Showing papers on "S-Nitrosylation published in 2022"

OUP accepted manuscript

Abstract: The thiol group of cysteine (Cys) residues, often present in the active center of the protein, is of particular importance to protein function, which is significantly determined by the redox state of a protein's environment. Our knowledge of different thiol-based oxidative posttranslational modifications (oxiPTMs), which compete for specific protein thiol groups, has increased over the last 10 years. The principal oxiPTMs include S-sulfenylation, S-glutathionylation, S-nitrosation, persulfidation, S-cyanylation and S-acylation. The role of each oxiPTM depends on the redox cellular state, which in turn depends on cellular homeostasis under either optimal or stressful conditions. Under such conditions, the metabolism of molecules such as glutathione, NADPH (reduced nicotinamide adenine dinucleotide phosphate), nitric oxide, hydrogen sulfide and hydrogen peroxide can be altered, exacerbated and, consequently, outside the cell's control. This review provides a broad overview of these oxiPTMs under physiological and unfavorable conditions, which can regulate the function of target proteins.

Phytohormonal Regulation Through Protein S-Nitrosylation Under Stress

Abstract: The liaison between Nitric oxide (NO) and phytohormones regulates a myriad of physiological processes at the cellular level. The interaction between NO and phytohormones is mainly influenced by NO-mediated post-translational modifications (PTMs) under basal as well as induced conditions. Protein S-nitrosylation is the most prominent and widely studied PTM among others. It is the selective but reversible redox-based covalent addition of a NO moiety to the sulfhydryl group of cysteine (Cys) molecule(s) on a target protein to form S-nitrosothiols. This process may involve either direct S-nitrosylation or indirect S-nitrosylation followed by transfer of NO group from one thiol to another (transnitrosylation). During S-nitrosylation, NO can directly target Cys residue (s) of key genes involved in hormone signaling thereby regulating their function. The phytohormones regulated by NO in this manner includes abscisic acid, auxin, gibberellic acid, cytokinin, ethylene, salicylic acid, jasmonic acid, brassinosteroid, and strigolactone during various metabolic and physiological conditions and environmental stress responses. S-nitrosylation of key proteins involved in the phytohormonal network occurs during their synthesis, degradation, or signaling roles depending upon the response required to maintain cellular homeostasis. This review presents the interaction between NO and phytohormones and the role of the canonical NO-mediated post-translational modification particularly, S-nitrosylation of key proteins involved in the phytohormonal networks under biotic and abiotic stresses.

Thiol-based Oxidative Posttranslational Modifications (OxiPTMs) of Plant Proteins

Abstract: Abstract The thiol group of cysteine (Cys) residues, often present in the active center of the protein, is of particular importance to protein function, which is significantly determined by the redox state of a protein’s environment. Our knowledge of different thiol-based oxidative posttranslational modifications (oxiPTMs), which compete for specific protein thiol groups, has increased over the last 10 years. The principal oxiPTMs include S-sulfenylation, S-glutathionylation, S-nitrosation, persulfidation, S-cyanylation and S-acylation. The role of each oxiPTM depends on the redox cellular state, which in turn depends on cellular homeostasis under either optimal or stressful conditions. Under such conditions, the metabolism of molecules such as glutathione, NADPH (reduced nicotinamide adenine dinucleotide phosphate), nitric oxide, hydrogen sulfide and hydrogen peroxide can be altered, exacerbated and, consequently, outside the cell’s control. This review provides a broad overview of these oxiPTMs under physiological and unfavorable conditions, which can regulate the function of target proteins.

The emerging roles of nitric oxide in ferroptosis and pyroptosis of tumor cells

Abstract: Tumor cells can develop resistance to cell death which is divided into necrosis and programmed cell death (PCD). PCD, including apoptosis, autophagy, ferroptosis, pyroptosis, and necroptosis. Ferroptosis and pyroptosis, two new forms of cell death, have gradually been of interest to researchers. Boosting ferroptosis and pyroptosis of tumor cells could be a potential cancer therapy. Nitric oxide (NO) is a ubiquitous, lipophilic, highly diffusible, free-radical signaling molecule that plays various roles in tumorigenesis. In addition, NO also has regulatory mechanisms through S-nitrosylation that do not depend on the classic NO/sGC/cGMP signaling. The current tumor treatment strategy for NO is to promote cell death through promoting S-nitrosylation-induced apoptosis while multiple drawbacks dampen this tumor therapy. However, numerous studies have suggested that suppression of NO is perceived to active ferroptosis and pyroptosis, which could be a better anti-tumor treatment. In this review, ferroptosis and pyroptosis are described in detail. We summarize that NO influences ferroptosis and pyroptosis and infer that S-nitrosylation mediates ferroptosis- and pyroptosis-related signaling pathways. It could be a potential cancer therapy different from NO-induced apoptosis of tumor cells. Finally, the information shows the drugs that manipulate endogenous production and exogenous delivery of NO to modulate the levels of S-nitrosylation.

