Detection of S-nitrosated nuclear proteins in pathogen-treated <em>Arabidopsis</em> cell cultures using biotin switch technique.

Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively

Analysis of recombinant protein S-Nitrosylation using the biotin-switch technique

Prod 2018-357 SPE IPM INRA UB CNRSInternational audienceNitric oxide is regarded as a key signaling messenger in several organisms. Its physiological relevance is partly due to its capacity to induce posttranslational modifications of proteins through its direct or indirect reaction with specific amino acid residues. Among them, S-nitrosylation has been shown to be involved in a broad range of cellular signaling pathways both in animals and plants. The identification of S-nitrosylated proteins has been made possible by the development of the Biotin-Switch Technique (BST) in the early 2000s. Here, we describe the BST protocol we routinely use to check in vitro S-nitrosylation of recombinant proteins induced by NO donors

Identification of nitrosylated proteins (SNO) and applications in plants

Over the last decade, due to its broad biological effects, nitric oxide (NO) has triggered a huge interest in the plant science too. Nitrosylation of cystein thiol residues (SNO) in proteins has been shown to be the main target of endogenously produced NO or in a biological sample exposed to this gas. This chapter summarizes the hitherto 18 different methods developed to identify and quantify nitrosylated proteins. These methods derive mostly from the original “Biotin-Switch” technique (BS) published in 2001 but new approaches try to circumvent BS weaknesses. Surprisingly, out of this bloomy panel only a couple of methods have been used in plants. By collecting all the plant published data up to now, we “blasted” them against the proteome of the plant model Arabidopsis and identified 373 nonredundant nitrosylated proteins. We then provide the first overview of plant nitrosylated proteome showing a wide range of functions and cellular compartments involved in NO signaling/targeting. This plant nitrosylated proteome resource expands our current understanding on NO-targeted proteins and facilitates comparisons with new nitrosylated protein data