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G Spyrou

Publications and source records attributed to G Spyrou.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa.

Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member of the thioredoxin family in humans, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx open reading frame encodes for a protein of 486 amino acids composed of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx mRNA is only expressed in human testis, specifically in round and elongating spermatids. Immunostaining of human testis sections identified Sptrx protein in spermatids, while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the postmeiotic phase of spermatogenesis, when the sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.

Amino Acid Sequence↗
PubMed2000

Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity.

Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors. Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues. Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor. However, the biological reducing agent for the 1-Cys Prx has not yet been identified. We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p. Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions. We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor. In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity. This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity. Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.

Amino Acid Sequence↗
PubMed2000

Expression of novel antioxidant thioredoxin-2 in the rat brain.

Thioredoxins are a class of small redox-regulating proteins that have been implicated in the control of various aspects of cellular functions and seem to be one of the key regulators of signalling in the cellular responses to various stresses. Thioredoxin-2 (Trx2) is a novel mammalian thioredoxin which, in contrast to previously known cytosolic thioredoxin (Trx1), has been localized to the mitochondria. Trx2 is abundantly expressed in skeletal muscle, heart and adrenal gland, as well as in some other peripheral tissues with high metabolic activity. Using in situ hybridization and immunohistochemistry, we have studied the distribution and regulation of Trx2 expression in the rat brain. Trx2 mRNA and protein are highly expressed in the neurons in several brain regions, including the olfactory bulb, frontal cortex, hippocampus, some hypothalamic and thalamic nuclei, cerebellum and numerous brainstem nuclei. In addition, the Trx2 mRNA expression in paraventricular hypothalamic nucleus and reticular thalamic nucleus was found to be sensitive to peripheral glucocorticoids, as dexamethasone treatment caused significant elevation of Trx2 mRNA level in this area. No changes in other brain areas were observed after dexamethasone treatment. These findings implicate a significant regulatory and/or protective function of Trx2 in the nervous system.

Animals↗
PubMed2000

The mitochondrial thioredoxin system.

Eukaryotic organisms from yeast to human possess a mitochondrial thioredoxin system composed of thioredoxin and thioredoxin reductase, similar to the cytosolic thioredoxin system that exists in the same cells. Yeast and mammalian mitochondrial thioredoxins are monomers of approximately 12 kDa and contain the typical conserved active site WCGPC. However, there are important differences between yeast and mammalian mitochondrial thioredoxin reductases that resemble the differences between their cytosolic counterparts. Mammalian mitochondrial thioredoxin reductase is a selenoprotein that forms a homodimer of 55 kDa/subunit; while yeast mitochondrial thioredoxin reductase is a homodimer of 37 kDa/subunit and does not contain selenocysteine. A function of the mitochondrial thioredoxin system is as electron donor for a mitochondrial peroxiredoxin, an enzyme that detoxifies the hydrogen peroxide generated by the mitochondrial metabolism. Experiments with yeast mutants lacking both the mitochondrial thioredoxin system as well as the mitochondrial peroxiredoxin system suggest an important role for mitochondrial thioredoxin, thioredoxin reductase, and peroxiredoxin in the protection against oxidative stress.

Animals↗
PubMed2000

Genomic structure and chromosomal localization of human thioredoxin-like protein gene (txl).

Human thioredoxin-like protein (txl) is a novel member of the expanding thioredoxin superfamily whose main characteristic is the presence, after the thioredoxin domain, of a C-terminal extension of 184 residues with no homology with any other protein in the databases. Txl is a cytosolic ubiquitously expressed protein and it has been copurified with a kinase of the STE20 family, which is proteolytically activated by caspases in apoptosis. However, no cellular function has yet been assigned to this protein. In the present study we report the genomic organization of the txl gene which encompasses approximately 36 kb organized in eight exons ranging from 96 bp to 303 bp. In contrast, intron sizes are much bigger ranging from 1.5 kb to 12 kb. Chromosomal localization of txl gene revealed that it maps at position 18q21, a region frequently affected in different human tumours. Furthermore, we have identified the putative homologues of txl in both Drosophila melanogaster and Caenorhabditis elegans that display much closer homology to the known thioredoxins than the human txl protein. Indeed, critical residues for optimal thioredoxin activity are present in both Drosophila and Caenorhabditis txl but absent in the human protein suggesting that txl might have evolved to carry out a function different from the general disulfide reductase typical of thioredoxins.

Amino Acid Sequence↗
PubMed1999

cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1).

Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.

Amino Acid Sequence↗
PubMed1999

Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae.

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The Deltatrr2 mutant was more sensitive to H2O2, whereas the Deltatrx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae.

Amino Acid Sequence↗
PubMed1999

Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes.

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

Animals↗
PubMed1999

The orphan nuclear receptor SHP inhibits agonist-dependent transcriptional activity of estrogen receptors ERalpha and ERbeta.

SHP (short heterodimer partner) is an unusual orphan nuclear receptor that contains a putative ligand-binding domain but lacks a conserved DNA-binding domain. Although no conventional receptor function has yet been identified, SHP has been proposed to act as a negative regulator of nuclear receptor signaling pathways, because it interacts with and inhibits DNA binding and transcriptional activity of various nonsteroid receptors, including thyroid hormone and retinoid receptors. We show here that SHP interacts directly with agonist-bound estrogen receptors, ERalpha and ERbeta, and inhibits ER-mediated transcriptional activation. SHP specifically targets the ligand-regulated activation domain AF-2 and competes for binding of coactivators such as TIF2. Thus, SHP may represent a new category of negative coregulators for ligand-activated nuclear receptors. SHP mRNA is widely expressed in rat tissues including certain estrogen target tissues, and subcellular localization studies demonstrate that SHP is a nuclear protein, suggesting a biological significance of the SHP interactions with ERs. Taken together, these results identify ERs as novel SHP targets and suggest that competition for coactivator-binding is a novel mechanism by which SHP may inhibit nuclear receptor activation.

Animals↗
PubMed1999

Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II).

The thioredoxin (TRX) system, composed of nicotinamide adenine dinucleotide phosphate (reduced form), TRX, and TRX reductase (TRXR), has multiple biologic functions via thiol-mediated redox control. In this study, we investigated the relationship between intracellular TRXR levels and cellular sensitivity to cis-diamminedichloroplatinum (II) (CDDP). HeLa, a human cervical carcinoma cell line, cultured with CDDP showed a time- and dose-dependent reduction of intracellular TRXR activity, which was well correlated with the decrease in cell viability after exposure to CDDP. In a cell-free system, CDDP was found to directly inactivate the reduced form of purified human TRXR. The CDDP-resistant variants of HeLa cells, established by continuous exposure to CDDP, exhibited an increased expression and activity of TRXR as well as TRX compared with the parental cells. In addition, sodium selenate, an inhibitor of TRXR, was found to increase the susceptibility to CDDP in the CDDP-resistant cells. Moreover, the HeLa cells transfected with an antisense TRXR RNA expression vector to reduce the intracellular enzyme activity displayed an enhanced sensitivity to CDDP. Taken together with previous reports on TRX, these results indicate the possible involvement of TRXR as well as TRX in the cellular sensitivity and resistance to CDDP.

Antineoplastic Agents↗
PubMed1999

Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization.

We have isolated a 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa. It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD-binding and NADPH-binding domains. However, human TrxR2 differs from human TrxR1 by the presence of a 33-amino acid extension at the N-terminus which has properties characteristic of a mitochondrial translocation signal. Northern-blot analysis identified one mRNA species of 2.2 kb with highest expression in prostate, testis and liver. We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria. Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation. Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q11.2.

3' Untranslated Regions↗
PubMed1999

Cloning and sequencing of mouse glutaredoxin (grx) cDNA.

Glutaredoxins are small proteins (12 kDa) with a conserved active sequence Cys-Pro-Tyr(-Phe)-Cys that catalyse GSH-disulfide oxidoreduction reactions in the presence of NADPH and glutathione reductase. Many mammalian glutaredoxins have been characterized and human and pig cDNA sequence determined. However, no mouse glutaredoxin cDNA or protein sequence has yet been reported. We have cloned a cDNA from a mouse liver library that encodes the putative mouse glutaredoxin homologue. The deduced polypeptide sequence encodes a 107 amino acid protein displaying a high degree of homology with other members of the glutaredoxin family.

Amino Acid Sequence↗
PubMed1998

Transcriptional control of the galanin gene. Tissue-specific expression and induction by NGF, protein kinase C, and estrogen.