Mechanistic insight into female predominance in Alzheimer’s disease based on aberrant protein S-nitrosylation of C3

Abstract: Protein S-nitros(yl)ation (SNO) is a posttranslational modification involved in diverse processes in health and disease and can contribute to synaptic damage in Alzheimer’s disease (AD). To identify SNO proteins in AD brains, we used triaryl phosphine (SNOTRAP) combined with mass spectrometry (MS). We detected 1449 SNO proteins with 2809 SNO sites, representing a wide range of S-nitrosylated proteins in 40 postmortem AD and non-AD human brains from patients of both sexes. Integrative protein ranking revealed the top 10 increased SNO proteins, including complement component 3 (C3), p62 (SQSTM1), and phospholipase D3. Increased levels of S-nitrosylated C3 were present in female over male AD brains. Mechanistically, we show that formation of SNO-C3 is dependent on falling β-estradiol levels, leading to increased synaptic phagocytosis and thus synapse loss and consequent cognitive decline. Collectively, we demonstrate robust alterations in the S-nitrosoproteome that contribute to AD pathogenesis in a sex-dependent manner.

GSNOR regulates ganoderic acid content in Ganoderma lucidum under heat stress through S-nitrosylation of catalase

Abstract: As a master regulator of the balance between NO signaling and protein S-nitrosylation, S-nitrosoglutathione (GSNO) reductase (GSNOR) is involved in various developmental processes and stress responses. However, the proteins and specific sites that can be S-nitrosylated, especially in microorganisms, and the physiological functions of S-nitrosylated proteins remain unclear. Herein, we show that the ganoderic acid (GA) content in GSNOR-silenced (GSNORi) strains is significantly lower (by 25%) than in wild type (WT) under heat stress (HS). Additionally, silencing GSNOR results in an 80% increase in catalase (CAT) activity, which consequently decreases GA accumulation via inhibition of ROS signaling. The mechanism of GSNOR-mediated control of CAT activity may be via protein S-nitrosylation. In support of this possibility, we show that CAT is S-nitrosylated (as shown via recombinant protein in vitro and via GSNORi strains in vivo). Additionally, Cys (cysteine) 401, Cys642 and Cys653 in CAT are S-nitrosylation sites (assayed via mass spectrometry analysis), and Cys401 may play a pivotal role in CAT activity. These findings indicate a mechanism by which GSNOR responds to stress and regulates secondary metabolite content through protein S-nitrosylation. Our results also define a new S-nitrosylation site and the function of an S-nitrosylated protein regulated by GSNOR in microorganisms.

Protein S-Nitrosation: Biochemistry, Identification, Molecular Mechanisms, and Therapeutic Applications.

Abstract: Protein S-nitrosation (SNO), a posttranslational modification (PTM) of cysteine (Cys) residues elicited by nitric oxide (NO), regulates a wide range of protein functions. As a crucial form of redox-based signaling by NO, SNO contributes significantly to the modulation of physiological functions, and SNO imbalance is closely linked to pathophysiological processes. Site-specific identification of the SNO protein is critical for understanding the underlying molecular mechanisms of protein function regulation. Although careful verification is needed, SNO modification data containing numerous functional proteins are a potential research direction for druggable target identification and drug discovery. Undoubtedly, SNO-related research is meaningful not only for the development of NO donor drugs but also for classic target-based drug design. Herein, we provide a comprehensive summary of SNO, including its origin and transport, identification, function, and potential contribution to drug discovery. Importantly, we propose new views to develop novel therapies based on potential protein SNO-sourced targets.

