair Protein Ogg1 University of Birmingham School of Biosciences AN INVESTIGATION INTO THE LOCALISATION AND POSSIBLE PROTEIN PARTNERS OF THE BASE EXCISION REPAIR PROTEIN OGG1 And THE ROLE OF SRC-LIKE ADAPTOR PROTEINS IN REGULATING GPVI SIGNALLING A research project report submitted by Sarah Akbar as part of the requirement for the degree of MRes in Molecular and Cellular Biology This project was carried out at: The University of Birmingham Under the supervision of: Dr N J Hodges Dr M G Tomlinson August 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract A common theme among the two research projects is the dichotomy of biological molecules or pathways contributing to both physiological and pathophysiological processes in vivo. Reactive oxygen species (ROS) are involved in redox signalling and phagocytosis, however when present in excess ROS attack cellular structures contributing to the progression of a number of degenerative pathologies such as Alzheimer’s and cardiovascular disease. Project 1 investigates the DNA repair protein OGG1 which counteracts the reactive species damage by repairing oxidised DNA base lesions. The DNA glycosylase OGG1 acts via the base excision repair (BER) pathway to mediate the nucleophilic excision of oxidised guanine residues 7,8-dihydro-8-oxoguanine (8-oxoG). The localisation of OGG1 is investigated in the cellular contexts of oxidative stress, apoptosis and mitosis. The protein partners of OGG1 were identified and compared to that of the single-nucleotide polymorphism variant Ser326Cys-OGG1to determine whether reduced repair capacity of the variant is due to impaired protein interactions. Platelets mediate the physiological process of haemostasis and the pathophysiological process of thrombosis. GPVI is a glycoprotein platelet receptor integral to stable collagen binding at sites of vascular damage to activate haemostasis. As GPVI shares considerable homology to ITAM-receptors, it was hypothesised that GPVI is regulated by Src-like adaptor proteins (SLAP) which are the key negative regulators of ITAM-containing receptors of B cells and T cells. Project 2 investigates the regulation of GPVI by SLAP1 and SLAP2 in a DT40 cell line model using the NFAT/AP1-luciferase assay. Furthermore the contribution of each SLAP2 domain was investigated to elucidate a potential mechanism of SLAP2 mediated inhibition. SLAP1 and SLAP2 significantly inhibit collagen-stimulated GPVI signalling without altering GPVI expression levels indicating intrinsic inhibition of GPVI signalling. As SLAP proteins are expressed in platelets these are potential regulators of GPVI signalling in vivo. Elucidation of GPVI regulation could have implications in the development of anti-thrombosis therapy. Acknowledgements I would like to especially thank Dr Nik Hodges and Dr Mike Tomlinson for their support and patience. I would also like to thank the 4th floor and the 8th floor for the great time I have had during laboratory work. I would like to thank Dr Dafforn and Dr Lodge for the continuous support throughout this course. I would like to thank the various friends who have helped me maintain some sanity during this course. I thank my parents for their encouragement and provision of tea, and I would also like to thank my siblings for supporting me and letting me feel clever every now and then. Contents Project 1. AN INVESTIGATION INTO THE LOCALISATION AND POSSIBLE PROTEIN PARTNERS OF THE BASE EXCISION REPAIR PROTEIN OGG1 Chapter 1. Introduction ...................................................................................................................... 1 Chapter 2 Materials and Methods .................................................................................................... 18 2.1 Chemicals .................................................................................................................................... 18 2.2 Cell Culture ................................................................................................................................. 18 2.3 Oxidation Treatment ................................................................................................................... 20 2.4. EGFP-OGG1 Vectors ................................................................................................................. 21 2.5 Transfection ................................................................................................................................. 22 2.6 Immunoprecipitation of OGG1 ................................................................................................... 23 2.7 Protein Analysis .......................................................................................................................... 27 2.8 Mass Spectrometry ............................................................................................................. 29 2.9 Confocal microscopy................................................................................................................... 29 Chapter 3 Results ............................................................................................................................. 30 3.1 Cell Viability Assay .................................................................................................................... 30 3.2 Flow Cytometry Analysis of Transfection Efficiency ................................................................ 31 3.3 Optimisation of Immunoprecipitation protocol ........................................................................... 33 3.4 Mass Spectrometry Results ......................................................................................................... 46 Chapter 4. Discussion ....................................................................................................................... 54 4.1 Cell Viability on BSO treatment ................................................................................................. 54 4.2 Transfection Efficiency ............................................................................................................... 55 4.3 Immunoprecipitation ................................................................................................................... 55 4.4 Mass Spectrometry ...................................................................................................................... 57 4.5 Confocal Microscopy .................................................................................................................. 62 4.6 Conclusion ................................................................................................................................... 68 Appendix .......................................................................................................................................... 70 Appendix II ...................................................................................................................................... 75 References ........................................................................................................................................ 88 Project 2. THE ROLE OF SRC-LIKE ADAPTOR PROTEINS IN REGULATING GPVI SIGNALLING…………………………………………………100 Chapter 1. Introduction ...................................................................................................................... 1 Chapter 2 Material and Methods ...................................................................................................... 22 2.1 Materials ...................................................................................................................................... 22 2.2.1 Cell Culture .............................................................................................................................. 22 2.3 DT40Transfection ....................................................................................................................... 23 2.4 NFAT-Luciferase Assay ............................................................................................................. 24 2.5 β-galactosidase assay .................................................................................................................. 25 2.6 Flow cytometry ........................................................................................................................... 25 2.7 Western Blot ................................................................................................................................ 25 Chapter 3 Results ............................................................................................................................. 28 3.1.1 SLAP1 and SLAP2 inhibit GPVI/FcR-γ signalling in a cell line model .................................. 28 3.2.1 Optimisation of the NFAT/AP-1 Assay for analysis of GPVI/FcR-γ signalling ..................... 30 3.3.1 SLAP-mediated inhibition at optimised GPVI/FcR-γ concentration ......................................
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