Sptrx-2, a fusion protein composed of one thioredoxin and three tandemly repeated NDP-kinase domains is expressed in human testis germ cells

42 páginas, 9 figuras.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.This work was supported by grants from the Swedish Medical Research Council (Projects 03P-14096-01A, 03X-14041-01A and 13X-10370), the Åke Wibergs Stiftelse, the Karolinska Institutet, the Södertörns Högskola and the Medical Research Fund of Tampere University Hospital.Peer reviewe

Induction of Cell Membrane Protrusions by the N-terminal Glutaredoxin Domain of a Rare Splice Variant of Human Thioredoxin Reductase 1

18 páginas, 5 figuras, 1 esquema, 1 película.The human thioredoxin system has a wide range of functions in cells including regulation of cell proliferation and differentiation, immune system modulation, antioxidant defense, redox control of transcription factor activity, and promotion of cancer development. A key component of this enzymatic system is the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene. Transcription of TXNRD1 involves alternative splicing, leading to a number of transcripts also encoding isoforms of TrxR1 that differ from each other at their N-terminal domains. Here we have studied the TXNRD1_v3 isoform containing an atypical N-terminal glutaredoxin (Grx) domain. Expression of the transcript of this isoform was found predominantly in testis but was also detected in ovary, spleen, heart, liver, kidney, and pancreas. By immunohistochemical analysis in human testis with antibodies specific for the Grx domain of TXNRD1_v3, the protein was found to be predominantly expressed in the Leydig cells. Expression of the TXNRD1_v3 transcript was also found in several cancer cell lines (HCC1937, H23, A549, U1810, or H157), and in HeLa cells, it was induced by estradiol or testosterone treatments. Surprisingly, green fluorescent protein fusions with the complete TXNRD1_v3 protein or with only its Grx domain localized to distinct cellular sites in proximity to actin, and furthermore, had a potent capacity to rapidly induce cell membrane protrusions. Analyses of these structures suggested that the Grx domain of TXNRD1_v3 localizes first in the emerging protrusion and is then followed into the protrusions by actin and subsequently by tubulin. The results presented thus reveal that TXNRD1_v3 has a unique and distinct expression pattern in human cells and suggest that the protein can guide actin polymerization in relation to cell membrane restructuring.This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council (Medicine), the Åke Wibergs Foundation and Karolinska Institutet.Peer reviewe

Members of the murine Pate family are predominantly expressed in the epididymis in a segment-specific fashion and regulated by androgens and other testicular factors

<p>Abstract</p> <p>Background</p> <p>Spermatozoa leaving the testis are not able to fertilize the egg <it>in vivo</it>. They must undergo further maturation in the epididymis. Proteins secreted to the epididymal lumen by the epithelial cells interact with the spermatozoa and enable these maturational changes, and are responsible for proper storage conditions before ejaculation. The present study was carried out in order to characterize the expression of a novel <it>Pate </it>(prostate and testis expression) gene family, coding for secreted cysteine-rich proteins, in the epididymis.</p> <p>Methods</p> <p>Murine genome databases were searched and sequence comparisons were performed to identify members of the <it>Pate </it>gene family, and their expression profiles in several mouse tissues were characterized by RT-PCR. Alternate transcripts were identified by RT-PCR, sequencing and Northern hybridization. Also, to study the regulation of expression of <it>Pate </it>family genes by the testis, quantitative (q) RT-PCR analyses were performed to compare gene expression levels in the epididymides of intact mice, gonadectomized mice, and gonadectomized mice under testosterone replacement treatment.</p> <p>Results</p> <p>A revised family tree of <it>Pate </it>genes is presented, including a previously uncharacterized <it>Pate </it>gene named <it>Pate-X</it>, and the data revealed that <it>Acrv1 </it>and <it>Sslp1 </it>should also be considered as members of the <it>Pate </it>family. Alternate splicing was observed for <it>Pate-X, Pate-C </it>and <it>Pate-M</it>. All the <it>Pate </it>genes studied are predominantly expressed in the epididymis, whereas expression in the testis and prostate is notably lower. Loss of androgens and/or testicular luminal factors was observed to affect the epididymal expression of several <it>Pate </it>genes.</p> <p>Conclusions</p> <p>We have characterized a gene cluster consisting of at least 14 expressed <it>Pate </it>gene members, including <it>Acrv1, Sslp1 </it>and a previously uncharacterized gene which we named <it>Pate-X</it>. The genes code for putatively secreted, cysteine-rich proteins with a TFP/Ly-6/uPAR domain. Members of the <it>Pate </it>gene cluster characterized are predominantly expressed in the murine epididymis, not in the testis or prostate, and are regulated by testicular factors. Similar proteins are present in venoms of several reptiles, and they are thought to mediate their effects by regulating certain ion channels, and are thus expected to have a clinical relevance in sperm maturation and epididymal infections.</p

Identification and functional characterization of novel thioredoxin systems

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. Tbioredoxins 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. Tbioredoxins 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

The Mitochondrial Thioredoxin System

30 páginas.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.This work was supported by grants from the Swedish Medical Research Council (Project 13X-10370), Karolinska Institutet, Åke Winbergs Stiftelse, the TMR Marie Curie Research Training Grants (contract ERBFMBICT972824) and the Spanish Ministerio de Educación y Cultura.Peer reviewe

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510-54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.Funding Agencies|Swedish Research Council [2004-5057]; Medical Faculty of Linkoping University</p

Induction of Cell Membrane Protrusions by the N-terminal Glutaredoxin Domain of a Rare Splice Variant of Human Thioredoxin Reductase 1

18 páginas, 5 figuras, 1 esquema, 1 película.The human thioredoxin system has a wide range of functions in cells including regulation of cell proliferation and differentiation, immune system modulation, antioxidant defense, redox control of transcription factor activity, and promotion of cancer development. A key component of this enzymatic system is the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene. Transcription of TXNRD1 involves alternative splicing, leading to a number of transcripts also encoding isoforms of TrxR1 that differ from each other at their N-terminal domains. Here we have studied the TXNRD1_v3 isoform containing an atypical N-terminal glutaredoxin (Grx) domain. Expression of the transcript of this isoform was found predominantly in testis but was also detected in ovary, spleen, heart, liver, kidney, and pancreas. By immunohistochemical analysis in human testis with antibodies specific for the Grx domain of TXNRD1_v3, the protein was found to be predominantly expressed in the Leydig cells. Expression of the TXNRD1_v3 transcript was also found in several cancer cell lines (HCC1937, H23, A549, U1810, or H157), and in HeLa cells, it was induced by estradiol or testosterone treatments. Surprisingly, green fluorescent protein fusions with the complete TXNRD1_v3 protein or with only its Grx domain localized to distinct cellular sites in proximity to actin, and furthermore, had a potent capacity to rapidly induce cell membrane protrusions. Analyses of these structures suggested that the Grx domain of TXNRD1_v3 localizes first in the emerging protrusion and is then followed into the protrusions by actin and subsequently by tubulin. The results presented thus reveal that TXNRD1_v3 has a unique and distinct expression pattern in human cells and suggest that the protein can guide actin polymerization in relation to cell membrane restructuring.This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council (Medicine), the Åke Wibergs Foundation and Karolinska Institutet.Peer reviewe

Characterization of Sptrx, a Novel Member of the Thioredoxin Family Specifically Expressed in Human Spermatozoa

35 páginas, 8 figuras.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.This work was supported by grants from the Swedish Medical Research Council (Projects 03P-14096-01A, 03X-14041-01A, and 13X-10370), the Åke Wibergs Stiftelse, the Karolinska Institutet, the Södertörns Högskola, the Marie Curie Research Training Grants (contract ERBFMBICT972824), the Medical Research Fund of Tampere University Hospital, and Natural Sciences and Engineering Research Council of Canada (to R. O.).Peer reviewe

An Alternative Splicing Variant of the Selenoprotein Thioredoxin Reductase Is a Modulator of Estrogen Signaling

9 páginas, 10 figuras.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.This work was supported by Swedish Medical Research Council Project Grants 13-X10370 and 19X-11622-03C, the Karolinska Institutet, Södertörns Högskola, and Swedish Medical Research Council Project Grants 038-14096-01A and 03X-14041-01A.Peer reviewe

Human Mitochondrial Thioredoxin. Involvement in mitochondrial membrane potential and cell death

9 páginas, 8 figuras.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, ΔΨm. 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.This work was supported by grants from the Swedish Medical Research Council (Project 13X-10370 and 19X-11622-03C), the Karolinska Institutet, Södertörns Högskola, Swedish Medical Research Council (Projects 038-14096-01A, 03X-14041-01A), and Åke Wibergs stiftelse (to A .M -V.).Peer reviewe