RESUMEN
MYH7 gene mutations are associated with wide clinical and genetic heterogeneity. We report a novel founder mutation in MYH7 in Southern Spain (Andalucía). We studied two index patients and 24 family members from two apparently independent families by physical examination, serum creatine-kinase, muscle MRI, sequencing studies and genetic linkage analysis. Sixteen individuals were heterozygous for a (p.R1560P) variant in the MYH7 gene. Haplotype was consistent with a common ancestor for the two families. The patients displayed the classic Laing distal myopathy phenotype, with hanging first toe as the initial presentation, even in mildly affected patients who declared themselves asymptomatic, although neck flexor weakness was revealed as an early sign in some cases. MRI showed that the sartorius was the first muscle involved, even in two out of three asymptomatic carriers. Our findings support the novel variant p.R1560P in MYH7 as a founder mutation in Andalucía. The early involvement of the sartorius muscle in MRI may be useful as an indicator of affection status.
Asunto(s)
Miosinas Cardíacas/genética , Miopatías Distales/genética , Mutación , Cadenas Pesadas de Miosina/genética , Adolescente , Adulto , Anciano , Niño , Diagnóstico Diferencial , Miopatías Distales/diagnóstico por imagen , Miopatías Distales/patología , Miopatías Distales/fisiopatología , Familia , Femenino , Haplotipos , Heterocigoto , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/patología , Fenotipo , Polimorfismo de Nucleótido Simple , España , Adulto JovenRESUMEN
Strong evidence indicates oxidative stress in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Abeta) has been implicated in both oxidative stress mechanisms and in neuronal apoptosis. Glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) are antioxidants that can inhibit apoptosis signal-regulating kinase (ASK1). We examined levels of GRX1 and TRX1 in AD brain as well as their effects on Abeta neurotoxicity. We show an increase in GRX1 and a decrease in neuronal TRX1 in AD brains. Using SH-SY5Y cells, we demonstrate that Abeta causes an oxidation of both GRX1 and TRX1, and nuclear export of Daxx, a protein downstream of ASK1. Abeta toxicity was inhibited by insulin-like growth factor-I (IGF-I) and by overexpressing GRX1 or TRX1. Thus, Abeta neurotoxicity might be mediated by oxidation of GRX1 or TRX1 and subsequent activation of the ASK1 cascade. Deregulation of GRX1 and TRX1 antioxidant systems could be important events in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/fisiología , Oxidorreductasas/metabolismo , Tiorredoxinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacología , Apoptosis , Encéfalo/metabolismo , Encéfalo/patología , Catalasa/metabolismo , Línea Celular Tumoral , Proteínas Co-Represoras , Elafina/metabolismo , Glutarredoxinas , Glutatión/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , MAP Quinasa Quinasa Quinasa 5/metabolismo , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Oxidación-Reducción , Fragmentos de Péptidos/farmacología , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Mitocondrias/enzimología , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , Expresión Génica , Riñón/metabolismo , Masculino , Ratones , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Datos de Secuencia Molecular , Alineación de Secuencia , Testículo/metabolismo , Reductasa de Tiorredoxina-Disulfuro/químicaRESUMEN
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.
Asunto(s)
Mitocondrias/metabolismo , Tiorredoxinas/metabolismo , Animales , Humanos , Mutación , Oxidación-Reducción , Estrés Oxidativo , Peroxidasas/metabolismo , Peroxirredoxinas , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
BACKGROUND: Prostaglandin E1 (PGE1) treatment of humans and rodents during acute hepatic failure ameliorates different parameters of hepatic dysfunction. PURPOSE: To investigate whether prevention of acute liver injury induced by D-galactosamine (D-GalN) with preadministration of PGE1 is correlated with a change in the concentration of two proinflammatory cytokines, as tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1alpha, and/or nitrite+nitrate (NOx), as nitric oxide-related end products in serum. RESULTS: D-GalN significantly increased alanine aminotransferase (ALT) and TNF-alpha concentration in serum 5 and 10 mins, respectively, after treatment compared with the control group (P< or =0.05). D-GalN did not change the IL-1alpha concentration at any time during the study. Preadministration of PGE1 to D-GalN-treated rats significantly reduced the ALT content and increased significantly the TNF-alpha concentration in serum 1, 2.5, 5 and 10 mins after D-GalN treatment compared with the D-GalN group (P< or =0.05). Nitric oxide was not involved in either the toxic effect due to D-GalN or the protection observed with PGE1 against D-GalN toxicity. CONCLUSIONS: Acute liver injury induced by D-GalN is correlated with an increased TNF-alpha release. Preadministration of PGE1 to D-GalN-treated rats exerted a priming effect on inflammatory cells to release enhanced levels of TNF-alpha but not IL-1alpha. These findings indicate that stimulation of TNF-alpha release may be involved in the acute D-GalN-induced liver injury and also in PGE1 protection from hepatotoxicity in clinical and experimental studies.
Asunto(s)
Alprostadil/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Galactosamina , Interleucina-1/farmacología , Hígado/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Alanina Transaminasa/sangre , Animales , Humanos , Masculino , Óxido Nítrico/farmacología , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
Genes/genética , Tiorredoxinas/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Mapeo Cromosómico , Cromosomas Humanos Par 18/genética , ADN/química , ADN/genética , Drosophila melanogaster/genética , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Oxidorreductasas , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Etiquetas de Secuencia Expresada , Glutarredoxinas , Humanos , Mamíferos/genética , Ratones , Datos de Secuencia Molecular , Proteínas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.
