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Identification of S-Nitrosylated Nuclear Proteins in Rat Cortical Neurons

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Identification of S-Nitrosylated Nuclear Proteins in Rat Cortical Neurons Identification of S-nitrosylated nuclear proteins in rat cortical neurons Jacob Anderson Thesis submitted for the degree of Doctor of Philosophy Riccio Lab MRC Laboratory for Molecular Cell Biology University College London January 2016 I, Jacob Anderson, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Abstract The ability of neurons to modulate gene expression in response to changing environmental conditions, both during development and throughout life, is necessary for proper brain function. Although the mechanisms responsible for transducing extracellular signals into changes in gene expression remain poorly characterized, S- nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols, has been shown to be critical. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is essential for gene expression during neuronal development, however few nuclear targets have been identified to date. Progress in this field of research has been hampered by the technical difficulties associated with the detection of S-nitrosylated nuclear proteins. Here, I took a novel approach by enriching for nuclear proteins of rat cortical neurons before inducing S-nitrosylation with nitrosocysteine (CysNO) and isolating S-nitrosylated proteins using S- nitrosothiol Resin Assisted Capture (SNORAC). Mass spectrometry analysis identified 614 S-nitrosylated nuclear proteins, including potential site(s) of S- nitrosylation for 67% of targets. 612 of these proteins are novel potential targets of S- nitrosylation in cortical neurons and 131 have not been shown to be S-nitrosylated in any system. Two hits, the transcription factor CREB and the histone binding protein RBBP7, were further studied and I confirmed endogenous S-nitrosylation in depolarized cortical neurons for both. In addition, I showed that CREB is S- nitrosylated at cysteines 300/310/337 located within the DNA binding domain, whereas RBBP7 is S-nitrosylated at cysteine 166, which is in a WD-repeat region that regulates protein-protein interactions. Overall, this work comprehensively identifies, for the first time, the nuclear proteins that undergo S-nitrosylation in neurons and highlights S-nitrosylation of CREB and RBBP7 as a candidate mechanism by which NO regulates gene expression in mammalian cells. Acknowledgements I thank Dr. Antonella Riccio for her expert guidance and support through an extremely interesting and challenging project. I extend my gratitude to Marco Gaspari at the University of Catanzaro (Italy) for his highly skilled work on mass spectrometry that formed the bedrock of this PhD project and also for providing me with an extremely positive first experience of scientific collaboration. Next, I would like to thank the students I have supervised (Andreas Pavlou, Sarah Aldous, Marta Iannicelli). In particular, Sarah Aldous deserves special recognition for her effort towards confirming the site of S-nitrosylation of RBBP7. The Riccio Lab has provided an excellent environment in which to develop scientifically and a genuinely enjoyable place to work. Many thanks to the members of the lab who have been here during my time at UCL: Sarah Aldous, Catia Andreassi, Emily Brookes, Hamish Crerar, Luca Crepaldi, Aniko Ludanyi, Justyna Nitarska, Cristina Policarpi, William Sherlock and Carola Zimmermann. Thanks for reading and editing drafts of this thesis go to Antonella Riccio, Emily Brookes, Hamish Crerar and Lucie Van Emmenis. Many thanks to my friends and family for their support. Contents 1. Introduction .............................................................................................. 10 1.1 The role of post-translational modifications in cellular signalling ........... 11 1.2 The discovery of nitric oxide as a cellular messenger ............................... 12 1.3 Nitric oxide synthases ................................................................................... 13 1.4 NO signalling: S-nitrosylation ...................................................................... 16 1.4.1 Mechanisms .............................................................................................. 17 1.4.2 Transnitrosylation ...................................................................................... 18 1.4.3 Temporal regulation .................................................................................. 19 1.4.4 Specificity .................................................................................................. 20 1.5 Neuronal nitric oxide synthase ..................................................................... 21 1.6 Roles of NO in the adult brain ...................................................................... 22 1.7 Roles of NO in neurological diseases .......................................................... 25 1.8 NO signalling during cortical development ................................................. 27 1.8.1 S-nitrosylation of HDAC2 .......................................................................... 30 1.9 S-nitrosylation of additional transcriptional regulators ............................. 32 1.9.1 Transcription factors .................................................................................. 32 1.9.2 Epigenetic modifiers .................................................................................. 34 1.9.3 ATP-dependent chromatin remodelers ..................................................... 36 1.10 Identification of S-nitrosylated proteins .................................................... 38 1.10.1 Previously published screens ................................................................. 39 Aims ...................................................................................................................... 42 2. Materials and Methods ............................................................................ 43 4.1 Preparation of E17 cortical neurons ............................................................ 43 4.2 HEK293T cell culture ..................................................................................... 43 4.3 Transfection conditions and plasmids ........................................................ 44 4.4 Nuclear and cytoplasmic protein extractions ............................................. 44 4.5 Working with S-nitrosylation ........................................................................ 44 4.6 Detection of S-nitrosylation using the biotin switch technique ................ 44 4.7 Detection of S-nitrosylation: SNORAC ........................................................ 46 4.8 Silver staining and western .......................................................................... 48 4.9 Antibodies ...................................................................................................... 48 4.10 Gene Ontology Analysis ............................................................................. 48 4.11 Motif-X analysis ............................................................................................ 48 4.12 Mutagenesis ................................................................................................. 48 4.13 Software ........................................................................................................ 48 4.14 Mass Spectrometry: CysNO-treated HEK293T cell lysates ..................... 48 4.15 Mass Spectrometry: CysNO-treated neuronal extracts ........................... 51 4.16 Direct Detection of S-nitrosylation ............................................................. 55 3. Results ...................................................................................................... 57 Chapter 1: Identification of S-nitrosylated proteins using SNORAC .............. 57 Validation of SNORAC in CysNO-treated HEK293T cell lysates ....................... 58 Validation of SNORAC in CysNO-treated HEK293T nuclear extracts ............... 61 Identification of novel nuclear targets of S-nitrosylation in neurons ................... 64 Comparative analysis of nuclear and cytoplasmic S-nitrosylated proteins ........ 70 Protein targets of S-nitrosylation are involved in transcriptional regulation ....... 73 Identification of S-nitrosylated nuclear peptides ................................................ 74 Amino acid composition of SNO-peptides .......................................................... 77 Motifs containing acidic or basic amino acids are associated with S-nitrosylated cysteines ............................................................................................................ 78 Summary: Chapter 1 .......................................................................................... 79 Chapter 2: Endogenous S-nitrosylation of nuclear factors in cortical neurons ............................................................................................................................... 81 CREB is S-nitrosylated in cortical neurons ........................................................ 82 Serine 133 phosphorylation is unaffected in the bZIP cysteine triple mutant .... 91 RBBP7 is S-nitrosylated in cortical neurons ....................................................... 97 Summary: Chapter 2 ........................................................................................ 100 Chapter 3: Direct detection of S-nitrosylated cysteines by mass spectrometry .....................................................................................................
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