硫氧还蛋白与氧化还原反应

硫氧还蛋白是生物体调节体内氧化还原系统的一种重要蛋白质,它参与了生物体内众多的氧化还原反应,其活性位点是-Cys-Gly-Pro-Cys-,在众多的生命过程中,通过构象的改变行使其调节功能。     

Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys⁷³ only after the formation of a Cys³²-Cys³⁵ disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1^C32S/C35S (a mutant Trx1 with both Cys³² and Cys³⁵ replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys¹⁷³ and Cys⁸³ and protects it from H₂O₂-induced overoxidation. Functionally, we found that Cys⁷³-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.Nitric oxide (NO) is an important second messenger for signal transduction in cells. The production of cGMP by guanylyl cyclase, enabled by the binding of NO onto heme, is considered the primary mechanism responsible for the plethora of functions exerted by NO (1). However, S-nitrosylation, the covalent addition of the NO moiety onto cysteine thiols, is increasingly recognized as an important post-translational modification for regulating protein functions (for reviews, see Refs. 2 and 3). S-Nitrosylation is dynamic, reversible, site-specific, and modulated by selected cellular stimuli (4–7). With improved detection sensitivity, an increasing number of S-nitrosylated proteins have been identified by proteomics technologies (5, 8–13). Among the known modified proteins, nitrosylation occurs only on selected cysteines (4, 6, 14–17). Non-enzymatic mechanisms proposed to determine S-nitrosylation specificity include the availability of specific NO donors and protein microenvironments that stabilize the pK_a of acidic target cysteines (18). Furthermore, several enzymes, including hemoglobin (19, 20), superoxide dismutase 1 (21, 22), S-nitrosoglutathione reductase (23–25), and protein-disulfide isomerase (26), have been shown to possess either transnitrosylase or denitrosylase activities. However, an enzymatic system that governs site-specific transnitrosylation and denitrosylation, analogous to the kinase/phosphatase paradigm for regulating protein phosphorylation, has remained largely uncharacterized.Trx1¹ is an important antioxidant protein with protein reductase activity (27, 28). It has been characterized as an antiapoptotic protein because of its ability to suppress proapoptotic proteins, including apoptosis signal-regulating kinase 1 via disulfide reduction and Casp3 via transnitrosylation of Cys¹⁶³ (14, 29). Conversely, Trx1 can denitrosylate Casp3 at Cys¹⁶³, resulting in Casp3 activation (7). Trx1 appears to govern site-specific reversible nitrosylation of selected protein targets (14, 15), but what are the underlying mechanisms that regulate Trx1 transnitrosylation and denitrosylation activities? Are there additional Trx1-mediated transnitrosylation or denitrosylation targets that have not yet been identified? In this study, we used ESI-Q-TOF mass spectrometry (MS) to analyze the nitrosylation of Trx1 and a Casp3 peptide (Casp3p) under different redox conditions. Because of the labile nature of the S–NO bond, direct identification of S-nitrosylated proteins and their specific nitrosylation sites by MS remains challenging (8). A biotin switch method that is based on the derivatization of protein S–NO with a biotinylating agent is typically used for such analyses (8). However, like any indirect method, both false positive and negative identifications have been reported (30). Recently, we developed a method for direct analysis of protein S-nitrosylation by ESI-Q-TOF MS without prior chemical derivatization (31). Here we applied the same technique to determine the regulation of Trx1 by stepwise oxidative and nitrosative modifications of distinct cysteines and its subsequent ability to transnitrosylate target proteins. Nitrosative modification at Cys⁷³ of Trx1 cannot occur without prior attenuation of the Trx1 disulfide reductase and denitrosylase activities via either disulfide bond formation between Cys³² and Cys³⁵ or their mutation to serines. This is a key observation that has never been previously reported. Consequently, we designed a proteomics approach and discovered over 40 putative Trx1 transnitrosylation target proteins. We further characterized the Trx1 transnitrosylation proteome and identified three consensus motifs surrounding the putative Trx1 transnitrosylation sites, suggesting a protein-protein interaction mechanism for determining transnitrosylation specificity.     

硫氧化还原蛋白与细胞内的氧化还原调控

细胞内氧化还原状态直接影响细胞的生存、活化和增殖。硫氧化还原蛋白是一个具有氧化还原活性的小分子蛋白质,它和NADPH以及硫氧化还原蛋白还原酶一起协同作用,组成蛋白质的一个重要的还原体系。这个还原体系还与谷胱甘肽等共同控制细胞的氧化还原状态,对维持和调节细胞的氧化还原内环境有着重要的作用。而另一方面,细胞的氧化还原状态又可以通过对多种信号分子的作用直接影响细胞的多种生理功能。     

The Zinc Center Influences the Redox and Thermodynamic Properties of Escherichia coli Thioredoxin 2

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK_a of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK_a of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (ΔΔG°_H2O = 9 kJ/mol and ΔT_m = 7. 4 °C) than for Trx1 (ΔΔG°_H2O = 15 kJ/mol and ΔT_m = 13 °C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn²⁺-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK_a of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn²⁺ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn²⁺-center of Trx2 fine-tunes the properties of this unique thioredoxin.     

