Thioredoxin and Thioredoxin Reductase in Relation to Reversible S -Nitrosylation

Abstract

Significance: Nitric oxide (NO) regulates a diverse range of cellular processes, including vasodilation, neurotransmission, and antimicrobial and anti-tumor activities. S-nitrosylation with the formation of S-nitrosothiols (RSNOs) is an important feature of NO signaling regulating protein function. In mammalian cells, glutathione (GSH), S-nitrosoglutathione reductase (GSNOR), and thioredoxin (Trx) have been identified as the major protein denitrosylases. Recent Advances: Human cytosolic/nuclear Trx1 in the disulfide form can be nitrosylated at Cys73 and transnitrosylate target proteins, including caspase 3. Thus, similar to GSH, which by forming S-nitrosoglutathione (GSNO) can transnitrosylate proteins, Trx can either denitrosylate or nitrosylate proteins depending on its oxidation state. Critical Issues: In this review, we discuss the regulation of cellular processes by reversible S-nitrosylation and Trx-mediated cellular homeostasis of RSNOs and S-nitrosoproteins. Future Directions: Functions of RSNOs in vivo and their pharmacological uses have not yet been fully studied. Further investigations on the role of Trx systems in relation to biologically relevant RSNOs, their functions, and the mechanisms of denitrosylation will facilitate the development of drugs and therapies. Antioxid. Redox Signal. 18, 259–269.

Introduction

N itric oxide (NO) is an important messenger molecule that is involved in many physiological and pathological processes within the mammalian body which are both beneficial and detrimental (10, 43, 53). It is involved in various cellular signal transduction pathways through protein S-nitrosylation (25, 26, 31, 35), a ubiquitous redox modification of a cysteine thiol, which transduces NO bioactivity. S-nitrosylation (or S-nitrosation) and the formation of S-nitrosothiols (RSNOs) has emerged as an important feature of NO signaling. The RSNOs range in size from low molecular mass (LMM), such as S-nitrosoglutathione (GSNO), S-nitrosocysteine (L-CysNO), and S-nitrosohomocysteine (HCysNO), to high molecular mass S-nitrosylated proteins (e.g., albumin-SNO). More than a thousand proteins, till date, have been shown to be S-nitrosylated in vitro and in vivo. While elevated levels of RSNOs (the condition referred to as “nitrosative stress”) have been detected in patients with inflammatory and autoimmune diseases, many S-nitrosylated proteins have been directly implicated in human diseases—arginase in asthma, parkin in Parkinson's disease (PD), hemoglobin in diabetes, ryanodine receptors (RyRs) in heart failure, matrix metalloproteases (MMPs) in stroke, and so on (25, 26, 63) (Table 1).

Table 1.

Effects of S-Nitrosylation on Enzyme Activity

Proteins	Effect of S-nitrosylation
Hemoglobin	SNO-Hb releases vasoactive NO (49, 97)
Albumin	HAS-SNO showed antibacterial and antitumor activities (44, 52, 96)
HIF-1	Inhibition of activity (56)
NF-κB	Inhibition of activity (69)
Ras GTPases	Activation of GTPase activity (34, 42)
Akt/PKB	Inhibition of activity (15, 114)
Ryanodine receptor	Activation of the calcium release channel (14, 99, 112)
Parkin	Inhibition of ubiquitin E3 ligase activity (17, 113)
MMP-9	Activation of enzyme activity (30)
GAPDH	Initiation of apoptotic cell death by nuclear translocation (32)
Caspases	Inhibition of enzyme activity and apoptosis (57, 90 –93)

See text for details.

HAS-SNO, S-nitrosylated human serum albumin; SNO-Hb, S-nitrosohemoglobin; NF-κB, nuclear factor-Kappa B; MMP, matrix metalloproteinases.

The thioredoxin system comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH is the major protein-disulfide reductase of the cells (36 –40). Trxs are crucial proteins, and the cells' major disulfide reductases with Trx1 in the cytosol and Trx2 in mitochondria (61) regulate the activities of several transcription factors, redox regulation of different metabolic pathways, oxidative stress defense, DNA synthesis by being a hydrogen donor for ribonucleotide reductase, apoptosis, and NO signaling. The active site disulfide form of Trx undergoes NADPH-dependent reduction by TrxR and, in turn, reduces oxidized cysteine groups (disulfide or sulfenic acid) on substrate proteins by utilizing the two cysteinyl residues in its Cys-Gly-Pro-Cys active site. TrxR is a homodimeric, FAD-containing selenoenzyme with three isoforms: TrxR1 (cytosol), TrxR2 (mitochondrial), and TrxR3 (present primarily in testis) (2). Mammalian cytosolic TrxR has a redox center, consisting of Cys59/Cys64 adjacent to the flavin ring of FAD and another redox center consisting of Cys497/SeCys498 close to the C-terminus (71, 120). This selenothiol is the proper active site that can reduce many of the potential substrates of the enzyme, and the essential role SeCys residue makes it the target of several drugs in cancer treatment (2, 77, 120). The structure and function of Trx1 is regulated by structural SH-groups that can be oxidized to an extra disulfide or nitrosylated (33). Trx plays a major role in protein S-denitrosylation but, under certain conditions, it can also trans-S-nitrosylate other proteins (33, 72, 93). Taking account of the recent progress in the field of research, this review focuses on the regulation of cellular processes by reversible S-nitrosylation and Trx-mediated cellular homeostasis of RSNOs and S-nitrosoproteins.

Trx-TrxR System

Trxs are a family of small proteins (12 kDa or larger) catalyzing thiol-disulfide oxidoreductions by using redox-active cysteine residues that are present in a Trp–Cys-Gly-Pro-Cys sequence motif (36 –40, 51). In oxidized Trx (Trx-S₂), the active site is a disulfide and in reduced Trx [Trx-(SH)₂], it is a corresponding dithiol. Trx-(SH)₂ reduces protein disulfides (Reaction 1) and forms Trx-S₂, which, in turn, is reduced by TrxR and NADPH (Reaction 2) (38, 39, 51). \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm Trx}\hbox {-} ({\rm SH})_2 + {\rm Protein}\hbox{-}{\rm S}_{2} \longrightarrow {\rm Protein}\hbox{-}({\rm SH} )_{2} + {\rm Trx}\hbox{-}S_{2} & ({\rm Reaction} \ 1) \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm Trx}\hbox {-}{\rm S}_{2} + {\rm NADPH} + {\rm H}^{+} \mathop{\longrightarrow}\limits^{\rm TrxR} {\rm Trx}\hbox {-}({\rm SH })_{2} + {\rm NADP}^{+} & {( \rm Reaction \ 2 )} \end{align*} \end{document}

TrxR from bacteria, fungi, and plants are FAD-containing homodimers of 70 kDa with active site cysteine residues (2). In contrast, mammalian TrxRs are larger dimeric selenoenzymes (114 kDa or larger) with a catalytically active selenocysteine residue located in the C-terminus (2, 28, 120). The catalytic mechanism of mammalian TrxR homodimer involves the transfer of electrons from NADPH to the enzyme-bound FAD, which reduces the disulfide within the CVNVGC motif in the N-terminal domain that subsequently reduces the selenylsulfide motif to a selenothiol at the C-terminal Sec-containing GCUG motif of the neighboring subunit (2). The selenothiol is the proper active site that can reduce many of the potential substrates of the enzyme.

