Mixed Disulfide Research Articles

Forcing the reversibility of a mechanochemical reaction

Mechanical force modifies the free-energy surface of chemical reactions, often enabling thermodynamically unfavoured reaction pathways. Most of our molecular understanding of force-induced reactivity is restricted to the irreversible homolytic scission of covalent bonds and ring-opening in polymer mechanophores. Whether mechanical force can by-pass thermodynamically locked reactivity in heterolytic bimolecular reactions and how this impacts the reaction reversibility remains poorly understood. Using single-molecule force-clamp spectroscopy, here we show that mechanical force promotes the thermodynamically disfavored SN2 cleavage of an individual protein disulfide bond by poor nucleophilic organic thiols. Upon force removal, the transition from the resulting high-energy unstable mixed disulfide product back to the initial, low-energy disulfide bond reactant becomes suddenly spontaneous, rendering the reaction fully reversible. By rationally varying the nucleophilicity of a series of small thiols, we demonstrate how force-regulated chemical kinetics can be finely coupled with thermodynamics to predict and modulate the reversibility of bimolecular mechanochemical reactions.

Thiol disulfide exchange reactions in human serum albumin: the apparent paradox of the redox transitions of Cys34.

Human serum albumin (HSA) is characterized by 17 disulfides and by only one unpaired cysteine (Cys34 ), which can be free in the reduced albumin or linked as a mixed disulfide with cysteine, or in minor amount with other natural thiols, in the oxidized albumin. In healthy subjects, the level of the oxidized form is about 35%, but it rises up to 70% after oxidative insults or in patients with kidney diseases. Oxidized albumin is therefore considered a short-term biomarker of oxidative stress as its level may increase or decrease under appropriate redox inputs in discrete temporal spans. This paper defines, for the first time, the kinetic properties of reduced and oxidized Cys34 of HSA in their reactions with natural disulfides and thiols. Kinetic constants support the evidence that the Cys34 redox oscillations observed in vivo are mainly due to the interaction with cysteine and cystine without the involvement of any enzymatic support. This study gives also a plausible explanation for the absence of involvement of the 17 disulfides naturally present in HSA in these redox transitions. This inert behavior toward cysteine is marginally due to solvent accessibility or flexibility factors of these bonds but mainly to their strong thermodynamic stability, which is caused essentially by a proximity effect. A similar mechanism is likely at play in the many proteins that maintain disulfide bridges in a reducing medium like the cytosol.

THz absorption spectrum employed for characterize the mixed Gallium selenide-Tin disulfide saturable absorber and its application for passively Q-switched solid state laser

Due to the ultra-short recovery time and the wider absorption range, graphene-like 2D material have been widely employed as saturable absorbers (SAs) in passively Q-switched and mode-locked lasers. Due to the low photon energy, noninvasive and high resolution, THz wave was widely used to characterize samples. In this paper, the nonlinear saturable absorption property and THz absorption spectrum of Gallium selenide/Tin disulfide (GaSe/SnS2) is reported. Passively Q-switched Nd:GdTaO4 laser with GaSe/SnS2 SA is demonstrated. The maximum output power of 551 mW under the pump power of 5 W. The minimum pulse width is 365 ns at the pulse repetition rate of 89 kHz. Our results indicate that GaSe/SnS2 nanosheets could be used in Q-switched lasers. The GaSe/SnS2 SA with good nonlinear absorption properties and easy fabricating process will promote the ultrafast photonics applications in solid state lasers.

Nanowires of conjugated polymer prepared by tuning the interaction between the solvent and polymer

The nanowire of conjugated polymer with higher density is desirable for its improved charge transport. However, at a higher solution concentration, the conjugated polymer chain entanglement is severe and not beneficial for the nucleation to grow nanowires. In this paper, we control the equilibrium of entanglement ↔ disentanglement ↔ nucleation and growth by tuning the radius of interaction (Ra) between the solvent and conjugated polymer. At the critical Ra, the disentangled polymer chain could nucleate to form nanowires because its content is moderately supersaturated. Thus, the equilibrium of entanglement ↔ disentanglement is broken and the entangled polymer chain is further continuously disentangled to provide easier diffusivity of chains for nanowire formation. Accordingly, the dense nanowires of poly[2,5-bis(3-tetradecylthiophen-2-yl)thieno-[3,2-b]thiophene] (PBTTT-C14) were formed in the mixed solvent carbon disulfide (CS2): cyclohexylbenzene (ChB) = 3:7 (Ra = 2.56) at a higher concentration of 1 mg/ml by aging. The density of the nanowire increased with increasing concentration and aging time. The nanowires were more than 10 μm in length and about 20 nm in width. The contrast was obvious in transmission electron microscopy which indicated the high electronic density and the high crystallinity of the nanowires.

Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding.

Oxidative protein folding in the ER is driven mainly by oxidases of the endoplasmic reticulum oxidoreductin 1 (Ero1) family. Their action is regulated to avoid cell stress, including hyperoxidation. Previously published regulatory mechanisms are based on the rearrangement of active site and regulatory disulfides. In this study, we identify two novel regulatory mechanisms. First, both human Ero1 isoforms exist in a dynamic mixed disulfide complex with protein disulfide isomerase, which involves cysteines (Cys166 in Ero1α and Cys165 in Ero1β) that have previously been regarded as being nonfunctional. Second, our kinetic studies reveal that Ero1 not only has a high affinity for molecular oxygen as the terminal acceptor of electrons but also that there is a high cooperativity of binding (Hill coefficient >3). This allows Ero1 to maintain high activity under hypoxic conditions, without compromising cellular viability under hyper-hypoxic conditions. These data, together with novel mechanistic details of differences in activation between the two human Ero1 isoforms, provide important new insights into the catalytic cycle of human Ero1 and how they have been fine-tuned to operate at low oxygen concentrations.

OP-32 - Oxidation of disulfide bonds: a novel pathway to protein glutathionylation

Disulfide bonds, formed from two cysteine (Cys) residues, play a key role in defining and stabilizing protein structures. Whilst the reactions of Cys are well characterized, little is known about oxidation of disulfides, despite their high abundance in some proteins. Recent data indicate that some disulfides are rapidly oxidized by a range of oxidants, with rate constants, k, 105–107 M-1 s-1. Here we show that these reactions yield intermediates, potentially thiosulfinates [RSS(=O)R•], that can undergo further reaction, including disulfide bond cleavage and reaction with another thiol. Thus, oxidation of alpha-lactalbumin, which contains no free Cys residues, by HOCl or ONOOH gives long-lived reactive thiosulfinates that can react with GSH to give a glutathionylated protein, which can be detected by both anti-GSH antibodies and mass spectrometry. The thiosulfinate has a lifetime of many hours allowing glutathionylation to occur hours after initial oxidation. Disulfide oxidation can therefore give rise to long-lived oxidants on proteins that can undergo further reaction with thiols, including GSH and other proteins, to give mixed disulfides and protein dimers. These reactions may play a key role in GSH-dependent cell signaling and tissue damage.

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) is a key enzyme connecting carbon and nitrogen metabolism in all living organisms. Despite extensive studies on GDHs from both prokaryotic and eukaryotic organisms in the last 40 years, the structural basis of the catalytic features of this enzyme remains incomplete. This study reports the structural basis of the GDH catalytic mechanism and allosteric behavior. We determined the first high-resolution crystal structures of glutamate dehydrogenase from the fungus Aspergillus niger (AnGDH), a unique NADP+-dependent allosteric enzyme that is forward-inhibited by the formation of mixed disulfide. We determined the structures of the active enzyme in its apo form and in binary/ternary complexes with bound substrate (α-ketoglutarate), inhibitor (isophthalate), coenzyme (NADPH), or two reaction intermediates (α-iminoglutarate and 2-amino-2-hydroxyglutarate). The structure of the forward-inhibited enzyme (fiAnGDH) was also determined. The hexameric AnGDH had three open subunits at one side and three partially closed protomers at the other, a configuration not previously reported. The AnGDH hexamers having subunits with different conformations indicated that its α-ketoglutarate-dependent homotropic cooperativity follows the Monod-Wyman-Changeux (MWC) model. Moreover, the position of the water attached to Asp-154 and Gly-153 defined the previously unresolved ammonium ion-binding pocket, and the binding site for the 2'-phosphate group of the coenzyme was also better defined by our structural data. Additional structural and mutagenesis experiments identified the residues essential for coenzyme recognition. This study reveals the structural features responsible for positioning α-ketoglutarate, NADPH, ammonium ion, and the reaction intermediates in the GDH active site.

Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress.

Aims: Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation.Results: The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H2O2) or NaOCl in vitro. Treatment with H2O2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes.Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 28, 410–430.

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

Profiling of dalcetrapib metabolites in human plasma by accelerator mass spectrometry and investigation of the free phenothiol by derivatisation with methylacrylate

Dalcetrapib, a thioester prodrug, undergoes rapid and complete conversion in vivo to its phenothiol metabolite M1 which exerts the targeted pharmacological response in human. In clinical studies, M1 has been quantified together with its dimer and mixed disulfide species that represent the ‘dalcetrapib active form’ in plasma. In this article, we describe the determination of the free phenothiol M1 by derivatisation with methylacrylate as a percentage of ‘dalcetrapib active form’. Pharmacokinetic profiles of M1 after oral administration of dalcetrapib to humans could be established, underscoring the validity to use a composite measure of ‘dalcetrapib active form’ as a surrogate marker for pharmacodynamic evaluations. ‘Dalcetrapib active form’ and M1 made up 8.9% and 3.6% of total drug-related material, respectively. In addition, complete metabolite profiling of 14C-labeled dalcetrapib was conducted after two-dimensional HPLC using fast fractionation into 384-well plates and ultrasensitive determination of the 14C-content by accelerator mass spectrometry. M1 underwent further biotransformation to its S-methyl metabolite M3, which was further oxidized to its sulfoxide and sulfone. Another metabolic pathway was the formation of the S-glucuronide. All of these species underwent further oxidation in the ethylbutyl cyclohexyl moiety leading to a multitude of hydroxyl and keto metabolites undergoing further conjugation to O-glucuronides. More than 80 metabolites were identified, demonstrating extensive metabolism. However, it was unambiguously demonstrated that none of these metabolites were major according to the MIST guideline (exceeding 10% of drug related material in circulation). The combination of accelerator mass spectrometry with HPLC together with high resolution mass spectrometry allowed for structural characterization of the most relevant human metabolites.

Nucleoredoxin-Dependent Targets and Processes in Neuronal Cells.

Nucleoredoxin (Nrx) is an oxidoreductase of the thioredoxin family of proteins. It was shown to act as a signal transducer in some pathways; however, so far, no comprehensive analysis of its regulated substrates and functions was available. Here, we used a combination of two different strategies to fill this gap. First, we analyzed the thiol-redox state of the proteome of SH-SY5Y neuroblastoma cells depleted of Nrx compared to control cells using a differential thiol-labeling technique and quantitative mass spectrometry. 171 proteins were identified with an altered redox state; 161 of these were more reduced in the absence of Nrx. This suggests functions of Nrx in the oxidation of protein thiols. Second, we utilized the active site mutant Cys208Ser of Nrx, which stabilizes a mixed disulfide intermediate with its substrates and therefore trapped interacting proteins from the mouse brain (identifying 1710 proteins) and neuronal cell culture extracts (identifying 609 proteins). Profiling of the affected biological processes and molecular functions in cells of neuronal origin suggests numerous functions of Nrx in the redox regulation of metabolic pathways, cellular morphology, and signal transduction. These results characterize Nrx as a cellular oxidase that itself may be oxidized by the formation of disulfide relays with peroxiredoxins.

