S-nitrosylation Of Cysteine Residues Research Articles

Nitric oxide and thioredoxin modulate the activity of caspase 9 in HepG2 cells

The interdependent and finely tuned balance between the well-established redox-based modification, S-nitrosylation, and its counteractive mechanism of S-nitrosothiol degradation, i.e., S-denitrosylation of biological protein or non-protein thiols defines the cellular fate in the context of redox homeostasis. S-nitrosylation of cysteine residues by S-nitrosoglutathione, S-nitroso-L-cysteine-like physiological and S-nitroso-L-cysteine ethyl ester-like synthetic NO donors inactivate Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-denitrosylation of S-nitrosocaspases into their reduced, free thiol active states, aided by the members of the ubiquitous cellular redoxin (thioredoxin/ thioredoxin reductase/ NADPH) and low molecular weight dithiol (lipoic acid/ lipoamide dehydrogenase/ dihydrolipoic acid/ NADPH) systems imply a direct relevance to their proteolytic activities and further downstream signaling cascades. Additionally, our previous and current findings offer crucial insight into the concept of redundancy between thioredoxin and lipoic acid systems, and the redox-modulated control of the apoptotic and proteolytic activity of caspases, triggering their cyto- and neurotoxic effects in response to nitro-oxidative stress. Thus, this might lay the foundation for the exogenous introduction of precise and efficient NO or related donor drug delivery systems that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.

PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

Protein disulfide isomerase (PDI) is a redox-active enzyme and also serves as a nitric oxide donor causing S-nitrosylation of cysteine residues in various proteins. Although PDI knockdown reduces α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)-mediated neuronal activity, the underlying mechanisms are largely unknown. In the present study, we found that under physiological condition PDI knockdown increased CaMKII activity (phosphorylation) in the mouse hippocampus. However, PDI siRNA inhibited protein phosphatase (PP) 2A-mediated GluA2 S880 dephosphorylation by increasing PP2A oxidation, independent of S-nitrosylation. PDI siRNA also enhanced glutamate ionotropic receptor AMPA type subunit 1 (GluA1) S831 and GluA2 S880, but not GluA1 S845 and GluA2 Y869/Y873/Y876 phosphorylations, concomitant with the enhanced protein interacting with C kinase 1 (PICK1)-mediated AMPAR internalization. Furthermore, PDI knockdown attenuated seizure activity and neuronal damage in response to kainic acid (a non-desensitizing agonist of AMPAR). Therefore, these findings suggest that PDI may regulate surface AMPAR expression through PP2A-GluA2-PICK1 signaling pathway, and that PDI may be one of the therapeutic targets for epilepsy via AMPAR internalization without altering basal neurotransmission.

NO and Heme Proteins: Cross-Talk between Heme and Cysteine Residues

Heme proteins are a diverse group that includes several unrelated families. Their biological function is mainly associated with the reactivity of the heme group, which-among several other reactions-can bind to and react with nitric oxide (NO) and other nitrogen compounds for their production, scavenging, and transport. The S-nitrosylation of cysteine residues, which also results from the reaction with NO and other nitrogen compounds, is a post-translational modification regulating protein activity, with direct effects on a variety of signaling pathways. Heme proteins are unique in exhibiting this dual reactivity toward NO, with reported examples of cross-reactivity between the heme and cysteine residues within the same protein. In this work, we review the literature on this interplay, with particular emphasis on heme proteins in which heme-dependent nitrosylation has been reported and those for which both heme nitrosylation and S-nitrosylation have been associated with biological functions.

Nitric Oxide Sensing by a Blue Fluorescent Protein.

