Anderson, J; (2016) Identification of S-nitrosylated nuclear proteins in rat cortical neurons. Doctoral thesis , UCL (University College London).

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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.

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