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J Biol Chem, Vol. 274, Issue 35, 24522-24530, August 27, 1999
,
,, and§§
From the Department of Biochemistry, University of
Nebraska, Lincoln, Nebraska 68588, the § Laboratory of
Molecular Microbiology, NIAID, National Institutes of Health, Bethesda,
Maryland 20892, the ¶ Laboratory of Molecular Structure, NIAID,
National Institutes of Health, Rockville, Maryland 20852, the
** Laboratory of Molecular Genetics, Institute for Molecular Biology and
Genetics, Seoul National University, Seoul 151-742, Korea, and the
Basic Research Laboratory, NCI, National
Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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The intracellular generation of reactive oxygen
species, together with the thioredoxin and glutathione systems, is
thought to participate in redox signaling in mammalian cells. The
activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a
selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in
addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at
the carboxyl terminus and which is encoded by a UGA codon. The
generation of reactive oxygen species in a human carcinoma cell line
was shown to result in both the oxidation of the selenocysteine in TR1
and a subsequent increase in the expression of this enzyme. These
observations identify the carboxyl-terminal selenocysteine of TR1
as a cellular redox sensor and support an essential role for
mammalian TR isozymes in redox-regulated cell signaling.
Reactive oxygen species
(ROS),1 such as hydrogen
peroxide, the superoxide anion radical, and the hydroxyl radical, have
been thought of as toxic by-products of cellular oxygen metabolism. Excessive production of ROS or an insufficiency of antioxidant defenses
has been implicated in apoptosis, aging, and cancer (1). However,
recent evidence indicates that, at low concentrations, ROS mediate
regulatory events and are essential participants in cell signaling (2).
Redox signaling is thought to be achieved through the coupling of ROS
with oxidation-reduction processes that involve essential thiol groups
in proteins, resulting in the modulation of tyrosine or
serine-threonine phosphorylation of target proteins (3, 4). The redox
state of essential thiol groups is controlled by two cellular redox
systems: the thioredoxin (thioredoxin, thioredoxin reductase
(designated TR herein, but it should be noted that TrxR has also been
used, e.g. see Ref. 5-8), and thioredoxin peroxidase) and
glutathione (glutathione, glutathione reductase, glutaredoxin, and
glutathione peroxidase (GPX)) systems (9). Although the role of ROS in
intracellular signaling has been demonstrated (2, 10), the direct
targets of these molecules remain poorly characterized, and the
specific mechanisms through which ROS are sensed within mammalian cells are unclear (3, 4, 11).
Several proteins, including apoptosis signal-regulating kinase 1 (ASK1;
this protein belongs to a family of mitogen-activated protein kinase
kinase kinases) (12), protein-tyrosine phosphatase 1B (PTP1B) (13), and
thioredoxin peroxidase (14), have been identified as components of
redox signaling pathways that act downstream of the generation of
intracellular ROS. The activities of these proteins are regulated by
thioredoxin; reduced thioredoxin activates the phosphatase and
peroxidase, and inactivates the kinase (12-14). Thioredoxin is a
12-kDa protein that catalyzes the reduction of protein disulfides and
regulates a variety of processes including reduction of ribonucleotide
reductase, protein-DNA interactions, and cellular growth (9). The redox
state and activity of thioredoxin are in turn controlled by TR, a
selenocysteine-containing flavoprotein composed of two identical 57-kDa
subunits. The selenocysteine residue is located at the penultimate
COOH-terminal position of TR, is encoded by a UGA codon (15-17), and
is essential for enzyme activity (6, 18). TR is a member of the
pyridine nucleotide-disulfide oxidoreductase family of enzymes, but
other members of this family lack the COOH-terminal extension
containing the selenocysteine residue (15, 16). The reactivity of TR
with various alkylating agents, quinones, and gold-containing organic
compounds is about 3 orders of magnitude greater than that of these
other enzymes (for example, glutathione reductase and lipoamide
dehydrogenase) (5, 7, 19). The inactivation of TR by such compounds
has been attributed to the irreversible modification of the
selenocysteine residue (6, 7, 18, 19).
