A sleigh ride through the SNO: regulation of plant immune function by protein S-nitrosylation

S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO) is rapidly emerging as a prototypic, redox-based post-translational modification during plant immune function. Here we review recently identified targets for S-nitrosylation and the consequences of these modifications in relation to the control of plant disease resistance.     

Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus–host protein–protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification–mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus–host protein–protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus–host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.     

S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability

S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K_M of nitrosylated PtpA was similar to its unmodified form, but the V_max was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K_M or V_max. These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.     

The O-linked N-acetylglucosamine modification in cellular signalling and the immune system. 'Protein modifications: beyond the usual suspects' review series

The intracellular modification of proteins by the addition of a single O-linked N-acetylglucosamine (O-GlcNAc) molecule is a ubiquitous post-translational modification in eukaryotic cells. It is catalysed by O-linked N-acetylglucosaminyltransferase, which attaches O-GlcNAc to serine/threonine residues, and it is counter-regulated by β-N-acetylglucosaminidase, which is the antagonistic glycosidase that removes the O-GlcNAc group. O-GlcNAc modification competes with phosphorylation by protein kinases at similar sites, thereby affecting important signalling nodes. Accumulating evidence supports a central role for O-GlcNAc modifications and the corresponding enzymes in the regulation of immune cells, particularly in the activation processes of T and B lymphocytes. Here, we discuss recent advances in the field of O-GlcNAc modifications, focusing on the cells of the immune system.     

Regulation of Protein Function and Signaling by Reversible Cysteine S-Nitrosylation

NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications.     

S-nitrosylation-regulated GPCR signaling

G protein-coupled receptors (GPCRs) are the most numerous and diverse type of cell surface receptors, accounting for about 1% of the entire human genome and relaying signals from a variety of extracellular stimuli that range from lipid and peptide growth factors to ions and sensory inputs. Activated GPCRs regulate a multitude of target cell functions, including intermediary metabolism, growth and differentiation, and migration and invasion. The GPCRs contain a characteristic 7-transmembrane domain topology and their activation promotes complex formation with a variety of intracellular partner proteins, which form basis for initiation of distinct signaling networks as well as dictate fate of the receptor itself. Both termination of active GPCR signaling and removal from the plasma membrane are controlled by protein post-translational modifications of the receptor itself and its interacting partners. Phosphorylation, acylation and ubiquitination are the most studied post-translational modifications involved in GPCR signal transduction, subcellular trafficking and overall expression. Emerging evidence demonstrates that protein S-nitrosylation, the covalent attachment of a nitric oxide moiety to specified cysteine thiol groups, of GPCRs and/or their associated effectors also participates in the fine-tuning of receptor signaling and expression. This newly appreciated mode of GPCR system modification adds another set of controls to more precisely regulate the many cellular functions elicited by this large group of receptors. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.     

Membrane traffic: controlling membrane fusion by modifying NSF

Recent studies show that NSF, isolated over 15 years ago as a protein required for membrane fusion in vitro, can be reversibly inactivated by both S-nitrosylation and tyrosine phosphorylation. Different cell types use distinct post-translational modifications of NSF for localized regulation of membrane fusion.     

S-nitrosylation and S-glutathionylation of GAPDH: Similarities,differences, and relationships

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation.Incubation of HEK 293 T cells in the presence of H₂O₂ or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H₂O₂ or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.     

Biological selectivity and functional aspects of protein tyrosine nitration

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.     

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.     

蛋白质巯基亚硝基化参与调控一氧化氮介导的心肌缺血预适应

一氧化氮（nitricoxide,NO）作为一种重要的信使分子参与缺血预适应（ischemic preconditioning,IPC）心肌保护。目前普遍认为NO通过经典的NO／cGMP依赖的信号转导途径调节线粒体ATP敏感性钾（ATP-sensitive potassium,KATP通道来发挥其保护作用,然而越来越多的数据表明NO还可能通过蛋白质巯基亚硝基化（S-nitrosylation）来发挥生理功能。蛋白质巯基亚硝基化,即蛋白质半胱氨酸巯基与NO基团形成共价键,是一种氧化还原依赖的蛋白质翻译后可逆修饰。蛋白质巯基亚硝基化不仅可以改变蛋白质的结构和功能,而且还可以阻抑目标半胱氨酸的进一步氧化修饰。IPC增加S-亚硝基硫醇（S-nitrosothi01）含量,引起蛋白质巯基亚硝基化。S-亚硝基硫醇还能发挥药理性预适应作用,抵抗心肌缺血,再灌注损伤。因此,蛋白质巯基亚硝基化是IPC心肌保护的一种重要途径,参与抵抗细胞内氧化应激和亚硝化应激（nitrosative stress）。     

Characterisation of protein microheterogeneity and protein complexes using on-chip immunoaffinity purification-mass spectrometry.