S-Glutathionylation and S-Nitrosylation in Mitochondria: Focus on Homeostasis and Neurodegenerative Diseases

Abstract: Redox post-translational modifications are derived from fluctuations in the redox potential and modulate protein function, localization, activity and structure. Amongst the oxidative reversible modifications, the S-glutathionylation of proteins was the first to be characterized as a post-translational modification, which primarily protects proteins from irreversible oxidation. However, a growing body of evidence suggests that S-glutathionylation plays a key role in core cell processes, particularly in mitochondria, which are the main source of reactive oxygen species. S-nitrosylation, another post-translational modification, was identified >150 years ago, but it was re-introduced as a prototype cell-signaling mechanism only recently, one that tightly regulates core processes within the cell’s sub-compartments, especially in mitochondria. S-glutathionylation and S-nitrosylation are modulated by fluctuations in reactive oxygen and nitrogen species and, in turn, orchestrate mitochondrial bioenergetics machinery, morphology, nutrients metabolism and apoptosis. In many neurodegenerative disorders, mitochondria dysfunction and oxidative/nitrosative stresses trigger or exacerbate their pathologies. Despite the substantial amount of research for most of these disorders, there are no successful treatments, while antioxidant supplementation failed in the majority of clinical trials. Herein, we discuss how S-glutathionylation and S-nitrosylation interfere in mitochondrial homeostasis and how the deregulation of these modifications is associated with Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and Friedreich’s ataxia.

GSNOR regulates ganoderic acid content in Ganoderma lucidum under heat stress through S-nitrosylation of catalase

Abstract: As a master regulator of the balance between NO signaling and protein S-nitrosylation, S-nitrosoglutathione (GSNO) reductase (GSNOR) is involved in various developmental processes and stress responses. However, the proteins and specific sites that can be S-nitrosylated, especially in microorganisms, and the physiological functions of S-nitrosylated proteins remain unclear. Herein, we show that the ganoderic acid (GA) content in GSNOR-silenced (GSNORi) strains is significantly lower (by 25%) than in wild type (WT) under heat stress (HS). Additionally, silencing GSNOR results in an 80% increase in catalase (CAT) activity, which consequently decreases GA accumulation via inhibition of ROS signaling. The mechanism of GSNOR-mediated control of CAT activity may be via protein S-nitrosylation. In support of this possibility, we show that CAT is S-nitrosylated (as shown via recombinant protein in vitro and via GSNORi strains in vivo). Additionally, Cys (cysteine) 401, Cys642 and Cys653 in CAT are S-nitrosylation sites (assayed via mass spectrometry analysis), and Cys401 may play a pivotal role in CAT activity. These findings indicate a mechanism by which GSNOR responds to stress and regulates secondary metabolite content through protein S-nitrosylation. Our results also define a new S-nitrosylation site and the function of an S-nitrosylated protein regulated by GSNOR in microorganisms.

S‐Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S‐Nitrosylation and Preeclampsia

Abstract: Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase (GSNOR-⁄-), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR-⁄- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR-⁄- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

Phytochrome B enhances seed germination tolerance to high temperature by reducing S‐nitrosylation of HFR1

Abstract: Light and ambient high temperature (HT) have opposite effects on seed germination. Light induces seed germination through activating the photoreceptor phytochrome B (phyB), resulting in the stabilization of the transcription factor HFR1, which in turn sequesters the suppressor PIF1. HT suppresses seed germination and triggers protein S‐nitrosylation. Here, we find that HT suppresses seed germination by inducing the S‐nitrosylation of HFR1 at C164, resulting in its degradation, the release of PIF1, and the activation of PIF1‐targeted SOMNUS (SOM) expression to alter gibberellin (GA) and abscisic acid (ABA) metabolism. Active phyB (phyBY276H) antagonizes HFR1 S‐nitrosylation and degradation by increasing S‐nitrosoglutathione reductase (GSNOR) activity. In line with this, substituting cysteine‐164 of HFR1 with serine (HFR1C164S) abolishes the S‐nitrosylation of HFR1 and decreases the HT‐induced degradation of HFR1. Taken together, our study suggests that HT and phyB antagonistically modulate the S‐nitrosylation level of HFR1 to coordinate seed germination, and provides the possibility to enhance seed thermotolerance through gene‐editing of HFR1.