Asunto(s)
Miocardio/metabolismo , Filogenia , Tiorredoxinas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
ADN Complementario/genética , Tiorredoxinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Cartilla de ADN/genética , Expresión Génica , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Tiorredoxinas/química , Distribución TisularRESUMEN
Thioredoxin (Trx) and glutaredoxin (Grx1) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. The viability of a double mutant lacking both Trx and Grx1 implies the presence of a third, unknown hydrogen donor. This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grx1 (delta trxA, grx::kan). Affinity chromatography was used to bind glutaredoxin on a glutathione-containing thiol-Sepharose column. The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000. Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys. The proteins are therefore referred to as Grx2 and Grx3. The low hydrogen donor activity for ribonucleotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive. As a hydrogen donor for E. coli ribonucleotide reductase, Grx3 showed approximately the same Km value (0.35 microM) as Grx1, whereas its Vmax value was only 5% that of Grx1. The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a delta trxA, grx double mutant provides an explanation for its viability and deoxyribonucleotide biosynthesis. The physiological functions of Grx2 and Grx3 remain to be determined.
Asunto(s)
Escherichia coli/metabolismo , Mutación , Oxidorreductasas , Proteínas/metabolismo , Ribonucleótido Reductasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Desoxirribonucleótidos/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Glutarredoxinas , Cinética , Datos de Secuencia Molecular , Peso Molecular , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Proteínas/aislamiento & purificación , Ribonucleótido Reductasas/biosíntesis , Ribonucleótido Reductasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.
Asunto(s)
Oxidorreductasas , Proteínas/genética , Seudogenes/genética , Tiorredoxinas/genética , Animales , Secuencia de Bases , Cromosomas Humanos/genética , Genoma Humano , Glutarredoxinas , Humanos , Filogenia , Mapeo Físico de Cromosoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Mitocondrias/enzimología , Proteínas de Neoplasias , Peroxidasas/química , Peroxidasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Cinética , Datos de Secuencia Molecular , Mutación , Estrés Oxidativo , Peroxidasas/genética , Peróxidos/metabolismo , Peróxidos/farmacología , Peroxirredoxinas , Proteínas Recombinantes , Tiorredoxinas/metabolismoRESUMEN
Thioredoxin (Trx) is a small ubiquitous protein that displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel thioredoxin in Escherichia coli as well as the expression and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2 contains two distinct domains: an N-terminal domain of 32 amino acids including two CXXC motifs and a C-terminal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce the interchain disulfide bridges of insulin. The apparent Km value of Trx2 for thioredoxin reductase is similar to that of the previously characterized E. coli thioredoxin (Trx1). The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with dithiothreitol, suggesting that oxidation of cysteine residues other than the ones in the active site might regulate its activity. A truncated form of the protein, lacking the N-terminal domain, is insensitive to the presence of dithiothreitol, further confirming the involvement of the additional cysteine residues in modulating Trx2 activity. In addition, the presence of the N-terminal domain appears to confer heat sensitivity to Trx2, unlike Trx1. Finally, Trx2 is present normally in growing E. coli cells as shown by Western blot analysis.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli , Proteínas de la Membrana/química , Tiorredoxinas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/química , Escherichia coli/química , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Ribonucleótido Reductasas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Tiorredoxinas/biosíntesis , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
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.
Asunto(s)
Glándulas Suprarrenales/enzimología , Isoenzimas/genética , Mitocondrias/enzimología , Reductasa de Tiorredoxina-Disulfuro/genética , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 22/genética , Clonación Molecular , Secuencia Conservada , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Isoenzimas/química , Microscopía Fluorescente , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Reductasa de Tiorredoxina-Disulfuro/químicaRESUMEN
BACKGROUND: Thioredoxins (Trx) are small redox proteins that function as general protein disulphide reductases and regulate several cellular processes such as transcription factor DNA binding activity, apoptosis and DNA synthesis. In mammalian organisms, thioredoxins are generally ubiquitously expressed in all tissues, with the exception of Sptrx-1 which is specifically expressed in sperm cells. RESULTS: We report here the identification and characterization of a novel member of the thioredoxin family, the second with a tissue-specific distribution in human sperm, termed Sptrx-2. The Sptrx-2 ORF (open reading frame) encodes for a protein of 588 amino acids with two different domains: an N-terminal thioredoxin domain encompassing the first 105 residues and a C-terminal domain composed of three repeats of a NDP kinase domain. The Sptrx-2 gene spans about 51 kb organized in 17 exons and maps at locus 7p13-14. Sptrx-2 mRNA is exclusively expressed in human testis, mainly in primary spermatocytes, while Sptrx-2 protein expression is detected from the pachytene spermatocytes stage onwards, peaking at round spermatids stage. Recombinant full-length Sptrx-2 expressed in bacteria displayed neither thioredoxin nor NDP kinase enzymatic activity. CONCLUSIONS: The sperm specific expression of Sptrx-2, together with its chromosomal assignment to a position reported as a potential locus for flagellar anomalies and male infertility phenotypes such as primary ciliary dyskinesia, suggests that it might be a novel component of the human sperm axonemal organization.
Asunto(s)
Espermatozoides/metabolismo , Tiorredoxinas/química , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Humanos , Masculino , Datos de Secuencia Molecular , Nucleósido-Difosfato Quinasa/química , Estructura Terciaria de Proteína , ARN Mensajero/análisis , Espermátides/metabolismo , Distribución TisularRESUMEN
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.
Asunto(s)
Proteínas de la Membrana , Espermatozoides/metabolismo , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 18 , Clonación Molecular , ADN Complementario , Genoma Humano , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.