Trxs exist in all living cells and in multiple forms such as approximately 19 isoforms in plants (89). Humans have two classical Trxs, including Trx1 (cytosolic/nuclear) and Trx2 (mitochondrial), both of which are essential and embryonically lethal (61). The structures of Trxs from many species, determined by X-ray crystallography and multidimensional NMR, showed the Trx fold comprising a central core of β-sheet surrounded by α-helices (36 –40, 61, 108) (Fig. 1). The active site of Escherichia coli and human Trx1 comprises Cys32 and Cys35, which are located at the end of the second β-sheet and in the beginning of α helix2. The pKa value of Cys32 is lowered to 7, and this is due to hydrogen bonding within the active site. The pKa value of Cys35 is high in Trx-(SH)₂. The mechanism of Trx-catalyzed reduction involves docking and formation of a transient mixed disulfide with the substrate after a nucleophilic attack by the depronated N-terminal Cys residue in the active site (38, 61). Kinetically, Trx is known to be four to six orders of magnitude faster as a dithiol reductant compared with a strong chemical reductant such as dithiothreitol. Recently, the mechanism of Trx-catalysis has been probed by single molecule techniques (109). The Trx fold is found in a large number of proteins, including glutaredoxins, glutathione (GSH) peroxidases, GSH transferases, protein disulfide isomerases (PDIs), and many other proteins (70). The structure and function is regulated by structural SH-groups that can be oxidized to an extra disulfide or nitrosylated. Moreover, depending on the redox states of the active site dithiols, Trx1 catalyzes either trans-S-nitrosylation or S-denitrosylation of its target proteins (33, 93, 110).

FIG. 1.

The human thioredoxin (Trx). Reduced (A) and oxidized (B) form of human Trx. The figures are adopted from Ref. (108). See text for details. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars).

Formation of RSNOs and S-Nitrosoproteins

Nitric oxide synthases (NOSs) are the family of enzymes that catalyze the production of NO from L-arginine in the presence of NADPH and O₂ (1, 45, 62). In mammalian cells, NOSs are the major sources of endogenous NO and the three mammalian NOS isoforms (inducible NOS [iNOS], endothelial NOS [eNOS], and neuronal NOS [nNOS]) exhibit a similar catalytic profile and composition. However, iNOS was found to generate a much greater extent of NO, to more than 100 fold, compared with that produced by constitutive NOSs (eNOS and nNOS) (55). Moreover, iNOS and nitrosative stress have been implicated in many human diseases, including atherosclerosis, insulin resistance, inflammation, and neurodegenerative diseases (21, 60, 79).

S-nitrosylation, the covalent attachment of the NO function to the thiol group of cysteine, is a ubiquitous redox modification and an important mechanism for post-translational regulation of different proteins. The products of S-nitrosylation, RSNOs, have been suggested to play crucial roles in human health and diseases (25). Several mechanisms, such as trans-S-nitrosylation, acidic S-nitrosylation, and catalysis by metalloproteins, have been proposed for the formation of RSNOs (31, 35). However, some of the pathways can be predominant over others depending on the biological context of cells; such as the acidic S-nitrosylation under inflammatory conditions (31, 83) and metal-catalyzed S-nitrosylation in the presence of ceruloplasmin (101).

NO forms metal-nitrosyl complexes and N₂O₃, which can act as S-nitrosylating agents in cells (11, 54). Activated mouse macrophage and vascular smooth muscle cells have been reported to produce NO₂ ⁻ at a rate of 50–70 nmol/mg of protein/h for approximately 20 h (4, 105). The formation of GSNO is expected to follow the production of endogeneous NO due to the presence of GSH, the most abundant intracellular thiol. GSNO has been detected in the brain (54) and liver (98) of healthy rats in amounts of 15–34 pmol/mg protein. Thus, the formation of low-molecular-weight RSNOs could be predicted in both the intra- and extracellular environments, as NO can S-nitrosylate extracellular thiols on diffusion across plasma membranes. In another study, on lipopolysaccharide (LPS) stimulation, RAW 264.7 cells were found to be generated 17.4±1.0 pmol/mg of S-nitrosoproteins, which decayed with a half life of about 3 h (118). Moreover, S-nitrosoalbumin and S-nitrosohemoglobin (SNO-Hb) were found to be increased by ∼3.4- and ∼25-fold, respectively, in response to the LPS challenge in rats (50). The administration of LPS to the experimental animals resulted in an upregulation of iNOS and sustained overproduction of NO that contributed to the increased level of circulating RSNOs. The overproduction of NO has been proposed to contribute to circulatory failure, myocardial dysfunction, organ injury, and multiple organ dysfunction syndrome associated with endotoxic shock (76, 103). It was further demonstrated that plasma albumin became saturated with NO at physiological NO concentrations and catalyzed the RSNO formation involving a micellar catalysis mechanism (79). The albumin-mediated S-nitrosylation and its vasodilatory effect were found to be directly dependent on the concentration of circulating low-molecular-weight thiols. The transmembrane movement of RSNOs from the plasma albumin-SNO reservoir was suggested to be mediated by cysteine, which serves as a carrier (58, 59, 119). The mechanism involves trans-S-nitrosylation of free cysteine from S-nitrosoalbumin (or S-nitrosylated albumin) and stereoselective uptake of L-CysNO by two major members of the ubiquitous amino acid transporter system L (LAT1 and LAT2) (58, 59, 119).

Functional Alteration of S-Nitrosoproteins

Till date, more than a thousand of S-nitrosylated cellular proteins have been identified (25) (Table 1). The S-nitrosylated proteins have been suggested to be involved in a variety of cellular processes/diseases, including signal transduction, insulin resistance, apoptosis, and cancer. There are several proteins containing multiple cysteine residues, while the S-nitrosylation of specific cysteine depends on the concentration of S-nitrosylating species at the modification site, pKa values of thiols, hydrophobic environment, and several other factors (20). The S-nitrosylated human serum albumin (HAS-SNO) was suggested to act as a circulating reservoir for NO produced by endothelial cells (44, 82, 96). HAS-SNO showed potent antibacterial activity and inhibited the proliferation of cultured human vascular smooth muscle cells. Furthermore, antitumor activity of HAS-SNO was examined in murine colon 26 carcinoma cells (C26) and also in C26 tumor-bearing mice (52). SNO-Hb was found in the systemic circulation, at concentrations ∼10 times higher in the arterial than the venous blood (49, 97). SNO-Hb has been suggested to act as an endogenous NO donor and a physiological regulator of blood flow.

