Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases. We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot. Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition. In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC. In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody.     

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.     

Influence of microtubule-associated proteins on the differential effects of paclitaxel and docetaxel

Microtubules are complex structures arising in part from the polymerization of tubulin dimers. Tubulin binds to a wide range of drugs which have been used as probes for tubulin conformation and assembly properties. There is some evidence that taxol and taxotere have differing effects on tubulin conformation. Previous work has shown that MAP2 and Tau, although they both induce microtubule assembly, have qualitatively different effects on tubulin's behavior. Since most microtubulesin vivo are likely to be associated with MAPs, we decided to characterize the differential effects of MAP2, Tau, taxol, and taxotere on tubulin polymerization with the aim of understanding the mechanisms through which these agents stimulate microtubule assembly. Furthermore, the inhibitive effect of calcium has been used to elucidate the ability of the two drugs to force tubulin assembly. These observations suggest that docetaxel, in addition to its greater efficiency in tubulin assembly, may have the capacity to differently alter certain classes of microtubules. Tau and MAP2 accessory proteins may represent important cofactors modulating the effects of taxoids.     

Dysfunction of microtubule-associated proteins of MAP2/tau family in Prion disease

The aggregation of PrP^Sc is thought to be crucial for the neuropathology of prion diseases. A growing body of evidence demonstrates that the perturbation of the microtubule network contributes to PrP^Sc-mediated neurodegeneration. Microtubules are a component of the cytoskeleton and play a central role in organelle transport, axonal elongation and cellular architecture in neurons. The polymerization, stabilization, arrangement of microtubules can be modulated by interactions with a series of microtubule-associated proteins (MAPs). Recent studies have proposed the abnormal alterations of two major microtubule-associated proteins, tau and MAP2, in the brain tissues of naturally occurred and experimental human and animal prion diseases. Increased total tau protein and hyperphosphorylation of tau at multiple residues are observed at the terminal stage of prion disease. The abnormal aggregation of tau protein disturbs its binding ability to microtubules and affects the microtubule dynamic. Significantly downregulated MAP2 is detected in the brain tissues of scrapie-infected hamsters and PrP106–126 treated cells, which corresponds well with the remarkably low levels of tubulin. In conclusion, dysfunction of MAP2/tau family leads to disruption of microtubule structure and impairment of axonal transport, and eventually triggers apoptosis in neurons, which becomes an essential pathway for prion to induce the neuropathology.     

Dysfunction of microtubule-associated proteins of MAP2/tau family in Prion disease

The aggregation of PrP^Sc is thought to be crucial for the neuropathology of prion diseases. A growing body of evidence demonstrates that the perturbation of the microtubule network contributes to PrP^Sc-mediated neurodegeneration. Microtubules are a component of the cytoskeleton and play a central role in organelle transport, axonal elongation and cellular architecture in neurons. The polymerization, stabilization, arrangement of microtubules can be modulated by interactions with a series of microtubule-associated proteins (MAPs). Recent studies have proposed the abnormal alterations of two major microtubule-associated proteins, tau and MAP2, in the brain tissues of naturally occurred and experimental human and animal prion diseases. Increased total tau protein and hyperphosphorylation of tau at multiple residues are observed at the terminal stage of prion disease. The abnormal aggregation of tau protein disturbs its binding ability to microtubules and affects the microtubule dynamic. Significantly downregulated MAP2 is detected in the brain tissues of scrapie-infected hamsters and PrP106–126 treated cells, which corresponds well with the remarkably low levels of tubulin. In conclusion, dysfunction of MAP2/tau family leads to disruption of microtubule structure and impairment of axonal transport, and eventually triggers apoptosis in neurons, which becomes an essential pathway for prion to induce the neuropathology.     

Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.     

A strongly basic protein of the MAP2 family copolymerizes with tubulin and induces polymerization

The family of microtubuli-associated proteins of approximately 300 kD molecular weight (MAP2) from porcine brain was fractionated into components of neutral isoelectric point and one polypeptide of strongly basic nature. Both fractions are able to induce the polymerization of purified porcine brain tubulin. In the case of the fractions of an isoelectric point of 7.2, thick and short tubular structures result. Under the influence of the basic protein, extremely long tubules of normal diameter of microtubules are produced. This basic MAP2 copolymerizes with tubulin.     

Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.     

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.     

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.     

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H₂O₂ concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H₂O₂ concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H₂O₂, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H₂O₂ and ABA-induced activities of APX and GR. ABA-induced accumulation of H₂O₂ was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H₂O₂ is involved in ABA-induced APX and GR activities in rice roots.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Targeting the intersubunit cavity of Plasmodium falciparum glutathione reductase by a novel natural inhibitor: Computational and experimental evidence

Glutathione reductase (GR), a homodimeric FAD-dependent disulfide reductase, is essential for redox homeostasis of the malaria parasite Plasmodium falciparum and has been proposed as an antimalarial drug target. In this study we performed a virtual screening against PfGR, using the structures of about 170,000 natural compounds. Analysis of the two top-scoring molecules, TTB and EPB, indicated that these ligands are likely to interact with the homodimer intersubunit cavity of PfGR with high binding energy scores of −9.67 and −9.60 kcal/mol, respectively. Both compounds had a lower affinity for human GR due to differences in structure and electrostatic properties. In order to assess the putative interactions in motion, molecular dynamics simulations were carried out for 30 ns, resulting in TTB being more dynamically and structurally favored than EPB. A closely related compound MDPI 21618 was tested on recombinant PfGR and hGR, resulting in IC₅₀ values of 11.3 ± 2.5 μM and 10.2 ± 1.7 μM, respectively. Kinetic characterization of MDPI 21618 on PfGR revealed a mixed-type inhibition with respect to glutathione disulfide (K_i = 9.7 ± 2.3 μM) and an uncompetitive inhibition with respect to NADPH. Furthermore, MDPI 21618 was found to inhibit the growth of the chloroquine-sensitive P. falciparum strain 3D7 with an IC₅₀ of 3.2 ± 1.9 μM and the chloroquine-resistant Dd2 strain with an IC₅₀ of 3.2 + 1.6 μM. In drug combination assays with chloroquine, artemisinin, or mefloquine MDPI 21618 showed an antagonistic action, which might suggest partially overlapping routes of action. This study further substantiates research on PfGR as a potential antimalarial drug target.     

