An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.     

Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.

Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.     

Role of glutaredoxin in metabolic oxidative stress. Glutaredoxin as a sensor of oxidative stress mediated by H2O2

Epitope-tagged glutaredoxin (GRX) was utilized to determine the role of GRX in oxidative stress-induced signaling and cytotoxicity in glucose-deprived human cancer cells (MCF-7/ADR and DU-145). GRX-overexpressing cells demonstrated resistance to glucose deprivation-induced cytotoxicity and decreased activation of c-Jun N-terminal kinase (JNK1). Deletion mutants showed the C-terminal portion of apoptosis signal-regulating kinase 1 (ASK1) bound GRX, and glucose deprivation disrupted binding. Treatment with l-buthionine-(S,R)-sulfoximine reduced glutathione content by 99% and prevented glucose deprivation-induced dissociation of GRX from ASK1. A thiol antioxidant, N-acetyl-l-cysteine, or overexpression of an H(2)O(2) scavenger, catalase, inhibited glucose deprivation-induced dissociation of GRX from ASK1. GRX active site cysteine residues (Cys(22) and Cys(25)) were required for dissociation of GRX from ASK1 during glucose deprivation. Kinase assays revealed that SEK1 and JNK1 were regulated in an ASK1-dependent fashion during glucose deprivation. Overexpression of GRX or catalase inhibited activation of ASK1-SEK1-JNK1 signaling during glucose deprivation. These results demonstrate that GRX is a negative regulator of ASK1 and dissociation of GRX from ASK1 activates ASK1-SEK1-JNK1 signaling leading to cytotoxicity during glucose deprivation. These results support the hypothesis that the GRX-ASK1 interaction is redox sensitive and regulated in a glutathione-dependent fashion by H(2)O(2).     

Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.     

Essential 110Cys in active site of membrane-associated prostaglandin E synthase-2

The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence (110Cys-x-x-113Cys). The mutation from 110Cys to Ser or the double mutation from 110Cys and 113Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from 113Cys to Ser did not affect the enzyme activity. These results indicate that 110Cys, but not 113Cys, is the essential amino acid in the active site of mPGE synthase-2. 110Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells.     

Meics, a novel zinc-finger protein which relocates from nuclei to the central meiotic spindle during Drosophila spermatogenesis

PEBP2 beta/Cbf beta is the beta subunit of PEBP2/Cbf, which has been demonstrated to have important biological activities in hematopoiesis and osteogenesis. However, PEBP2 beta is ubiquitously expressed, suggesting that PEBP2 has other additionally important physiological activities. In an effort to elucidate other possible functions for PEBP2, we have isolated a novel gene that encodes a PEBP2 beta-interacting protein from a mouse cDNA library. We have called this gene Crl-1 for charged amino acid rich leucine zipper-1 (Crl-1) because it is rich in charged amino acids and contains a putative leucine zipper region. Expression studies in a 17.5 days post-coitum mouse embryo demonstrated Crl-1 expression mainly in the olfactory bulb and cerebral cortex. Post-natally, Crl-1 expression was additionally observed in the cerebellar cortex with strong expression in the hippocampus. These findings show that this novel PEBP2 beta-interacting protein is expressed mainly in subsets of neuronal cells, suggesting that Crl-1 plays some role in the developing mouse brain.     

Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.     

Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH(2)-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only approximately 10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys(48)) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys(48) is the site of oxidation by peroxides and that oxidized Cys(48) reacts with the sulfhydryl group of Cys(152) to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys(48) or Cys(152) showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys(73) had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys(48) mutant, in NIH 3T3 cells inhibited H(2)O(2) accumulation and activation of c-Jun NH(2)-terminal kinase induced by tumor necrosis factor-alpha.     

Isolation and characterization of human TR3 receptor: a member of steroid receptor superfamily

Complementary DNAs (cDNAs) encoding a member of steroid receptor super-family, named TR3 receptor, were isolated from a human prostate lambda gt11 cDNA library on the basis of homology of oligonucleotide probes to the DNA-binding domain common to members of the steroid receptor super-family. Expression of TR3 receptor cDNA produced a 64 kDa DNA-binding protein in a rabbit reticulocyte lysate. Nucleotide sequence analysis showed that TR3 receptor cDNA contains two regions of sequences which correspond to the DNA- and hormone-binding domains of members of the steroid receptor super-family. The amino acid sequences in the hormone-binding domain of the TR3 receptor shares about 20% homology with estrogen receptor and less than 15% homology with other known steroid receptors. The DNA-binding domain of the TR3 receptor has about 55% homology with all other known steroid receptors. TR3 receptor had 86% nucleotide and 91% amino acid sequence homology with mouse NUR/77, suggesting that TR3 receptor may be a human homologue of mouse NUR/77 gene product.     

Localization of glutaredoxin (thioltransferase) in the rat brain and possible functional implications during focal ischemia.

We investigated the distribution of glutaredoxin (GRX, thioltransferase) in the rat brain using the in situ hybridization and immunohistochemical methods. GRX mRNA and GRX were expressed widely in the rat brain. The endothelial cell, tanycyte and ependymal cell expressed GRX mRNA and GRX protein. Neurons in various regions also showed GRX mRNA and GRX. Among them, pyramidal neurons in hippocampal CA3 region expressed a higher level of GRX mRNA. In addition, GRX mRNA signals were reduced after middle cerebral artery occlusion. Immunohistochemical analysis for GRX also revealed that GRX was reduced after ischemia. Northern blot analysis also showed that GRX mRNA from ischemic hemispheres decreased after ischemia. This reduction was parallel with the neuronal damage. This observation indicated that the maintenance of GRX and the redox regulating system was important for neuronal survival against oxidative stress.     

Production, purification and characterization of biologically active recombinant human nerve growth factor

The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.