Inês M. Araújo

Regulation of neurogenesis by nitric oxide: a role for S-nitrosylation in neurogenic signaling

Inês M. Araújo
Centre for Biomedical Research (CBMR), Algarve Biomedical Center (ABC) and Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal
Nitric oxide (NO) has been established as an important regulator of neurogenesis. NO enhances proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. Several pathways may be activated by NO that result in increased neurogenic signaling, such as the extracellular regulated kinase (ERK/MAPK) pathway, and the canonical NO pathway that is triggered by increased levels of cyclic GMP produced by guanylate cyclase. However, non-canonical NO signaling by post-translational modification of proteins has emerged in the last years as a main mechanism of NO signaling regarding neuronal plasticity. Thus, such post-translational modifications may have a substantial role in the activation or inhibition of several proteins involved in the neurogenic process. S-nitrosylation is a post-translational modification that consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. The aim of this work was to identify proteins modified by S-nitrosylation in conditions that promote cell proliferation in NSC derived from the subventricular zone, and that could take part in non-classical NO signaling.

Treatment with S-nitroso-L-cysteine (CysSNO), a physiological permeable nitrosothiol, increased protein cysteine oxidation and S-nitrosylation in NSC. Separation by two-dimensional electrophoresis and analysis by mass spectrometry resulted in the identification of several proteins that were modified by treatment with CysSNO. From those, p21Ras, PEBP-1, PCNA, 14-3-3 proteins and hnRNP K, were further validated due to their relevance in the neurogenic context, including their involvement in the ERK/MAPK pathway. By using the biotin switch technique, we show a strong increase in S-nitrosylation of p21Ras, PEBP-1, PCNA, 14-3-3 and hnRNP K in the presence of CysSNO. Several of these proteins were also evaluated using NO-insensitive constructs and neural stem cell proliferation was analysed. Overall, this work identified several proteins as targets of S-nitrosylation in NSC and suggests new candidates for NO-induced regulation of neurogenesis.

Supported by FCT, Portugal, COMPETE and FEDER (grants PTDC/SAU-OSD/0473/2012, PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013; fellowships SFRH/BD/77903/2011 and SFRH/BD/79308/2011), by a Spanish-Portuguese Ação Integrada (PRI-AIBPT-2011-1015/E-10/12), and by ISCIII (Spain, PS09/00101 and PI12/00875). Protein Identification by LC/MS/MS and Identification of posttranslational modifications was carried out in the ‘Centro de Biología Molecular Severo Ochoa PROTEIN CHEMISTRY FACILITY’, a member of ProteoRed network. Imaging was performed in the Light Microscopy Facility at CBMR, a member of the Portuguese Platform for BioImaging (PPBI).
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