Nadzeya V. Marozkina

S-Nitrosylation signaling regulates cellular protein interactions

BACKGROUND S-Nitrosothiols are made by nitric oxide synthases and other metalloproteins. Unlike nitric oxide, S-nitrosothiols are involved in localized, covalent signaling reactions in specific cellular compartments. These reactions are enzymatically regulated. SCOPE S-Nitrosylation affects interactions involved in virtually every aspect of normal cell biology. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. MAJOR CONCLUSIONS AND SIGNIFICANCE S-Nitrosylation is a regulated signaling reaction.

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product ΔF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in ΔF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of ΔF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on ΔF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects ΔF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies—using differing mechanisms of action—may have additive benefits in treating CF.

Direct Regulation of Striated Muscle Myosins by Nitric Oxide and Endogenous Nitrosothiols

Background Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. Principal Findings Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. Significance These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.

Breath formate is a marker of airway S-nitrosothiol depletion in severe asthma

Background Children with severe asthma have poor symptom control and elevated markers of airway oxidative and nitrosative stress. Paradoxically, they have decreased airway levels of S-nitrosothiols (SNOs), a class of endogenous airway smooth muscle relaxants. This deficiency results from increased activity of an enzyme that both reduces SNOs to ammonia and oxidizes formaldehyde to formic acid, a volatile carboxylic acid that is more easily detected in exhaled breath condensate (EBC) than SNOs. We therefore hypothesize that depletion of airway SNOs is related to asthma pathology, and breath formate concentration may be a proxy measure of SNO catabolism. Methods and Findings We collected EBC samples from children and adolescents, including 38 with severe asthma, 46 with mild-to-moderate asthma and 16 healthy adolescent controls, and the concentration of ionic constituents was quantified using ion chromatography. The concentrations of EBC components with volatile conjugates were log-normally distributed. Formate was the principal ion that displayed a significant difference between asthma status classifications. The mean EBC formate concentration was 40% higher in samples collected from all asthmatics than from healthy controls (mean = 5.7 µM, mean±standard deviation = 3.1−10.3 µM vs. 4.0, 2.8−5.8 µM, p = 0.05). EBC formate was higher in severe asthmatics than in mild-to-moderate asthmatics (6.8, 3.7−12.3 µM vs. 4.9, 2.8−8.7 µM, p = 0.012). In addition, formate concentration was negatively correlated with methacholine PC20 (r = −0.39, p = 0.002, asthmatics only), and positively correlated with the NO-derived ion nitrite (r = 0.46, p<0.0001) as well as with total serum IgE (r = 0.28, p = 0.016, asthmatics only). Furthermore, formate was not significantly correlated with other volatile organic acids nor with inhaled corticosteroid dose. Conclusions We conclude that EBC formate concentration is significantly higher in the breath of children with asthma than in those without asthma. In addition, amongst asthmatics, formate is elevated in the breath of those with severe asthma compared to those with mild-to-moderate asthma. We suggest that this difference is related to asthma pathology and may be a product of increased catabolism of endogenous S-nitrosothiols.

S-Nitrosoglutathione Reductase in Human Lung Cancer

S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

Essential role of hemoglobin beta-93-cysteine in posthypoxia facilitation of breathing in conscious mice

When erythrocyte hemoglobin (Hb) is fully saturated with O2, nitric oxide (NO) covalently binds to the cysteine 93 residue of the Hb β-chain (B93-CYS), forming S-nitrosohemoglobin. Binding of NO is allosterically coupled to the O2 saturation of Hb. As saturation falls, the NO group on B93-CYS is transferred to thiols in the erythrocyte, and in the plasma, forming circulating S-nitrosothiols. Here, we studied whether the changes in ventilation during and following exposure to a hypoxic challenge were dependent on erythrocytic B93-CYS. Studies were performed in conscious mice in which native murine Hb was replaced with human Hb (hB93-CYS mice) and in mice in which murine Hb was replaced with human Hb containing an alanine rather than cysteine at position 93 on the Bchain (hB93-ALA). Both strains expressed human γ-chain Hb, likely allowing a residual element of S-nitrosothiol-dependent signaling. While resting parameters and initial hypoxic (10% O2, 90% N2) ventilatory responses were similar in hB93-CYS mice and hB93-ALA mice, the excitatory ventilatory responses (short-term potentiation) that occurred once the mice were returned to room air were markedly diminished in hB93-ALA mice. Further, short-term potentiation responses were virtually absent in mice with bilateral transection of the carotid sinus nerves. These data demonstrate that hB93-CYS plays an essential role in mediating carotid sinus nerve-dependent short-term potentiation, an important mechanism for recovery from acute hypoxia.

