Thioredoxin-related protein-1 induced by prostaglandin E2

3 páginas, 2 figuras. The definitive version is available at www3.interscience.wiley.comPeer reviewe

Functional characterization of thioredoxin 3 (TRX-3), a Caenorhabditis elegans intestine-specific thioredoxin

Jiménez-Hidalgo, María et al.Thioredoxins are a class of evolutionarily conserved proteins that have been demonstrated to play a key role in many cellular processes involving redox reactions. We report here the genetic and biochemical characterization of Caenorhabditis elegans TRX-3, the first metazoan thioredoxin with an intestine-specific expression pattern. By using green fluorescent protein reporters we have found that TRX-3 is expressed in both the cytoplasm and the nucleus of intestinal cells, with a prominent localization at the apical membrane. Although intestinal function, reproductive capacity, longevity, and resistance of trx-3 loss-of-function mutants to many stresses are indistinguishable from those of wild-type animals, we have observed a slight reduction in size and a minor reduction in the defecation cycle timing of trx-3 mutants. Interestingly, trx-3 is induced upon infection by Photorhabdus luminescens and Candida albicans, and TRX-3 overexpression provides a modest protection against these pathogens. Together, our data indicate that TRX-3 function in the intestine is dispensable for C. elegans development but may be important to fight specific bacterial and fungal infections. © 2013 Elsevier Inc.Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440) and the Japanese National Bioresource Project of the MEXT, Japan. We thank Howard Baylis, Keith Nehrke, Bart Braeckman, and Jim McGhee for sharing worm strains and plasmids. We are grateful to María Jesús Rodriguez-Palero and Fernando Calahorro for excellent technical assistance, to Cristina Méndez-Vidal for help with qPCR, to Helen M. Crook-McMahon for help with PRDX-2 blots, and to Britta Spanier for critical reading of the manuscript. A.M.-V. was supported by the Instituto de Salud Carlos III (Projects PI050065 and PI080557, cofinanced by the Fondo Social Europeo) and Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629), Spain. The work in the laboratory of P.S., a member of the NordForsk Nordic C. elegans Network, was supported by a grant from the Swedish Research Council. C.L.K. was supported by the INSERM, the CNRS, and the French Ministry of Research. E.L.B. and E.A.V. were supported by the MRC. J.R.P. was supported by the Plan de Apoyo a la Investigación, Desarrollo Tecnológico e Investigación de la Universidad de Jaén (Project UJA2011/12/55). J.C. was funded by the Spanish Ministry of Science and Innovation (Grant BFU2010-21794) and the Rioja Salud Foundation. E.L. and A.G.S. were supported by grants from the Fondo de Investigaciones Sanitarias (PI080642 and PI110120) and Ramón y Cajal Program to E.L. (Spanish government).Peer Reviewe

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death. © 2014 Elsevier Inc.A.P.F. was supported by Stiftelsen Lars Hiertas Minne and Karolinska Institutet research grants. Research in the Swoboda laboratory was supported by the Swedish Research Council (VR) and the NordForsk Nordic network for C. elegans research. A.M.-V. was supported by the Instituto de Salud Carlos III (Projects PI050065 and PI080557, cofinanced by the Fondo Social Europeo, FEDER) and Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629), Spain. Some C. elegans strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).Peer Reviewe

Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date. After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International).Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Secincorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.This work was supported by Universidad de la República, Uruguay (Grant Number 418 to G.S., PhD fellowship to L.O.); Asociación Española de Cooperación Internacional (C/7646/07 to A.M.-V. and G.S.; A/016083/08 to A.M.-V. and G.S.); Asociación Universitaria Iberoamericana de Posgrado and Agencia Nacional de Innovación e Investigación (BE_POS_2009_183 and BE_POS_2010_2160 to L.O.), and was partially funded by FOCEM (MERCOSUR Structural Convergence Fund), [COF 03/11].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

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

Redox-dependent and redox-independent functions of Caenorhabditis elegans thioredoxin 1

Thioredoxins (TRX) are traditionally considered as enzymes catalyzing redox reactions. However, redox-independent functions of thioredoxins have been described in different organisms, although the underlying molecular mechanisms are yet unknown. We report here the characterization of the first generated endogenous redox-inactive thioredoxin in an animal model, the TRX-1 in the nematode Caenorhabditis elegans. We find that TRX-1 dually regulates the formation of an endurance larval stage (dauer) by interacting with the insulin pathway in a redox-independent manner and the cGMP pathway in a redox-dependent manner. Moreover, the requirement of TRX-1 for the extended longevity of worms with compromised insulin signalling or under calorie restriction relies on TRX-1 redox activity. In contrast, the nuclear translocation of the SKN-1 transcription factor and increased LIPS-6 protein levels in the intestine upon trx-1 deficiency are strictly redox-independent. Finally, we identify a novel function of C. elegans TRX-1 in male food-leaving behaviour that is redox-dependent. Taken together, our results position C. elegans as an ideal model to gain mechanistic insight into the redox-independent functions of metazoan thioredoxins, overcoming the limitations imposed by the embryonic lethal phenotypes of thioredoxin mutants in higher organisms.NIH Office of Research Infrastructure P40 OD010440Spanish Ministry of Economy and Competitiveness BFU2015- 64408-PFondo Social Europeo BFU2015- 64408-PNational Institute of Allergy and Infectious Diseases of the National Institutes of Health R01AI07640

Cleavage of Disulfide Bonds in Mouse Spermatogenic Cell-Specific Type 1 Hexokinase Isozyme Is Associated with Increased Hexokinase Activity and Initiation of Sperm Motility1

During epididymal transit, sperm acquire the ability to initiate rapid forward progressive motility on release into the female reproductive tract or physiological media. Glycolysis is the primary source of the ATP necessary for this motility in the mouse, and several novel glycolytic enzymes have been identified that are localized to the principal piece region of the flagellum. One of these is the spermatogenic cell-specific type 1 hexokinase isozyme (HK1S), the only member of the hexokinase enzyme family detected in sperm. Hexokinase activity was found to be lower in immotile sperm immediately after removal from the cauda epididymis (quiescent) than in sperm incubated in physiological medium for 5 min and showing rapid forward progressive motility (activated). However, incubating sperm in medium containing diamide, an inhibitor of disulfide bond reduction, resulted in lower motility and HK activity than in controls. HK1S was present in dimer and monomer forms in extracts of quiescent sperm but mainly as a monomer in motile sperm. A dimer-size band detected in quiescent sperm with phosphotyrosine antibody was not detected in activated sperm, and the monomer-size band was enhanced. In addition, the general protein oxido-reductase thioredoxin-1 was able to catalyze the in vitro conversion of HK1S dimers to the monomeric form. These results strongly suggest that cleavage of disulfide bonds in HK1S dimers contributes to the increases in HK activity and motility that occur when mouse sperm become activated

Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.Instituto de Salud Carlos III PI15/00895 PI16/01898European Regional Development Fund/FEDERNetherlands Organization for Scientific Research 711.014.005Sociedad Española de Oncología MédicaMinisterio de Economía y Competitividad BFU2007-67123 BFU2015-64408-PEuropean Social Fund BFU2015-64408-