S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Aequorea victoria Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology. We have examined the response to nitric oxide of fluorescence emission by the blue-emitting fluorescent protein mTagBFP2. To our surprise, upon exposure to micromolar concentrations of nitric oxide, we observed a roughly 30% reduction in fluorescence quantum yield and lifetime. Recovery of fluorescence emission is observed after treatment with Na-dithionite. Experiments on related fluorescent proteins from different families show similar nitric oxide sensitivity of their fluorescence. We correlate the effect with S-nitrosylation of Cys residues. Mutation of Cys residues in mTagBFP2 removes its nitric oxide sensitivity. Similarly, fluorescent proteins devoid of Cys residues are insensitive to nitric oxide. We finally show that mTagBFP2 can sense exogenously generated nitric oxide when expressed in a living mammalian cell. We propose mTagBFP2 as the starting point for a new class of genetically encoded nitric oxide sensors based on fluorescence lifetime imaging.

Nitric Oxide-Dependent Protein Post-Translational Modifications Impair Mitochondrial Function and Metabolism to Contribute to Neurodegenerative Diseases.

Significance: Most brains affected by neurodegenerative diseases manifest mitochondrial dysfunction as well as elevated production of reactive oxygen species and reactive nitrogen species (RNS), contributing to synapse loss and neuronal injury. Recent Advances: Excessive production of RNS triggers nitric oxide (NO)-mediated post-translational modifications of proteins, such as S-nitrosylation of cysteine residues and nitration of tyrosine residues. Proteins thus affected impair mitochondrial metabolism, mitochondrial dynamics, and mitophagy in the nervous system. Critical Issues: Identification and better characterization of underlying molecular mechanisms for NO-mediated mitochondrial dysfunction will provide important insights into the pathogenesis of neurodegenerative disorders. In this review, we highlight recent discoveries concerning S-nitrosylation of the tricarboxylic acid cycle enzymes, mitochondrial fission GTPase dynamin-related protein 1, and mitophagy-related proteins Parkin and phosphatase and tensin homolog-induced putative kinase protein 1. We delineate signaling cascades affected by pathologically S-nitrosylated proteins that diminish mitochondrial function in neurodegenerative diseases. Future Directions: Further elucidation of the pathological events resulting from aberrant S-nitrosothiol or nitrotyrosine formation may lead to new therapeutic approaches to ameliorate neurodegenerative disorders.

PDI Knockdown Inhibits Seizure Activity in Acute Seizure and Chronic Epilepsy Rat Models via S-Nitrosylation-Independent Thiolation on NMDA Receptor.

Redox modulation and S-nitrosylation of cysteine residues are the post-translational modifications of N-methyl-D-aspartate receptor (NMDAR) to regulate its functionality. Recently, we have reported that protein disulfide isomerase (PDI) reduces disulfide bond (S-S) to free thiol (-SH) on NMDAR. Since PDI is a modulator of S-nitrosylation on various proteins, it is noteworthy whether PDI affects S-nitrosylation of NMDAR in acute seizure and chronic epilepsy models. In the present study, we found that acute seizures in response to pilocarpine and spontaneous seizures in chronic epilepsy rats led to the reduction in S-nitrosylated thiol (SNO-thiol)-to-total thiol ratio on NMDAR, while they elevated nitric oxide (NO) level and S-nitrosylation on NMDAR. N-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor) did not affect seizure activities in both models, although it decreased SNO-thiol levels on NMDAR. However, PDI knockdown effectively inhibited pilocarpine-induced acute seizures and spontaneous seizures in chronic epilepsy rats, accompanied by increasing the SNO-thiol-to-total thiol ratio on NMDAR due to diminishing the amounts of total thiols on GluN1 and GluN2A. Therefore, these findings indicate that PDI may not be a NO donor or a denitrosylase for NMDAR, and that PDI knockdown may inhibit seizure activity by the S-nitrosylation-independent thiolation on NMDAR.

Climate Change and the Impact of Greenhouse Gasses: CO2 and NO, Friends and Foes of Plant Oxidative Stress.