Mycobacterium tuberculosis Phosphoenolpyruvate Carboxykinase Is Regulated by Redox Mechanisms and Interaction with Thioredoxin

Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.     

Redox Biochemistry of Hydrogen Sulfide

H₂S, the most recently discovered gasotransmitter, might in fact be the evolutionary matriarch of this family, being both ancient and highly reduced. Disruption of γ-cystathionase in mice leads to cardiovascular dysfunction and marked hypertension, suggesting a key role for this enzyme in H₂S production in the vasculature. However, patients with inherited deficiency in γ-cystathionase apparently do not present vascular pathology. A mitochondrial pathway disposes sulfide and couples it to oxidative phosphorylation while also exposing cytochrome c oxidase to this metabolic poison. This report focuses on the biochemistry of H₂S biogenesis and clearance, on the molecular mechanisms of its action, and on its varied biological effects.     

Thioredoxin and Redox Control within the New Concept of Oxidative Signaling

During the last decade, plant thioredoxins (TRX) h-type have been shown to be implicated in several new roles like the protection against the oxidative stress by their ability to reduce some antioxidant proteins as peroxiredoxins (PRX) or methionine-sulphoxide-reductases (MSR). However, the concept of the oxidative stress is changing and this fact raises the question of the TRX roles in this new context. In the January issue of Plant Physiology, we have presented two TRXsh from Pisum sativum differently involved in the control of the redox status. PsTRXh1 is an h-type TRX that acts by reducing classical antioxidant proteins. PsTRXh2 seems to be also involved in redox control, however it could act contrary to its counterpart h1. Both proteins may play antagonistic roles in pea in order to lead a better control of the redox status.Key Words: abiotic stress, oxidative signalling, thioredoxins, Pisum sativum, ROSHigh concentration of reactive oxygen species (ROS) in plant cells involve the activation of different antioxidant systems which reestablish the redox status leading to better physiological conditions. On the other hand, it has been well established that at certain levels, the ROS act as second messengers in signal transduction cascades in several processes in plant cells.¹ At the light of these events, it has been proposed the reevaluation of the concept of oxidative stress towards “oxidative signalling.”² This concept involves all the cellular mechanisms that let the plant cells sense and act in response to modified environmental conditions. Several cellular systems are involved in such role, and in these last years, plant TRXs have been shown to be involved in several number of metabolic pathways linked to the regulation of the redox imbalance,³ mainly for the case of the h-type cluster of the TRXs.^4–6In our last work,⁷ we have described two pea TRXs of the h-type cluster, PsTRXh1 and PsTRXh2 that are differentially, and even antagonistically, involved in the redoxregulation control, probably through their interaction with different target proteins. We proposed that PsTRXh1 might be involved in the control of the ROS levels in pea tissues due to its ability to interact with several antioxidant proteins in vivo. It is now very well known that some members of the TRX family reduce PRX,^8–10 an antioxidant enzyme involves in the direct deactivation of some oxidant agents or the MSR,^11,14,15 in charge of the recovery of the oxidized methionine, both in a very specific manner. Due to the increase of PsTRXh1 both at gene expression and protein levels in plant heterotrophic tissues in response to the H₂O₂ treatment, and because it is also capable of conferring resistance towards hydrogen peroxide when produced in a yeast trx1Δ trx2Δ strain,¹⁶ one function of this TRX member could be the reduction in vivo of some PRX and/or MSRA counterparts in Pea tissues in the context of the oxidative signalling.Interestingly, PsTRXh2 gene and its corresponding protein showed very different behaviours to that presented by its homologous h1, reinforcing the idea that some TRX isoforms in plants are capable of functional specificity in vivo. PsTRXh2 is expressed in all tissues assays, mainly in roots, but at an extremely low level compared with that of PsTRXh1. Its divergent functional behaviour was confirmed both in Pea plantlets and yeast. In fact, contrary to PsTRXh1, PsTRXh2 provides hypersensitivity in the yeast trx1Δ trx2Δ mutant. We explained the different behaviour by suggesting that PsTRXh2 might interact with some target(s) involved either directly or indirectly in hydrogen peroxide detoxification, either by compromising the target function in resistance to the ROS or by reinforcing the target function in producing sensitivity to H₂O₂. Most probably, PsTRXh1 and PsTRXh2 interact with very different partners, and the characterization of such targets may help in the deciphering of PsTRX isoforms. As short-term future experiments, using the TRX-specific two-hybrid system that was published recently,⁸ comparative efficiencies of PsTRX isoforms could be performed to reduce some putative target involved in H₂O₂ detoxification, including PsTRXh3 and PsTRXh4.¹⁷ Unravelling Pea TRX interactome should help in deciphering the function of each isoform.However, we have also considered the possibility that PsTRXh2 could interact with the same target that PsTRXh1, but producing an opposite effect. In the yeast context, the protein targeted by the heterologous plant TRXs responsible of this complementation is a type-II PRX.⁵ We think that PsTRXh2 could interact with this yeast PRX blocking it and producing the hypersensitivity. In fact, we have found a similar effect in other protein targeted in vitro by TRXs. In the (Fig. 1), we present the in vitro ability of PsTRXh1 and PsTRXh2 to reduce and activate the pea chloroplastic fructose-1,6-bisphosphatase (FBPase). In the presence of PsTRXh1, FBPase presents TRX-dependent activity but lower than that found when chloroplastic f and m1 isoforms are used.¹² On the opposite, PsTRXh2 presents no only FBPase activation capability but its presence induces the FBPase inhibition, as the enzymatic activity was lower than that exhibited by this enzyme without TRX.Open in a separate windowFigure 1TRX-dependent chloroplastic FBPase activity two-step procedure.¹³ , PsTRXf; ▴, PsTRXm1; •, PsTRXh1; , PsTRXh2. The negative control (no TRX) is represented by a symbol-less line. Numbers in each line represent maximum enzymatic velocity as OD₃₄₀/min.Considering all data, we think that the behaviour showed by both pea h-type TRXs is due to their interactions with several protein-targets, as the PRXs: when the ROS levels increase drastically, cells develop high redox imbalances or even undergo oxidative stress. In this situation, all antioxidant mechanisms must be activated, including the increase of PsTRXh1 expression and protein quantities, giving rise to a more efficient cell detoxification. Under nonimbalance conditions of the redox status, PsTRXh2 could act by interacting (activating or inhibiting) with some protein targets. However, the physiological target for PsTRXh2 are not yet described nor supposed. Our results suggest that its role in the redox control is by producing sensitivity to oxidant agents, maybe by allowing physiological ROS levels in cells.     