Structurally, selenocysteine differs from cysteine by the single
substitution of selenium for sulfur. Selenium is a better nucleophile
than sulfur and, under physiological conditions, selenocysteine residues are ionized whereas cysteines are typically protonated (20,
21). These properties render selenocysteine a promising candidate for
an ROS sensor in proteins. Indeed, several selenoproteins (including
TR) and selenium compounds exhibit affinity for hydroperoxides and
oxygen radicals (22-24).
We have tested whether selenocysteine in mammalian thioredoxin
reductase may be involved in redox regulation of cell signaling. We
initially identified two new thioredoxin reductases, TR2 and TR3, and
found that all three mammalian thioredoxin reductases conserve the
COOH-terminal penultimate selenocysteine residue. We further found
that, when cells generated ROS, selenocysteine in TR1 was oxidized
while TR1 expression was elevated. The data suggest that the
selenocysteine residue in thioredoxin reductases serves as a cellular
redox sensor.
Materials--
Recombinant human thioredoxin was obtained from
American Diagnostica; recombinant Escherichia coli
thioredoxin, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (DNCB) and
NADPH from Sigma; 5-IAF and anti-fluorescein antibodies from Molecular
Probes; pre-cast NuPAGE polyacrylamide gels and immunoblot system from
Novex; ADP-Sepharose, phenyl-Sepharose, and DEAE-Sepharose from
Amersham Pharmacia Biotech; C18 and phenyl-high performance
liquid chromatography (HPLC) columns from TosoHaas; mouse and human
expressed sequence tag clones for TR1, TR2, and TR3 from Research
Genetics, Genome Systems, and ATCC; mouse tissues from Pel-Freez; and
mouse multiple tissue Northern blot from CLONTECH.
Other reagents were of the highest grade available.
Isolation of TR1, TR2, and TR3--
To obtain
75Se-labeled tissues, we injected mice with 0.2 mCi of
[75Se]selenite (specific radioactivity 1000 Ci/mmol;
University of Missouri Research Reactor) 2 days before tissue removal
as described (25). Thioredoxin reductases were purified from
75Se-labeled mouse liver and testis according to the
previously published procedure (16), which consisted of the subsequent anion-exchange (DEAE-Sepharose), affinity (ADP-Sepharose), and hydrophobic-interaction (phenyl-Sepharose or phenyl-HPLC column) chromatographic procedures. To distinguish between TR isozymes, we
assumed that, on the basis of the deduced protein sequences, TR3 is
more basic than is either TR1 or TR2, and that the molecular mass of
TR2 is higher than that of TR1 or TR3. Indeed, during purification,
TR1, TR2, and TR3 had different affinities for DEAE-Sepharose and
phenyl-Sepharose columns. Purification yielded homogenous preparations
of 75Se-labeled liver TR1, testis TR1, testis TR2, and
liver TR3. The enzymes were detected during purification by their TR
catalytic activities as described by Tamura and Stadtman (17), by the presence of 75Se in fractions analyzed with a 5-detector
Wallac Alkylation of TR1 with 5-IAF--
The procedure for alkylation
by 5-IAF of highly reactive, ROS-sensitive thiol or selenol groups in
cell extracts was adapted from Wu et al. (26). Nearly
confluent A431 cells were incubated for 16 h in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 0.1% fetal bovine
serum (FBS), washed once with DMEM containing 0.1% FBS, and then
incubated either for 0-45 min with EGF (500 ng/ml) in the same medium
(Fig. 3C) or for 4 h in the same medium containing 100 µCi of [75Se]selenite (Fig. 3D). The cells
were washed four times with phosphate-buffered saline and frozen in
liquid nitrogen. They were subsequently harvested at 4 °C by
scraping, under anaerobic conditions, into an oxygen-free lysis buffer
containing 50 mM Tris-HCl (pH 7.5), leupeptin (2 µg/ml),
aprotinin (2 µg/ml), 0.1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 1% Nonidet P-40 detergent,
and 0.04% NaN3. Freshly prepared 5-IAF was added to a
final concentration of 20 µM before scraping. The cell
lysate was incubated under anaerobic conditions at 37 °C for 20 min,
after which the alkylation reaction was quenched by the addition of 1 mM dithiothreitol.
