| 1. |
- Andersson, M., et al.
(författare)
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NK-lysin, a disulfide-containing effector peptide of T-lymphocytes, is reduced and inactivated by human thioredoxin reductase. Implication for a protective mechanism against NK-lysin cytotoxicity
- 1996
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 271:17, s. 10116-10120
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Tidskriftsartikel (refereegranskat)abstract
- The cytotoxic and antibacterial polypeptide NK-lysin has a molecular mass of approximately 9 kDa and contains three disulfide bonds. The activity was highly dependent on intact disulfides, because the bactericidal effect on Escherichia coli and the cytolytic effect on human 3B6 lymphocytes was inhibited when NK-lysin was treated with dithiothreitol prior to incubation with the cells. NK-lysin was a direct substrate for human or calf thymus thioredoxin reductase and preincubation of the peptide with mammalian thioredoxin reductase, and NADPH abolished its antibacterial and cytolytic activities. The addition of human thioredoxin further enhanced the inhibitory effect of thioredoxin reductase and NADPH. In contrast, e. coli thioredoxin reductase showed no direct disulfide reductase activity with NK-lysin in agreement with previous data showing large differences in structure and substrate specificity between the mammalian and E. coli enzymes. NK-lysin is the first identified macromolecular disulfide substrate for human thioredoxin reductase apart from human thioredoxin. When 3B6 cells were incubated with NADPH, thioredoxin, and thioredoxin reductase prior to addition of NK-lysin, cytotoxicity was markedly reduced. These data suggest that thioredoxin reductase inactivates NK-lysin and provides a mechanism by which the cytotoxic activity of NK-lysin is regulated.
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| 2. |
- Bohm, S., et al.
(författare)
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Cooperating nonconsensus cAMP-responsive elements are mediators of adrenocorticotropin-induced VL30 transcription in steroidogenic adrenal cells
- 1993
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 268:6, s. 3952-3963
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Tidskriftsartikel (refereegranskat)abstract
- Pituitary-derived trophic hormones regulate cell-type-specific expression of VL30 retrotransposons in tissues that are engaged in steroidogenesis. We show that adrenocorticotropic hormone and forskolin induced VL30 transcription in the steroidogenic adrenal cell line Y1 and that the transcriptional activation was cell type- and protein kinase A-dependent. Three novel cAMP-responsive elements (CREs), within the VL30 long terminal repeat, were identified and shown to activate transcription synergistically when templates bearing multiple sites were compared with templates bearing a single site. This type of regulation was evident only in forskolin-treated cells, and the response elements were found to be inactive as mediators of constitutive transcription. In vitro binding analyses indicated that a consensus CRE and the nonconsensus VL30 CREs differ with respect to binding affinity and specificity to a number of nuclear factors that were identified to be related to proteins within the CREB, Jun, and C/EBP families of transcription factors. The relatively low affinity and/or a restricted binding specificity of the VL30 CREs made it possible to detect forskolin-induced binding of CREB- and Jun-related proteins to these sequences. We suggest that cAMP-induced transcription, specific for steroidogenic cells, can be mediated by a novel type of nonconsensus CREs and that the mechanism for this type of gene regulation is distinct from that mediated through a consensus CRE. We also report the identification of a novel factor, distinct from previously characterized CRE-binding proteins, that constitutively binds to the identified CREs.
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| 3. |
- Cunnea, Paula M, et al.
(författare)
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ERdj5, an endoplasmic reticulum (ER)-resident protein containing DnaJ and thioredoxin domains, is expressed in secretory cells or following ER stress.
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:2, s. 1059-66
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Tidskriftsartikel (refereegranskat)abstract
- A complex array of chaperones and enzymes reside in the endoplasmic reticulum (ER) to assist the folding and assembly of and the disulfide bond formation in nascent secretory proteins. Here we characterize a novel human putative ER co-chaperone (ERdj5) containing domains resembling DnaJ, protein-disulfide isomerase, and thioredoxin domains. Homologs of ERdj5 have been found in Caenorhabditis elegans and Mus musculus. In vitro experiments demonstrated that ERdj5 interacts via its DnaJ domain with BiP in an ATP-dependent manner. ERdj5 is a ubiquitous protein localized in the ER and is particularly abundant in secretory cells. Its transcription is induced during ER stress, suggesting potential roles for ERdj5 in protein folding and translocation across the ER membrane.
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| 4. |
- Damdimopoulos, Anastasios E., et al.
(författare)
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An alternative splicing variant of the selenoprotein thioredoxin reductase is a modulator of estrogen signaling
- 2004
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 279:37, s. 38721-38729
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Tidskriftsartikel (refereegranskat)abstract
- The selenoprotein thioredoxin reductase (TrxR1) is an integral part of the thioredoxin system. It serves to transfer electrons from NADPH to thioredoxin leading to its reduction. Interestingly, recent work has indicated that thioredoxin reductase can regulate the activity of transcription factors such as p53, hypoxia-inducible factor, and AP-1. Here, we describe that an alternative splicing variant of thioredoxin reductase (TrxR1b) containing an LXXLL peptide motif, is implicated in direct binding to nuclear receptors. In vitro interaction studies revealed direct interaction of the TrxR1b with the estrogen receptors alpha and beta. Confocal microscopy analysis showed nuclear colocalization of the TrxR1b with both estrogen receptor alpha and beta in estradiol-17beta-treated cells. Transcriptional studies demonstrated that TrxR1b can affect estrogen-dependent gene activation differentially at classical estrogen response elements as compared with AP-1 response elements. Based on these results, we propose a model where thioredoxin reductase directly influences the estrogen receptor-coactivator complex assembly on non-classical estrogen response elements such as AP-1. In summary, our results suggest that TrxR1b is an important modulator of estrogen signaling.
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| 5. |
- Damdimopoulos, Anastasios E., et al.
(författare)
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Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death
- 2002
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 277:36, s. 33249-33257
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Tidskriftsartikel (refereegranskat)abstract
- Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, DeltaPsi(m). Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.
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| 6. |
- Johansson, L., et al.
(författare)
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The orphan nuclear receptor SHP inhibits agonist-dependent transcriptional activity of estrogen receptors ERalpha and ERbeta
- 1999
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 274:1, s. 345-353
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Miranda-Vizuete, Antonio, et al.
(författare)
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Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa
- 2001
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 276:34, s. 31567-31574
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Miranda-Vizuete, A., et al.
(författare)
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Cloning, expression, and characterization of a novel Escherichia coli thioredoxin
- 1997
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 272:49, s. 30841-30847
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Nalvarte, Ivan, et al.
(författare)
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Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells : Effect on cell growth and differentiation
- 2004
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 279:52, s. 54510-54517
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Pedrajas, José R., et al.
(författare)
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Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae
- 1999
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 274:10, s. 6366-6373
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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