Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.     

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (K_m, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn²⁺ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys₃₇, His₇₀, and Glu₇₁, while the structural zinc site is probably composed of Cys₁₀₀, Cys₁₀₃, Cys₁₀₆, and Cys₁₁₄. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.     

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.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

Inhibition of Thioredoxin Reductase by Curcumin Analogs

Curcumin analogs were first investigated for their inhibitory effects on thioredoxin reductase (TrxR). Most of them were more potent TrxR inhibitors than natural curcumin. The structure-activity relationship was summarized, and the curcumin analog was found to inhibit TrxR irreversibly in a time-dependent manner. The action was caused by covalent modification of the redox-active residues Cys⁴⁹⁷ and Sec⁴⁹⁸ in TrxR.     

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.     

Characterizing Roles for the Glutathione Reductase,Thioredoxin Reductase and Thioredoxin Peroxidase-Encoding Genes of Magnaporthe oryzae during Rice Blast Disease

Understanding how pathogenic fungi adapt to host plant cells is of major concern to securing global food production. The hemibiotrophic rice blast fungus Magnaporthe oryzae, cause of the most serious disease of cultivated rice, colonizes leaf cells asymptomatically as a biotroph for 4–5 days in susceptible rice cultivars before entering its destructive necrotrophic phase. During the biotrophic growth stage, M. oryzae remains undetected in the plant while acquiring nutrients and growing cell-to-cell. Which fungal processes facilitate in planta growth and development are still being elucidated. Here, we used gene functional analysis to show how components of the NADPH-requiring glutathione and thioredoxin antioxidation systems of M. oryzae contribute to disease. Loss of glutathione reductase, thioredoxin reductase and thioredoxin peroxidase-encoding genes resulted in strains severely attenuated in their ability to grow in rice cells and that failed to produce spreading necrotic lesions on the leaf surface. Glutathione reductase, but not thioredoxin reductase or thioredoxin peroxidase, was shown to be required for neutralizing plant generated reactive oxygen species (ROS). The thioredoxin proteins, but not glutathione reductase, were shown to contribute to cell-wall integrity. Furthermore, glutathione and thioredoxin gene expression, under axenic growth conditions, was dependent on both the presence of glucose and the M. oryzae sugar/ NADPH sensor Tps1, thereby suggesting how glucose availability, NADPH production and antioxidation might be connected. Taken together, this work identifies components of the fungal glutathione and thioredoxin antioxidation systems as determinants of rice blast disease that act to facilitate biotrophic colonization of host cells by M. oryzae.     

NADPH依赖性硫氧还蛋白还原酶C在植物质体中的作用

硫氧还蛋白的氧化还原调节作用在生物界中普遍存在。它能够还原目标蛋白的二硫键,而自身的活性位点则被氧化。因此,对于新的催化循环,则需要由相应的还原酶将其再次还原成活性形式。硫氧还蛋白对维持高等植物的光合效率同样具有重要意义。叶绿体中的硫氧还蛋白分别由铁氧还蛋白依赖性硫氧还蛋白还原酶和NADPH依赖性硫氧还蛋白还原酶C(NTRC)两种酶还原。NTRC的本质是一种黄素蛋白,除了具有还原酶活性外,还整合了一个硫氧还蛋白结构域,在叶绿体和淀粉体的氧化还原调节中处于核心地位。这种特殊的双功能酶在卡尔文-本森循环、氧化戊糖磷酸途径、抗过氧化、四吡咯代谢、ATP和淀粉合成、生长素和光周期调控中扮演了多重角色。本综述总结了NTRC的生理功能,并讨论了该蛋白质对植物质体氧化还原稳态的调节机制。     

融合蛋白ICOSIg的三维结构模建的研究

目的:利用结构相似性的序列比对来模建ICOSIg的三维结构,分析其可能的结合位点,为改造ICOSIg的突变体,提高其结合活性提供理论基础。方法:利用生物信息学手段分析ICOS所属CD28家族各成员分子的结构域,通过基于结构相似的序列比对,以空间结构已经得到解析的CTLA4为模板,利用同源模建的方法,模建ICOS膜外区的空间结构。进一步地以人IgG2和CTLA4为模板,模建了ICOSIg全长的空间结构。在此基础上,结合氨基酸特性,分析其可能的功能位点。结果:FDPPPF及KTKGSGN基序可能是ICOSIg的功能结合位点。结论:模建了ICOSIg的空间结构,分析了其可能结合位点,为突变ICOSIg提高其亲和力提供了线索。     

Expression,Purification and Molecular Structure Modeling of Thioredoxin (Trx) and Thioredoxin Reductase (TrxR) from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.     