After reaction with 5-IAF, the cell lysate was dialyzed against a
solution containing 25 mM Tris-HCl (pH 7.5), leupeptin (2 µg/ml), and aprotinin (2 µg/ml), and TR1 was isolated from the dialysate by chromatography on ADP-Sepharose and DEAE-Sepharose columns. The purified enzyme was characterized by SDS-PAGE,
PhosphorImager analysis of 75Se, and immunoblot analysis
with antibodies to TR1 or to fluorescein. The 5-IAF-modified enzyme (50 µg) was then further reduced with dithiothreitol in the presence of
guanidine HCl, reacted with iodoacetamide, and digested with
trypsin, and the resulting peptides were separated by reversed-phase
HPLC on a C18 narrow-bore column with a gradient of
acetonitrile as described previously (16). The protein mixture
contained in the ADP-Sepharose flow-through fraction, which was
obtained during TR1 purification, was also digested with trypsin in
parallel with TR1, and the resulting peptides were separated on a
C18 column under conditions identical to those used for
fractionation of the TR1 tryptic digest. This TR1-deficient sample
served as a control for the TR1 sample. The eluted peptides from the
TR1 sample (Fig. 3D) and the TR1-deficient sample were
detected by measurement of absorbance at 214 and 440 nm; absorbance at
the latter wavelength was used to identify fluorescein-containing peptides. Only one major fluorescein-containing peak (fractions 56 and
57) was detected for the TR1 tryptic digest. This peak was not present
in the fractions obtained from the control digest of the TR1-deficient
sample, whereas several smaller peaks of absorbance at 440 nm present
in the TR1 digest (Fig. 3D) were also apparent in the digest
of the TR1-deficient sample. Fractions from the TR1 sample were
analyzed for the presence of 75Se with a Regulation of TR1 Expression--
A431 cells were cultured in
the presence of 10% FBS to near-confluence and then incubated in DMEM
containing 0.1% FBS for 16 h. The serum-deprived cells were
washed once with DMEM containing 0.1% FBS, and then incubated for
4 h in DMEM containing 0.1% FBS and 20 µCi of
[75Se]selenite, and in the absence (control) or presence
of either EGF (500 ng/ml), 0.2 mM
H2O2, or 10-30 µM DNCB. Cells
were then washed four times with phosphate-buffered saline and
harvested by scraping in a lysis buffer containing 50 mM
Tris-HCl (pH 7.5), leupeptin (2 µg/ml), aprotinin (2 µg/ml), 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, sodium
vanadate (2 mg/ml), 1% Nonidet P-40, and 0.04% NaN3. The
cells were sonicated and the resulting lysate was centrifuged at
15,000 × g for 30 min, after which the supernatant (3 µg of protein) was subjected to SDS-PAGE and either immunoblot
analysis with antibodies specific for TR1 or PhosphorImager analysis.
Conservation of Selenocysteine in Mammalian Thioredoxin
Reductases--
Only one mammalian TR isoform (TR1) has been
identified to date (15). We searched the human and mouse dbEST data
bases with human and rat TR1 sequences and found several homologous
human and mouse sequences. We determined the sequences of the
corresponding cDNAs by nucleotide sequencing of expressed sequence
tag clones, and identified two new TRs (TR2 and TR3) in humans and mice
(Fig. 1, A and B).