Nuclear factor-Kappa B (NF-κB) is a versatile transcription factor that regulates a wide variety of cellular processes, including inflammation and survival. NO has been suggested to play an anti-inflammatory role through the S-nitrosylation of components of the NF-κB pathway. Marshall and Stamler reported the inhibition of NF-κB activity by the S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit (69). Under normoxic condition, HIF-1α protein was found to be S-nitrosylated, which prevented its degradation (56). Similar to many other transcription factors, the GTPases were found to be S-nitrosylated. S-nitrosylation of Ras resulted in an increase of cellular Ras-GTP levels in vivo, which activated the downstream signaling pathways (34). In many cancers, Ras GTPases (H, N, or KRas) are mutated to remain in the active GTP-bound oncogenic state, while eNOS enhances the S-nitrosylation and activation of endogenous wild-type Ras, required throughout tumorigenesis (42, 45, 62). One of the important substrates of S-nitrosylation that regulate cardiac function is the ion channel RyR. It was reported that NO and RSNO-mediated oxidation of critical thiols in both skeletal (RyR1) and cardiac (RyR2) isoforms of the RyR resulted in channel activation and subsequent addition of a sulfhydryl reducing agent, resulting in channel closure (6, 14, 99, 112). Later, knocking out nNOS was reported to reduce S-nitrosylation of the RyR on the sarcoplasmic reticulum, which leads to diastolic Ca²⁺ leak with a negative impact on cardiac electrical stability and contractility in cardiac myocytes (29). The S-nitrosylation of RyR1 depleted the channel complex of FKBP12 (calstabin-1, for calcium channel stabilizing binding protein) and resulted in “leaky” channels. It was proposed that the sarcoplasmic reticulum Ca²⁺ leak via RyR1 was due to the hypernitrosylation of the channel, and calstabin1 depletion contributed to muscle weakness in muscular dystrophy (5).

NO is known to regulate apoptosis through the S-nitrosylation of many apoptosis-regulating proteins, including caspases that play an essential role in the initiation and execution of apoptosis. S-nitrosylation of the critical catalytic cysteine residue by basal NO production inhibits caspase activity and apoptosis in resting cells (22, 68). In vivo experiments were presented for the S-nitrosylation-mediated inhibition of caspase-3 (85). In addition to caspase 3, the activities of six other caspases were inhibited through S-nitrosylation in vitro, including caspase 8 and 9 (57). FLIP (FADD-like IL-1β-converting enzyme-inhibitory protein) and B-cell lymphoma-2 (Bcl-2) are the key regulatory proteins in the mitochondrial death receptor pathway. S-nitrosylation of FLIP was found to inhibit its degradation through the ubiquitin pathway (46). Similar to FLIP, S-nitrosylation of Bcl-2 was reported, which resulted in the inhibition of its ubiquitination and subsequent degradation (3, 46). Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine protein kinase that functions as an MAPK kinase kinase in the JNK and p38MAPK signaling pathways. The results suggested that NO mediated the interferon-γ-induced inhibition of ASK1 in L929 cells through the S-nitrosylation of Cys869 (77). Furthermore, Hara et al. reported a cell death cascade whereby cellular stressors activated NO formation, leading to the S-nitrosylation of GAPDH, which triggered its binding to Siah1 (an E3 ubiquitin ligase), translocation of GAPDH/Siah complex to the nucleus, and apoptotic cell death (32).

Akt/PKB is a serine/threonine protein kinase that plays a central role in the metabolic functions of insulin, while the genetic disruption of Akt2/PKBβ leads to insulin resistance in mice (15). NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells (15, 114). S-nitrosylation of Cys224 was identified as a mechanism for the inactivation of Akt/PKB (114). Moreover, it was further identified that S-nitrosylation of Akt/PKB was increased in the skeletal muscle of diabetic mice compared with wild-type mice. Carvalho-Filho et al. demonstrated that insulin resistance was associated with iNOS induction in two different models of obesity, and it was mediated by the S-nitrosylation of insulin receptor β subunit, insulin receptor substrate 1, and Akt (12). Abnormal S-nitrosylation plays an important role in the process of neurodegeneration. MMPs, which are involved in remodeling extracellular matrix, have been suggested in the pathogenesis of neurodegenerative diseases and stroke. During cerebral ischemia, MMPs were found to be colocalized with nNOS, and S-nitrosylation was found to activate MMPs (30). Furthermore, the S-nitrosylation of parkin was reported in vitro, as well as in vivo in the mouse model of PD and in brains of patients with PD (17, 113). Parkin is an E3 ubiquitin ligase which is involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in PD. The S-nitrosylation resulted in the inhibition of parkin's ubiquitin E3 ligase activity, which was suggested to contribute in the degenerative process of the disorder by impairing the ubiquitination of parkin substrates (17, 113).

Here, we have discussed some of the S-nitrosylated proteins with relation to signal transduction, insulin resistance, neurodegeneration, apoptosis, and cancer. It is noteworthy to mention that the Trx system represents a major antioxidant defense line in most of the eukaryotes and prokaryotes, and it is known to be involved in the redox regulation of many transcription factors. Thus, further characterization of the role of Trx will enhance our knowledge about the regulation of S-nitrosylation in different cellular processes. Researchers have documented the protective effects of Trx system on autoimmune diabetes, which suggests its possible therapeutic application (41, 67). Furthermore, Trx was found to be a physiological inhibitor of ASK1, and the anti-apoptotic role of Trx was further correlated with the NO-signaling and S-nitrosylation of protein thiols (86, 102).

Trx-Catalyzed S-Denitrosylation

Many S-nitrosoproteins are denitrosylated by GSH (84), while four enzyme systems have been characterized to catabolize GSNO: alcohol dehydrogenase class III (ADH) (48), carbonyl reductase (5, 95), PDI (94), and Trx (E. coli and human Trx) (75, 93, 100). GSNO was found to be an active substrate of rat ADH class III, and GSH sulphinamide was suggested to be the major stable product of the GSNO processing, with minor yields of GSSG and NH₃ (48). Later, a GSH-dependent formaldehyde dehydrogenase (GS-FDH), purified from E. coli, Saccharomyces cerevisiae, and mouse macrophage, was found to catabolize GSNO (65). Peptide-mass analysis of the purified GSNO reductase (GSNOR) from RAW 264.7 identified the protein as mouse GS-FDH or ADH III, the homolog of the bacterial GSNOR. The enzyme was found to be specific for GSNO (65). However, it was found that S-nitroso-N-acetylpenicillamine (SNAP) and HAS-SNO were not the substrates for ADH-catalyzed denitrosylation reaction. Further studies suggested GSNOR as an important regulator in human asthma (16, 27, 47, 64, 73, 80, 81, 83, 111). Carbonyl reductase was also reported to reduce GSNO; however, the catalytic efficency (K _cat) value was found to be lower than that of GS-FDH (5). S-denitrosylation activity of recombinant human PDI was previously characterized, and a mechanism of PDI-catalyzed GSNO denitrosylation, involving a nitroxyl (HNO) disulfide intermediate stabilized by imidazole residues of the subunits, has been proposed (94). It is noteworthy to mention that most PDIs contain more than one active site, and a combination of active and inactive Trx-like domains (19).