Physiological Properties of a Mutant of Pachysolen tannophilus Deficient in NADPH-Dependent d-Xylose Reductase

A d-xylose reductase mutant of Pachysolen tannophilus was isolated on the basis of its poor growth on d-xylose but normal growth on xylitol and d-glucose. Fractionation of cell extracts indicated that the mutant was deficient in d-xylose reductase activity that used NADPH exclusively as a cofactor, but not in activity that used both NADH and NADPH. Mutant cultures grown on d-xylose as the sole carbon source exhibited some properties that would be desired in improved strains. Growth rate, growth yield, and d-xylose consumption rate of the mutant were less sensitive than those of the wild type to changes in aeration rate. d-Xylose was utilized more efficiently in that less of a by-product, xylitol, was produced. In addition, under low aeration conditions, more ethanol was produced. A disadvantage was a relatively slow rate of d-xylose utilization.     

Thioredoxin Reductase 1 Expression and Castration-recurrent Growth of Prostate Cancer

INTRODUCTION: Many genes are differentially expressed between androgen-dependent and androgen-independent prostate cancer (CaP). Differential expression analysis and subtractive hybridization previously identified nine genes expressed in intact mice bearing CWR22 tumors and castrated mice bearing recurrent CWR22 tumors but not in regressed tumors. The objectives of this study were to develop an immunostaining method to dual-label foci of proliferating tumor cells [the origin of castration-recurrent CaP (CR-CaP)], to determine which of the nine candidate proteins were differentially expressed in proliferating versus nonproliferating cells at the onset of growth after castration, and to test preclinical findings using clinical specimens of androgen-stimulated benign prostate (AS-BP) and CaP (AS-CaP) and CR-CaP. METHODS: Paraffin-embedded, bromodeoxyuridine-injected CWR22 tumors were hydrated, antigen-retrieved using high heat and high pressure, labeled for each of the nine antigens of interest, visualized using peroxidase, and counterstained with hematoxylin. Mean optical density was calculated for proliferating and nonproliferating areas using automated (nuclear staining) or manual (cytoplasmic staining) image analysis. Prostate tissue microarray sections were immunostained and visually scored. RESULTS: Immunohistochemistry revealed higher nuclear expression of thioredoxin reductase 1 (TrxR1) in proliferating cells than nonproliferating cells (P < .005). There were no statistical differences between cell types in the expression of other proteins. TrxR1 expression was higher (P < .01) in CR-CaP compared with AS-BP or AS-CaP. CONCLUSIONS: Increased TrxR1 expression in CR-CaP was consistent with increased TrxR1 and BrdU expression at the onset of growth in the CWR22 model. Thioredoxin reductase 1 should be targeted in an attempt to delay or prevent CaP recurrence after castration.     

NADPH Thioredoxin Reductase C Is Involved in Redox Regulation of the Mg-Chelatase I Subunit in Arabidopsis thaliana Chloroplasts

Dear Editor,
The synthesis of tetrapyrroles, including chlorophylls, is central for chloroplast function. The metabolic pathway of tetrapyrrole biosynthesis in Arabidopsis is initiated with the formation of amino levulinic acid （ALA）, which is con- verted by a series of common reactions to protoporphy- tin IX （Proto IX） （Tanaka et al., 2011）. Then the pathway diverges into two branches： the synthesis of heme/bilin and chlorophylls. The insertion of Mg2＋ into Proto IX, cata- lyzed by Mg-chelatase, is the first committed reaction of the chlorophyll branch and is considered a key step for the regulation of the whole pathway. Mg-chelatase is a het- erotrimeric enzyme composed of subunits CHLI, CHLD, and CHLH, the reaction mechanism of which has been estab- lished. It is a two-step process consisting in the Mg-ATP- dependent activation of the enzyme, which implies the formation of a ternary complex of subunits CHLI and CHLD with ATP-Mg2＋, and Mg2＋ chelation, which is catalyzed by CHLH driven by ATP hydrolysis, CHLI providing ATPase activity to the complex （Tanaka et al., 2011）. In Arabidopsis, CHLH and CHLD are encoded by single genes, whereas two genes, CHLI-I and CHLI-2, encode the two isoforms of CHLI.     

Prevalent Mutations of Human Prion Protein: A Molecular Modeling and Molecular Dynamics Study

Abstract

Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrP^C to PrP^Sc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144–152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species.     

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys⁵⁹/Cys⁶⁴), and a C-terminal cysteine/selenocysteine motif (Cys⁴⁹⁷/Sec⁴⁹⁸). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO^•) to the hydroxyl radical adduct (DEPMPO/HO^•). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O. Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys⁵⁹/Cys⁶⁴ dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys⁵⁹/Cys⁶⁴ dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys⁴⁹⁷/Sec⁴⁹⁸ active site. Although the bulk of the DEPMPO/HO^• signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO^• could be generated by the enzyme in an H₂O₂-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.     

Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism

Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH-dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate `leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant.     

NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum Dimerizes at High Protein Concentration and Exclusively Receives Electrons from Thioredoxin Reductase

NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10⁶ and 5.6 × 10⁶ m⁻¹ min⁻¹, respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pK_a lower than 6.5 for nucleophilic attack, whereas the pK_a of the C-terminal cysteine is ∼10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum.