Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis>

Nitric oxide (NO) is a key signaling molecule in plants, being its biological effects mainly mediated through S-nitrosylation of cysteine thiols. Using the biotin switch method combined with mass spectrometry analysis we have identified 127 targets of S-nitrosylation in Arabidopsis cell suspension cultures and leaves challenged with virulent and avirulent isolates of Pseudomonas syringae pv. tomato. The NO targets are proteins associated with carbon, nitrogen, and sulpfur metabolism, photosynthesis, the cytoskeleton, stress-, pathogen- and redox-related and signaling proteins. Some proteins were previously identified in plants and mammals, while others (63%) represent novel targets of S-nitrosylation. Our data suggest that NO might be orchestrating the whole plant physiology, presumably through covalent modification of proteins.     

亚硝基化在植物细胞死亡及防御反应中的作用

亚硝基化是近年来新发现的不依赖于环磷酸鸟苷的一氧化氮信号转导途径, 是一氧化氮分子通过共价结合修饰靶蛋白的半胱氨酸残基从而改变其功能的过程。该文重点综述了近年来亚硝基化在细胞死亡和抗病反应这两个紧密关联的生物学过程中的最新研究成果, 总结了亚硝基化通过修饰和调控靶蛋白从而促进或抑制细胞死亡和抗病反应, 并对现有研究结果中某些不一致之处提出自己的观点。最后根据动物学领域的最新研究进展对植物学领域未来亚硝基化的研究方向进行了展望。     

蛋白亚硝基化研究进展及其在植物抗病中的作用

蛋白亚硝基化（S-nitrosylation）是一种在一氧化氮作用下与蛋白半胱氨酸巯基共价结合,使巯基-SH转化为-SNO的反应。作为一种氧化还原依赖的翻译后调控形式,蛋白亚硝基化对多种蛋白的功能具有调节作用,越来越多的证据表明蛋白亚硝基化在植物抗病中发挥重要的作用。简要介绍了蛋白巯基亚硝基化的特点、检测方法、功能研究以及在植物抗病调节方面的最新进展。     

Protein S‐Nitrosylation in plants: Current progresses and challenges

Nitric oxide(NO) is an important signaling molecule regulating diverse biological processes in all living organisms. A major physiological function of NO is executed via protein S-nitrosylation, a redox-based the past decade, significant progress has been made in functional characterization of S-nitrosylated proteins Inviteposttranslational modification by covalently adding a NO molecule to a reactive cysteine thiol of a target protein.S-nitrosylation is an evolutionarily conserved mechanism modulating multiple aspects of cellular signaling. Duringin plants. Emerging evidence indicates that protein Snitrosylation is ubiquitously involved in the regulation of plant development and stress responses. Here we review current understanding on the regulatory mechanisms of protein S-nitrosylation in various biological processes in plants and highlight key challenges in this field.     

Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response

Nitric oxide (NO) has a fundamental role in the plant hypersensitive disease resistance response (HR), and S-nitrosylation is emerging as an important mechanism for the transduction of its bioactivity. A key step toward elucidating the mechanisms by which NO functions during the HR is the identification of the proteins that are subjected to this PTM. By using a proteomic approach involving 2-DE and MS we characterized, for the first time, changes in S-nitrosylated proteins in Arabidopsis thaliana undergoing HR. The 16 S-nitrosylated proteins identified are mostly enzymes serving intermediary metabolism, signaling and antioxidant defense. The study of the effects of S-nitrosylation on the activity of the identified proteins and its role during the execution of the disease resistance response will help to understand S-nitrosylation function and significance in plants.     

Differential inhibition of Arabidopsis methionine adenosyltransferases by protein S-nitrosylation

In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, whereas MAT2 and MAT3 were not significantly affected. Cys-114 of MAT1 was identified as the most promising target of NO-induced inhibition of MAT1, because this residue is absent in MAT2 and MAT3. Structural analysis of MAT1 revealed that Cys-114 is located nearby the putative substrate binding site of this enzyme. Furthermore, Cys-114 is flanked by S-nitrosylation-promoting amino acids. The inhibitory effect of GSNO was drastically reduced when Cys-114 of MAT1 was replaced by arginine, and mass spectrometric analyses of Cys-114-containing peptides obtained after chymotryptic digestion demonstrated that Cys-114 of MAT1 is indeed S-nitrosylated. Because MAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling.     

植物细胞一氧化氮信号转导研究进展

一氧化氮(nitric oxide, NO)作为重要的信号分子, 调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程, 并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道, 精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基, 通过金属亚硝基化、巯基亚硝基化和Tyr-硝基化等化学修饰方式, 调节靶蛋白的活性, 并影响cGMP和Ca²⁺信使系统等下游信号途径, 调控相应的生理过程。最新的一些研究结果也显示, MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外, 作为活跃的小分子信号, NO和活性氧相互依赖并相互影响, 共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展, 对NO与活性氧信号途径间的交叉作用等也作了简要介绍。     

Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.     

Protein S-nitrosylation: What's going on in plants?

Nitric oxide (NO) is now recognized as a key regulator of plant physiological processes. Understanding the mechanisms by which NO exerts its biological functions has been the subject of extensive research. Several components of the signaling pathways relaying NO effects in plants, including second messengers, protein kinases, phytohormones, and target genes, have been characterized. In addition, there is now compelling experimental evidence that NO partly operates through posttranslational modification of proteins, notably via S-nitrosylation and tyrosine nitration. Recently, proteome-wide scale analyses led to the identification of numerous protein candidates for S-nitrosylation in plants. Subsequent biochemical and in silico structural studies revealed certain mechanisms through which S-nitrosylation impacts their functions. Furthermore, first insights into the physiological relevance of S-nitrosylation, particularly in controlling plant immune responses, have been recently reported. Collectively, these discoveries greatly extend our knowledge of NO functions and of the molecular processes inherent to signal transduction in plants.     