Trx is a ubiquitous protein, while ADH and PDI are expressed mainly in the liver and kidney (66) and in the lumen of the endoplasmic reticulum (8), respectively, (Human Protein Reference Database, www.hprd.org). Furthermore, it has been shown that NADPH is the universal reducing equivalent in vivo rather than NADH; and, thus, NADPH-dependent enzymes (Trx, etc.) generally operate in a reducing direction (104, 115). Trx system was reported to catalyze the denitrosylation of low-molecular-mass (LMM) RSNOs (GSNO, CysNO, HCysNO, and SNAP) and S-nitrosoproteins (S-nitrosoalbumin, S-nitrosometallothionenin, S-nitroso RhoA, S-nitrosocaspase 3, and S-nirosocaspase 8) (75, 90 –93, 100, 121) (Figs. 2 and 3). It was further identified that all the HepG2 cells-derived S-nitrosoproteins with a molecular mass of 23–30 kDa were catabolized by the Trx1/TrxR system (93). The results were further supported by the observation that Trx-catalyzed denitrosylation resulted in a 72% reduction of protein L-CysNO levels in isolated liver homogenates, and 72 proteins of the liver S-nitrosoproteome were found to be the substrate of Trx-catalyzed denitrosylation (23).

FIG. 2.

Trx-catalyzed S -denitrosylation and trans-S-nitrosylation reactions. The Trx-catalyzed S-denitrosylation of S-nitrosothiols (RSNOs) (Protein SNOs and low-molecular-mass RSNOs) involves trans-S-nitrosylation of the low-pKa thiol (Cys32 of Trx) [1→2]. Due to the presence of vicinal thiol, (Cys35 of Trx) 2 undergoes rapid ring closure to disulfide 3 with the concomitant release of nitroxyl (HNO). 3 may undergo S-nitrosylation at Cys 62, 69 or 73 to form 4. Both 3 and 4 can be reduced to 1 in the presence of TrxR and NADPH. On inhibition of disulfide reduction or S-denitrosylation activity, 4 can catalyze trans-S-nitrosylation of target proteins (87, 102). Thioredoxin-interacting protein (Txnip) binds directly to the active site Cys of Trx, inhibits the reducing activity of Trx, and facilitates protein S-nitrosylation (24, 72, 83, 90, 93, 100, 106, 110). The figure is adopted and modified from Refs. (90, 93).

FIG. 3.

Trx-catalyzed denitrosylation of intracellular RSNOs. MMTS/MTSEA-TAMRA-stained intracellular RSNOs before (A) and after treatment of TrxnR and mTrxnR HeLa cells with S-nitroso-L-cysteine ethyl ester (SNCEE) (B, 5 min; C, 30 min). The interaction of Trx with intracellular RSNOs was assessed using Trx/TrxR wild-type HeLa cells (TrxR) and HeLa cells transfected with a cysteine-to-serine mutant expressing plasmid (mTrxR) that rendered cells with dysfunctional TrxR and impaired Trx activity (100). SNCEE treatment resulted in a marked accumulation of RSNOs in cells (B, C; red stains). Clearance of intracellular RSNOs (A, B and C; red stains) occurred at an increased rate and more completely in the TrxR versus mTrxR HeLa cells. NO donors and lipoic acid (LA) modulated the caspase 3 activity in HepG2 cells (D and E). Caspase 3 activity was stimulated with TNF-α (15 nM) plus cycloheximide (40 μM); the cells were incubated with 50 μM SNCEE for 15 min at 37°C, washed with PBS, and assayed for caspase 3 activity either immediately or after an additional incubation for 60 min with NO donor-free medium in the absence and in the presence of 50 μM LA or 5 mM dithiothreitol (DTT) (90, 93). Treatment of the NO donor led to the inhibition of the caspase 3 activity, while the regeneration of caspase 3 activity was insignificant in TrxR-deficient cells (E) compared with the control cells (D). However, LA markedly reactivated caspase 3 activitiy in TrxR-deficient cells (E). The figures are adopted and modified from Refs. (90, 93, 100). (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

The interaction of Trx with intracellular RSNOs was assessed using Trx/TrxR wild-type HeLa cells (TrxR) and HeLa cells transfected with a cysteine-to-serine mutant expressing plasmid (mTrxR) that rendered cells with dysfunctional TrxR and impaired Trx activity (100). S-nitroso-L-cysteine ethyl ester (SNCEE) treatment resulted in a marked accumulation of RSNOs in cells (Fig. 3B, C; red stains). Clearance of intracellular RSNOs (Fig. 3; red stains) occurred at an increased rate and more completely in the TrxR versus mTrxR HeLa cells. The background intracellular level of RSNOs in wild-type TrxnR HeLa cells was approximately two fold lower than in mTrxR cells (Fig. 3C), indicating that decreased Trx activity was associated with less effective catabolism of intracellular RSNOs. Similarly, in HepG2 cells, it was observed that NO donor treatment resulted in the inhibition of caspase-3 and caspase-8 activities (Fig. 3D, E) (90, 91, 93). However, a subsequent incubation of the cells in RSNO-free medium resulted in the reconstitution of caspase activities, due to endogeneous denitrosylation of the S-nitrosocaspases (90 –93). The latter process was markedly inhibited in TrxR-deficient HepG2 cells, suggesting that the Trx/TrxR system tends to maintain intracellular caspases in a reduced state. It was further observed that lipoic acid (LA) markedly reactivated the caspases in TrxR-deficient HepG2 cells (80 –93) (Fig. 3). LA or (R)-5-(1,2-dithiolan-3-yl)pentanoic acid, a cyclic disulfide, is an essential prosthetic group in the dihydrolipoyl transacetylase component of the α-keto acid dehydrogenase complex in mitochondria. In cells, LA is reduced to dihydrolipoic acid (6,8-dimercaptooctanoic acid) by lipoamide dehydrogenase (LD) with consumption of NADH and by NADPH-dependent reductases. The LA/LD/NADH system was shown to catalyze the denitrosylation of both GSNO and S-nitrosylated caspase 3 (100). These results suggested the possible role of low-molecular-mass dithiols that mimic the activity of Trx in reactions of S-denitrosylation of proteins.

The S-nitrosylated proteins are expected to be denitrosylated by GSH, as it is the most abundant intracellular thiol. Chase experiments conducted in rat spinal cord slices were preincubated with GSNO (84), and the results revealed the intracellular GSH-mediated denitrosylation of protein RSNOs in intact cells by rapid trans-S-nitrosylation reactions. GSH-dependent S-denitrosylation resulted in an ∼75% decrease in high-molecular-mass RSNOs from Jurkat cells. It is noteworthy that S-nitrosocaspase 3, which was relatively stable in the presence of 5–10 mM GSH (117), was readily denitrosylated by the Trx/TrxR system (90, 93). Recently, using Jurkat cells, Stamler and coworkers identified 46 proteins as the substrates of the Trx 1-catalyzed denitrosylation reaction (9). The substrates are involved in a wide range of cellular functions, including cytoskeletal organization, cellular metabolism, signal transduction, and redox homeostasis. The results pointed to Trx1 as a major protein denitrosylase in mammalian cells. In another study, Trx2 has been found to mediate the denitrosylation of mitochondria-associated S-nitrosocaspase (7). However, further studies are needed to better understand the Trx-dependent catabolism of RSNOs in cells.

