| 1. |
- Cunnea, Paula M, et al.
(author)
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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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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:2, s. 1059-66
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Journal article (peer-reviewed)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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| 2. |
- Damdimopoulos, Anastasios E., et al.
(author)
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An alternative splicing variant of the selenoprotein thioredoxin reductase is a modulator of estrogen signaling
- 2004
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 279:37, s. 38721-38729
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Journal article (peer-reviewed)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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| 3. |
- Damdimopoulos, Anastasios E., et al.
(author)
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Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death
- 2002
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 277:36, s. 33249-33257
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Journal article (peer-reviewed)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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| 4. |
- Damdimopoulos, Anastasios E
(author)
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Identification and functional characterization of novel thioredoxin systems
- 2003
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Doctoral thesis (other academic/artistic)abstract
- Thioredoxins (Trx) are a class of small multifunctional 12-kDa proteins that are characterized by the redox active site Trp-Cys-Gly-Pro-Cys (WCGPC). In the oxidized (inactive) form of Trx (Trx-S2), the two cysteines at the active site form a disulfide bond. This can then be reduced by thioredoxin reductase (TrxR) and NADPH, the so-called thioredoxin system, to a dithiol (Trx-(SH)2), which can then act as a general protein disulfide reductase. Thioredoxins are present in all living organisms and have been isolated and characterized from a wide variety of prokaryotic and eukaryotic cells. In this thesis we describe the identification and functional characterization of novel members of the thioredoxin superfamily. We present evidence for a novel Trx (Trx2) in Escherichia coli. The E. coli Trx2 contains two domains: an N-terminal domain of 32 amino acids containing two CXXC motifs and a C-terminal domain with high homology to the prokaryotic thioredoxins, containing the conserved active site. Trx2 together with TrxR and NADPH can reduce ribonucleotide reductase as well as the interchain disulfide bridges of insulin. Thioredoxins are ubiquitously expressed in an tissues within the same organism. We have identified the first tissue specific Trx (Sptrx1) exclusively expressed in human spermatozoa. Sptrx1 is an active thioredoxin which under native conditions appears to have a multimeric structure. We also identify and characterize a complete thioredoxin system (Trx2, TrxR2) located in mitochondria. We show that Trx2 overexpressing cells have a higher mitochondrial membrane potential that is dependent on the function of the ATP synthase complex. Furthermore, overexpression of Trx2 was found to protect cells against the cytotoxic effects of etoposide, a drug commonly used in anticancer treatment. In addition, we showed that the second compound of the mitochondrial thioredoxin system, TrxR2, is capable of reducing cytochrome c and could protect cells against the cytotoxic effects of antimycin and myxothiazol, chemicals that inhibit the function of complex III in the mitochondrial electron transport chain. Furthemore, we identified an alternative splicing variant of cytosolic thioredoxin reductase (TrxR1b) that could bind to the Estrogen Receptors (ER) alpha and beta. As a result of this binding, a distinct subnuclear localization of TrxR1b was observed, co localizing with both ER alpha and beta. TrxR1b can act as a coactivator and enhance the transcriptional activity of ER in the classical activation pathway, which relies on the binding of the ER to an ER response element on the DNA. By contrast, TrxR1b is a co-repressor in the alternative pathway where ER activates AP-I transcription independently of its DNA binding activity. In summary, the results presented in this thesis give a better understanding of Thioredoxin systems in both prokaryotes and eukaryotes, with the introduction of new members in this redox superfamily of proteins. This study, which shows a wide spectrum of functions for these Thioredoxin systems in influencing various redox mechanisms and processess in biological systems, indicates that there is still a great deal of work yet to be done in this expanding field of research.
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| 5. |
- Miranda-Vizuete, Antonio, et al.
(author)
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Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa
- 2001
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 276:34, s. 31567-31574
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Journal article (peer-reviewed)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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| 6. |
- Miranda-Vizuete, Antonio, et al.
(author)
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The mitochondrial thioredoxin system
- 2000
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In: Antioxidants and Redox Signaling. - : Mary Ann Liebert. - 1523-0864 .- 1557-7716. ; 2:4, s. 801-810
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Nalvarte, Ivan, et al.
(author)
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Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition
- 2004
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In: Free Radical Biology & Medicine. - : Elsevier. - 0891-5849 .- 1873-4596. ; 36:10, s. 1270-1278
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Journal article (peer-reviewed)abstract
- The ubiquitously expressed mammalian thioredoxin reductases are selenoproteins that together with NADPH regenerate active reduced thioredoxins and are involved in diverse actions mediated by redox control. Two main forms of mammalian thioredoxin reductases have been isolated, one cytosolic (TrxR1) and one present in mitochondria (TrxR2). Although the principal target for TrxRs is thioredoxin, the cytosolic form can regenerate several important antioxidants such as ascorbic acid, lipoic acid, and ubiquinone. In this study we demonstrate that cytochrome c is a substrate for both TrxR1 and TrxR2. In addition, cells overexpressing TrxR2 are more resistant to impairment of complex III in the mitochondrial respiratory chain upon both antimycin A and myxothiazol treatments, suggesting a complex III bypassing function of TrxR2. Furthermore, we show that cytochrome c is reduced by TrxR2 in vitro, not only by using NADPH as an electron donor but also by using NADH, pointing at TrxR2 as an important redox protein on complex III impairment. These findings may be valuable in understanding respiratory disorders in mitochondrial diseases.
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| 8. |
- Nalvarte, Ivan, et al.
(author)
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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
-
In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 279:52, s. 54510-54517
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Journal article (peer-reviewed)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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| 9. |
- Sadek, Christine M., et al.
(author)
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Characterization of human thioredoxin-like 2. A novel microtubule-binding thioredoxin expressed predominantly in the cilia of lung airway epithelium and spermatid manchette and axoneme
- 2003
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 278:15, s. 13133-13142
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Journal article (peer-reviewed)abstract
- We describe here the cloning and characterization of a novel member of the thioredoxin family, thioredoxin-like protein 2 (Txl-2). The Txl-2 open reading frame codes for a protein of 330 amino acids consisting of two distinct domains: an N-terminal domain typical of thioredoxins and a C-terminal domain belonging to the nucleoside-diphosphate kinase family, separated by a small interface domain. The Txl-2 gene spans approximately 28 kb, is organized into 11 exons, and maps at locus 3q22.3-q23. A splicing variant lacking exon 5 (Delta 5Txl-2) has also been isolated. By quantitative real time PCR we demonstrate that Txl-2 mRNA is ubiquitously expressed, with testis and lung having the highest levels of expression. Unexpectedly, light and electron microscopy analyses show that the protein is associated with microtubular structures such as lung airway epithelium cilia and the manchette and axoneme of spermatids. Using in vitro translated proteins, we demonstrate that full-length Txl-2 weakly associates with microtubules. In contrast, Delta 5Txl-2 specifically binds with very high affinity brain microtubule preparations containing microtubule-binding proteins. Importantly, Delta 5Txl-2 also binds to pure microtubules, proving that it possesses intrinsic microtubule binding capability. Taken together, Delta 5Txl-2 is the first thioredoxin reported to bind microtubules and might therefore be a novel regulator of microtubule physiology.
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