Identification of NO-Sensitive Cysteine Residues Using Cysteine Mutants of Recombinant Proteins.

Nitric oxide (NO) is a free radical gas regulating a wide range of biological processes in plants. Proteins are the main reaction target of NO inside the cells. The relevance of S-nitrosation as one of the NO-mediated protein posttranslational modifications has been studied in detail. S-nitrosylation causes alterations of the activity/function, sub-cellular localization or interaction partners of proteins. Up to present, a large number of S-nitrosation candidates have been detected in plants. Recombinant proteins are widely used to show or confirm the protein posttranslational modifications. Here, using recombinant proteins subjected to biotin switch assay, the S-nitrosation of some nuclear candidates of Arabidopsis is verified. Proteins usually contain several cysteine residues which each might involve in structure of protein active sites. So, an important question is: which cysteine residue is the target of S-nitrosation and does it belong to an active site? Here, using the approach of substitution of cysteines by serines on recombinant proteins, the NO-sensitive cysteine residue of an Arabidopsis nuclear protein is identified. The next step could be to investigate the effect of S-nitrosation on protein activity/function and further to test the role of target cysteines and S-nitrosation of them in protein activity/function.

Detection of S-Nitrosated Nuclear Proteins in Pathogen-Treated Arabidopsis 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.

Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures.

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation--the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus.

Effect of nitric oxide on gene transcription - S-nitrosylation of nuclear proteins.

Nitric oxide (NO) plays an important role in many different physiological processes in plants. It mainly acts by post-translationally modifying proteins. Modification of cysteine residues termed as S-nitrosylation is believed to be the most important mechanism for transduction of bioactivity of NO. The first proteins found to be nitrosylated were mainly of cytoplasmic origin or isolated from mitochondria and peroxisomes. Interestingly, it was shown that redox-sensitive transcription factors are also nitrosylated and that NO influences the redox-dependent nuclear transport of some proteins. This implies that NO plays a role in regulating transcription and/or general nuclear metabolism which is a fascinating new aspect of NO signaling in plants. In this review, we will discuss the impact of S-nitrosylation on nuclear plant proteins with a focus on transcriptional regulation, describe the function of this modification and draw also comparisons to the animal system in which S-nitrosylation of nuclear proteins is a well characterized concept.