Solution structure and interaction with copper in vitro and in living cells of the first BIR domain of XIAP

The X-chromosome linked inhibitor of apoptosis (XIAP) is a multidomain metalloprotein involved in caspase inhibition and in copper homeostasis. It contains three zinc-binding baculoviral IAP repeats (BIR) domains, which are responsible for caspase interaction. Recently, it has been suggested that the BIR domains can bind copper, however high resolution data on such interaction is missing. Here we characterize by NMR the structural properties of BIR1 in solution, and the effects of its interaction with copper both in vitro and in physiological environments. BIR1 is dimeric in solution, consistent with the X-ray structure. Cysteine 12, located in the unfolded N-terminal region, has a remarkably low redox potential, and is prone to oxidation even in reducing physiological environments. Interaction of BIR1 with copper(II) results in the oxidation of cysteine 12, with the formation of either an intermolecular disulfide bond between two BIR1 molecules or a mixed disulfide bond with glutathione, whereas the zinc binding site is not affected by the interaction.

Reduction potentials of protein disulfides and catalysis of glutathionylation and deglutathionylation by glutaredoxin enzymes.

Glutaredoxins (Grxs) are a class of GSH (glutathione)-dependent thiol-disulfide oxidoreductase enzymes. They use the cellular redox buffer GSSG (glutathione disulfide)/GSH directly to catalyze these exchange reactions. Grxs feature dithiol active sites and can shuttle rapidly between three oxidation states, namely dithiol Grx(SH)2, mixed disulfide Grx(SH)(SSG) and oxidized disulfide Grx(SS). Each is characterized by a distinct standard reduction potential [Formula: see text] The [Formula: see text] values for the redox couple Grx(SS)/Grx(SH)2 are available, but a recent estimate differs by over 100 mV from the literature values. No estimates are available for [Formula: see text] for the mixed disulfide couple Grx(SH)(SSG)/(Grx(SH)2 + GSH). This work determined both [Formula: see text] and [Formula: see text] for two representative Grx enzymes, Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The empirical approaches were verified rigorously to overcome the sensitivity of these redox-labile enzymes to experimental conditions. The classic method of acid 'quenching' was demonstrated to shift the thiol-disulfide redox equilibria. Both enzymes exhibit an [Formula: see text] (vs. SHE) at a pH of 7.0. Their [Formula: see text] values (-213 and -230 mV for EcGrx1 and HsGrx1, respectively) are slightly less negative than that ([Formula: see text]) of the redox buffer GSSG/2GSH. Both [Formula: see text] and [Formula: see text] vary with log [GSH], but the former more sensitively by a factor of 2. This confers dual catalytic functions to a Grx enzyme as either an oxidase at low [GSH] or as a reductase at high [GSH]. Consequently, these enzymes can participate efficiently in either glutathionylation or deglutathionylation. The catalysis is demonstrated to proceed via a monothiol ping-pong mechanism relying on a single Cys residue only in the dithiol active site.

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.

Glutaredoxin 1 Deficiency Causes Late-Age Onset Metabolic Disease in Female C57BL/6 Mice, Associated with Monocyte and Macrophage Dysfunction

Glutaredoxin 1 (Grx1) reduces mixed disulfides between glutathione and protein thiols thereby controlling thiol-redox regulated cellular functions, including intracellular signaling, migration, angiogenesis, autophagy and apoptosis. Grx1 protects monocytes and macrophages from metabolic-stress-induced “priming’ and dysfunction. Here we report that female but not male Grx1-/- mice on a C57BL/6 background fed a normal chow diet become obese and hyperglycemic by 6 and 8 months of age respectively. At 18 months female Grx1-/- mice developed spontaneous atherosclerosis and late-age onset associated metabolic disease, characterized by mild hyperlipidemia, hyperglycemia and obesity. Atherosclerotic lesions in 18 month female Grx1-/- mice, measured by en-face analysis, were localized to both the ascending arch and abdominal aortic regions. In aged female Grx1-/- mice, monocyte priming was also measured in vivo using the Matrigel plug assay and ex vivo in multi-well Boyden chambers using peritoneal macrophages isolated from female Grx1-/- mice and age-matched WT mice. Furthermore, aged Grx1-/- female mice possessed increased numbers of circulating Ly6Chi inflammatory blood monocytes compared to male Grx1-/- and WT mice. Macrophages isolated from female Grx1-/- mice also displayed increased protein-S-glutathionylation, increased expression of pro-inflammatory “M1”-associated genes, increased susceptibility to ketocholesterol-induced apoptosis, and impaired autophagy. Together, these data underscore the critical role of protein-S-glutathionylation in monocyte macrophage dysfunction and the development of atherosclerosis and obesity and identify a protective role for Grx1 in these processes. Monocytic Grx1 and metabolic stress-induced protein-S glutathionylation may therefore represent a new mechanistic link between metabolic disorders and chronic inflammatory diseases.

93 - Protein S-Nitrosylation: Possible Links between Psychophysiological Stress and Neurodegeneration

Psychophysiological stress has long been recognized as a risk factor for diverse pathologies including cardiovascular disease, psychiatric illnesses, and aging-related neurodegenerative disorders but molecular events linking stress to disease remain ill-defined. Mounting evidence supports a role for nitro-oxidative modifications of protein thiols in cellular signaling and toxicity. In light of numerous reports that acute stress can increase the activities and/or expression levels of multiple nitric oxide synthase isoforms in the brains of rodent models, we investigated the possibility that subjecting rats to acute restraint stress may promote S-nitrosylation of proteins from the brain measured by the classical biotin-switch method. Our results indeed show that restraint stress increased S-nitrosylation of brain proteins albeit transiently. S-nitrosylation was highest following 2 hours of restraint but returned to levels at or below the control by six hours. Among the S-nitrosylated proteins identified by LC-MS/MS were, from highest to lowest abundance, glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin-2, protein phosphatase 2B, and the ADP/ATP translocase all of which have immediate implications for cellular pathology. As protein nitrosthiols are generally unstable and readily convert to disulfides, efforts were also made to measure the impacts of restraint stress on disulfides involving proteins. Levels of protein-glutathione mixed disulfides appeared to be inversely related to S-nitrosylation but changes across groups were not statistically-significant. In addition, unique challenges to the trapping of the in vivo redox states of protein vicinal thiols and the determination of the effects of restraint stress on oxidations of these, resulting in protein disulfides, were noted and linked to the very high reactivity of the catalytic thiols of peroxiredoxins. These findings provide initial support for the notion that protein S-nitrosylation may link psychophysiological stress to neural insults and open the door for further investigations of nitro-oxidative modifications of protein thiols as potential links between life stresses and disease.

2 - The microenvironment of the sulfenic acid of human serum albumin: In search of clues for explaining its spontaneous decay

Human serum albumin (HSA) is the most abundant protein in plasma. It consists of a single, non-glycosylated polypeptide chain of 585 residues. Only one free cysteine, namely Cys34, is present in HSA structure. With a concentration of ∼500 μM, Cys34 constitutes more than 80 % of the plasma free thiol pool. HSA has many functions, such as transporting small molecules, maintaining the oncotic pressure and scavenging oxidant/electrophilic species. The latter is associated with Cys34 thiol, hereto identified as HSA-SH. Reaction of HSA with a two-electron oxidant (e.g. H2O2, HOONO, HOCl) leads to formation of a sulfenic acid, HSA−SOH, with a relatively long half-life [1]. HSA−SOH can follow three possible destinations: (a) reaction with a free thiol (e.g. free Cys) to give a mixed disulfide (HSA-SS-R); (b) reaction with a two-electron oxidant to yield a sulfinic acid (HSA−SO2H); and (c) spontaneously decay to an uncharacterized product, named HSA−SX, with a rate constant of ca. 10−3 s−1 (37 °C, pH 7.4).[2]

DrFrnE Represents a Hitherto Unknown Class of Eubacterial Cytoplasmic Disulfide Oxido-Reductases.