Here, we review information on how plants face redox imbalance caused by climate change, and focus on the role of nitric oxide (NO) in this response. Life on Earth is possible thanks to greenhouse effect. Without it, temperature on Earth’s surface would be around -19°C, instead of the current average of 14°C. Greenhouse effect is produced by greenhouse gasses (GHG) like water vapor, carbon dioxide (CO2), methane (CH4), nitrous oxides (NxO) and ozone (O3). GHG have natural and anthropogenic origin. However, increasing GHG provokes extreme climate changes such as floods, droughts and heat, which induce reactive oxygen species (ROS) and oxidative stress in plants. The main sources of ROS in stress conditions are: augmented photorespiration, NADPH oxidase (NOX) activity, β-oxidation of fatty acids and disorders in the electron transport chains of mitochondria and chloroplasts. Plants have developed an antioxidant machinery that includes the activity of ROS detoxifying enzymes [e.g., superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PRX)], as well as antioxidant molecules such as ascorbic acid (ASC) and glutathione (GSH) that are present in almost all subcellular compartments. CO2 and NO help to maintain the redox equilibrium. Higher CO2 concentrations increase the photosynthesis through the CO2-unsaturated Rubisco activity. But Rubisco photorespiration and NOX activities could also augment ROS production. NO regulate the ROS concentration preserving balance among ROS, GSH, GSNO, and ASC. When ROS are in huge concentration, NO induces transcription and activity of SOD, APX, and CAT. However, when ROS are necessary (e.g., for pathogen resistance), NO may inhibit APX, CAT, and NOX activity by the S-nitrosylation of cysteine residues, favoring cell death. NO also regulates GSH concentration in several ways. NO may react with GSH to form GSNO, the NO cell reservoir and main source of S-nitrosylation. GSNO could be decomposed by the GSNO reductase (GSNOR) to GSSG which, in turn, is reduced to GSH by glutathione reductase (GR). GSNOR may be also inhibited by S-nitrosylation and GR activated by NO. In conclusion, NO plays a central role in the tolerance of plants to climate change.

To eat, or NOt to eat: S-nitrosylation signaling in autophagy.

Autophagy is the main catabolic cellular process through which cells adapt their needs (e.g., growth and proliferation) to environmental availability of nutrients (e.g., amino acid and glucose) and growth factors. The rapid activation of the autophagy response essentially depends on protein post-translational modifications (PTMs), which act as molecular switches triggering signaling cascades. Deregulation of autophagy contributes to pathological conditions, such as cancer and neurodegeneration. Therefore, understanding how PTMs affect the occurrence of autophagy is of the highest importance for clinical applications. Besides phosphorylation and ubiquitylation, which represent the best known examples of PTMs, redox-based modifications are also emerging as contributing to the regulation of intracellular signaling. Of note, S-nitrosylation of cysteine residues is a redox PTM and is the principal mechanism of nitric oxide-based signaling. Results emerging in recent years suggest that NO has a role in modulating autophagy. However, the function of S-nitrosylation in autophagy regulation remains still unveiled. By this review, we describe the upstream events regulating autophagy activation focusing on recently published evidence implying a S-nitrosylation-dependent regulation.

Tumor Suppressor Roles of the Denitrosylase GSNOR.

Nitric oxide (NO) is a gaseous pleiotropic molecule that can both induce irreversible oxidative damages and modulate physiological signal transductions by transient protein modifications, the most important of which is the S-nitrosylation of cysteine residues. Being noxious and healthy, the role of NO in cancer is seemingly contradictory, as at low concentrations it mediates tumor growth and proliferation whereas at high concentrations it promotes apoptosis and cancer growth inhibition. However, it is becoming evident that when endogenously produced, such as upon inducible nitric oxide synthase (NOS) activation, NO acts to sustain tumorigenesis. Similarly, although less explored, defects and deficiency in the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) have been associated with the development and malignancy of liver and breast cancers, suggesting a primary role for NO signaling-that is, S-nitrosylation, being deeply involved in neoplastic transformation and progression. In this review, we summarize past and recent evidence on the role of S-nitrosylation and GSNOR in different processes that contribute to cell transformation when deregulated, such as DNA damage repair, energetic metabolism, and cell death. We also outline possible S-nitrosylation-targeted proteins that might contribute to tumorigenesis, and, finally, we speculate on the role of GSNOR in regulating the oncogenic effects induced downstream.

Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration.

Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that-amongst the 10 Tyr residues present in MSD1-Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.

Regulation of Actin Isoforms by Nitric Oxide

Nitric oxide is a known regulator of cellular function that can act indirectly via activation of guanylyl cyclase, or directly via the S-nitrosylation of cysteine residues. However, little is known about the breadth of proteins that are regulated. Actin is present in nearly all eukaryotic cells and in a variety of isoforms that differ slightly in amino acid sequence, and more specifically in the number and placement of cysteine residues. It has been shown by others that different cysteines in the various isoforms can be modified by a number of oxidation reactions including S-nitrosylation. We therefore sought to determine the effect of S-nitrosylation on the interactions of skeletal, smooth, and non-muscle actins with myosin. We measured skeletal, smooth, and non-muscle (β and γ) actin nitrosylation in response to in vitro treatment with nitroso-L-cysteine - an endogenous nitrosothiol and NO donor - as well as actin filament velocity over heavy meromyosin (HMM), and the actin activated ATPase rates of HMM. A coumarin switch assay showed that all three isoforms could be nitrosylated by nitroso-L-cysteine. Nitrosylation of each actin isoform significantly decreased the in vitro velocity over HMM by approximately 38% with 50 μM donor compared to untreated controls. In contrast, there was no difference observed in the α-skeletal actin activated ATPase rates of the control and treated filaments. Further studies are being conducted to determine which cysteine residues in skeletal, smooth, and non-muscle actin are modified by nitroso-L-cysteine. Together these data suggest that nitrosylation of actin affects the attached time of actomyosin crossbridges. Given the ubiquitous nature of actin it is possible that nitrosylation is a common regulatory scheme for muscle contraction and various forms of cell motility.

Nitric Oxide Signaling and Nitrosative Stress in Neurons: Role forS-Nitrosylation

Nitric oxide (NO) mediates cellular signaling pathways that regulate a plethora of physiological processes. One of the signaling mechanisms mediated by NO is through S-nitrosylation of cysteine residues in target proteins, which is now regarded as an important redox-based physiological action. Deregulation of the protein S-nitrosylation upon nitrosative stress, however, has also been linked to various human diseases, such as neurodegenerative disorders. Between these physiological and pathophysiological roles, there are mechanisms whereby a milder level of nitrosative stress provides S-nitrosylation of some proteins that counteracts the pathological processes, serving as a negative feedback mechanism. In addition, NO has recently emerged as a mediator of epigenetic gene expression and chromatin changes. In this review, these molecular mechanisms, especially those in the central nervous system and neurodegenerative disorders, are described.

Abstract 1708: Decreased S-Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

Vascular aging is marked by an increase in vascular stiffness. Tissue-type transglutaminase (tTG) enzymatically forms crosslinks between extracellular matrix proteins and may contribute to this pathobiology. Recently, it has been demonstrated that s-nitrosylation of cysteine residues within the enzyme leads to its inhibition. We hypothesize that tTG is more active in the aging vasculature due to decreased bioavailability of nitric oxide (NO) and impaired S-nitrosylation. tTG activity in aorta measured by the incorporation of biotin labeled pentylamine, a tTG substrate, into crosslinks was markedly increased in old (O) animals compared to young (Y) (3.2 fold increase, n = 6, p = 0.0018). S-nitrosylation of tTG, as determined by the biotin switch assay, was significantly reduced in O vs. Y rat aorta. tTG specific (ϵ-(γ-glutamyl)lysine) crosslinks measured by immunofluorescent staining was significantly increased in O vs Y rat aortic rings. Denitrosylation of tTG in Y rats with DTT increased tTG activity by 41% as did the NOS-inhibitor L-NAME. Treatment of O rats with cystamine (tTG inhibitor) lead to a decrease in vascular stiffness as measured by pulse wave velocity (5.55 ± 0.11 m/s to 4.99 ± 0.22 m/s, n = 6, p = 0.038). Given that tTG nitrosylation is likely NOS-3 dependent, we measured tTG activity and function in NOS-3 −/− mice. tTG activity was significantly increased in NOS-3 −/− vs. WT mice (18.94 ± 0.70 RU vs. 31.63 ± 2.06 RU, n = 4, p = 0.015). Treatment of NOS-3 mice with cystamine decreased aortic stiffness and increased carotid artery compliance. Finally, tTG activity in old human aortic tissue (collected post-mortem) is increased by 30% compared to young despite unchanged protein abundance. These data suggest that NOS-3 dependent NO constrains the activity of tTG by S-nitrosylation of the enzyme. Furthermore decreased NO bioavailability contributes to tTG mediated increases in vascular stiffness. Finally tTG promises to be a critical target in age-related vascular stiffness.

