Inactivation of thioredoxin reductases reveals a complex interplay between thioredoxin and glutathione pathways in Arabidopsis development

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.     

Similarities between rat liver mitochondrial and cytosolic glutathione reductases and their apoenzyme accumulation in riboflavin deficiency

Glutathione reductase has been purified 5,500-fold from rat liver mitochondrial matrix in a yield of 30%. The mitochondrial enzyme was immunochemically indistinguishable from that of the cytosol and the subunit molecular weight was apparently similar to that of the cytosolic enzyme, that is, 50,000 daltons. The optimum pH and kinetic properties investigated were not significantly different from those of the cytosolic enzyme. When rats were fed a riboflavin-deficient diet, the enzyme activity in the mitochondria decreased to a greater extent than that in the cytosol, and greater accumulation of apo-enzyme in the former than that in the latter was confirmed by the amount of immunoprecipitable protein, activation by FAD addition in vitro, and the enzyme activity recovery after injection of riboflavin, into riboflavin-deficient rats.     

Transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase,mitochondrial thioredoxin reductase and glutathione peroxidase 4

Selenium, as an integral part of selenoproteins, is essential for mammals. Unequivocal evidence had been provided more than a decade ago when it was proven that mice incapable of producing any of the 24 selenoproteins failed to develop beyond the gastrulation stage (E6.5). Since then, more specific attempts have been made to unmask novel and essential functions of individual selenoproteins in mice. Genetic disruption of glutathione peroxidase 4 (GPx4; also referred to as phospholipid hydroperoxide glutathione peroxidase, PHGPx) in mice showed for the first time that a specific selenoenzyme is in fact required for early embryonic development. Later on, systemic ablation of cytosolic thioredoxin reductase (Txnrd1) or mitochondrial thioredoxin reductase (Txnrd2) yielded embryonic lethal phenotypes. Beside those three, no other selenoproteins have been found being indispensable for murine development so far. This review aims at summarizing mainly the in vivo findings on these important mammalian selenoenzymes, which have not only common attributes of being required for embryogenesis, but that they are also instrumental in the regulation of cellular redox metabolism.     

Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.     

Differential effect of calcium ions on the cytosolic and mitochondrial thioredoxin reductase

The effect of calcium ions has been studied on three different isoforms of thioredoxin reductase. The cytosolic (TrxR1), mitochondrial (TrxR2), and the Escherichia coli enzymes were examined and compared. In our condition, TrxR1 appears extremely sensitive to Ca2+ showing an IC50 of about 160 nM, while Ca2+ exerts only a weak inhibitory effect on the mitochondrial isoform. The thioredoxin reductase purified from E. coli is almost completely insensitive to calcium ions. Circular dichroism analysis of highly purified mitochondrial and cytosolic thioredoxin reductases reveals that Ca2+ induces conformational alterations that are particularly relevant only in the cytosolic isoform. These observations are discussed with reference to the physiological role and, in particular, to the regulatory functions of the thioredoxin system.     

Comparative analysis of cytosolic and solubilized mitochondrial menadione reductases from rat liver]

The kinetic properties of cytosolic and solubilized mitochondrial menadione reductases (EC 1.6.99.2) from rat liver were compared. The mechanism of the reaction of cytosolic and mitochondrial menadione reductases with NADH and 4-anilino-5-methoxy-1,2-benzoquinone (AMOBQ) as substrates obeys the "ping-pong" kinetics. AMOBQ is a competitive inhibitor of cytosolic menadione reductase (Ki = 219 microM). Both menadione reductases have similar or identical values of true and effective kinetic constants and similar electrophoretic mobilities.     

Substrate and inhibitor specificities for human monoamine oxidase A and B are influenced by a single amino acid

Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines. It exists as two isoforms, A and B, which have a 70% amino acid identity and different substrate and inhibitor specificities. This study reports the identification of residues responsible for conferring this specificity in human MAO A and B. Using site-directed mutagenesis we reciprocally interchanged three pairs of corresponding nonconserved amino acids within the central portion of human MAO. Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for beta-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor). The reciprocal mutant MAO B-Y326I exhibited an increased preference for 5-hydroxytryptamine, a decreased preference for beta-phenylethylamine, and, similar to MAO A, was more sensitive to clorgyline than to deprenyl. These mutants also showed a distinct shift in sensitivity for the MAO A- and B-selective inhibitors Ro 41-1049 and Ro 16-6491. Mutant pair MAO A-T245I and MAO B-I236T and mutant pair MAO A-D328G and MAO B-G319D reduced catalytic activity but did not alter specificity. Our results indicate that Ile-335 in MAO A and Tyr-326 in MAO B play a critical role in determining substrate and inhibitor specificities in human MAO A and B.     

