Identification of a Novel Thioredoxin-related Transmembrane Protein*

Yoshiyuki MatsuoDagger , Nobutake AkiyamaDagger , Hajime Nakamura§, Junji Yodoi§, Makoto NodaDagger , and Shinae Kizaka-KondohDagger

From the Dagger  Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501 and the § Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan

Received for publication, December 7, 2000, and in revised form, December 27, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We recently identified a series of transforming growth factor-beta -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, designated TMX, 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 the Caenorhabditis 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. The TMX 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thioredoxin (Trx)1 is a small and ubiquitously expressed protein originally identified in Escherichia coli and is evolutionarily conserved from prokaryotes to higher eukaryotes (1-3). 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 (4). 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 (2). Reduced Trx (Trx-(SH)2) contains two thiol groups and can catalyze the reduction of disulfide bonds in multiple substrate proteins (2, 3).

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 (2). Trx also modulates the DNA binding activity of transcription factors such as AP-1, nuclear factor-kappa B, glucocorticoid receptor, and estrogen receptor (5-8). 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 (9, 10). In these cases, Trx was found to be involved in cell activation and growth promotion (11-13).

Recently, several mammalian proteins of the Trx superfamily have been reported, which include Trx2 (14), nucleoredoxin (15), and TRP32 (16). 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 (17). The ER also functions as a mobilizable calcium store that sequesters excess cytosolic calcium and acts as a reservoir for calcium signaling (18). 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 (19), the ER stands against relatively mild stresses by the unfolded protein response (20), suppression of translation (21), induction of Golgi-ER backward transport (22), and activation of the accumulating protein transport to proteasome (23, 24). 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 (25, 26).

In this study, we have characterized a novel protein, TMX, encoded by a gene previously isolated as a transforming growth factor (TGF)-beta -responsive gene (27). 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- A549 human lung adenocarcinoma cell line and human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). The concentration and source of reagents added to the medium were as follows: 0.1-1 µg/ml brefeldin A (BFA) (Nacalai), 0.1-2 µg/ml thapsigargin (Nacalai), and 0.1-1 µg/ml calcium ionophore A23187 (CalBiochem).

Cloning of TMX cDNA-- Gene trap screening and isolation of TMX cDNA fragment by 5'-RACE were performed as described previously (27). Poly(A)+ RNA prepared from A549 cells treated with TGF-beta was used for construction of a cDNA library with a ZAP Express cDNA synthesis kit (Stratagene) according to the manufacturer's instructions. Using 32P-labeled 5'-RACE products (193 bp) as a probe, ~2 × 106 plaques were screened, and positive clones were isolated. The nucleotide sequences were determined using the Dye Terminator Cycle Sequencing kit with a model 373S automated sequencer (Applied Biosystems).

Expression Vectors-- TMX/CS mutant was made by converting the two conserved cysteine residues (Cys-56 and Cys-59) in the Trx-like domain into serine by using the synthetic oligonucleotides 5'-GTGGTCCCCTGCTTCTCAAAATCTTCAACC-3' and 5'-AGATTTTGAGAAGCAGGGGACCACGGGGCA-3'. For mammalian expression, the full-length TMX coding sequence was amplified by polymerase chain reaction and inserted into pcDNA3.1(-)/Myc-His A vector (Invitrogen) to generate a plasmid encoding TMX or TMX/CS with a Myc-tag at the C terminus (pcDNA3.1-TMX·Myc, pcDNA3.1-TMX/CS·Myc). The same cDNA with the Myc-tag was inserted into the pTRE vector (CLONTECH) to generate the tet-inducible expression vector tet-TMX and tet-TMX/CS. To generate the bacterial expression plasmids pGEX-TMX-(27-180) and pGEX-TMX/CS-(27-180), a cDNA fragment encoding a part of TMX or the TMX/CS mutant (amino acids 27-180) was amplified by polymerase chain reaction and ligated into the pGEX-6P-1 vector (Amersham Pharmacia Biotech).

Northern Blot Analysis-- Human Multiple Tissue Northern blot (CLONTECH) was hybridized with the full-length TMX cDNA labeled with [alpha -32P]dCTP using ready-to-go DNA-labeling beads (Amersham Pharmacia Biotech) followed by purification using a MicroSpin Column S300-HR (Amersham Pharmacia Biotech). Hybridization was performed at 65 °C in ExpressHyb buffer (CLONTECH). Filters were washed once with 2× SSC, 0.1% SDS for 20 min at room temperature and three times with 0.5× SSC, 0.1% SDS for 15 min each at 65 °C and then subjected to autoradiography.

