From the 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
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ABSTRACT |
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We recently identified a series of transforming
growth factor- 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- 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)- 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 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( Northern Blot Analysis--
Human Multiple Tissue Northern blot
(CLONTECH) was hybridized with the full-length TMX
cDNA labeled with [ 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.
Cloning of TMX cDNA--
Our previous gene trap screening had
identified a series of TGF- 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).
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).
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).
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.
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).
In this study, we have characterized a novel Trx-related protein
encoded by a gene identified as one of the TGF- 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--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.
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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).
-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.
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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).
)/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).
-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.
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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-
-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.
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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.
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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 -actin was used as a
control to determine the relative amount of RNA from each tissue.
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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.
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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);
, 50 µg/ml TMX-(27-180);
, 100 µg/ml
TMX/CS-(27-180);
, 50 µg/ml TMX/CS-(27-180);
, 25 µg/ml
recombinant human Trx;
, negative control. The experiments were
repeated twice with essentially the same results.
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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.
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DISCUSSION
REFERENCES
-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.
treatment (27).
The functional relationship between TGF-
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-
signal transduction pathway. Further study is needed to
test the interesting possibility that TMX serves as an essential target
for TGF-
signaling and mediator for some of its biological effects.
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ACKNOWLEDGEMENTS |
---|
We thank Emi Nishimoto and Naoko Murakami for technical assistance.
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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
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ABBREVIATIONS |
---|
The abbreviations used are:
Trx, thioredoxin;
ER, endoplasmic reticulum;
PDI, protein disulfide isomerase;
TGF-, transforming growth factor-
;
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.
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