Mass spectrometry analysis of S-nitrosylation of proteins and its role in cancer, cardiovascular and neurodegenerative diseases

Abstract: Mass spectrometry (MS) has become an indispensable tool in the study of protein identification, protein interactions, and post-translational modifications (PTMs). S-nitrosylation is a major nitric oxide-dependent covalent PTM that targets specific cysteine residues and affects a variety of processes in diverse biological systems. Nowadays, the significance of protein S-nitrosylation in cellular physiology and pathophysiology is increasingly recognized. To deeply understand the formation, selectivity, and regulatory roles of S-nitrosylation, global identifications of S-nitrosylated proteins and their modification sites are highly required. This review summarizes the currently available approaches combining with MS for the analysis of protein S-nitrosylation. Sample preparation techniques specific to S-nitrosylated proteins are evaluated. Meanwhile, we also demonstrate the MS-based qualitative and quantitative proteomic strategies for S-nitrosylation measurement. Roles of protein S-nitrosylation in human diseases are further discussed with emphasis on cancer, cardiovascular and neurodegenerative diseases. In the end, current limitations and future research directions are presented.

Focus on Nitric Oxide Homeostasis: Direct and Indirect Enzymatic Regulation of Protein Denitrosation Reactions in Plants

Abstract: Protein cysteines (Cys) undergo a multitude of different reactive oxygen species (ROS), reactive sulfur species (RSS), and/or reactive nitrogen species (RNS)-derived modifications. S-nitrosation (also referred to as nitrosylation), the addition of a nitric oxide (NO) group to reactive Cys thiols, can alter protein stability and activity and can result in changes of protein subcellular localization. Although it is clear that this nitrosative posttranslational modification (PTM) regulates multiple signal transduction pathways in plants, the enzymatic systems that catalyze the reverse S-denitrosation reaction are poorly understood. This review provides an overview of the biochemistry and regulation of nitro-oxidative modifications of protein Cys residues with a focus on NO production and S-nitrosation. In addition, the importance and recent advances in defining enzymatic systems proposed to be involved in regulating S-denitrosation are addressed, specifically cytosolic thioredoxins (TRX) and the newly identified aldo-keto reductases (AKR).

S-Denitrosylation: A Crosstalk between Glutathione and Redoxin Systems

Abstract: S-nitrosylation of proteins occurs as a consequence of the derivatization of cysteine thiols with nitric oxide (NO) and is often associated with diseases and protein malfunction. Aberrant S-nitrosylation, in addition to other genetic and epigenetic factors, has gained rapid importance as a prime cause of various metabolic, respiratory, and cardiac disorders, with a major emphasis on cancer and neurodegeneration. The S-nitrosoproteome, a term used to collectively refer to the diverse and dynamic repertoire of S-nitrosylated proteins, is relatively less explored in the field of redox biochemistry, in contrast to other covalently modified versions of the same set of proteins. Advancing research is gradually unveiling the enormous clinical importance of S-nitrosylation in the etiology of diseases and is opening up new avenues of prompt diagnosis that harness this phenomenon. Ever since the discovery of the two robust and highly conserved S-nitrosoglutathione reductase and thioredoxin systems as candidate denitrosylases, years of rampant speculation centered around the identification of specific substrates and other candidate denitrosylases, subcellular localization of both substrates and denitrosylases, the position of susceptible thiols, mechanisms of S-denitrosylation under basal and stimulus-dependent conditions, impact on protein conformation and function, and extrapolating these findings towards the understanding of diseases, aging and the development of novel therapeutic strategies. However, newer insights in the ever-expanding field of redox biology reveal distinct gaps in exploring the crucial crosstalk between the redoxins/major denitrosylase systems. Clarifying the importance of the functional overlap of the glutaredoxin, glutathione, and thioredoxin systems and examining their complementary functions as denitrosylases and antioxidant enzymatic defense systems are essential prerequisites for devising a rationale that could aid in predicting the extent of cell survival under high oxidative/nitrosative stress while taking into account the existence of the alternative and compensatory regulatory mechanisms. This review thus attempts to highlight major gaps in our understanding of the robust cellular redox regulation system, which is upheld by the concerted efforts of various denitrosylases and antioxidants.