Cell Fractionation-- HEK293 cells were transfected with pcDNA3.1- TMX·Myc by electroporation using Gene Pulser (Bio-Rad). After 24 h incubation, the cells (5 × 106/fraction) were subjected to the following subcellular fractionation procedures at 4 °C. The method for preparing the nuclear fraction was described previously (28). Briefly, cells were homogenized in 0.25 M sucrose-TKM (20 mM Tris, pH 7.6, 50 mM KCl, 2 mM MgCl2). The homogenate was overlaid on 2.4 M sucrose-TKM solution and centrifuged for 30 min at 14000 × g. The precipitates (nuclear fraction) were collected and sonicated. To prepare cytosolic and microsomal fractions, we followed the methods described by Hogeboom et al. (29). Briefly, cells were suspended in 0.25 M sucrose-TKM solution and homogenized. Lysates were serially centrifuged at 1,000, 12,000, and 105,000 × g. The supernatant (cytosolic fraction) and precipitate (microsomal fraction) of the final centrifugation were collected. To prepare the plasma membrane fraction, we followed the methods of Koizumi et al. (30). Briefly, cells were suspended in 1 mM NaHCO3, 0.5 mM CaCl2 to prepare the cell ghost and applied to serial sucrose gradient centrifugation (42-68% sedimentation and 42-48-68% sedimentation).

Immunofluorescence Microscopy-- A549 cells were transfected with pcDNA3.1-TMX·Myc using LipofectAMINE (Life Technologies, Inc.) and plated on a multiwell chamber slides (Nunc). After incubation at 37 °C for 48 h, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and then permeabilized with 0.2% Triton X-100 in PBS at room temperature for 4 min. After blocking in 5% bovine serum albumin in PBS at room temperature for 30 min, the cells were stained with anti-Myc monoclonal antibody (9E10, CLONTECH) for 1 h, followed by incubation with secondary antibody, Cy3-conjugated anti-mouse IgG antibody (Amersham Pharmacia Biotech) for 1 h. After rinsing, the slides were analyzed using a confocal microscope (Micro Radiance, Bio-Rad).

Purification of Recombinant Protein-- Either pGEX-TMX-(27-180) or pGEX-TMX/CS-(27-180) was introduced into E. coli, and the expression of glutathione S-transferase (GST) fusion proteins was induced with 0.1 mM IPTG at 20 °C. GST fusion proteins were bound to Glutathione-Sepharose beads (Amersham Pharmacia Biotech) and cleaved with PreScission Protease (Amersham Pharmacia Biotech) at 4 °C in the cleavage buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT. The treated beads were transferred to the column and the flow-through was collected. The solution containing the GST-free protein was concentrated by Ultrafree-MC Centrifugal Filter Units (Millipore).

Insulin Disulfide Reduction Assay-- The insulin disulfide reduction assay was performed as described previously (31) with slight modifications. Briefly, aliquots of TMX-(27-180), TMX/CS-(27-180), or purified recombinant human Trx (rhTrx) was preincubated in a 90-µl reaction mixture (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.3 mM DTT) at room temperature for 15 min. The reaction was started by adding 10 µl of 10 mg/ml bovine insulin (Sigma), and the change in the absorbance at 595 nm was recorded at room temperature. The nonenzymatic reduction of insulin by DTT was recorded as a control. rhTrx was produced and provided by Ajinomoto Co. Inc., Basic Research Laboratory (32).

Establishment of rtTA/tet-TMX and rtTA/tet-TMX/CS Cells and Response to ER Stress Inducers-- 293rtTA cells, a subline of HEK293 cells expressing the Tet-controlled transcriptional activator (rtTA) gene, were transfected with a tet-inducible expression vector, tet-TMX or tet-TMX/CS, together with pUCSV-BSD plasmid (Kaken) and selected with 8 µg/ml blasticidin-S (BLA-S) (Kaken). The pooled Bla-S-resistant cells are referred to as rtTA/tet-TMX and rtTA/tet-TMX/CS, respectively. The rtTA/tet-TMX or rtTA/tet-TMX/CS cells (500/well) were seeded in a 24-well plate in the presence or absence of 2 µg/ml doxycycline (Dox), and on the following day, BFA (0.1 µg/ml), thapsigargin (0.1 µg/ml) or A23187 (0.5 µg/ml) was added to the culture. Twenty-four hours later, the morphology of the cells were observed and photographed under an inverted microscope.

Western Blot Analysis-- To detect Dox-induced proteins, rtTA/TMX or rtTA/TMX/CS cells (5 × 106) were seeded onto 100-mm dishes in the presence or absence of 2 µg/ml Dox, and 48 h later the cells were washed with PBS twice and lysed with 500 µl radioimmune precipitation assay buffer. Ten µg of each sample was separated by SDS-polyacrylamide gel electrophoresis, followed by transfer to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech). After blocking in TBS containing 5% skim milk, the filter was incubated with anti-Myc monoclonal antibody at room temperature for 1 h. To detect TMX in cell fractions described above, a blotted filter prepared by the same method was incubated with anti-Myc or anti-histone H1 monoclonal antibody (Santa Cruz Biotechnologies) at room temperature for 1 h. The filter was then washed with TBST and incubated with horseradish peroxidase-conjugated anti-mouse Ig (Amersham Pharmacia Biotech). The signals were detected using ECL detection system (Amersham Pharmacia Biotech).

Detection of Apoptosis-- rtTA/tetTMX or rtTA/tetTMX/CS cells (1 × 104/well) were seeded onto a 6-well plate in the presence or absence of 2 µg/ml Dox. On the following day, the cells were cultured in the presence or absence of BFA (0.1 and 0.2 µg/ml) for an additional 20 h. Then the cells were washed twice with PBS and incubated with the binding buffer containing FITC-conjugated annexin V (MBL) for 10 min. The fluorescence was observed and photographed under an inverted fluorescence microscope.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of TMX cDNA-- Our previous gene trap screening had identified a series of TGF-beta responsive genes in A549 human lung adenocarcinoma cell line (27). One such gene, A83, was found to encode a previously undescribed protein and therefore was subjected to further characterization. A short fragment (193 bp) of A83 cDNA was initially recovered from a trap line by 5'-RACE. Using this 5'-RACE product as a probe, a cDNA library of TGF-beta -treated A549 cells was screened, and a cDNA clone of 1.6 kb was isolated. This cDNA contained an open reading frame of 840 bp, an inframe stop codon located at 81 bp upstream of the first ATG, and the Kozak consensus sequence for mammalian translation initiation (33) around the first ATG (Fig. 1). The predicted open reading frame encodes a protein of 280 amino acids with the calculated molecular mass of 31.8 kDa and isoelectric point of 4.77. 


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Fig. 1.   Nucleotide sequence and deduced amino acid sequence of TMX. The predicted active site sequence is boxed. The putative N-terminal signal sequence and transmembrane domain are underlined and double underlined, respectively. The stop codons are marked with asterisks.

TMX Encodes a Novel Member of the Trx Family-- The A83-encoded protein was identical to none of the known proteins in the public databases, except for the putative product of a human cDNA fragment of unknown function (see "Discussion"). We could also detect substantial sequence similarity with two members of the Trx family found in the Caenorhabditis elegans (F46E10.9, accession no. AAD14719) and Drosophila (CG5554, accession no. AAF47072) genomes (34, 35). The hydropathy plot and a motif analysis using Simple Modular Architecture Research Tool (SMART) (36, 37) indicated that the A83 protein may contain a cleavable signal sequence (amino acids 1-26) and a transmembrane domain (amino acids 183-203) (Figs. 1 and 2A).


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Fig. 2.   Primary structures of TMX and its homologs. A, domain structure of human TMX. Positions of the N-terminal signal sequence (SS), TRX-like domain with an active site sequence (CPAC), and transmembrane domain (TM) are indicated. The numbers above the bar represent amino acid positions. B, alignment of human TMX and the homologs from C. elegans and Drosophila. The conserved active site is boxed. Identical amino acids between the three proteins are marked with asterisks.

The Trx-like domain present in the N-terminal half of this protein contains an atypical active site sequence, CPAC (Figs. 1 and 2A), which is conserved in the homologs in C. elegans and Drosophila. The amino acid sequence around the potential active site (amino acids 36-109) shares 63.5% identity and 89% similarity with the C. elegans protein, and 63.5% identity and 85% similarity with the Drosophila protein (Fig. 2B). Moreover, they also share significant homology outside the Trx-like domain (Fig. 2B), whereas the similarities between A83 and other members of the mammalian Trx family are confined to this domain. Based on these structural features, we named this protein TMX (transmembrane Trx-related protein).

Tissue Distribution of TMX mRNA-- The tissue distribution of TMX mRNA was examined by RNA blot hybridization using the full-length TMX cDNA as a probe. The TMX mRNA was detected as a single band of 2.5 kb in all the tissues examined. The expression seems to be relatively high in kidney, liver, placenta, and lung (Fig. 3).


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Fig. 3.   Distribution of TMX mRNA in human tissues. RNA blot containing poly(A)+ RNA from multiple human tissues was hybridized with the full-length TMX cDNA as a probe. Human beta -actin was used as a control to determine the relative amount of RNA from each tissue.

Subcellular Localization of TMX-- To determine the subcellular localization of TMX protein, HEK293 cells were transfected with a plasmid expressing a Myc-tagged TMX (TMX·Myc), and subcellular fractions were prepared by sucrose density gradient centrifugation. Each fraction was analyzed by immunoblotting using anti-Myc antibody (Fig. 4A). Successful preparation of the nuclear fraction was confirmed by anti-histone H1 antibody. The TMX protein was detected mainly in the microsomal fraction (M). A smaller amount of TMX protein was present in the plasma membrane fraction (P), but it was undetectable in the nuclear (N) and cytosolic fractions (Fig. 4A and data not shown). TMX·Myc was transiently expressed in A549 cells and observed with confocal microscopy. The cells expressing TMX·Myc showed ER-like staining pattern characterized by a diffuse network-like labeling of the cytoplasm and nuclear rim (Fig. 4B, left panel). This result is consistent with the result of the above subcellular fractionation study. In A549 cells coexpressing TMX·Myc and the glycosylphosphatidylinositol (GPI)-anchored EGFP known to be localized on the plasma membrane (Ref. 38 and Fig. 4B, middle panel), the localization of these two proteins differ considerably from each other (Fig. 4B, right panel).


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Fig. 4.   Subcellular localization of TMX. A, subcellular fractionation of TMX·Myc transfectants. Cell fractions were analyzed by immunoblotting with anti-Myc and anti-histone H1 antibodies. N, nuclear fraction; M, microsomal fraction; P, plasma membrane fraction. B; left, immunostaining for TMX·Myc using Cy3-conjugated anti-mouse Ig as a secondary antibody; middle; expression of GPI-anchored EGFP; right, merged image.

Reductase Activity of TMX-- The putative active site sequence (CPAC) of TMX does not completely match those of classical Trx and other related proteins. To examine whether TMX indeed shows a Trx-like reducing activity, an insulin disulfide reducing assay was carried out using recombinant TMX proteins (Fig. 5). In this assay, disulfide reductase activity is monitored by an increase in the turbidity of reaction mixtures because of the formation of fine precipitates of the dissociated insulin B chain (31). Because intact TMX containing the signal sequence and transmembrane domain was found to be toxic to the bacterial cells, a part of TMX containing the catalytic domain, TMX-(27-180), was expressed as a GST fusion protein in E. coli and cleaved from GST. The TMX/CS-(27-180) mutant in which two cysteines in the putative active site (Cys-56 and Cys-59) were substituted to serines, was also prepared. In the negative control with DTT and insulin alone, no precipitation was observed up to 55 min. The addition of 100 µg/ml TMX-(27-180) resulted in a rapid increase in turbidity, as did the addition of the control Trx protein. The reaction was dose-dependent: TMX-(27-180) showed a longer latency period at 50 µg/ml than at 100 µg/ml. Importantly, the TMX/CS-(27-180) mutant failed to reduce insulin, indicating that the CPAC motif constitutes the active site of TMX as expected, and the two cysteine residues are essential for the disulfide reductase activity.


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Fig. 5.   Reductase activity of TMX. Bacterially expressed TMX and TMX/CS proteins were incubated with insulin and their ability to reduce insulin disulfide bonds was measured. The absorbance at 595 nm was monitored every 2.5 min. , 100 µg/ml TMX-(27-180); open circle , 50 µg/ml TMX-(27-180); black-triangle, 100 µg/ml TMX/CS-(27-180); triangle , 50 µg/ml TMX/CS-(27-180); , 25 µg/ml recombinant human Trx; cross , negative control. The experiments were repeated twice with essentially the same results.