Role of S-Nitrosothioredoxin

Unlike Trxs from lower species, mammalian Trx1 contains additional cysteines at 62, 69, and 73 positions (107). The nitrosylation of either Cys69 or Cys73 leads to the formation of S-nitrosothioredoxin (ONS-Trx-S₂), which has been suggested to impede apoptosis via trans-S-nitrosylation of caspase 3 (33, 72, 110) (Fig. 2). However, experimental evidence was provided for the denitrosylation of ONS-Trx-S₂ in the presence of either reduced Trx [Trx(SH)₂] or GSH (93). The results suggest that both S-nitrosothioredoxin and S-nitrosocaspase 3, if formed in cells, will undergo GSH and Trx/TrxR system-dependent denitrosylation (Fig. 2). Recently, an optimized mass spectrometric method has been used to demonstrate the GSNO-mediated nitrosylation at Cys73 of Trx after the formation of a Cys32/Cys35 disulfide bond, on which Trx1 disulfide reductase and denitrosylase activities were attenuated (110). Moreover, the overexpression of Trx1C32S/C35S mutant in HeLa cells promoted the S-nitrosylation of specific target proteins, and 47 novel Trx1 trans-S-nitrosylation target proteins were identified. The results demonstrated that Trx1 can catalyze either trans-S-nitrosylation or S-denitrosylation reactions depending on the redox state of the cell. The subcellular localization and stability of the S-nitrosothioredoxin should be tightly regulated by different reductants (reduced Trx1 or GSH) (93, 110). Thus, redox states of the active site dithiols of Trx are critical regulators that determine the S-nitrosylation status of the additional cysteines (33, 93, 110). On inhibition of disulfide reduction or S-denitrosylation activity, ONS-Trx-S₂ can catalyze trans-S-nitrosylation of target proteins (Fig. 2).

Trx-Interacting Protein

Trx-interacting protein (Txnip; also called “vitamin-D3-upregulated protein-1” and “thioredoxin-binding protein 2”), a 50 kDa ubiquitous protein, binds directly to the active site Cys of Trx and inhibits the reducing activity of Trx (78, 106) (Fig. 2). Studies suggest major physiological roles for Txnip in glucose metabolism and cell differentiation (18, 106). The expression of Txnip was induced by a variety of stresses, including UV light, γ-rays, heat shock, and H₂O₂, as well as glucose treatments. Furthermore, overexpression of Txnip renders cells more susceptible to oxidative stress and promotes apoptosis (13, 116). Schulze et al. reported that the expression of the gene encoding Txnip was suppressed by NO in rat pulmonary artery smooth muscle cells (88). The net effect of these transcriptional effects was suggested to increase the Trx activity. Another study implicated Txnip as a regulator of S-nitrosylation by interfering with the Trx-catalyzed denitrosylation (Fig. 2) (24). Endogenously synthesized NO was suggested to repress Txnip to facilitate Trx-mediated denitrosylation. In the study, HEK293 cells transfected with iNOS exhibited increased intracellular levels of S-nitrosylated proteins, while the cotransfection of Txnip with iNOS further resulted in an increase in protein S-nitrosylation by ∼40%. This increase corresponds to the groups of S-nitrosylated proteins that are the substrates for Trx1-catalyzeded denitrosylation. Further characterization of the Trx-Txnip system will enhance our knowledge of the development of drugs and therapies.

Conclusion

Delivery of Trx1 as a new therapeutic agent has been documented (74) and recently, it has been shown that the impairment of angiogenesis and myocardial dysfunction can be regulated by Trx1 gene therapy in diabetic rats (87). On the other hand, the involvement of Trx system in the regulation of cellular RSNOs and RSNO signaling has been studied by several researchers (74). However, the dynamics of protein S-nitrosylation and cellular homeostasis of RSNOs remain poorly understood. Further investigations on the role of the Trx system in relation to biologically relevant RSNOs and their functions will facilitate the development of drugs and therapies.

Footnotes

Abbreviations Used

References

Alderton

, Cooper

, Knowles

. Nitric oxide synthases: structure, function and inhibition. Biochem J, 357:593–615. 2001.

Arnér

. Focus on mammalian thioredoxin reductases-important selenoproteins with versatile functions. Biochim Biophys Acta, 1790:495–526. 2009.

Azad

, Vallyathan

, Wang

, Tantishaiyakul

, Stehlik

, Leonard

, Rojanasakul

. S-nitrosylation of Bcl-2 inhibits its ubiquitin-proteasomal degradation. A novel antiapoptotic mechanism that suppresses apoptosis. J Cell Biol, 281:34124–34134. 2006.

Bani

, Failli

, Bello

, Thiemermann

, Bani Sacchi

, Bigazzi

, Masini

. Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension, 31:1240–1247. 1998.

Bateman

, Rauh

, Tavshanjian

, Shokat

. Human carbonyl reductase 1 is an S-nitrosoglutathione reductase. J Biol Chem, 283:35756–35762. 2008.

Bellinger

, Reiken

, Carlson

, Mongillo

, Liu

, Rothman

, Matecki

, Lacampagne

, Marks

. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med, 15:325–330. 2009.

Benhar

, Forrester

, Hess

, Stamler

. Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science, 320:1050–1054. 2008.

Benhar

, Forrester

, Stamler

. Nitrosative stress in the ER: a new role for S-nitrosylation in neurodegenerative diseases. ACS Chem Biol, 1:355–358. 2006.

Benhar

, Thompson

, Moseley

, Stamler

. Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach. Biochemistry, 49:6963–6969. 2010.

10.

Bian

, Murad

. Nitric oxide (NO)—biogeneration, regulation, and relevance to human diseases. Front Biosci, 8:264–278. 2003.

11.

Boese

, Mordvintcev

, Vanin

, Busse

, Mülsch

. S-nitrosation of serum albumin by dinitrosyl-iron complex. J Biol Chem, 270:29244–29249. 1995.

12.

Carvalho-Filho

, Ueno

, Hirabara

, Seabra

, Carvalheira

, de Oliveira

, Velloso

, Curi

, Saad

. S-nitrosation of the insulin receptor, insulin receptor substrate 1, and protein kinase B/Akt: a novel mechanism of insulin resistance. Diabetes, 54:959–967. 2005.

13.

Chen

, Fontes

, Saxena

, Poitout

, Shalev

. Lack of TXNIP protects against mitochondria-mediated apoptosis but not against fatty acid-induced ER stress-mediated beta-cell death. Diabetes, 59:440–447. 2010.

14.

Cheong

, Tumbev

, Stoyanovsky

, Salama

. Effects of pO2 on the activation of skeletal muscle ryanodine receptors by NO: a cautionary note. Cell Calcium, 38:481–488. 2005.

15.