Regulation of the Catalytic Activity and Structure of Human Thioredoxin 1 via Oxidation and S-Nitrosylation of Cysteine Residues

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys(32)-Gly-Pro-Cys(35) thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys(62)-Cys(69) disulfide as well as dimers and multimers. We have discovered that after incubation with H(2)O(2) only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys(69) and Cys(73) were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys(73), which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154-158). The reversible inhibition of human Trx1 activity by H(2)O(2) and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.

Identification of the Tyrosine Nitration Sites in Human Endothelial Nitric Oxide Synthase by Liquid Chromatography-Mass Spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.

Protein Phosphatase 2B Inhibitor, Cyclosporine A, Stimulates Endothelial Cell Exocytosis of Ultra-Large VWF Multimeric Strings by Regulating Munc18c Phosphorylation.

Exocytosis of Weibel Palade bodies (WPBs) containing ultra-large Von Willebrand factor (ULVWF) plays an essential role in the processes of inflammation and thrombosis. The exocytosis of ULVWF involves the fusion of WPBs to the endothelial cell plasma membrane via vesicular trafficking families of proteins. Reversible post-translational modification, including the phosphorylation of tyrosine (Tyr) and/or serine/threonine (Ser/Thr) residues or S-nitrosylation of cysteine residues on one or more vesicular trafficking proteins tightly regulates the exocytotic process. The phosphorylation of any trafficking protein is determined by the interplay between protein kinases and protein phosphatases and any alteration in the activity of either enzyme can affect exocytosis. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate exocytosis in endothelial cells, there is scant information about any possible role for the regulation of protein phosphatases in this process. Inhibition of protein phosphatase 2B (PP2B) also called calcineurin by the immunosuppressive drug, cyclosporine (CsA), is associated with thrombotic microangiopathy in a subset of transplant patients. Calcineurin is the target of a cyclophilin-CsA complex. We detected both calcineurin and cyclophilin A in resting human umbilical vein endothelial cells (HUVECs). Because CsA is a potent inhibitor of PP2B, we hypothesized that CsA induces ULVWF secretion and thrombotic events at least in part, by facilitating the phosphorylation of vesicle trafficking proteins that promote the exocytosis of ULVWF from endothelial cells. In vitro, we found that nanomolar concentrations of CsA or a cell permeable PP2B auto inhibitory peptide stimulated the release of ULVWF from HUVECs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assays revealed that PP2B inhibitors did not damage the HUVECs. In vivo, plasma levels of VWF were increased after intraperitoneal injection of CsA. These studies suggest that PP2B negatively regulate ULVWF exocytosis. Microscopic studies revealed that the ULVWF strings secreted by HUVECs treated with CsA or PP2B auto-inhibitory peptide induced the adhesion of platelets. CsA-treated HUVECs exhibited an increased serine phosphorylation of Munc18c, a vesicle trafficking associated protein that promotes granule exocytosis. These findings support a mechanism whereby inhibition of PP2B by CsA enhances cell secretion, including endothelial cell secretion of ULVWF by facilitating Munc18c phosphorylation.