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network.     

Towards a DNA-Like Duplex Without Hydrogen Bonds

Abstract

The inverse quadrupolar moments of the phenyl and pentafluorophenyl residues in the base pair P-F⁵ promotes strong intramolecular stacking interactions in DNA duplexes. The more natural base pairs are replaced by this novel pair the higher the thermodynamic stability of the resulting duplex if they are arranged in an alternating fashion.     

Artificial Electron Carriers for Preparative Biocatalytic Redox Reactions Forming Reversibly Carbon Hydrogen Bonds with Enzymes Present in Strict or Facultative Anaerobes

The application of various artificial electron mediators in combination with redox enzymes present in anaerobically grown cells is described. Examples include viologens of different redox potential, cobalt complexes, anthraquinones, phenoxazines, phenazines and others. The regeneration of the reduced or oxidised mediators by various methods is discussed, with hydrogen gas, carbon monoxide, formate or a cathode as electron donors, and dimethylsulphoxide, quinones, oxygen or an anode as electron acceptors. The enzymes used, usually in the form of resting cells or crude cell extracts of clostridia or Proteus species are mostly reversible. Examples for preparative reductions as well as for dehydrogenations are given.     

Tuning Plasmonic Properties of Truncated Gold Nanospheres by Coating

The influence of TiO₂ coating on resonant properties of gold nanoisland films deposited on silica substrates was studied numerically and in experiments. The model describing plasmonic properties of a metal truncated nanosphere placed on a substrate and covered by a thin dielectric layer has been developed. The model allows calculating a particle polarizability spectrum and, respectively, its surface plasmon resonance (SPR) wavelength for any given cover thickness, particle radius and truncation parameter, and dielectric functions of the particle, the substrate, the coating layer, and the surrounding medium. Dependence of the SPR position calculated for truncated gold nanospheres has coincided with the measured one for the gold nanoisland films covered with titania of different thicknesses. In the experiments, gold films with thickness of 5 nm were deposited on a silica glass substrate, annealed at 500 °C to form nanoislands of 20 nm in diameter, and covered with amorphous titania layers using atomic layer deposition technique. The resulting structures were characterized with scanning electron microscopy and optical absorption spectroscopy. The measured dependence of the SPR position on titania film thickness corresponded to the one calculated for truncated sphere-shaped nanoparticles with the truncation angle of ~50°. We demonstrated the possibility of tuning the SPR position within ~100 nm range by depositing to 30 nm thick titania layer.