Biological Function of TMX-- To explore biological activities of TMX, we established HEK293 lines expressing TMX or TMX/CS under the control of Dox. Because TMX was predominantly localized in ER, we first examined the effects of TMX expression on the cells under ER stresses (Fig. 6). The cells were cultured in the presence or absence of Dox for 24 h and then placed for 16 h in the medium containing a reagent known to induce ER stress and apoptosis: namely, thapsigargin, calcium ionophore A23187, or BFA (19). When the cells were treated with thapsigargin, or calcium ionophore A23187 (both disrupt intracellular calcium homeostasis), apoptosis was induced equally in the Dox-treated and untreated cells (data not shown). Interestingly, the cells exhibited significant resistance to the apoptosis-inducing activity of BFA (an inhibitor of ER-Golgi transport) only when the TMX transgene was switched on by Dox-treatment (Fig. 6A, top and middle panels). Such an effect was not evident when the enzymatically inactive TMX/CS mutant was expressed. The BFA-induced apoptosis could be confirmed by fluorescence-conjugated annexin V (FITC-AV) on the cell surface. Although the BFA (0.1 µg/ml)-treated rtTA/TMX/CS cells were stained equally with FITC-AV regardless of Dox treatment, the BFA (0.2 µg/ml)-treated rtTA/TMX cells were hardly stained with FITC-AV when TMX expression was induced (Fig. 6A, bottom panels). This apoptosis-suppressing effect of TMX was observed in the presence of up to 0.2 µg/ml of BFA for more than 20 h, but at higher concentrations the suppression was hardly observed (data not shown). Immunoblot assay confirmed the Dox-dependent expression of TMX and TMX/CS (Fig. 6B).


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Fig. 6.   Suppression of BFA-mediated apoptosis by TMX. A, top and middle panels, HEK293 cells harboring either tet-inducible TMX gene (rtTA/tet-TMX) or tet-inducible TMX/CS gene (rtTA/tet-TMX/CS) were cultured in the presence (+) or absence (-) of Dox and then treated with BFA (0.1 µg/ml) for 16 h. Morphology were examined and photographed under an inverted microscope. Bottom panels, to detect apoptosis, rtTA/tet-TMX and rtTA/tet-TMX/CS cells were cultured in the presence (+) or absence (-) of Dox and then treated with BFA (0.1 and 0.2 µg/ml) for 20 h. Apoptotic cells were stained in a 10-min incubation with FITC-conjugated annexin V. The figure shows rtTA/tet-TMX cells treated with 0.2 µg/ml BFA and rtTA/tet-TMX/CS cells treated with 0.1 µg/ml BFA. B, induction of TMX and TMX/CS protein by Dox was confirmed by immunoblotting with anti-Myc antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have characterized a novel Trx-related protein encoded by a gene identified as one of the TGF-beta -responsive genes isolated by a retrovirus-mediated gene trap screening (27). 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 and Drosophila 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. 2B). 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 (31). 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. 4B), where another protein family with Trx-like domains, PDI, also exists. PDI contains two Trx-like domains and catalyzes the disulfide bond formation (25, 26). The retention of some proteins in the ER is known to depend on the presence of the C-terminal sorting signals such as KDEL (39) and the double lysine motif (KKXX or KXKXX) (40). There is a KDEL motif at the C terminus of PDI, and it resides in the lumen of the ER (26). 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. 4A). 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. 4B 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 (41). BFA is an effective ER stress inducer and was shown to induce apoptotic cell death in several human tumor cell lines (42, 43). 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 (44, 45). In this study, we showed that the overexpression of TMX could relieve the ER stress induced by BFA (Fig. 6A). 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) (45). 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 (46, 47) and ATF6 (48, 49), 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 TMX mRNA was increased by about 2-fold after TGF-beta treatment (27). The functional relationship between TGF-beta 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 influence TMX 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-beta signal transduction pathway. Further study is needed to test the interesting possibility that TMX serves as an essential target for TGF-beta signaling and mediator for some of its biological effects.