Galanin is a neuropeptide widely expressed in the central and peripheral nervous system where it acts as a neurotransmitter/neuromodulator and possibly an immunoregulator and growth factor. Galanin gene expression is highly regulated during development and by certain hormones and injury situations. We have examined transcriptional control mechanisms for this gene using chimeric bovine galanin/luciferase reporter genes. These were analyzed in cultured cells and in transgenic mice. The studies reveal that enhancer and silencer sequences are involved in conferring cell- and tissue-specific expression, and that specific elements close to the promoter are responsible for nerve growth factor and protein kinase C induction. So far, the studies have not revealed sequences on the bovine gene that mediate the action of estrogen.

Animals↗
PubMed1998

Characterization of phorbolester-inducible human neuronal factors involved in trans-activation of the galanin gene.

The expression of the neuropeptide galanin (GAL) is elevated in vivo upon nerve stimulation, injury, and in vitro by phorbol 12-myristate-13-acetate (PMA), suggesting that a signal pathway involving protein kinase C activation may be involved in GAL-gene activation. When plasmids containing a different length of the bovine GAL-promoter fused to luciferase were transfected into the human neuroblastoma cell line (SK-N-SH subclone SH-SY5Y), a PMA-responsive element was identified in the promoter-region -68 to -46 base pairs (bp). Co-transfection experiments with plasmids expressing cJun and cFos revealed that they could act alone, as well as synergistically with PMA to induce luciferase activity. Electrical mobility shift assays revealed that a cAMP response element (CRE)-like sequence (TGACGCGG; -59 to -52 bp) bound PMA-inducible nuclear proteins present in SH-SY5Y cells. These proteins appear to bind mainly as CRE-binding protein/activating-transcription-factor (CREB/ATF) and Jun/ATF heterodimers. In addition, an apparent PMA-inducible protein(s) not recognized by CREB/ATF and Jun antibodies bound to the CRE-like containing probe.

Activating Transcription Factor 2↗
PubMed1998

Molecular cloning and expression of a cDNA encoding a human thioredoxin-like protein.

This report describes the cloning of a human cDNA that encodes a new protein (Txl, Thioredoxin-like) that belongs to the expanding family of thioredoxins based on sequence comparison of the deduced amino acid sequence. This cDNA, with a total length of 1,278 bp, consists of 205 bp of 5'-untranslated sequence (including an in frame stop codon), an open reading frame of 870 bp and a 203 bp fragment of 3'-untranslated sequence. The coding sequence predicts a protein of 289 amino acids with two distinct domains: an N-terminal domain of 105 residues homologous to the rest of mammalian thioredoxins containing the conserved active site (CGPC) and a C-terminal domain of 184 residues with no homology with any other protein in the database. Northern blot analysis indicates that the txl probe hybridizes to a 1.3 Kb mRNA and is ubiquitously expressed in human tissues with the highest expression in stomach, testis and bone marrow.

Amino Acid Sequence↗
PubMed1998

Inhibition of AP-1 DNA binding by nitric oxide involving conserved cysteine residues in Jun and Fos.

Nitric oxide (NO), which has diverse biological effects, can modulate AP-1 activity. Since DNA binding of Jun-Jun and Jun-Fos dimers is regulated in vitro by redox control involving conserved cysteines, we hypothesized that the action of NO is mediated via these residues. We performed electrophoretic mobility-shift analyses using Jun and Fos recombinant proteins and NO solutions. Cysteine-to-serine mutants showed that the inhibition of AP-1 activity following NO treatment was dependent on the presence of Cys7272 and Cys154 in the DNA binding domain of Jun and Fos, respectively. The inhibitory effect of NO was reversed by DTT and the thioredoxin system. Our results demonstrate that NO mediates its inhibitory effect by reacting specifically with the conserved cysteine residues in Jun and Fos.

Amino Acid Sequence↗
PubMed1998

Monte Carlo generated mammograms: development and validation.

We have developed a model using Monte Carlo methods to simulate x-ray mammography. All possible physical processes of interaction of x-rays with matter have been taken into account. A simplified geometry of the mammographic apparatus has been considered along with a software phantom of compressed breast. The phantom may contain inhomogeneities of various compositions and sizes. We have used this model to produce Monte Carlo mammograms under realistic conditions. The validation of the simulation includes both the modelling of physical processes and the production of Monte Carlo mammograms. The first part is accomplished by the demonstration of the coincidence between Monte Carlo and theoretical data, whereas the second is accomplished by the comparison of real mammograms, taken from irradiation of a simplified breast phantom that we have constructed, and Monte Carlo mammograms taken from simulation of the above phantom under the corresponding exposure conditions. The limitations of the model as well as the future use of Monte Carlo mammograms are discussed.

Biophysical Phenomena↗