S-Nitrosylation-Mediated Reduction of Ca _V 1.2 Surface Expression and Open Probability Underlies Attenuated Vasoconstriction Induced by Nitric Oxide

Abstract: Background: L-type Ca V 1.2 calcium channel, the primary gateway for Ca 2+ influx in smooth muscles, is widely regulated by multiple posttranslational modifications, such as protein kinase-mediated phosphorylation and nitric oxide-induced S-nitrosylation. However, the effect of S-nitrosylation on Ca V 1.2 channel function and its role in arterial contractility are not well understood. Methods: Electrophysiological recordings, Ca 2+ and confocal imaging, and biochemical assays were used to functionally characterize S-nitrosylated Ca V 1.2 channels in vitro, while pressure myography and tail-cuff blood pressure measurement were conducted to evaluate the physiological effects of Ca V 1.2 S-nitrosylation ex vivo and in vivo. Results: S-nitrosylation significantly reduced the Ca V 1.2 current density by promoting lysosomal degradation that leads to decreased levels of total and surface Ca V 1.2 channel proteins in a Ca V β-independent manner and reducing the open probability of Ca V 1.2 channel. Mechanistically, the Cys1180 and Cys1280 residues within Ca V 1.2 channel have been determined as the molecular targets for S-nitrosylation as substitution of either Cys1180 or Cys1280 for serine resulted in substantial reduction of S-nitrosylation levels. Of note, Ca V 1.2 S-nitrosylation levels were significantly reduced in arteries isolated from both spontaneously hypertensive rats and patients with pulmonary hypertension. Moreover, mouse resistance arteries incubated with S-nitrosocysteine displayed much lower contractility and spontaneously hypertensive rats injected with S-nitrosocysteine also showed significantly reduced blood pressure, suggesting that reduced S-nitrosylation contributes to the upregulation of Ca V 1.2 channel activity in hypertensive arteries. Conclusions: This study provides strong evidence that S-nitrosylation-mediated downregulation of Ca V 1.2 channels is via 2 distinctive mechanisms and the findings offer potential pathways for therapeutic inventions in hypertension.

The role of activation of two different sGC binding sites by NO‐dependent and NO‐independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes

Abstract: The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+‐sensitive nonselective cation current, an analog of ISAC, while in a stretched cell it eliminates ISAC. The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC, this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation.

S-Nitrosylation of OTUB1 Alters Its Stability and Ubc13 Binding.

Abstract: S-Nitrosylation is a reversible post-translational modification that regulates protein function involving the covalent attachment of the nitric oxide (NO) moiety to sulfhydryl residues of the protein. It is an important regulator in the cell signaling process under physiological conditions. However, the release of an excess amount of NO due to dysregulated NOS machinery causes aberrant S-nitrosylation of proteins, which affects protein folding, localization, and activity. Here, we have shown that OTUB1, a deubiquitinating enzyme, undergoes S-nitrosylation under redox stress conditions in vivo and in vitro. Previously, we have shown that OTUB1 forms an amyloid-like structure that promotes phosphorylation of α-synuclein and neuronal toxicity. However, the mechanistic insight into OTUB1 aggregation remains elusive. Here, we identified that OTUB1 undergoes S-nitrosylation in SH-SY5Y neuroblastoma cells under rotenone-induced stress, as well as excitotoxic conditions, and in rotenone-treated mouse brains. The in vitro S-nitrosylation of OTUB1 followed by mass-spectrometry analysis has identified cysteine-23 and cysteine-91 as S-nitrosylation sites. S-Nitrosylated OTUB1 (SNO-OTUB1) diminished its catalytic activity, impaired its native structure, promoted amyloid-like aggregation, and compromised its binding with Ubc13. Thus, our results demonstrated that nitrosylation of OTUB1 might play a crucial role in regulating the ubiquitin signaling and Parkinson's disease pathology.

TRAP1 S-nitrosylation as a model of population-shift mechanism to study the effects of nitric oxide on redox-sensitive oncoproteins

