Abstract
Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated α- and β-tubulin isoforms in primary neurons after fixation. The approach was based on in situ replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.
Highlights
Nitric oxide (NO) is a well-established neuromodulator and neurotransmitter in the central and peripheral nervous systems [1] and has been shown to be involved in the modulation of synaptic efficacy, pain perception and neuronal damage/protection [2]
Functional consequences of S-nitrosylation have been demonstrated for a small number of proteins, including caspases [8], parkin [9], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [10], tubulin [11], microtubule-associated protein MAP1B [12], histone deacetylase-2 (HDAC2) [13], PSD-95 [14] and AMPA receptors [15]
We reasoned that proteins containing Snitrosylated cysteine residues in live cells could be fixed, tagged in situ by biotin-HPDP and detected and localized by antibodies specific for the tagged cysteine residue in the context of the polypeptide chain
Summary
Nitric oxide (NO) is a well-established neuromodulator and neurotransmitter in the central and peripheral nervous systems [1] and has been shown to be involved in the modulation of synaptic efficacy, pain perception and neuronal damage/protection [2]. A major obstacle in the analysis of protein S-nitrosylation is the low stability of this posttranslational modification in reducing environments and upon exposure to light [16]. This problem was partially overcome by the development of the biotin-switch procedure [5]. In this biochemical assay the unstable nitroso moiety of S-nitrosylated cysteine residues is replaced by a stable biotin tag.
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