Glutaredoxin 2 prevents H2O2-induced cell apoptosis by protecting complex I activity in the mitochondria

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200 μM hydrogen peroxide (H₂O₂) for 24 h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H₂O₂-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H₂O₂ treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H₂O₂-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H₂O₂-induced apoptosis is likely associated with its ability to preserve complex I.     

Oxidative aldehyde deformylation catalyzed by NADPH-cytochrome P450 reductase and the flavoprotein domain of neuronal nitric oxide synthase

We report here the unexpected finding that recombinant or hepatic microsomal NADPH-cytochrome P450 reductase catalyzes the oxidative deformylation of a model xenobiotic aldehyde, 2-phenylpropionaldehyde, to the n-1 alcohol, 1-phenylethanol, in the absence of cytochrome P450. The flavoprotein and NADPH are absolute requirements, and the reaction displays a dependence on time and on NADPH and reductase concentration. Not surprisingly, the hydrophobic tail of the flavoprotein is not required for catalytic competence. The reductase domain of neuronal nitric oxide synthase is about 30% more active than P450 reductase, and neither flavoprotein catalyzes conversion of the aldehyde to the carboxylic acid, by far the predominant metabolite with P450s in a reconstituted system. Reductase-catalyzed deformylation is unaffected by metal ion chelators and oxygen radical scavengers, but is strongly inhibited by catalase, and the catalase-mediated inhibition is prevented by azide. These results, together with observed parallel increases in 1-phenylethanol and H(2)O(2) formation as a function of NADPH concentration, are evidence that free H(2)O(2) is rate-limiting in aldehyde deformylation by the flavoprotein reductases. This contrasts sharply with the P450-catalyzed reaction, which is brought about by iron-bound peroxide that is inaccessible to catalase.     

Inhibition of glutathione reductase by cadmium ion in some rabbit tissues and the protective role of dietary selenium

This study is aimed at investigating the inhibitory effect of cadmium ion on glutathione reductase activity of rabbit brain and liver and the relationship of this effect with dietary selenium. For this purpose, one group of New Zealand rabbits were fed a selenium-deficient diet, another group was fed a selenium-rich diet, and the control group was fed a normal diet. The brain and liver tissues of these groups were investigated for the in vitro inhibitory effects of Cd²⁺ on glutathione reductase activity. For liver, the percentage inhibition of glutathione reductase by 40 nmol/mg protein of Cd²⁺ was similar for selenium-deficient and control groups, but significantly lower in the selenium-rich group. For brain tissues, there was no difference with respect to cadmium inhibition of glutathione reductase in all three groups.     

The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria

In rat heart mitochondria, auranofin, arsenite, diamide, and BCNU increase H2O2 formation, further stimulated by antimycin. However, in submitochondrial particles, H2O2 formation and oxygen uptake are not affected, indicating that these substances do not alter respiration. Mitochondria are also able to rapidly metabolize added H2O2 in a process partially prevented by BCNU or auranofin. Calcium does not modify the production of H2O2 and the mitochondrial thioredoxin system is not affected by calcium ions. Auranofin, arsenite, and diamide determine a large mitochondrial permeability transition, while BCNU and acetoacetate are ineffective. Thiols and glutathione are modified only by BCNU and diamide. However, all the compounds tested cause the release of cytochrome c that occurs also in the absence of mitochondrial swelling. In conclusion, the compounds utilized share the common feature of shifting the mitochondrial thiol-linked redox balance towards a more oxidized condition that is responsible of the observed effects.     

二氧化硫对小麦的氧化胁迫及其某些信号分子的调节

通过在密闭的培养箱中一次性通入不同体积浓度的SO₂气体,研究了小麦幼苗超氧自由基O2-含量和3种抗氧化酶活性的变化,探讨了信号分子水杨酸、乙烯和过氧化氢对SO₂氧化胁迫的调节作用.结果表明,当通入10和40 μl·L^-1 SO₂时,小麦叶片中O2-含量递增,过氧化物酶(POD)和过氧化氢酶(CAT)活性增强,但超氧化物歧化酶(SOD)活性降低.当SO₂浓度达到50 μl·L^-1时,POD和CAT活性也开始降低,此时叶片尖端出现坏死,叶片绿色部位滋生大量真菌.用1 mmol·L^-1水杨酸(SA)(pH6.5)浸泡小麦干种子6 h,或用10 mmol·L^-1 H₂O₂浸泡幼苗,O2-含量低于对照植株,而3种酶的活性高于对照植株,均可有效地减轻SO₂的氧化胁迫.在SO₂熏蒸下,乙烯显著抑制3种酶的活力,提高O2-的形成速率.SA与乙烯同时使用时,SA几乎完全消除了乙烯的负面作用.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.