Post-translational modification of proteins may influence their interactions with other plasma proteins, as well as having an effect on many aspects of the metabolism of the protein, such as receptor binding, tissue uptake, degradation and excretion. Many post-translational modifications occur in a physiological context, while others are specific for certain diseases, which is why they are of diagnostic importance in clinical proteomics. Analytical approaches to the study of post-translational modifications and protein complexes through the combined use of on-chip immunological affinity purification on a surface-enhanced laser desorption/ionisation platform and subsequent mass spectrometry are illustrated in the author's own work relating to plasma transthyretin (TTR) and retinol-binding protein (RBP). In those studies, both the aspects of post-translational modifications of TTR and the formation of a protein complex between TTR and RBP have been discussed. Such aspects are of diagnostic interest in clinical proteomics, especially with regard to the modification of TTR in relation to the occurrence of amyloidotic diseases.     

S-nitrosylation of Bcl-2 inhibits its ubiquitin-proteasomal degradation. A novel antiapoptotic mechanism that suppresses apoptosis

Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway whose function is dependent on its expression levels. Although Bcl-2 expression is controlled by various mechanisms, post-translational modifications, such as ubiquitination and proteasomal degradation, have emerged as important regulators of Bcl-2 function. However, the underlying mechanisms of this regulation are unclear. We report here that Bcl-2 undergoes S-nitrosylation by endogenous nitric oxide (NO) in response to multiple apoptotic mediators and that this modification inhibits ubiquitin-proteasomal degradation of Bcl-2. Inhibition of NO production by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and by NO synthase inhibitor aminoguanidine effectively inhibited S-nitrosylation of Bcl-2, increased its ubiquitination, and promoted apoptotic cell death induced by chromium (VI). In contrast, the NO donors dipropylenetriamine NONOate and sodium nitroprusside showed opposite effects. The effect of NO on Bcl-2 stability was shown to be independent of its dephosphorylation. Mutational analysis of Bcl-2 further showed that the two cysteine residues of Bcl-2 (Cys158 and Cys229) are important in the S-nitrosylation process and that mutations of these cysteines completely inhibited Bcl-2 S-nitrosylation. Treatment of the cells with other stress inducers, including Fas ligand and buthionine sulfoxide, also induced Bcl-2 S-nitrosylation, suggesting that this is a general phenomenon that regulates Bcl-2 stability and function under various stress conditions. These findings indicate a novel function of NO and its regulation of Bcl-2, which provides a key mechanism for the control of apoptotic cell death and cancer development.     

S-nitrosylation-induced conformational change in blackfin tuna myoglobin

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.     

Protein S-nitrosylation and denitrosylation in the mouse spinal cord upon injury of the sciatic nerve

Nitric oxide is a pain signaling molecule and exerts its influence through two primary pathways: by stimulation of soluble guanylylcyclase and by direct S-nitrosylation (SNO) of target proteins. We assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME). After nerve injury, SNO-DIGE revealed 30 proteins with increased and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 proteins. After sham surgery only 3 proteins were up-nitrosylated. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7days showed that SNO modifications of protein disulfide isomerase, glutathione synthase and peroxiredoxin-6 had returned to baseline within 7days whereas S-nitrosylation of mitochondrial aconitase 2 was further increased. The identified SNO modified proteins are involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. The data show that nitric oxide mediated S-nitrosylation contributes to the nerve injury-evoked pathology in nociceptive signaling pathways.     

S-Nitrosylation in plants: Pattern and function

During the last two decades nitric oxide (NO) has emerged as a new chemical messenger in plant biology, which is involved in many different physiological processes, such as plant defense, transpiration and gas exchange, seed germination, and root development. Protein S-nitrosylation, the post-translational modification of thiol residues, has been suggested to be the most important mechanism for transduction of the bioactivity of NO. The characterization of protein S-nitrosylation as well as the physiological relevance of this type of modification is essential information, which is necessary to understand the function of NO in plants. In this review we focus on the formation of nitrosothiols and describe the chemistry of NO and thiol groups. Furthermore, different methods for detection of S-nitrosothiols are highlighted and the function of S-nitrosylation in plants is discussed.     

Protein S-nitrosylation: purview and parameters

S-nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine, has emerged as an important mechanism for dynamic, post-translational regulation of most or all main classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of nitric oxide (NO) on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.     

Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies

Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.     

S-nitrosylation of PTEN Invovled in Ischemic Brain Injury in Rat Hippocampal CA1 Region

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is not only a protein, but also a lipid phosphatase that can negatively regulate the serine/threonine kinase Akt. It has been reported that PTEN can be regulated by means of phosphorylation. However, whether PTEN can be regulated by another post-translational protein modification (S-nitrosylation) was not fully elucidated. In this study, we investigated the S-nitrosylation of PTEN during transient cerebral ischemia/reperfusion in rat hippocampus. Transient brain ischemia was induced by the four-vessel occlusion in Sprague–Dawley rats. Our data show that S-nitrosylation of PTEN was increased significantly after 12 h of reperfusion compared with sham control. Pretreatment with the inhibitor of nNOS (7-NI) and the inhibitor of iNOS could inhibit PTEN’s activity and decrease S-nitrosylation of PTEN. Taken together, these results indicate that nitric oxide could regulate PTEN’s activity via S-nitrosylation during transient global ischemia in rat hippocampus. The authors D.-S. Pei, Y.-F. Sun contribute equally to this work.