Cho

, Mu

, Kim

, Thorvaldsen

, Chu

, Crenshaw

3rd , Kaestner

, Bartolomei

, Shulman

, Birnbaum

. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta) Science, 292:1728–1731. 2001.

16.

Choudhry

, Que

, Yang

, Liu

, Eng

, Kim

, Kumar

, Thyne

, Chapela

, Rodriguez-Santana

, Rodriguez-Cintron

, Avila

, Stamler

, Burchard

. GSNO reductase and beta2-adrenergic receptor gene-gene interaction: bronchodilator responsiveness to albuterol. Pharmacogenet Genomics, 20:351–358. 2010.

17.

Chung

, Thomas

, Li

, Pletnikova

, Troncoso

, Marsh

, Dawson

. S-nitrosylation of parkin regulates ubiquitination and compromises Parkin's protective function. Science, 304:1328–1331. 2004.

18.

Chutkow

, Patwari

, Yoshioka

, Lee

. Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production. J Biol Chem, 283:2397–2406. 2008.

19.

Clissold

, Bicknell

. The thioredoxin-like fold: hidden domains in protein disulfide isomerases and other chaperone proteins. Bioessays, 25:603–611. 2003.

20.

Derakhshan

, Hao

, Gross

. Balancing reactivity against selectivity: the evolution of protein S-nitrosylation as an effector of cell signaling by nitric oxide. Cardiovasc Res, 75:210–219. 2007.

21.

Detmers

, Hernandez

, Mudgett

, Hassing

, Burton

, Mundt

, Chun

, Fletcher

, Card

, Lisnock

, Weikel

, Bergstrom

, Shevell

, Hermanowski-Vosatka

, Sparrow

, Chao

, Rader

, Wright

, Puré

. Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice. J Immunol, 165:3430–3435. 2000.

22.

Dimmeler

, Zeiher

. Nitric oxide and apoptosis: another paradigm for the double-edged role of nitric oxide. Nitric Oxide, 1:275–281. 1997.

23.

Doulias

, Greene

, Greco

, Tenopoulou

, Seeholzer

, Dunbrack

, Ischiropoulos

. Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation. Proc Natl Acad Sci U S A, 107:16958–16963. 2010.

24.

Forrester

, Seth

, Hausladen

, Eyler

, Foster

, Matsumoto

, Benhar

, Marshall

, Stamler

. Thioredoxin-interacting protein (Txnip) is a feedback regulator of S-nitrosylation. J Biol Chem, 284:36160–36166. 2009.

25.

Foster

, Hess

, Stamler

. Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med, 5:391–404. 2009.

26.

Foster

, McMahon

, Stamler

. S-nitrosylation in health and disease. Trends Mol Med, 9:160–168. 2003.

27.

Gaston

, Reilly

, Drazen

, Fackler

, Ramdev

, Arnelle

, Mullins

, Sugarbaker

, Chee

, Singel

, Loscalzo

, Stamler

. Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proc Natl Acad Sci U S A, 90:10957–10961. 1993.

28.

Gladyshev

, Jeang

, Stadtman

. Selenocysteine identified as the penulitimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc Natl Acad Sci U S A, 93:6146–6151. 1996.

29.

Gonzalez

, Beigi

, Treuer

, Hare

. Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes. Proc Natl Acad Sci U S A, 104:20612–20617. 2007.

30.

, Kaul

, Yan

, Kridel

, Cui

, Strongin

, Smith

, Liddington

, Lipton

. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science, 297:1186–1190. 2002.

31.

Guikema

, Lu

, Jourd'heuil

. Chemical considerations and biological selectivity of protein nitrosation: implications for NO-mediated signal transduction. Antioxid Redox Signal, 7:593–606. 2005.

32.

Hara

, Agrawal

, Kim

, Cascio

, Fujimuro

, Ozeki

, Takahashi

, Cheah

, Tankou

, Hester

, Ferris

, Hayward

, Snyder

, Sawa

. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat Cell Biol, 7:665–674. 2005.

33.

Hashemy

, Holmgren

. Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues. J Biol Chem, 283:21890–21898. 2008.

34.

Heo

, Prutzman

, Mocanu

, Campbell

. Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation. J Mol Biol, 346:1423–1440. 2005.

35.

Hogg

. The biochemistry and physiology of S-nitrosothiols. Annu Rev Pharmacol Toxicol, 42:585–600. 2002.

36.

Holmgren

. Thioredoxin: amino acid sequence of the protein from Escherichia coli. B Eur J Biochem, 6:475–484. 1968.

37.

Holmgren

. Reduction of disulfides by thioredoxin. Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action. J Biol Chem, 254:9113–9119. 1979.

38.

Holmgren

. Thioredoxin. Annu Rev Biochem, 54:237–271. 1985.

39.

Holmgren

, Lu

. Thioredoxin and thioredoxin reductase: current research with special reference to human disease. Biochem Biophys Res Commun, 396:120–124. 2010.

40.

Holmgren

, Söderberg

, Eklund

, Brändén

. Three-dimensional structure of Escherichia coli thioredoxin-S₂ to 2.8 Å resolution. Proc Natl Acad Sc U S A, 72:2305–2309. 1975.

41.

Hotta

, Tashiro

, Ikegami

, Niwa

, Ogihara

, Yodoi

, Miyazaki

. Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes. J Exp Med, 188:1445–1451. 1998.

42.

Ibiza

, Pérez-Rodríguez

, Ortega

, Martínez-Ruiz

, Barreiro

, García-Domínguez

, Víctor

, Esplugues

, Rojas

, Sánchez-Madrid

, Serrador

. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells. Proc Natl Acad Sci U S A, 105:10507–10512. 2008.

43.

Ignarro

. Nitric oxide as a unique signaling molecule in the vascular system: a historical overview. J Physiol Pharmacol, 53:503–514. 2002.

44.

Ishima

, Akaike

, Kragh-Hansen

, Hiroyama

, Sawa

, Maruyama

, Kai

, Otagiri

. Effects of endogenous ligands on the biological role of human serum albumin in S-nitrosylation. Biochem Biophys Res Commun, 364:790–795. 2007.

45.

Iwakiri

, Satoh

, Chatterjee

, Toomre

, Chalouni

, Fulton

, Groszmann

, Shah

, Sessa

. Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking. Proc Natl Acad Sci U S A, 103:19777–19782. 2006.

46.

Iyer

, Azad

, Wang

, Rojanasakul

. Role of S-nitrosylation in apoptosis resistance and carcinogenesis. Nitric Oxide, 19:146–151. 2008.

47.

Janocha

, Koch

, Tiso

, Ponchia

, Doctor

, Gibbons

, Gaston

, Beall

, Erzurum

. Nitric oxide during altitude acclimatization. N Engl J Med, 365:1942–1944. 2011.

48.

Jensen

, Belka

, Du Bois

. S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme. Biochem J, 331:659–668. 1998.

49.

Jia

, Bonaventura

, Stamler

. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature, 380:221–226. 1996.

50.

Jourd'heuil

, Gray

, Grisham

. S-nitrosothiol formation in blood of lipopolysaccharide-treated rats. Biochem Biophys Res Commun, 273:22–26. 2000.