    ACKNOWLEDGEMENTS

We thank Emi Nishimoto and Naoko Murakami for technical assistance.

    FOOTNOTES

* This work was supported by research grants from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB048246.

To whom correspondence should be addressed. Tel.: 81-75-751-4150; Fax: 81-75-751-4159; E-mail: mnoda@virus.kyoto-u.ac.jp.

Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M011037200

    ABBREVIATIONS

The abbreviations used are: Trx, thioredoxin; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; TGF-beta , transforming growth factor-beta ; BFA, brefeldin A; GST, glutathione S-transferase; DTT, dithiothreitol; Dox, doxycycline; GPI, glycosylphosphatidylinositol; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; bp, base pairs; FITC, fluorescein isothiocyanate; RACE, rapid amplification of cDNA ends.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Laurent, T. C., Moore, E. C., and Reichard, P. (1964) J. Biol. Chem. 239, 3436-3444[Free Full Text]
2. Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237-271[CrossRef][Medline] [Order article via Infotrieve]
3. Holmgren, A. (1989) J. Biol. Chem. 264, 13963-13966[Free Full Text]
4. Nakamura, H., and Yodoi, J. (1998) Curr. Trends Immunol. 1, 133-140
5. Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K., and Yodoi, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3633-3638[Abstract/Free Full Text]
6. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T. (1992) Nucleic Acids Res. 20, 3821-3830[Abstract]
7. Makino, Y., Okamoto, K., Yoshikawa, N., Aoshima, M., Hirota, K., Yodoi, J., Umesono, K., Makino, I., and Tanaka, H. (1996) J. Clin. Invest. 98, 2469-2477[Abstract/Free Full Text]
8. Hayashi, S., Hajiro-Nakanishi, K., Makino, Y., Eguchi, H., Yodoi, J., and Tanaka, H. (1997) Nucleic Acids Res. 25, 4035-4040[Abstract/Free Full Text]
9. Tagaya, Y., Maeda, Y., Mitsui, A., Kondo, N., Matsui, H., Hamuro, J., Brown, N., Arai, K., Yokota, T., Wakasugi, H., and Yodoi, J. (1989) EMBO J. 8, 757-764[Abstract]
10. Wollman, E. E., d'Auriol, L., Rimsky, L., Shaw, A., Jacquot, J.-P., Wingfield, P., Graber, P., Dessarps, F., Robin, P., Galibert, F., Bertoglio, J., and Fradelizi, D. (1988) J. Biol. Chem. 263, 15506-15512[Abstract/Free Full Text]
11. Wakasugi, N., Tagaya, Y., Wakasugi, H., Mitsui, A., Maeda, M., Yodoi, J., and Tursz, T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8282-8286[Abstract]
12. Oblong, J. E., Berggren, M., Gasdaska, P. Y., and Powis, G. (1994) J. Biol. Chem. 269, 11714-11720[Abstract/Free Full Text]
13. Nakamura, H., Nakamura, K., and Yodoi, J. (1997) Annu. Rev. Immunol. 15, 351-369[CrossRef][Medline] [Order article via Infotrieve]
14. Spyrou, G., Enmark, E., Miranda-Vizuete, A., and Gustafsson, J-A. (1997) J. Biol. Chem. 272, 2936-2941[Abstract/Free Full Text]
15. Kurooka, H., Kato, K., Minoguchi, S., Takahashi, Y., Ikeda, J., Habu, S., Osawa, N., Buchberg, A. M., Moriwaki, K., Shisa, H., and Honjo, T. (1997) Genomics 39, 331-339[CrossRef][Medline] [Order article via Infotrieve]
16. Lee, K.-K., Murakawa, M., Takahashi, S., Tsubuki, S., Kawashima, S., Sakamaki, K., and Yonehara, S. (1998) J. Biol. Chem. 273, 19160-19166[Abstract/Free Full Text]
17. Gething, M. J., and Sambrook, J. (1992) Nature 355, 33-45[CrossRef][Medline] [Order article via Infotrieve]
18. Pozzan, T., Rizzuto, R., Volpe, P., and Meldolesi, J. (1994) Physiol. Rev. 74, 595-636[Free Full Text]
19. Nakagawa, T., Zhu, H, Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103[CrossRef][Medline] [Order article via Infotrieve]
20. Sidrauski, C., Chapman, R., and Walter, P. (1998) Trends Cell Biol. 8, 245-249[CrossRef][Medline] [Order article via Infotrieve]
21. Harding, H. P., Zhang, Y., and Ron, D. (1999) Nature 397, 271-274[CrossRef][Medline] [Order article via Infotrieve]
22. Hammond, C., and Helenius, A. (1994) J. Cell Biol. 126, 41-52[Abstract]
23. Kopito, R. R. (1997) Cell 88, 427-430[Medline] [Order article via Infotrieve]
24. Zhou, M., and Schekman, R. (1999) Mol. Cell 4, 925-934[Medline] [Order article via Infotrieve]
25. Freedman, R. B., Hirst, T. R., and Tuite, M. F. (1994) Trends Biochem. Sci. 19, 331-336[CrossRef][Medline] [Order article via Infotrieve]
26. Noiva, R., and Lennarz, W. J. (1992) J. Biol. Chem. 267, 3553-3556[Free Full Text]
27. Akiyama, N., Matsuo, Y., Sai, H., Noda, M., and Kizaka-Kondoh, S. (2000) Mol. Cell. Biol. 20, 3266-3273[Abstract/Free Full Text]
28. Li, Q., Yoshioka, N., Yutsudo, M., Inafuku, S., Aozasa, K., Kitamura, Y., Aizawa, S., Nishimune, Y., Hakura, A., and Kondoh, G. (1998) Virology 252, 28-33[CrossRef][Medline] [Order article via Infotrieve]
29. Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) J. Biol. Chem. 172, 619[Free Full Text]
30. Koizumi, K., Ito, Y., Kojima, K., and Fujii, T. (1976) J. Biochem. (Tokyo) 79, 739-748[Abstract]
31. Holmgren, A. (1979) J. Biol. Chem. 254, 9627-9632[Abstract]
32. Mitsui, A., Hirakawa, T., and Yodoi, J. (1992) Biochem. Biophys. Res. Commun. 186, 1220-1226[Medline] [Order article via Infotrieve]
33. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148[Abstract]
34. Wilson, R., et al.. (1994) Nature 368, 32-38[CrossRef][Medline] [Order article via Infotrieve]
35. Adams, M. D., et al.. (2000) Science 287, 2185-2195[Abstract/Free Full Text]
36. Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5857-5864[Abstract/Free Full Text]
37. Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P., and Bork, P. (2000) Nucleic Acids Res. 28, 231-234[Abstract/Free Full Text]
38. Kondoh, G., Gao, X.-H., Nakano, Y., Koike, H., Yamada, S., Okabe, M., and Takeda, J. (1999) FEBS Lett. 458, 299-303[CrossRef][Medline] [Order article via Infotrieve]
39. Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907[Medline] [Order article via Infotrieve]
40. Jackson, M. R., Nilsson, T., and Peterson, P. A. (1990) EMBO J. 9, 3153-3162[Abstract]
41. Welihinda, A. A., Tirasophon, W., and Kaufman, R. J. (1999) Gene Expr. 7, 293-300[Medline] [Order article via Infotrieve]
42. Shao, R-G., Shimizu, T., and Pommier, Y. (1996) Exp. Cell Res. 227, 190-196[CrossRef][Medline] [Order article via Infotrieve]
43. Guo, H., Tittle, T. V., Allen, H., and Maziarz, R. T. (1998) Exp. Cell Res. 245, 57-68[CrossRef][Medline] [Order article via Infotrieve]
44. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080[Medline] [Order article via Infotrieve]
45. Chardin, P., and McCormick, F. (1999) Cell 97, 153-155[Medline] [Order article via Infotrieve]
46. Tirasophon, W., Welihinda, A. A., and Kaufman, R. J. (1998) Genes Dev. 12, 1812-1824[Abstract/Free Full Text]
47. Wang, X.-Z., Harding, H. P., Zhang, Y., Jolicoeur, E. M., Kuroda, M., and Ron, D. (1998) EMBO J. 17, 5708-5717[Abstract/Free Full Text]
48. Yoshida, H., Haze, K., Yanagi, H., Yura, T., and Mori, K. (1998) J. Biol. Chem. 273, 33741-33749[Abstract/Free Full Text]
49. Haze, K., Yoshida, H., Yanagi, H., Yura, T., and Mori, K. (1999) Mol. Biol. Cell 10, 3787-3799[Abstract/Free Full Text]


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