Endothelial Function and Dysfunction

Atherosclerosis begins in childhood, progresses silently through a long preclinical stage, and eventually manifests clinically, usually from middle age. Over the last 30 years, it has become clear that the initiation and progression of disease, and its later activation to increase the risk of morbid events, depends on profound dynamic changes in vascular biology.1 The endothelium has emerged as the key regulator of vascular homeostasis, in that it has not merely a barrier function but also acts as an active signal transducer for circulating influences that modify the vessel wall phenotype.2 Alteration in endothelial function precedes the development of morphological atherosclerotic changes and can also contribute to lesion development and later clinical complications.3 Appreciation of the central role of the endothelium throughout the atherosclerotic disease process has led to the development of a range of methods to test different aspects of its function, which include measures of both endothelial injury and repair. These have provided not only novel insights into pathophysiology, but also a clinical opportunity to detect early disease, quantify risk, judge response to interventions designed to prevent progression of early disease, and reduce later adverse events in patients. The present review summarizes current understanding of endothelial biology in health and disease, the strengths and weaknesses of current testing strategies, and their potential applications in clinical research and patient care. Although only a simple monolayer, the healthy endothelium is optimally placed and is able to respond to physical and chemical signals by production of a wide range of factors that regulate vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and vessel wall inflammation. The importance of the endothelium was first recognized by its effect on vascular tone. This is achieved by production and release of several vasoactive molecules that relax or constrict the vessel, as …

Coupling eNOS Uncoupling to the Innate Immune Response

The endothelial isoform of nitric oxide synthase (eNOS) has been well recognized for its central role in conserving vascular homeostasis by inducing quiescence of endothelial cells and adjacent vessel wall structures. For example, eNOS-derived NO can alter protein function by S-nitrosylation of cysteine residues and, in endothelial cells, patterns of S-nitrosylated transcriptional regulators have been shown to interrupt inflammatory signaling and induce cell cycle arrest.1 Likewise, by competing with oxygen as an electron acceptor at complex IV, NO can limit energy expenditure through reducing oxidative phosphorylation during episodes of hypoxic challenge.2 See page 2688 Yet eNOS has also been recognized as a source of non-nitrous reactive oxygen species that can elicit a coordinated proinflammatory response. This is related to the basic chemistry in the enzyme where NADPH-derived electrons flow from the reductase domain toward the oxygenase domain. With the cofactor calmodulin bound to the native homodimeric enzyme, and in the presence of tetrahydrobiopterin (BH4), these electrons react with oxygen and L-arginine and lead to the formation of L-citrulline and NO.3 However, if either one of these factors is (relatively) lacking, oxidation of oxygen occurs and the enzyme subsequently releases superoxide.3,4 This phenomenon, the so-called “uncoupling” of the eNOS, appears to be strongly conserved throughout evolution. It is therefore likely that this is not so much an anomaly of the enzyme but part of its physiological role in vascular homeostasis. As we recently …

S‐nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II

1. Nitric oxide (NO) is known to affect the properties of various proteins via the S-nitrosylation of cysteine residues. This study evaluated the direct effects of the NO donor sodium nitroprusside (SNP) on the pharmacological properties of the AT1 receptor for angiotensin II expressed in HEK-293 cells. 2. SNP dose-dependently decreased the binding affinity of the AT1 receptor without affecting its total binding capacity. This modulatory effect was reversed within 5 min of removing SNP. 3. The effect of SNP was not modified in the presence of the G protein uncoupling agent GTPgammaS or the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. 4. The binding properties of a mutant AT1 receptor in which all five cysteine residues within the transmembrane domains had been replaced by serine was not affected by SNP. Systematic analysis of mutant AT1 receptors revealed that cysteine 289 conferred the sensitivity to SNP. 5. These results suggest that NO decreased the binding affinity of the AT1 receptor by S-nitrosylation of cysteine 289. This modulatory mechanism may be particularly relevant in pathophysiological situations where the beneficial effects of NO oppose the deleterious effects of angiotensin II.

The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry

Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched to four different proteins: synaptosomal-associated protein, actin, immunoglobulin α Fc receptor, and cGMP-dependent protein kinase 2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility.