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Identification of a Novel Thioredoxin-related Transmembrane Protein

2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês

10.1074/jbc.m011037200

1083-351X

Yoshiyuki Matsuo, Nobutake Akiyama, Hajime Nakamura, Junji Yodoi, Makoto Noda, Shinae Kizaka‐Kondoh,

Heat shock proteins research

We recently identified a series of transforming growth factor-β-responsive genes in A549 human adenocarcinoma cell line by a gene trap screening method. Here we report the molecular cloning and characterization of one of these genes, designatedTMX, that encodes a novel protein of 280 amino acid residues. The TMX protein possesses an N-terminal signal peptide followed by one thioredoxin (Trx)-like domain with a unique active site sequence, Cys-Pro-Ala-Cys, and a potential transmembrane domain. There are putative TMX homologs with identical active site sequences in theCaenorhabditis elegans and Drosophila genomes. Using recombinant proteins expressed in Escherichia coli, we demonstrated the activity of the Trx domain of TMX to cleave the interchain disulfide bridges in insulin in vitro. TheTMX transcript is widely expressed in normal human tissues, and subcellular fractionation and immunostaining for an epitope-tagged TMX protein suggest that TMX is predominantly localized in the endoplasmic reticulum (ER). When TMX was expressed in HEK293 cells, it significantly suppressed the apoptosis induced by brefeldin A, an inhibitor of ER-Golgi transport. This activity was abolished when two Cys residues in the active site sequence were mutated to Ser, suggesting that the Trx-like activity of TMX may help relieve ER stress caused by brefeldin A.AB048246 We recently identified a series of transforming growth factor-β-responsive genes in A549 human adenocarcinoma cell line by a gene trap screening method. Here we report the molecular cloning and characterization of one of these genes, designatedTMX, that encodes a novel protein of 280 amino acid residues. The TMX protein possesses an N-terminal signal peptide followed by one thioredoxin (Trx)-like domain with a unique active site sequence, Cys-Pro-Ala-Cys, and a potential transmembrane domain. There are putative TMX homologs with identical active site sequences in theCaenorhabditis elegans and Drosophila genomes. Using recombinant proteins expressed in Escherichia coli, we demonstrated the activity of the Trx domain of TMX to cleave the interchain disulfide bridges in insulin in vitro. TheTMX transcript is widely expressed in normal human tissues, and subcellular fractionation and immunostaining for an epitope-tagged TMX protein suggest that TMX is predominantly localized in the endoplasmic reticulum (ER). When TMX was expressed in HEK293 cells, it significantly suppressed the apoptosis induced by brefeldin A, an inhibitor of ER-Golgi transport. This activity was abolished when two Cys residues in the active site sequence were mutated to Ser, suggesting that the Trx-like activity of TMX may help relieve ER stress caused by brefeldin A. AB048246 thioredoxin endoplasmic reticulum protein disulfide isomerase transforming growth factor-β brefeldin A glutathioneS-transferase dithiothreitol doxycycline glycosylphosphatidylinositol enhanced green fluorescent protein phosphate-buffered saline base pairs fluorescein isothiocyanate rapid amplification of cDNA ends Thioredoxin (Trx)1 is a small and ubiquitously expressed protein originally identified inEscherichia coli and is evolutionarily conserved from prokaryotes to higher eukaryotes (1Laurent T.C. Moore E.C. Reichard P. J. Biol. Chem. 1964; 239: 3436-3444Abstract Full Text PDF PubMed Google Scholar, 2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 3Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Trx is characterized by two cysteine residues within the conserved active site sequence, CGPC, and many Trx-like proteins are members of the Trx superfamily (4Nakamura H. Yodoi J. Curr. Trends Immunol. 1998; 1: 133-140Google Scholar). Trx shows various functions via reversible oxidation and reduction of these two cysteine residues. Oxidized Trx (Trx-S2) in which the two cysteine residues form an intramolecular disulfide bond is reduced by thioredoxin reductase and NADPH (2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Reduced Trx (Trx-(SH)2) contains two thiol groups and can catalyze the reduction of disulfide bonds in multiple substrate proteins (2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 3Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar).Trx is involved in many thiol-dependent cellular processes, including gene expression, signal transduction, and proliferation. Trx functions as a hydrogen donor for ribonucleotide reductase, an essential enzyme providing deoxyribonucleotides for DNA synthesis (2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Trx also modulates the DNA binding activity of transcription factors such as AP-1, nuclear