Transnitrosation Signals Oxyhemoglobin Desaturation

See related article, pages 545–553 Erythrocytes dilate peripheral blood vessels as a function of oxyhemoglobin desaturation.1 This effect increases regional blood flow to hypoxic tissues. The mechanisms underlying the peripheral vasodilatory effects of desaturating erythrocytes are incompletely understood but do not involve activation of local, endothelial NO synthase (eNOS). Indeed, eNOS-derived NO itself primarily relaxes large vessels and does that primarily only in the absence of blood. In this issue of Circulation Research , Diesen et al confirm that thiols carrying a nitrosonium (NO+) equivalent signal cyclic GMP-dependent vascular smooth muscle relaxation during erythrocytic oxyhemoglobin desaturation.2 These data support paradigm-changing work demonstrating that nitrogen oxides are transported by circulating erythrocytes to signal oxyhemoglobin desaturation through serial NO/NO+ thiol equilibria and transfer reactions (transnitrosation) and that these reactions normally take place at sites remote from NOS activity.1–3 These new data show clearly that this signaling is independent of local NOS activity, of cyclooxygenase, of ATP, and of the effects of hypoxia itself on vascular smooth muscle. Erythrocytes are endogenously “preloaded” with nitrogen oxides for delivery to vessels in conditions of oxyhemoglobin desaturation.1–3 This signaling links delivery of erythrocytic NO/NO+ groups to oxyhemoglobin desaturation, permitting augmented blood flow to hypoxic tissue. Three mechanisms have been proposed by which transitions in hemoglobin (Hb) conformation may result in nitrogen oxide transfer to blood vessels. (1) In the originally proposed mechanism, Hb deoxygenation (R-to-T transition) permits transnitrosation from Hb β-chain cysteine 93 (βCys93) to erythrocytic carrier thiols (Figure).1–3 This concept is supported by the data obtained by Diesen et al.2 (2) At one time, NO radical was proposed to diffuse away from the Fe (II) heme iron in T state Hb. Although there was an initial enthusiasm for this construct as an NO radical–based …

Nitrogen balance in the ecosystem of the cystic fibrosis lung

In this issue of the Journal, Grasemann and coworkers (pp. 1363–1368) report that levels of asymmetric dimethyl arginine (ADMA) are increased in cystic fibrosis (CF) airways (1). ADMA inhibits cellular arginine uptake and nitric oxide (NO) synthase (NOS) activity. Levels of ADMA decrease during antibiotic therapy in association with improved lung function. This observation may prove to have therapeutic relevance. However, it is important to note that increased ADMA in CF airways may be both beneficial (through inhibition of NO production) and harmful (through inhibition of S-nitrosothiol production). Nitric oxide in the concentrations measured in exhaled air (parts per billion) is generally irrelevant to normal lung physiology (2). However, NO can be relevant to lung pathology. It interacts with oxygen, superoxide, and other molecules to injure airway epithelium. Products of these reactions, such as nitrous acid and peroxynitrous acid, are potent cytotoxins downstream of inducible NOS activity (3). Nitric oxide can also affect bacteria in the CF airway (4, 5). Indeed, the CF airway is a complex ecosystem in which nitrogen oxides, oxygen, protons, and more complex chemical species are exchanged between prokaryotic and eukaryotic cells (Figure 1; 4–7). For example, levels of NO are lower than normal in the CF airway, in part because NO is consumed by Pseudomonas, Aspergillus, and other denitrifying organisms (4). NO can serve both as an electron acceptor (dissimilatory denitrification, ultimately forming ammonia) and as a precursor for amino acid formation (assimilatory denitrification). Airway NO levels rise and NH3 levels fall with antibiotic therapy in CF (4). Levels of oxidized NO in the form of nitrate are high in the CF airway (6); and nitrate, like NO, can feed denitrifying organisms. Together, the effects of NOS products to cause cytotoxicity and promote prokaryotic growth suggest that the high levels of ADMA should be advantageous for patients with CF, and that a decrease in ADMA levels with antibiotic therapy might be disadvantageous. Figure 1. Cystic fibrosis airway nitrogen redox ecology. Nitric oxide (NO) synthase (NOS) isoforms in the cystic fibrosis (CF) lung produce both NO and S-nitrosothiols. NO is oxidized to nitrite (NO2−) and nitrate (NO3−). In this issue of the Journal ... However, NOS also produces S-nitrosothiols in concentrations two log orders higher than NO (8–10). S-nitrosothiols are antimicrobial. They augment ciliary beat frequency (2). They relax human airway smooth muscle and prevent tachyphylaxis to β2-adrenergic agonists (2, 10, 11). They inhibit amiloride-sensitive sodium transport (12). They augment expression, maturation and function of delF508 CF transmembrane conductance regulator through inhibition of the expression of Hsp70/Hsp90-organizing protein (13). S-nitrosothiol levels are decreased in the CF airway (14), consistent with high ADMA levels (1); indeed, S-nitrosothiol replacement therapy improves oxygenation in CF (9). Prevention of S-nitrosothiol formation is therefore likely to be an important disadvantage of having high levels of ADMA in the CF airway (1). We do not know why ADMA levels are high in CF. One possible mechanism is based on what we know about the CF airway ecosystem. Anti-pseudomonal therapy decreases ADMA levels in patients with CF (1). Biochemically, ADMA levels are decreased by dimethylarginine dimethylaminohydrolases (DDAHs). DDAHs are inhibited by S-nitrosylation, or physiological protein modification by NO (15). S-nitrosylation is driven both by NOS activation and—in relatively acidic conditions in the CF airway ecosystem (5)—by nitrite protonation; at baseline, this should increase ADMA levels. Antimicrobial therapy can decrease bacterial conversion of abundant airway nitrate to nitrite. Nitrite depletion would decrease DDAH S-nitrosylation, thereby increasing ADMA breakdown during the course of therapy. However, this proposed mechanism is speculative. Much work remains to be done. The CF airway is dark, damp, and largely anaerobic (7). It is also surprisingly well-suited to denitrifying prokaryotic species, given that NOS expression is decreased and ADMA levels are increased. To understand why—and to learn how to use nitrogen oxide redox ecology to therapeutic advantage—we need to get beyond simply modeling NO radical diffusion (2, 3). The Cystic Fibrosis Foundation has shown the benefit of promoting interactions among scientists across disciplines. The work of Grasemann and coworkers suggests that there might be much to gain by organizing a small working group to bring together nitrogen balance ecologists, biochemists, CF airway epithelial biologists, and mathematicians experienced in biochemical modeling. This group could model prokaryotic and eukaryotic outcomes of specific CF interventions to identify strategies to optimize therapeutic development in CF. The elegant insights of Grasemann and coworkers serve to alert us that both the benefits and toxicities of airway nitrogen oxides need to be better understood to improve clinical outcomes in CF.

Lessons From Unilateral Loss of Cilia: Early Nasal Nitric Oxide Gas Mixing and the Role of Sinus Patency in Determining Nasal Nitric Oxide

Nasal nitric oxide (nNO) measurement is a diagnostic test for primary ciliary dyskinesia (PCD). Here, we have shown the development of unilateral PCD-like symptoms associated with low nNO. A 60-year-old man had been previously healthy but developed unilateral, severe pansinusitis. He required surgical drainage of all left sinuses, and biopsies showed loss of the ciliated epithelium. At 4 weeks, he had unilateral (left-sided), profuse, clear rhinorrhea characteristic of PCD, and his surgical ostia were all patent endoscopically. His left-sided nNO was less than the right side by 37 ± 1.2 nL/min; this difference decreased to 18 ± 0.87 nL/min at 5 weeks and was gone by 6 weeks when his symptoms resolved. Measurements of 2- and 10-second measurements, in addition to standard nNO measurements, identified this discordance. We conclude that nNO reflects, in part, the production of NO by the ciliated epithelium, not just in the absence or occlusion of sinuses. Early (nasal/sinus volume) measures may be better for diagnosing PCD in than standard, steady-state assays in certain populations.

Erratum: Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity (Eur Respir J (2015) 45 (87-97))

“Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity.” Nadzeya V. Marozkina, Xin-Qun Wang, Vitali Stsiapura, Anne Fitzpatrick, Silvia Carraro, Gregory A. Hawkins, Eugene Bleecker, Deborah Meyers, Nizar Jarjour, Sean B. Fain, Sally Wenzel, William Busse, Mario Castro, Reynold A. Panettieri Jr, Wendy Moore, Stephen J. Lewis, Lisa A. Palmer, Talissa Altes, Eduard E. de Lange, Serpil Erzurum, W. Gerald Teague and Benjamin Gaston. Eur Respir J 2015; 45: 87–97.