Characterization of alternative cytosolic forms and cellular targets of mouse mitochondrial thioredoxin reductase

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.     

Differential redox potential between the human cytosolic and mitochondrial branched-chain aminotransferase

The human branched-chain aminotransferase (hBCAT) isoenzymes are CXXC motif redox sensitive homodimers central to glutamate metabolism in the central nervous system. These proteins respond differently to oxidation by H(2)O(2), NO, and S-glutathionylation, suggesting that the redox potential is distinct between isoenzymes. Using various reduced to oxidized glutathione ratios (GSH:GSSG) to alter the redox environment, we demonstrate that hBCATc (cytosolic) has an overall redox potential that is 30 mV lower than hBCATm (mitochondrial). Furthermore, the CXXC motif of hBCATc was estimated to be 80 mV lower, suggesting that hBCATm is more oxidizing in nature. Western blot analysis revealed close correlations between hBCAT S-glutathionylation and the redox status of the assay environment, offering the hBCAT isoenzymes as novel biomarkers for cytosolic and mitochondrial oxidative stress.     

Enzymology of a carbonyl reduction clearance pathway for the HIV integrase inhibitor, S-1360: role of human liver cytosolic aldo-keto reductases

S-1360, a 1,3-diketone derivative, was the first HIV integrase inhibitor to enter human trials. Clinical data suggested involvement of non-cytochrome P450 clearance pathways, including reduction and glucuronidation. Reduction of S-1360 generates a key metabolite in humans, designated HP1, and constitutes a major clearance pathway. For characterization of subcellular location and cofactor dependence of HP1 formation, [(14)C]-S-1360 was incubated with commercially available pooled human liver fractions, including microsomes, cytosol, and mitochondria, followed by HPLC analysis with radiochemical detection. Incubations were performed in the presence and absence of the cofactors NADH or NADPH. Results showed that the enzyme system responsible for generation of HP1 in vitro is cytosolic and NADPH-dependent, implicating aldo-keto reductases (AKRs) and/or short-chain dehydrogenases/reductases (SDRs). A validated LC/MS/MS method was developed for investigating the reduction of S-1360 in detail. The reduction reaction exhibited sigmoidal kinetics with a K(m,app) of 2 microM and a Hill coefficient of 2. The ratio of V(max)/K(m) was approximately 1 ml/(min mg cytosolic protein). The S-1360 kinetic data were consistent with positive cooperativity and a single enzyme system. The relative contributions of AKRs and SDRs were examined through the use of chemical inhibitors. For these experiments, non-radiolabeled S-1360 was incubated with pooled human liver cytosol and NADPH in the presence of inhibitors, followed by quantitation of HP1 by LC/MS/MS. Quercetin and menadione produced approximately 30% inhibition at a concentration of 100 microM. Enzymes sensitive to these inhibitors include the carbonyl reductases (CRs), a subset of the SDR enzyme family predominantly located in the cytosol. Flufenamic acid and phenolphthalein were the most potent inhibitors, with > 67% inhibition at a concentration of 20 microM, implicating the AKR enzyme family. The cofactor dependence, subcellular location, and chemical inhibitor results implicated the aldo-keto reductase family of enzymes as the most likely pathway for generation of the major metabolite HP1 from S-1360.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

Substrate specific associations of epebionts on Middle Devonian brachiopods: Implications for paleoecology

A detailed study of over 2500 host brachiopods, from the Middle Devonian Hamilton Group of New York State, revealed distinct patterns of epibiont encrustation, that provide insight into taphonomy and paleoautecology of the host brachiopod shells and depositional environments. The concavo‐convex orthid, Tropidoleptus carinatus (Conrad), as well as strophomenid, and smooth athyrid brachiopods are among the most heavily encrusted. However, terebratulids of nearly identical size and shape are relatively clean of epibionts. This selective distribution strongly suggests that epibionts were discouraged from settling on punctate brachiopods. Brachiopods with small spines and frills were also nearly clean of epibionts, possibly because of entrapment of a mud layer, which made the outer layer of the host inhospitable for larval settling. Concavo‐convex taxa reveal high percent coverage and diversity of epibionts on the convex valve, which probably rested on the substrate during the life of brachiopod. This pattern is observed even on brachiopods that were buried with the convex valve downward. This implies complex post‐mortem histories involving multiple episodes of reorientation and colonization.     

Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.     

Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.     

Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins

Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.     

Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism

Swainsonine is a potent inhibitor of lysosomal alpha-D-mannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary alpha-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. The accumulated oligosaccharides were mainly Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, Man(alpha 1-3)[Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4) GlcNAc, and Man(alpha 1-3)[Man(alpha 1-6)]Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc. Analogous branched Man4 and Man5 structures are found in pig and sheep tissues, but they are N, N'-diacetylchitobiose derivatives. The substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases were investigated because in one type of hereditary alpha-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic alpha-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (I50 at 100 microM swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic alpha-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic alpha-D-mannosidase in glycoprotein degradation in the rat.     

Oral contraceptives as substrates and inhibitors for human cytosolic SULTs

Cytosolic sulfotransferases (SULTs) in mammals are involved in the biotransformation and homeostasis of various endogenous and xenobiotic compounds. The current study aimed to examine the sulfation of contraceptive compounds by various human cytosolic SULTs and to investigate the inhibitory effects and mode of action of these compounds on the sulfation of 17beta-estradiol, a major endogenous estrogen. A systematic study using all eleven known human cytosolic SULTs revealed the differential substrate specificity of these enzymes for the eight representative contraceptive compounds and two endogenous estrogens (estrone and 17beta-estradiol) tested as substrates. Activity data showed that SULT1A1 displayed the strongest activity toward 17alpha-ethynylestradiol. Kinetic studies revealed that the V (max) value of the sulfation of 17alpha-ethynylestradiol by SULT1A1 was 1.64 times that of the sulfation of 17beta-estradiol, while the K (m) values were almost equal for the two compounds. The inhibitory effects of three contraceptive compounds on the sulfation of 17beta-estradiol by SULT1A1 were examined. IC(50) values determined were 0.193, 1.84, and 2.98 mM, respectively, for 19-norethindrone acetate, ethynodiol diacetate and mifepristone. Kinetic analyses indicated that the mechanism underlying the inhibition by these contraceptives is of a mixed noncompetitive type. Metabolic labeling experiments confirmed the sulfation of contraceptive compounds and the release of their sulfated derivatives by HepG2 human hepatoma cells. Collectively, the results obtained suggest a role of sulfation in the metabolism of contraceptive compounds in vivo. Moreover, in view of their inhibitory effects on the sulfation of 17beta-estradiol, these compounds may potentially act to disrupt the homeostasis of endogenous estrogens.     

Substrate specificities of Cl- -activated arginine aminopeptidases from human and rat origin

The substrate specificities of four Cl^?-activated arginine aminopeptidases purified from the livers and inflammatory exudates of the rat, human fetal livers, and human erythrocytes were studied using peptides and N-l-aminoacyl-2-naphthylamides as substrates. With 2-naphthylamide substrates, these aminopeptidases showed similar substrate specificity; only the derivatives of Arg and Lys were measurably hydrolyzed. Di- and tripeptides with Arg or Lys as the N-terminal residue were readily split by the enzymes from the livers and inflammatory exudates of the rat and human fetal livers but oligopeptides were not hydrolyzed. Arg- and Lys-peptides were also hydrolyzed by the erythrocyte enzyme but this enzyme additionally split several other peptides, oligopeptides being hydrolyzed at internal bonds. The following properties were similar for all four arginine aminopeptidases: Dipeptides were preferred over tripeptides both in substrate binding and catalysis. The rat and human liver, rat exudate, and human erythrocyte enzymes revealed similar K_m values for the best substrates, the values increasing in the following order: ArgPhe, ArgTrp, ArgLys < ArgVal, ArgGly, Arg-2-naphthylamide < ArgGlyGly. The k_cat values were also similar for the four arginine aminopeptidases. Arg-2-naphthylamide was by far the most rapidly hydrolyzed substrate by all enzymes followed by ArgPhe and ArgTrp. With peptide substrates the highest Cl^? activation (10–20%) was found with ArgPhe and ArgTrp. With Arg-2-naphthylamide, however, the activating effect of 0.2 m Cl^? was severalfold. The hydrophobicity of the C-terminal residue of the substrate seemed to play an important role both in the Cl^? effect and substrate catalysis. Substrate binding, however, also depended on the charged groups of the substrate. Evidently Arg-2-naphthylamide and the peptides were hydrolyzed at the same active center but the mechanisms involved in the hydrolyses of chromogenic substrates and peptides may be different. It was also concluded that the less specific Cl^?-activated enzyme from human erythrocytes does not belong to the same group of Cl^?-activated arginine aminopeptidases that show a narrow substrate specificity.