Living cells employ thioredoxin and glutaredoxin disulfide oxido-reductases to protect thiol groups in intracellular proteins. FrnE protein of Deinococcus radiodurans (drFrnE) is a disulfide oxido-reductase that is induced in response to Cd2+ exposure and is involved in cadmium and radiation tolerance. The aim of this study is to probe structure, function, and cellular localization of FrnE class of proteins. Here, we show drFrnE as a novel cytoplasmic oxido-reductase that could be functional in eubacteria under conditions where thioredoxin/glutaredoxin systems are inhibited or absent. Crystal structure analysis of drFrnE reveals thioredoxin fold with an alpha helical insertion domain and a unique, flexible, and functionally important C-terminal tail. The C-tail harbors a novel 239-CX4C-244 motif that interacts with the active site 22-CXXC-25 motif. Crystal structures with different active site redox states, including mixed disulfide (Cys22-Cys244), are reported here. The biochemical data show that 239-CX4C-244 motif channels electrons to the active site cysteines. drFrnE is more stable in the oxidized form, compared with the reduced form, supporting its role as a disulfide reductase. Using bioinformatics analysis and fluorescence microscopy, we show cytoplasmic localization of drFrnE. We have found "true" orthologs of drFrnE in several eubacterial phyla and, interestingly, all these groups apparently lack a functional glutaredoxin system. Innovation and Conclusion: We show that drFrnE represents a new class of hitherto unknown intracellular oxido-reductases that are abundantly present in eubacteria. Unlike other well-known oxido-reductases, FrnE harbors an additional dithiol motif that acts as a conduit to channel electrons to the active site during catalytic turnover. Antioxid. Redox Signal. 28, 296-310.

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme

BackgroundGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. MethodsRabbit muscle GAPDH was S-glutathionylated in the presence of H2O2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. ResultsIncubation of GAPDH in the presence of H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD+ and thermal stability. ConclusionsThe mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. General significanceThe manuscript considers one of the possible mechanisms of redox regulation of cell functions.

Guanylyl cyclase sensitivity to nitric oxide is protected by a thiol oxidation-driven interaction with thioredoxin-1

Nitric oxide (NO) modulates many physiological events through production of cGMP from its receptor, the NO-sensitive guanylyl cyclase (GC1). NO also appears to function in a cGMP-independent manner, via S-nitrosation (SNO), a redox-based modification of cysteine thiols. Previously, we have shown that S-nitrosated GC1 (SNO-GC1) is desensitized to NO stimulation following prolonged NO exposure or under oxidative/nitrosative stress. In animal models of nitrate tolerance and angiotensin II-induced hypertension, decreased vasodilation in response to NO correlates with GC1 thiol oxidation, but the physiological mechanism that resensitizes GC1 to NO and restores basal activity is unknown. Because GC1 interacts with the oxidoreductase protein-disulfide isomerase, we hypothesized that thioredoxin-1 (Trx1), a cytosolic oxidoreductase, could be involved in restoring GC1 basal activity and NO sensitivity because the Trx/thioredoxin reductase (TrxR) system maintains thiol redox homeostasis. Here, by manipulating activity and levels of the Trx1/TrxR system and by using a Trx1-Trap assay, we demonstrate that Trx1 modulates cGMP synthesis through an association between Trx1 and GC1 via a mixed disulfide. A proximity ligation assay confirmed the endogenous Trx1–GC1 complex in cells. Mutational analysis suggested that Cys609 in GC1 is involved in the Trx1–GC1 association and modulation of GC1 activity. Functionally, we established that Trx1 protects GC1 from S-nitrosocysteine–induced desensitization. A computational model of Trx1–GC1 interaction illustrates a possible mechanism for Trx1 to maintain basal GC1 activity and prevent/rescue GC1 desensitization to NO. The etiology of some oxidative vascular diseases may very well be explained by the dysfunction of the Trx1–GC1 association.