51.

Kallis

, Holmgren

. Differential reactivity of the functional sulfhydryl groups of cysteine-32 and cysteine-35 present in the reduced form of thioredoxin from Escherichia coli. J Biol Chem, 255:10261–10265. 1980.

52.

Katayama

, Nakajou

, Komori

, Uchida

, Yokoe

, Yasui

, Yamamoto

, Kai

, Sato

, Nakagawa

, Takeya

, Maruyama

, Otagiri

. Design and evaluation of S-nitrosylated human serum albumin as a novel anticancer drug. J Pharmacol Exp Ther, 325:69–76. 2008.

53.

Kim

, Chung

, Simmons

, Billiar

. Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition. J Biol Chem, 275:10954–10961. 2000.

54.

Kluge

, Gutteck-Amsler

, Zollinger

, Do

. S-nitrosoglutathione in rat cerebellum: identification and quantification by liquid chromatography-mass spectrometry. J Neurochem, 69:2599–2607. 1997.

55.

Kuo

, Schroeder

. The emerging multifaceted roles of nitric oxide. Ann Surg, 221:220–235. 1995.

56.

, Sonveaux

, Rabbani

, Liu

, Yan

, Huang

, Vujaskovic

, Dewhirst

, Li

. Regulation of HIF-1alpha stability through S-nitrosylation. Mol Cell, 26:63–74. 2007.

57.

, Billiar

, Talanian

, Kim

. Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem Biophys Res Commun, 240:419–424. 1997.

58.

, Whorton

. Identification of stereoselective transporters for S-nitroso-L-cysteine: role of LAT1 and LAT2 in biological activity of S-nitrosothiols. J Biol Chem, 280:20102–20110. 2005.

59.

, Whorton

. Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells. Am J Physiol Cell Physiol, 292:C1263–C1271. 2007.

60.

Liberatore

, Jackson-Lewis

, Vukosavic

, Mandir

, Vila

, McAuliffe

, Dawson

, Przedborski

. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med, 5:1403–1409. 1999.

61.

Lillig

, Holmgren

. Thioredoxin and related molecules-from biology to health and disease. Antioxid Redox Signal, 9:25–47. 2007.

62.

Lim

, Ancrile

, Kashatus

, Counter

. Tumour maintenance is mediated by eNOS. Nature, 452:646–649. 2008.

63.

Lima

, Forrester

, Hess

, Stamler

. S-nitrosylation in cardiovascular signaling. Circ Res, 106:633–646. 2010.

64.

Lima

, Lam

, Xie

, Diesen

, Villamizar

, Nienaber

, Messina

, Bowles

, Kontos

, Hare

, Stamler

, Rockman

. Endogenous S-nitrosothiols protect against myocardial injury. Proc Natl Acad Sci U S A, 106:6297–6302. 2009.

65.

Liu

, Hausladen

, Zeng

, Que

, Heitman

, Stamler

. A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature, 410:490–494. 2001.

66.

Liu

, Yan

, Zeng

, Zhang

, Hanes

, Ahearn

, McMahon

, Dickfeld

, Marshall

, Que

, Stamler

. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell, 116:617–628. 2004.

67.

Luan

, Liu

, Yin

, Lau

, Wang

, Guo

, Wang

, Tao

. High glucose sensitizes adult cardiomyocytes to ischaemia/reperfusion injury through nitrative thioredoxin inactivation. Cardiovasc Res, 83:294–302. 2009.

68.

Mannick

, Schonhoff

, Papeta

, Ghafourifar

, Szibor

, Fang

, Gaston

. S-Nitrosylation of mitochondrial caspases. J Cell Biol, 154:1111–1116. 2001.

69.

Marshall

, Stamler

. Inhibition of NF-kappa B by S-nitrosylation. Biochemistry, 40:1688–1693. 2001.

70.

Martin

. Thioredoxin—a fold for all reasons. Structure, 3:245–250. 1995.

71.

Micke

, Schomburg

, Buentzel

, Kisters

, Muecke

. Selenium in oncology: from chemistry to clinics. Molecules, 14:3975–3988. 2009.

72.

Mitchell

, Marletta

. Thioredoxin catalyzes the S-nitrosation of the caspase-3 active site cysteine. Nat Chem Biol, 1:154–158. 2005.

73.

Moore

, Ryckman

, Williams

, Patel

, Summar

, Sheller

. Genetic variants of GSNOR and ADRB2 influence response to albuterol in African-American children with severe asthma. Pediatr Pulmonol, 44:649–654. 2009.

74.

Nakamura

, Hoshino

, Okuyama

, Matsuo

, Yodoi

. Thioredoxin 1 delivery as new therapeutics. Adv Drug Deliv Rev, 61:303–309. 2009.

75.

Nikitovic

, Holmgren

. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. J Biol Chem, 271:19180–19185. 1996.

76.

Nussler

, Billiar

. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol, 54:171–178. 1993.

77.

Park

, Yu

, Cho

, Kim

, Huh

, Ryoo

, Choi

. Inhibition of apoptosis signal-regulating kinase 1 by nitric oxide through a thiol redox mechanism. J Cell Biol, 279:7584–7590. 2004.

78.

Patwari

, Higgins

, Chutkow

, Yoshioka

, Lee

. The interaction of thioredoxin with Txnip. Evidence for formation of a mixed disulfide by disulfide exchange. J Biol Chem, 281:21884–21891. 2006.

79.

Perreault

, Marette

. Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Nat Med, 7:1138–1143. 2001.

80.

Que

, Liu

, Yan

, Whitehead

, Gavett

, Schwartz

, Stamler

. Protection from experimental asthma by an endogenous bronchodilator. Science, 308:1618–1621. 2005.

81.

Que

, Yang

, Stamler

, Lugogo

, Kraft

. S-nitrosoglutathione reductase: an important regulator in human asthma. Am J Respir Crit Care Med, 180:226–231. 2009.

82.

Rafikova

, Rafikov

, Nudler

. Catalysis of S-nitrosothiols formation by serum albumin: the mechanism and implication in vascular control. Proc Natl Acad Sci U S A, 99:5913–5918. 2002.

83.

Ricciardolo

, Gaston

, Hunt

. Acid stress in the pathology of asthma. J Allergy Clin Immunol, 113:610–619. 2004.

84.

Romero

, Bizzozero

. Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cord. J Neurosci Res, 87:701–709. 2009.

85.

Rössig

, Fichtlscherer

, Breitschopf

, Haendeler

, Zeiher

, Mülsch

, Dimmeler

. Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J Cell Biol, 274:6823–6826. 1999.

86.

Saitoh

, Nishitoh

, Fujii

, Takeda

, Tobiume

, Sawada

, Kawabata

, Miyazono

, Ichijo

. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J, 17:2596–2606. 1998.

87.

Samuel

, Thirunavukkarasu

, Penumathsa

, Koneru

, Zhan

, Maulik

, Sudhakaran

, Maulik

. Thioredoxin-1 gene therapy enhances angiogenic signaling and reduces ventricular remodeling in infarcted myocardium of diabetic rats. Circulation, 121:1244–1255. 2010.