Abstract: S -nitrosylation is a post-translational modification in which nitric oxide (NO) binds to the thiol group of cysteine, generating an S -nitrosothiol (SNO) adduct. S -nitrosylation has different physiological roles, and its alteration has also been linked to a growing list of pathologies, including cancer. SNO can affect the function and stability of different proteins, such as the mitochondrial chaperone TRAP1. Interestingly, the SNO site (C501) of TRAP1 is in the proximity of another cysteine (C527). This feature suggests that the S -nitrosylated C501 could engage in a disulfide bridge with C527 in TRAP1, resembling the well-known ability of S -nitrosylated cysteines to resolve in disulfide bridge with vicinal cysteines. We used enhanced sampling simulations and in-vitro biochemical assays to address the structural mechanisms induced by TRAP1 S- nitrosylation. We showed that the SNO site induces conformational changes in the proximal cysteine and favors conformations suitable for disulfide bridge formation. We explored 4172 known S -nitrosylated proteins using high-throughput structural analyses. Furthermore, we used a coarse-grained model for 44 protein targets to account for protein flexibility. This resulted in the identification of up to 1248 proximal cysteines, which could sense the redox state of the SNO site, opening new perspectives on the biological effects of redox switches. In addition, we devised two bioinformatic workflows ( https://github.com/ELELAB/SNO_investigation_pipelines ) to identify proximal or vicinal cysteines for a SNO site with accompanying structural annotations. Finally, we analyzed mutations in tumor suppressors or oncogenes in connection with the conformational switch induced by S -nitrosylation. We classified the variants as neutral, stabilizing, or destabilizing for the propensity to be S -nitrosylated and undergo the population-shift mechanism. The methods applied here provide a comprehensive toolkit for future high-throughput studies of new protein candidates, variant classification, and a rich data source for the research community in the NO field.

Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH

Abstract: S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

Nitric oxide negatively regulates gibberellin signaling to coordinate growth and salt tolerance in Arabidopsis

Abstract: In response to dynamically altered environments, plants must finely coordinate the balance between growth and stress responses for their survival. However, the underpinning regulatory mechanisms remain largely elusive. The phytohormone gibberellin promotes growth via a derepression mechanism by proteasomal degradation of the DELLA transcription repressors. Conversely, the stress-induced burst of nitric oxide (NO) enhances stress tolerance, largely relaying on NO-mediated S-nitrosylation, a redox-based posttranslational modification. Here, we show that S-nitrosylation of Cys-374 in the Arabidopsis RGA protein, a key member of DELLAs, inhibits its interaction with the F-box protein SLY1, thereby preventing its proteasomal degradation under salinity condition. The accumulation of RGA consequently retards growth but enhances salt tolerance. We propose that NO negatively regulates gibberellin signaling via S-nitrosylation of RGA to coordinate the balance of growth and stress responses when challenged by adverse environments.

Recovery of reduced thiol groups by superoxide-mediated denitrosation of nitrosothiols

Abstract: Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase. In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O2•-) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40-70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O2•- dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP+-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O2•- indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O2•- or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O2•- appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O2•- could represent a mechanism to regain reducing equivalents in situations of excess O2•- formation, e.g. in the reperfusion phase after ischemia.

Products of S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase: Relation between S-nitrosylation and oxidation

Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the major targets of NO in cells, especially in neurodegenerative diseases. S-Nitrosylation of GAPDH is accompanied by its translocation into the nucleus with subsequent apoptosis. The product of GAPDH modification by NO is considered to be S-nitrosylated GAPDH (GAPDH-SNO). However, this has not been confirmed by direct methods.Products of GAPDH modification in the presence of the NO donor diethylamine NONOate were analyzed by MALDI- and ESI- mass spectrometry methods.The adduct between GAPDH and dimedone was detected by MALDI-MS analysis after incubation of S-nitrosylated GAPDH with dimedone, which points to the formation of cysteine-sulfenic acid (GAPDH-SOH) in the protein. Analysis of the protein hydrolysate revealed the incorporation of dimedone into the catalytic residue Cys150. An additional peak that corresponded to GAPDH-SNO was detected by ESI-MS analysis in GAPDH after the incubation with the NO donor. The content of GAPDH-SNO and GAPDH-SOH in the modified GAPDH was evaluated by different approaches and constituted 2.3 and 0.7 mol per mol GAPDH, respectively. A small fraction of GAPDH was irreversibly inactivated after NO treatment, suggesting that a minor part of the products includes cysteine-sulfinic or cysteine-sulfonic acids.The main products of GAPDH modification by NO are GAPDH-SNO and GAPDH-SOH that is presumably formed due to the hydrolysis of GAPDH-SNO.The obtained results are important for understanding the molecular mechanism of redox regulation of cell functions and the role of GAPDH in the development of neurodegenerative disorders.