factor-κB, glucocorticoid receptor, and estrogen receptor (5Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (718) Google Scholar, 6Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (716) Google Scholar, 7Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar, 8Hayashi S. Hajiro-Nakanishi K. Makino Y. Eguchi H. Yodoi J. Tanaka H. Nucleic Acids Res. 1997; 25: 4035-4040Crossref PubMed Scopus (118) Google Scholar). Trx has also been discovered as an adult T-cell leukemia-derived factor produced by human T-cell leukemia virus-I-transformed T-cells, or as interleukin-1-like factor produced by Epstein-Barr virus-transformed cells (9Tagaya Y. Maeda Y. Mitsui A. Kondo N. Matsui H. Hamuro J. Brown N. Arai K. Yokota T. Wakasugi H. Yodoi J. EMBO J. 1989; 8: 757-764Crossref PubMed Scopus (514) Google Scholar, 10Wollman E.E. d'Auriol L. Rimsky L. Shaw A. Jacquot J.-P. Wingfield P. Graber P. Dessarps F. Robin P. Galibert F. Bertoglio J. Fradelizi D. J. Biol. Chem. 1988; 263: 15506-15512Abstract Full Text PDF PubMed Google Scholar). In these cases, Trx was found to be involved in cell activation and growth promotion (11Wakasugi N. Tagaya Y. Wakasugi H. Mitsui A. Maeda M. Yodoi J. Tursz T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8282-8286Crossref PubMed Scopus (254) Google Scholar, 12Oblong J.E. Berggren M. Gasdaska P.Y. Powis G. J. Biol. Chem. 1994; 269: 11714-11720Abstract Full Text PDF PubMed Google Scholar, 13Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (992) Google Scholar).Recently, several mammalian proteins of the Trx superfamily have been reported, which include Trx2 (14Spyrou G. Enmark E. Miranda-Vizuete A. Gustafsson J-A. J. Biol. Chem. 1997; 272: 2936-2941Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), nucleoredoxin (15Kurooka H. Kato K. Minoguchi S. Takahashi Y. Ikeda J. Habu S. Osawa N. Buchberg A.M. Moriwaki K. Shisa H. Honjo T. Genomics. 1997; 39: 331-339Crossref PubMed Scopus (99) Google Scholar), and TRP32 (16Lee K.-K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The active site sequences of Trx2 and TRP32 (CGPC) are identical to that of Trx. Trx2 and TRP32 are localized in the mitochondria and the cytoplasm, respectively. Nucleoredoxin is a nuclear protein with a modified active site sequence, CPPC. These proteins seem to be involved in the various redox regulations, but the precise biological functions are not well understood.The endoplasmic reticulum (ER) is well characterized as an organelle in which secretory proteins are folded and processed before export from the cell (17Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar). The ER also functions as a mobilizable calcium store that sequesters excess cytosolic calcium and acts as a reservoir for calcium signaling (18Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar). The ER undergoes stress responses when secretory proteins are misfolded or calcium balance is perturbed. Although severe stress in the ER can result in apoptosis through ER-specific caspase-12 (19Nakagawa T. Zhu H Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2926) Google Scholar), the ER stands against relatively mild stresses by the unfolded protein response (20Sidrauski C. Chapman R. Walter P. Trends Cell Biol. 1998; 8: 245-249Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), suppression of translation (21Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2478) Google Scholar), induction of Golgi-ER backward transport (22Hammond C. Helenius A. J. Cell Biol. 1994; 126: 41-52Crossref PubMed Scopus (392) Google Scholar), and activation of the accumulating protein transport to proteasome (23Kopito R.R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar,24Zhou M. Schekman R. Mol. Cell. 1999; 4: 925-934Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These quality control mechanisms in ER have been extensively studied, and many ER-associated proteins involved in such processes have been identified. Among them, protein disulfide isomerase (PDI), a member of the Trx superfamily, is well characterized as a foldase that assists disulfide bond formation (25Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar).In this study, we have characterized a novel protein, TMX, encoded by a gene previously isolated as a transforming growth factor (TGF)-β-responsive gene (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). TMX possesses one Trx-like domain with a unique potential active site sequence, CPAC, and bacterially expressed TMX indeed show Trx-like reducing activity in vitro. The sequence analysis also suggested that TMX has an N-terminal signal sequence and a transmembrane domain. A tagged TMX was predominantly localized in the ER and overexpression of TMX significantly reduced the ER stress induced by brefeldin A (BFA), an inhibitor of ER-Golgi transport. These data suggest that TMX is a novel member of the Trx family and may function to help relieve ER stresses.DISCUSSIONIn this study, we have characterized a novel Trx-related protein encoded by a gene identified as one of the TGF-β-responsive genes isolated by a retrovirus-mediated gene trap screening (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). This gene is widely expressed in normal human tissues (Fig. 3). Because the product of this gene contains one redox active site, a signal sequence, and a transmembrane domain, we named it transmembrane Trx-related protein (TMX). When TMX was tagged with FLAG epitope at its N terminus, the product was rarely detectable with anti-FLAG antibody (data not shown), supporting the prediction that the N terminus may serve as a signal peptide.The TMX cDNA was found to largely overlap with the human hypothetical cDNA clone DKFZp564E1962 whose sequence was previously deposited in the databases (GenBankTM/EBI accession no.AL080080). The 5′ region of this cDNA clone extends up to the 23rd nucleotide upstream of the translation initiation codon, and this segment lacks an inframe stop codon, which we found at 81 bases upstream of the initiation codon (Fig. 1). When homology protein searches were performed against databases of other species, two conceptual translation products in C. elegans andDrosophila were found with high identity scores. They share an identical active site sequence and structural similarity with TMX even outside of the Trx-like domain (Fig. 2 B). The strong sequence conservation in these distantly related species suggests the possibility that they compose a novel Trx family.Although the potential active site sequence of TMX, CPAC, has not been found in any other mammalian proteins with a Trx-domain, the sequence has Trx-like reducing activity when detected by the insulin disulfide reducing assay (Fig. 5), a classical spectrophotometric assay detecting the reduction of the two interchain disulfide bonds of insulin (31Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). Replacement of two cysteine residues in the redox active site to serines resulted in a complete loss of the reductase activity of TMX protein. These results suggest the potential function of TMX as an oxidoreductase with the novel active site sequence.The TMX amino acid sequence predicts that TMX may be a type I membrane protein; the Trx-like domain in the N-terminal half protrudes on the luminal side of the ER. Sequence analysis of TMX revealed no known motifs for subcellular localization. Analysis with the Myc-tagged protein revealed that TMX is probably localized primarily in the ER (Fig. 4 B), where another protein family with Trx-like domains, PDI, also exists. PDI contains two Trx-like domains and catalyzes the disulfide bond formation (25Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). The retention of some proteins in the ER is known to depend on the presence of the C-terminal sorting signals such as KDEL (39Munro S. Pelham H.R.B. Cell. 1987; 48: 899-907Abstract Full Text PDF PubMed Scopus (1561) Google Scholar) and the double lysine motif (KKXX or KXKXX) (40Jackson M.R. Nilsson T. Peterson P.A. EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (721) Google Scholar). There is a KDEL motif at the C terminus of PDI, and it resides in the lumen of the ER (26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). There is no such ER-retention motif at the C terminus of TMX, and the localization pattern of overexpressed protein did not change when the Myc epitope was inserted immediate to the C-terminal site of the predicted signal sequence (SS) cleavage site (SS-Myc·TMX, data not shown), suggesting that the ER retention mechanism for TMX is independent of the C-terminal sequence.The cell fractionation experiments suggest that a low level of TMX·Myc was expressed on the plasma membrane (Fig. 4 A). However, when the staining patterns of TMX·Myc or SS-Myc·TMX was compared with that of GPI-anchored EGFP, overlapped staining on the plasma membrane was not obvious (Fig. 4 B and data not shown). Therefore it has remained unclear whether some TMX protein can be localized on the plasma membrane. Because we could not exclude the possibility that the tagging of the Myc epitope might lead to the mislocalization of the protein, the localization of endogenous protein should be elucidated by using specific antibodies against TMX itself.The accumulation of unfolded or abnormal proteins and the disruption of ER calcium homeostasis give rise to ER stress, and excess or prolonged stress results in apoptosis (41Welihinda A.A. Tirasophon W. Kaufman R.J. Gene Expr. 1999; 7: 293-300PubMed Google Scholar). BFA is an effective ER stress inducer and was shown to induce apoptotic cell death in several human tumor cell lines (42Shao R-G. Shimizu T. Pommier Y. Exp. Cell Res. 1996; 227: 190-196Crossref PubMed Scopus (104) Google Scholar, 43Guo H. Tittle T.V. Allen H. Maziarz R.T. Exp. Cell Res. 1998; 245: 57-68Crossref PubMed Scopus (63) Google Scholar). BFA, a small fungal metabolite, has been shown to alter the function of the Golgi and trans-Golgi network, disrupt the traffic between endosomes and lysosomes, and inhibit protein secretion and synthesis because of impairment of vesicular transport (44Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1537) Google Scholar, 45Chardin P. McCormick F. Cell. 1999; 97: 153-155Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In this study, we showed that the overexpression of TMX could relieve the ER stress induced by BFA (Fig. 6 A). The apoptosis suppression by TMX was rather specific to BFA; no resistance was observed in the TMX-expressing HEK293 cells to other ER stress inducers such as thapsigargin and calcium ionophore A23187. BFA disrupts protein trafficking and Golgi morphology by inhibiting Golgi-associated guanine nucleotide exchange factors that activate ADP-ribosylation factors (ARFs) (45Chardin P. McCormick F. Cell. 1999; 97: 153-155Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). At the moment, it is unclear whether TMX interferes with the action of BFA or during later events leading to cell death. In the former case, TMX may bind and/or inactivate BFA itself or may by an unknown mechanism reactivate ARFs. In the latter case, an interesting possibility, among others, would be that TMX, like Ire1 (46Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (729) Google Scholar, 47Wang X.-Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (650) Google Scholar) and ATF6 (48Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar, 49Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1507) Google Scholar), functions as a stress sensor residing on the ER membrane, detecting the accumulation of unfolded proteins and activating downstream anti-stress response. Alternatively, TMX may modify certain proteins with its oxidoreductase activity thereby suppressing ER stress-induced cell death.Our previous results indicated that the level of TMXmRNA was increased by about 2-fold after TGF-β treatment (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). The functional relationship between TGF-β and TMX is presently unclear. RNA blot analysis revealed that BFA treatment (0.2 μg/ml) for 24 h did not increase the level of TMX mRNA (data not shown), indicating that ER stress itself does not influenceTMX expression. No alteration was observed in the interaction between Smad3 and Smad4 or in the expression of these proteins in the HEK293 cells coexpressing TMX and Smad3 and/or Smad4 (data not shown), suggesting that TMX may not have direct effects on the TGF-β signal transduction pathway. Further study is needed to test the interesting possibility that TMX serves as an essential target for TGF-β signaling and mediator for some of its biological effects. Thioredoxin (Trx)1 is a small and ubiquitously expressed protein originally identified inEscherichia coli and is evolutionarily conserved from prokaryotes to higher eukaryotes (1Laurent T.C. Moore E.C. Reichard P. J. Biol. Chem. 1964; 239: 3436-3444Abstract Full Text PDF PubMed Google Scholar, 2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 3Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Trx is characterized by two cysteine residues within the conserved active site sequence, CGPC, and many Trx-like proteins are members of the Trx superfamily (4Nakamura H. Yodoi J. Curr. Trends Immunol. 1998; 1: 133-140Google Scholar). Trx shows various functions via reversible oxidation and reduction of these two cysteine residues. Oxidized Trx (Trx-S2) in which the two cysteine residues form an intramolecular disulfide bond is reduced by thioredoxin reductase and NADPH (2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Reduced Trx (Trx-(SH)2) contains two thiol groups and can catalyze the reduction of disulfide bonds in multiple substrate proteins (2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 3Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Trx is involved in many thiol-dependent cellular processes, including gene expression, signal transduction, and proliferation. Trx functions as a hydrogen donor for ribonucleotide reductase, an essential enzyme providing deoxyribonucleotides for DNA synthesis (2Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Trx also modulates the DNA binding activity of transcription factors such as AP-1, nuclear factor-κB, glucocorticoid receptor, and estrogen receptor (5Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (718) Google Scholar, 6Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (716) Google Scholar, 7Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar, 8Hayashi S. Hajiro-Nakanishi K. Makino Y. Eguchi H. Yodoi J. Tanaka H. Nucleic Acids Res. 1997; 25: 4035-4040Crossref PubMed Scopus (118) Google Scholar). Trx has also been discovered as an adult T-cell leukemia-derived factor produced by human T-cell leukemia virus-I-transformed T-cells, or as interleukin-1-like factor produced by Epstein-Barr virus-transformed cells (9Tagaya Y. Maeda Y. Mitsui A. Kondo N. Matsui H. Hamuro J. Brown N. Arai K. Yokota T. Wakasugi H. Yodoi J. EMBO J. 1989; 8: 757-764Crossref PubMed Scopus (514) Google Scholar, 10Wollman E.E. d'Auriol L. Rimsky L. Shaw A. Jacquot J.-P. Wingfield P. Graber P. Dessarps F. Robin P. Galibert F. Bertoglio J. Fradelizi D. J. Biol. Chem. 1988; 263: 15506-15512Abstract Full Text PDF PubMed Google Scholar). In these cases, Trx was found to be involved in cell activation and growth promotion (11Wakasugi N. Tagaya Y. Wakasugi H. Mitsui A. Maeda M. Yodoi J. Tursz T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8282-8286Crossref PubMed Scopus (254) Google Scholar, 12Oblong J.E. Berggren M. Gasdaska P.Y. Powis G. J. Biol. Chem. 1994; 269: 11714-11720Abstract Full Text PDF PubMed Google Scholar, 13Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (992) Google Scholar). Recently, several mammalian proteins of the Trx superfamily have been reported, which include Trx2 (14Spyrou G. Enmark E. Miranda-Vizuete A. Gustafsson J-A. J. Biol. Chem. 1997; 272: 2936-2941Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), nucleoredoxin (15Kurooka H. Kato K. Minoguchi S. Takahashi Y. Ikeda J. Habu S. Osawa N. Buchberg A.M. Moriwaki K. Shisa H. Honjo T. Genomics. 1997; 39: 331-339Crossref PubMed Scopus (99) Google Scholar), and TRP32 (16Lee K.-K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The active site sequences of Trx2 and TRP32 (CGPC) are identical to that of Trx. Trx2 and TRP32 are localized in the mitochondria and the cytoplasm, respectively. Nucleoredoxin is a nuclear protein with a modified active site sequence, CPPC. These proteins seem to be involved in the various redox regulations, but the precise biological functions are not well understood. The endoplasmic reticulum (ER) is well characterized as an organelle in which secretory proteins are folded and processed before export from the cell (17Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar). The ER also functions as a mobilizable calcium store that sequesters excess cytosolic calcium and acts as a reservoir for calcium signaling (18Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar). The ER undergoes stress responses when secretory proteins are misfolded or calcium balance is perturbed. Although severe stress in the ER can result in apoptosis through ER-specific caspase-12 (19Nakagawa T. Zhu H Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2926) Google Scholar), the ER stands against relatively mild stresses by the unfolded protein response (20Sidrauski C. Chapman R. Walter P. Trends Cell Biol. 1998; 8: 245-249Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), suppression of translation (21Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2478) Google Scholar), induction of Golgi-ER backward transport (22Hammond C. Helenius A. J. Cell Biol. 1994; 126: 41-52Crossref PubMed Scopus (392) Google Scholar), and activation of the accumulating protein transport to proteasome (23Kopito R.R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar,24Zhou M. Schekman R. Mol. Cell. 1999; 4: 925-934Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These quality control mechanisms in ER have been extensively studied, and many ER-associated proteins involved in such processes have been identified. Among them, protein disulfide isomerase (PDI), a member of the Trx superfamily, is well characterized as a foldase that assists disulfide bond formation (25Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). In this study, we have characterized a novel protein, TMX, encoded by a gene previously isolated as a transforming growth factor (TGF)-β-responsive gene (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). TMX possesses one Trx-like domain with a unique potential active site sequence, CPAC, and bacterially expressed TMX indeed show Trx-like reducing activity in vitro. The sequence analysis also suggested that TMX has an N-terminal signal sequence and a transmembrane domain. A tagged TMX was predominantly localized in the ER and overexpression of TMX significantly reduced the ER stress induced by brefeldin A (BFA), an inhibitor of ER-Golgi transport. These data suggest that TMX is a novel member of the Trx family and may function to help relieve ER stresses. DISCUSSIONIn this study, we have characterized a novel Trx-related protein encoded by a gene identified as one of the TGF-β-responsive genes isolated by a retrovirus-mediated gene trap screening (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). This gene is widely expressed in normal human tissues (Fig. 3). Because the product of this gene contains one redox active site, a signal sequence, and a transmembrane domain, we named it transmembrane Trx-related protein (TMX). When TMX was tagged with FLAG epitope at its N terminus, the product was rarely detectable with anti-FLAG antibody (data not shown), supporting the prediction that the N terminus may serve as a signal peptide.The TMX cDNA was found to largely overlap with the human hypothetical cDNA clone DKFZp564E1962 whose sequence was previously deposited in the databases (GenBankTM/EBI accession no.AL080080). The 5′ region of this cDNA clone extends up to the 23rd nucleotide upstream of the translation initiation codon, and this segment lacks an inframe stop codon, which we found at 81 bases upstream of the initiation codon (Fig. 1). When homology protein searches were performed against databases of other species, two conceptual translation products in C. elegans andDrosophila were found with high identity scores. They share an identical active site sequence and structural similarity with TMX even outside of the Trx-like domain (Fig. 2 B). The strong sequence conservation in these distantly related species suggests the possibility that they compose a novel Trx family.Although the potential active site sequence of TMX, CPAC, has not been found in any other mammalian proteins with a Trx-domain, the sequence has Trx-like reducing activity when detected by the insulin disulfide reducing assay (Fig. 5), a classical spectrophotometric assay detecting the reduction of the two interchain disulfide bonds of insulin (31Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). Replacement of two cysteine residues in the redox active site to serines resulted in a complete loss of the reductase activity of TMX protein. These results suggest the potential function of TMX as an oxidoreductase with the novel active site sequence.The TMX amino acid sequence predicts that TMX may be a type I membrane protein; the Trx-like domain in the N-terminal half protrudes on the luminal side of the ER. Sequence analysis of TMX revealed no known motifs for subcellular localization. Analysis with the Myc-tagged protein revealed that TMX is probably localized primarily in the ER (Fig. 4 B), where another protein family with Trx-like domains, PDI, also exists. PDI contains two Trx-like domains and catalyzes the disulfide bond formation (25Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). The retention of some proteins in the ER is known to depend on the presence of the C-terminal sorting signals such as KDEL (39Munro S. Pelham H.R.B. Cell. 1987; 48: 899-907Abstract Full Text PDF PubMed Scopus (1561) Google Scholar) and the double lysine motif (KKXX or KXKXX) (40Jackson M.R. Nilsson T. Peterson P.A. EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (721) Google Scholar). There is a KDEL motif at the C terminus of PDI, and it resides in the lumen of the ER (26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). There is no such ER-retention motif at the C terminus of TMX, and the localization pattern of overexpressed protein did not change when the Myc epitope was inserted immediate to the C-terminal site of the predicted signal sequence (SS) cleavage site (SS-Myc·TMX, data not shown), suggesting that the ER retention mechanism for TMX is independent of the C-terminal sequence.The cell fractionation experiments suggest that a low level of TMX·Myc was expressed on the plasma membrane (Fig. 4 A). However, when the staining patterns of TMX·Myc or SS-Myc·TMX was compared with that of GPI-anchored EGFP, overlapped staining on the plasma membrane was not obvious (Fig. 4 B and data not shown). Therefore it has remained unclear whether some TMX protein can be localized on the plasma membrane. Because we could not exclude the possibility that the tagging of the Myc epitope might lead to the mislocalization of the protein, the localization of endogenous protein should be elucidated by using specific antibodies against TMX itself.The accumulation of unfolded or abnormal proteins and the disruption of ER calcium homeostasis give rise to ER stress, and excess or prolonged stress results in apoptosis (41Welihinda A.A. Tirasophon W. Kaufman R.J. Gene Expr. 1999; 7: 293-300PubMed Google Scholar). BFA is an effective ER stress inducer and was shown to induce apoptotic cell death in several human tumor cell lines (42Shao R-G. Shimizu T. Pommier Y. Exp. Cell Res. 1996; 227: 190-196Crossref PubMed Scopus (104) Google Scholar, 43Guo H. Tittle T.V. Allen H. Maziarz R.T. Exp. Cell Res. 1998; 245: 57-68Crossref PubMed Scopus (63) Google Scholar). BFA, a small fungal metabolite, has been shown to alter the function of the Golgi and trans-Golgi network, disrupt the traffic between endosomes and lysosomes, and inhibit protein secretion and synthesis because of impairment of vesicular transport (44Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1537) Google Scholar, 45Chardin P. McCormick F. Cell. 1999; 97: 153-155Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In this study, we showed that the overexpression of TMX could relieve the ER stress induced by BFA (Fig. 6 A). The apoptosis suppression by TMX was rather specific to BFA; no resistance was observed in the TMX-expressing HEK293 cells to other ER stress inducers such as thapsigargin and calcium ionophore A23187. BFA disrupts protein trafficking and Golgi morphology by inhibiting Golgi-associated guanine nucleotide exchange factors that activate ADP-ribosylation factors (ARFs) (45Chardin P. McCormick F. Cell. 1999; 97: 153-155Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). At the moment, it is unclear whether TMX interferes with the action of BFA or during later events leading to cell death. In the former case, TMX may bind and/or inactivate BFA itself or may by an unknown mechanism reactivate ARFs. In the latter case, an interesting possibility, among others, would be that TMX, like Ire1 (46Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (729) Google Scholar, 47Wang X.-Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (650) Google Scholar) and ATF6 (48Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar, 49Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1507) Google Scholar), functions as a stress sensor residing on the ER membrane, detecting the accumulation of unfolded proteins and activating downstream anti-stress response. Alternatively, TMX may modify certain proteins with its oxidoreductase activity thereby suppressing ER stress-induced cell death.Our previous results indicated that the level of TMXmRNA was increased by about 2-fold after TGF-β treatment (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). The functional relationship between TGF-β and TMX is presently unclear. RNA blot analysis revealed that BFA treatment (0.2 μg/ml) for 24 h did not increase the level of TMX mRNA (data not shown), indicating that ER stress itself does not influenceTMX expression. No alteration was observed in the interaction between Smad3 and Smad4 or in the expression of these proteins in the HEK293 cells coexpressing TMX and Smad3 and/or Smad4 (data not shown), suggesting that TMX may not have direct effects on the TGF-β signal transduction pathway. Further study is needed to test the interesting possibility that TMX serves as an essential target for TGF-β signaling and mediator for some of its biological effects. In this study, we have characterized a novel Trx-related protein encoded by a gene identified as one of the TGF-β-responsive genes isolated by a retrovirus-mediated gene trap screening (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). This gene is widely expressed in normal human tissues (Fig. 3). Because the product of this gene contains one redox active site, a signal sequence, and a transmembrane domain, we named it transmembrane Trx-related protein (TMX). When TMX was tagged with FLAG epitope at its N terminus, the product was rarely detectable with anti-FLAG antibody (data not shown), supporting the prediction that the N terminus may serve as a signal peptide. The TMX cDNA was found to largely overlap with the human hypothetical cDNA clone DKFZp564E1962 whose sequence was previously deposited in the databases (GenBankTM/EBI accession no.AL080080). The 5′ region of this cDNA clone extends up to the 23rd nucleotide upstream of the translation initiation codon, and this segment lacks an inframe stop codon, which we found at 81 bases upstream of the initiation codon (Fig. 1). When homology protein searches were performed against databases of other species, two conceptual translation products in C. elegans andDrosophila were found with high identity scores. They share an identical active site sequence and structural similarity with TMX even outside of the Trx-like domain (Fig. 2 B). The strong sequence conservation in these distantly related species suggests the possibility that they compose a novel Trx family. Although the potential active site sequence of TMX, CPAC, has not been found in any other mammalian proteins with a Trx-domain, the sequence has Trx-like reducing activity when detected by the insulin disulfide reducing assay (Fig. 5), a classical spectrophotometric assay detecting the reduction of the two interchain disulfide bonds of insulin (31Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). Replacement of two cysteine residues in the redox active site to serines resulted in a complete loss of the reductase activity of TMX protein. These results suggest the potential function of TMX as an oxidoreductase with the novel active site sequence. The TMX amino acid sequence predicts that TMX may be a type I membrane protein; the Trx-like domain in the N-terminal half protrudes on the luminal side of the ER. Sequence analysis of TMX revealed no known motifs for subcellular localization. Analysis with the Myc-tagged protein revealed that TMX is probably localized primarily in the ER (Fig. 4 B), where another protein family with Trx-like domains, PDI, also exists. PDI contains two Trx-like domains and catalyzes the disulfide bond formation (25Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). The retention of some proteins in the ER is known to depend on the presence of the C-terminal sorting signals such as KDEL (39Munro S. Pelham H.R.B. Cell. 1987; 48: 899-907Abstract Full Text PDF PubMed Scopus (1561) Google Scholar) and the double lysine motif (KKXX or KXKXX) (40Jackson M.R. Nilsson T. Peterson P.A. EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (721) Google Scholar). There is a KDEL motif at the C terminus of PDI, and it resides in the lumen of the ER (26Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). There is no such ER-retention motif at the C terminus of TMX, and the localization pattern of overexpressed protein did not change when the Myc epitope was inserted immediate to the C-terminal site of the predicted signal sequence (SS) cleavage site (SS-Myc·TMX, data not shown), suggesting that the ER retention mechanism for TMX is independent of the C-terminal sequence. The cell fractionation experiments suggest that a low level of TMX·Myc was expressed on the plasma membrane (Fig. 4 A). However, when the staining patterns of TMX·Myc or SS-Myc·TMX was compared with that of GPI-anchored EGFP, overlapped staining on the plasma membrane was not obvious (Fig. 4 B and data not shown). Therefore it has remained unclear whether some TMX protein can be localized on the plasma membrane. Because we could not exclude the possibility that the tagging of the Myc epitope might lead to the mislocalization of the protein, the localization of endogenous protein should be elucidated by using specific antibodies against TMX itself. The accumulation of unfolded or abnormal proteins and the disruption of ER calcium homeostasis give rise to ER stress, and excess or prolonged stress results in apoptosis (41Welihinda A.A. Tirasophon W. Kaufman R.J. Gene Expr. 1999; 7: 293-300PubMed Google Scholar). BFA is an effective ER stress inducer and was shown to induce apoptotic cell death in several human tumor cell lines (42Shao R-G. Shimizu T. Pommier Y. Exp. Cell Res. 1996; 227: 190-196Crossref PubMed Scopus (104) Google Scholar, 43Guo H. Tittle T.V. Allen H. Maziarz R.T. Exp. Cell Res. 1998; 245: 57-68Crossref PubMed Scopus (63) Google Scholar). BFA, a small fungal metabolite, has been shown to alter the function of the Golgi and trans-Golgi network, disrupt the traffic between endosomes and lysosomes, and inhibit protein secretion and synthesis because of impairment of vesicular transport (44Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1537) Google Scholar, 45Chardin P. McCormick F. Cell. 1999; 97: 153-155Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In this study, we showed that the overexpression of TMX could relieve the ER stress induced by BFA (Fig. 6 A). The apoptosis suppression by TMX was rather specific to BFA; no resistance was observed in the TMX-expressing HEK293 cells to other ER stress inducers such as thapsigargin and calcium ionophore A23187. BFA disrupts protein trafficking and Golgi morphology by inhibiting Golgi-associated guanine nucleotide exchange factors that activate ADP-ribosylation factors (ARFs) (45Chardin P. McCormick F. Cell. 1999; 97: 153-155Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). At the moment, it is unclear whether TMX interferes with the action of BFA or during later events leading to cell death. In the former case, TMX may bind and/or inactivate BFA itself or may by an unknown mechanism reactivate ARFs. In the latter case, an interesting possibility, among others, would be that TMX, like Ire1 (46Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (729) Google Scholar, 47Wang X.-Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (650) Google Scholar) and ATF6 (48Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar, 49Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1507) Google Scholar), functions as a stress sensor residing on the ER membrane, detecting the accumulation of unfolded proteins and activating downstream anti-stress response. Alternatively, TMX may modify certain proteins with its oxidoreductase activity thereby suppressing ER stress-induced cell death. Our previous results indicated that the level of TMXmRNA was increased by about 2-fold after TGF-β treatment (27Akiyama N. Matsuo Y. Sai H. Noda M. Kizaka-Kondoh S. Mol. Cell. Biol. 2000; 20: 3266-3273Crossref PubMed Scopus (40) Google Scholar). The functional relationship between TGF-β and TMX is presently unclear. RNA blot analysis revealed that BFA treatment (0.2 μg/ml) for 24 h did not increase the level of TMX mRNA (data not shown), indicating that ER stress itself does not influenceTMX expression. No alteration was observed in the interaction between Smad3 and Smad4 or in the expression of these proteins in the HEK293 cells coexpressing TMX and Smad3 and/or Smad4 (data not shown), suggesting that TMX may not have direct effects on the TGF-β signal transduction pathway. Further study is needed to test the interesting possibility that TMX serves as an essential target for TGF-β signaling and mediator for some of its biological effects. We thank Emi Nishimoto and Naoko Murakami for technical assistance.