     

Acidic Hydrolysis of the N-Glycosidic Bonds of Deoxyribo-nucleic Acid by Hydrogen Fluoride Stabilized in Pyridine

Abstract

Hydrolysis of the N-glycosidic bonds of normal and modified DNAs as well as of model compounds was achieved by a mild approach involving the use of hydrogen fluoride in pyridine at room temperature.     

NADPH Thioredoxin Reductase C Controls the Redox Status of Chloroplast 2-Cys Peroxiredoxins in Arabidopsis thaliana

Chloroplast 2-Cys peroxiredoxins （2-Cys Prxs） are efficiently reduced by NADPH Thioredoxin reductase C （NTRC）. To investigate the effect of light/darkness on NTRC function, the content of abundant plastidial enzymes, Rubisco, glutamine synthetase （GS）, and 2-Cys Prxs was analyzed during two consecutive days in Arabidopsis wild-type and ntrc mutant plants. No significant difference of the content of these proteins was observed during the day or the night in wildtype and mutant plants. NTRC deficiency caused a lower content of fully reduced 2-Cys Prxs, which was undetectable in darkness, suggesting that NTRC is the most important pathway for 2-Cys Prx reduction, probably the only one during the night. Arabidopsis contains two plastidial 2-Cys Prxs, A and B, for which T-DNA insertion lines were characterized showing the same phenotype as wild-type plants. Two-dimensional gel analysis of leaf extracts from these mutants allowed the identification of basic and acidic isoforms of 2-Cys Prx A and B. In-vitro assays and mass spectrometry analysis showed that the acidic isoform of both proteins is produced by overoxidation of the peroxidatic Cys residue to sulfinic acid. 2-Cys Prx overoxidation was lower in the NTRC mutant. These results show the important function of NTRC to maintain the redox equilibrium of chloroplast 2-Cys Prxs.     

Thioredoxin and Thioredoxin Reductase Control Tissue Factor Activity by Thiol Redox-dependent Mechanism

Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond. hTrx1-Cys-73 is absolutely required for hTrx1 to interfere with FVIIa binding to purified and cell-surface TF, consequently suppressing TF-dependent procoagulant activity and proteinase-activated receptor-2 activation. Moreover, hTrx1/TrxR plays an important role in sensing the alterations of NADPH/NADP⁺ states and transducing this redox-sensitive signal into changes in TF activity. With NADPH, hTrx1/TrxR readily facilitates the reduction of TF, causing a decrease in TF activity, whereas with NADP⁺, hTrx1/TrxR promotes the oxidation of TF, leading to an increase in TF activity. By comparison, TF is more likely to favor the reduction by hTrx1-TrxR-NADPH. This reversible reduction-oxidation reaction occurs in the TF extracellular domain that contains partially opened Cys-49/-57 and Cys-186/-209 disulfide bonds. The cell-surface TF procoagulant activity is significantly increased after hTrx1-knockdown. The response of cell-surface TF procoagulant activity to H₂O₂ is efficiently suppressed through elevating cellular TrxR activity via selenium supplementation. Our data provide a novel mechanism for redox regulation of TF activity. By modifying Cys residues or regulating Cys redox states in TF extracellular domain, hTrx1/TrxR function as a safeguard against inappropriate TF activity.     

Synergistically Tuning Water and Hydrogen Binding Abilities Over Co4N by Cr Doping for Exceptional Alkaline Hydrogen Evolution Electrocatalysis

Searching for highly efficient and cost‐effective electrocatalysts toward the hydrogen evolution reaction (HER) in alkaline electrolyte is highly desirable for the development of alkaline water splitting, but still remains a significant challenge. Herein, the rational design of Cr‐doped Co₄N nanorod arrays grown on carbon cloth (Cr–Co₄N/CC) that can efficiently catalyze the HER in alkaline media is reported. It displays outstanding performance, with the exceptionally small overpotential of 21 mV to obtain the current density of 10 mA cm^?2 and good stability in 1.0 m KOH, which is even better than the commercial Pt/C electrocatalyst, and much lower than most of the reported transition metal nitride‐based and other non‐noble metal‐based electrocatalysts toward the alkaline HER. Density functional theory (DFT) calculations and experimental results reveal that the Cr atoms not only act as oxophilic sites for boosting water adsorption and dissociation, but also modulate the electronic structure of Co₄N to endow optimized hydrogen binding abilities on Co atoms, thereby leading to accelerating both the alkaline Volmer and Heyrovsky reaction kinetics. In addition, this strategy can be extended to other metals (such as Mo, Mn, and Fe) doped Co₄N electrocatalysts, thus may open up a new avenue for the rational design of highly efficient transition metal nitride‐based HER catalysts and beyond.