S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata- ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition

Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.     

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.     

Nitric oxide signalling via cytoskeleton in plants

Nitric oxide (NO) in plant cell mediates processes of growth and development starting from seed germination to pollination, as well as biotic and abiotic stress tolerance. However, proper understanding of the molecular mechanisms of NO signalling in plants has just begun to emerge. Accumulated evidence suggests that in eukaryotic cells NO regulates functions of proteins by their post-translational modifications, namely tyrosine nitration and S-nitrosylation. Among the candidates for NO-downstream effectors are cytoskeletal proteins because of their involvement in many processes regulated by NO. This review discusses new insights in plant NO signalling focused mainly on the involvement of cytoskeleton components into NO-cascades. Herein, examples of NO-related post-translational modifications of cytoskeletal proteins, and also indirect NO impact, are discussed. Special attention is paid to plant α-tubulin tyrosine nitration as an emerging topic in plant NO research.     

Proteomic analysis of defense response of wildtype Arabidopsis thaliana and plants with impaired NO- homeostasis

In recent years, nitric oxide (NO) has been recognized as a signalling molecule of plants, being involved in diverse processes like germination, root growth, stomatal closing, and responses to various stresses. A mechanism of how NO can regulate physiological processes is the modulation of cysteine residues of proteins (S-nitrosylation) by S-nitrosoglutathione (GSNO), a physiological NO donor. The concentration of GSNO and the level of S-nitrosylated proteins are regulated by GSNO reductase, which seems to play a major role in NO signalling. To investigate the importance of NO in plant defense response, we performed a proteomic analysis of Arabidopsis wildtype and GSNO-reductase knock-out plants infected with both the avirulent and virulent pathogen strains of Pseudomonas syringae. Using 2-D DIGE technology in combination with MS, we identified proteins, which are differentially accumulated during the infection process. We observed that both lines were more resistant to avirulent infections than to virulent infections mainly due to the accumulation of stress-, redox-, and defense-related proteins. Interestingly, after virulent infections, we also observed accumulation of defense-related proteins, but no or low accumulation of stress- and redox-related proteins, respectively. In summary, we present here the first detailed proteomic analysis of plant defense response.     

Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases

The pathological processes of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases engender synaptic and neuronal cell damage. While mild oxidative and nitrosative (nitric oxide (NO)-related) stress mediates normal neuronal signaling, excessive accumulation of these free radicals is linked to neuronal cell injury or death. In neurons, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation and subsequent Ca(2+) influx can induce the generation of NO via neuronal NO synthase. Emerging evidence has demonstrated that S-nitrosylation, representing covalent reaction of an NO group with a critical protein thiol, mediates the vast majority of NO signaling. Analogous to phosphorylation and other posttranslational modifications, S-nitrosylation can regulate the biological activity of many proteins. Here, we discuss recent studies that implicate neuropathogenic roles of S-nitrosylation in protein misfolding, mitochondrial dysfunction, synaptic injury, and eventual neuronal loss. Among a growing number of S-nitrosylated proteins that contribute to disease pathogenesis, in this review we focus on S-nitrosylated protein-disulfide isomerase (forming SNO-PDI) and dynamin-related protein 1 (forming SNO-Drp1). Furthermore, we describe drugs, such as memantine and newer derivatives of this compound that can prevent both hyperactivation of extrasynaptic NMDARs as well as downstream pathways that lead to nitrosative stress, synaptic damage, and neuronal loss.     

Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.     

Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.     

Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue

Nitric oxide (NO) regulates a number of signaling functions in both animals and plants under several physiological and pathophysiological conditions. S-Nitrosylation linking a nitrosothiol on cysteine residues mediates NO signaling functions of a broad spectrum of mammalian proteins, including caspases, the main effectors of apoptosis. Metacaspases are suggested to be the ancestors of metazoan caspases, and plant metacaspases have previously been shown to be genuine cysteine proteases that autoprocess in a manner similar to that of caspases. We show that S-nitrosylation plays a central role in the regulation of the proteolytic activity of Arabidopsis thaliana metacaspase 9 (AtMC9) and hypothesize that this S-nitrosylation affects the cellular processes in which metacaspases are involved. We found that AtMC9 zymogens are S-nitrosylated at their active site cysteines in vivo and that this posttranslational modification suppresses both AtMC9 autoprocessing and proteolytic activity. However, the mature processed form is not prone to NO inhibition due to the presence of a second S-nitrosylation-insensitive cysteine that can replace the S-nitrosylated cysteine residue within the catalytic center of the processed AtMC9. This cysteine is absent in caspases and paracaspases but is conserved in all reported metacaspases.     

Effects of endogenous ligands on the biological role of human serum albumin in S-nitrosylation

Many proteins have been identified as targets for S-nitrosylation, including structural and signaling proteins, and ion channels. S-nitrosylation plays an important role in regulating their activity and function. We used human serum albumin (HSA), a major endogenous NO traffic protein, and studied the effect of mediators on S-nitrosylation processes which control NO bioactivity. By using NOC-7, S-nitrosoglutathione, and activated RAW264.7 cells as NO-donors we found that high-affinity binding of endogenous ligands (Cu²⁺, bilirubin and fatty acid) can affect these processes. It is likely that the same effects take place in many clinical situations characterized by increased fatty acid concentrations in plasma such as type II diabetes and the metabolic syndrome. Thus, endogenous ligands, changing their plasma concentrations, could be a novel type of mediator of S-nitrosylation not only in the case of HSA but also for other target proteins.