88.

Schulze

, Liu

, Choe

, Yoshioka

, Shalev

, Bloch

, Lee

. Nitric oxide-dependent suppression of thioredoxin-interacting protein expression enhances thioredoxin activity, Arterioscler. Thromb Vasc Biol, 26:2666–2672. 2006.

89.

Schurmann

, Buchanan

. The ferredoxin/thioredoxin system of oxygenic photosynthesis. Antioxid Redox Signal, 10:1235–1274. 2008.

90.

Sengupta

, Billiar

, Atkins

, Kagan

, Stoyanovsky

. Nitric oxide and dihydrolipoic acid modulate the activity of caspase 3 in HepG2 cells. FEBS Lett, 583:3525–3530. 2009.

91.

Sengupta

, Billiar

, Kagan

, Stoyanovsky

. Nitric oxide and thioredoxin type 1 modulate the activity of caspase 8 in HepG2 cells. Biochem Biophys Res Commun, 391:1127–1130. 2010.

92.

Sengupta

, Billiar

, Stoyanovsky

. Studies toward the analysis of S-nitrosoproteins. Org Biomol Chem, 7:232–234. 2009.

93.

Sengupta

, Ryter

, Zuckerbraun

, Tzeng

, Billiar

, Stoyanovsky

. Thioredoxin catalyzes the denitrosation of low-molecular mass and protein S-nitrosothiols. Biochemistry, 46:8472–8483. 2007.

94.

Sliskovic

, Raturi

, Mutus

. Characterization of the S-denitrosation activity of protein disulfide isomerase. J Biol Chem, 280:8733–8741. 2005.

95.

Staab

, Hartmanová

, El-Hawari

, Ebert

, Kisiela

, Wsol

, Martin

, Maser

. Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3. Chem Biol Interact, 191:95–103. 2011.

96.

Stamler

, Jaraki

, Osborne

, Simon

, Keaney

, Vita

, Singel

, Valeri

, Loscalzo

. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci U S A, 89:7674–7677. 1992.

97.

Stamler

, Jia

, Eu

, McMahon

, Demchenko

, Bonaventura

, Gernert

, Piantadosi

. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science, 276:2034–2037. 1997.

98.

Steffen

, Sarkela

, Gybina

, Steele

, Trasseth

, Kuehl

, Giulivi

. Metabolism of S-nitrosoglutathione in intact mitochondria. Biochem J, 356:395–402. 2001.

99.

Stoyanovsky

, Murphy

, Anno

, Kim

, Salama

. Nitric oxide activates skeletal and cardiac ryanodine receptors. Cell Calcium, 21:19–29. 1997.

100.

Stoyanovsky

, Tyurina

, Tyurin

, Anand

, Mandavia

, Gius

, Ivanova

, Pitt

, Billiar

, Kagan

. Thioredoxin and lipoic acid catalyze the denitrosation of low molecular weight and protein S-nitrosothiols. J Am Chem Soc, 127:15815–15823. 2005.

101.

Stubauer

, Giuffrè

, Sarti

. Mechanism of S-nitrosothiol formation and degradation mediated by copper ions. J Biol Chem, 274:28128–28133. 1999.

102.

Sumbayev

. S-nitrosylation of thioredoxin mediates activation of apoptosis signal-regulating kinase 1. Arch Biochem Biophys, 415:133–136. 2003.

103.

Thiemermann

. Nitric oxide and septic shock. Gen Pharmacol, 29:159–166. 1997.

104.

Veech

, Eggleston

, Krebs

. The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver. Biochem J, 115:609–619. 1969.

105.

Vodovotz

, Kwon

, Pospischil

, Manning

, Paik

, Nathan

. Inactivation of nitric oxide synthase after prolonged incubation of mouse macrophages with IFN-gamma and bacterial lipopolysaccharide. J Immunol, 152:4110–4118. 1994.

106.

Watanabe

, Nakamura

, Masutani

, Yodoi

. Anti-oxidative, anti-cancer and anti-inflammatory actions by thioredoxin 1 and thioredoxin-binding protein-2. Pharmacol Ther, 127:261–270. 2010.

107.

Watson

, Pohl

, Montfort

, Stuchlik

, Reed

, Powis

, Jones

. Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. J Biol Chem, 278:33408–33415. 2003.

108.

Weichsel

, Gasdaska

, Powis

, Montfort

. Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer. Structure, 4:735–751. 1996.

109.

Wiita

, Perez-Jimenez

, Walther

, Grater

, Berne

, Holmgren

, Sanchez-Ruis

, Fernandez

. Probing the chemistry of thioredoxin catalysis with force. Nature, 450:124–127. 2007.

110.

, Liu

, Chen

, Oka

, Fu

, Jain

, Parrott

, Baykal

, Sadoshima

, Li

. Redox regulatory mechanism of transnitrosylation by Thioredoxin. Mol Cell Proteomics, 9:2262–2275. 2010.

111.

, Romieu

, Sienra-Monge

, Estela Del Rio-Navarro

, Anderson

, Jenchura

, Li

, Ramirez-Aguilar

, Del Carmen Lara-Sanchez

, London

. Genetic variation in S-nitrosoglutathione reductase (GSNOR) and childhood asthma. J Allergy Clin Immunol, 120:322–328. 2007.

112.

, Eu

, Meissner

, Stamler

. Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science, 279:234–237. 1998.

113.

Yao

, Gu

, Nakamura

, Shi

, Ma

, Gaston

, Palmer

, Rockenstein

, Zhang

, Masliah

, Uehara

, Lipton

. Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity. Proc Natl Acad Sci U S A, 101:10810–10814. 2004.

114.

Yasukawa

, Tokunaga

, Ota

, Sugita

, Martyn

, Kaneki

. S-nitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance. J Cell Biol, 280:7511–7518. 2005.

115.

Ying

. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal, 10:179–206. 2008.

116.

Yoshihara

, Chen

, Matsuo

, Masutani

, Yodoi

. Thiol redox transitions by thioredoxin and thioredoxin-binding protein-2 in cell signaling. Methods Enzymol, 474:67–82. 2010.

117.

Zech

, Wilm

, van Eldik

, Brüne

. Mass spectrometric analysis of nitric oxide-modified caspase-3. J Biol Chem, 274:20931–20936. 1999.

118.

Zhang

, Hogg

. Formation and stability of S-nitrosothiols in RAW 264.7 cells. Am J Physiol Lung Cell Mol Physiol, 287:L467–L474. 2004.

119.

Zhang

, Hogg

. The mechanism of transmembrane S-nitrosothiol transport. Proc Natl Acad Sci U S A, 101:7891–7896. 2004.

120.

Zhong

, Arnér

ESJ

, Holmgren

. Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence. Proc Natl Acad Sci U S A, 97:5854–5859. 2000.

121.

Zuckerbraun

, Stoyanovsky

, Sengupta

, Shapiro

, Ozanich

, Rao

, Barbato

, Tzeng

. Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA. Am J Physiol Cell Physiol, 292:C824–C831. 2007.