The deduced sequences of the three TR isozymes share >50% identity
and show conservation of all functional motifs, including two
active-site redox cysteines in the NH2-terminal region, the
NADPH- and FAD-binding residues, and a dimer interface domain (Fig. 1,
A and B). The 3'-untranslated region sequences of
all human and mouse TR isozyme mRNAs contain selenocysteine insertion sequence elements (data not shown) that are characteristic of
all mammalian selenoproteins (27). The TR isozymes are predicted to
contain a selenocysteine residue encoded by UGA in a conserved COOH-terminal GCUG sequence (U, selenocysteine) (Fig. 1, B
and C). For human proteins, TR3 displays 52-53% identity
with either TR1 or TR2, while the latter two proteins are 70%
identical. This suggests a closer evolutional link between TR1 and TR2,
while TR3 is more distantly related to TR1 and TR2.
Computer search analysis of the non-redundant data base with the human
cDNA sequence revealed matches to the previously determined genomic
DNA sequences (accession numbers AC000078, AC000080, AC000090) and the
complete human TR3 gene sequence was derived from these sequences. The
human TR3 gene spans ~65 kilobases in the DiGeorge region on
chromosome 22q11.2 and has an unusual gene structure. The 5'-end of
this gene, including 5'-untranslated region and the beginning of the
open reading frame, overlaps in different orientations with the
5'-untranslated region of the membrane-bound catechol
O-methyltransferase gene (data not shown). The physiological
significance of this overlap and the possible relevance of TR3 to
DiGeorge syndrome will require further studies.
Northern (RNA) hybridization and analysis of 75Se-labeled
mouse proteins suggested that TR1 and TR3 are expressed in a variety of
tissues, being especially abundant in liver, whereas TR2 is preferentially expressed in testis (Fig.
2, A and B). We
therefore directly purified to homogeneity, from
75Se-labeled mouse tissues, TR1 from mouse liver and
testis, TR2 from mouse testis, and TR3 from mouse liver (Fig.
2C). The identities of all TR isozymes were confirmed by
immunoblot analysis with isozyme-specific antibodies to TR1, TR2, and
TR3, and by comparison of the sequences of tryptic peptides with the
predicted TR1, TR2, and TR3 amino acid sequences (Fig. 1B).
In contrast to TR1 and TR3 which migrated as ~57-kDa species on
SDS-PAGE gels, TR2 had a higher, ~65-kDa, molecular mass (Fig.
2C) that is consistent with the coding region derived from
the cDNA sequence.
TR1 exists in the cytoplasm (28). TR3 has a putative mitochondrial
signal (the enrichment of arginines in the NH2-terminal structure that forms an Redox Regulation of Cell Signaling by Selenocysteine in
TR1--
On the basis of the redox properties of the selenocysteine in
TR1 (5-7, 9, 18, 19, 22, 23) and on the basis of conservation of
selenocysteine in mammalian TRs, we investigated whether oxidation of
selenocysteine in TR isozymes by ROS might contribute to redox
signaling. Specifically, we examined the effect of ROS on the redox
state of TR1, which we monitored by alkylating thiol and selenol groups
in the enzyme with 5-IAF and subsequently detecting
fluorescein-modified protein by immunoblot analysis (26). We initially
confirmed that purified mouse liver TR1 did not react with 5-IAF,
whereas the NADPH-reduced form of the enzyme was alkylated by this
compound (Fig. 3A). Thus,
changes in the fluorescein content of 5-IAF-treated TR1 reflect the
redox state of the enzyme.
With this approach, we further tested whether incubation of
NADPH-reduced TR1 with hydrogen peroxide leads to oxidation of the
enzyme. In the presence of 5-IAF, oxidation of the enzyme would be
reflected by the decreased level of fluorescein in immunoblots with
anti-fluorescein antibodies. We found that an increasing concentration
of hydrogen peroxide from 0 to 500 µM indeed results in
lowering the fluorescein signal (Fig. 3B, bottom
panel), while not affecting the protein level (Fig.
3B, upper panel) or the amount of
75Se present in the protein (Fig. 3B,
middle panel). This suggests that hydrogen
peroxide oxidized the thiol and/or selenol groups in the enzyme in the
presence of NADPH.
To assess intracellular changes in the redox state of TR1, we used
human epidermoid carcinoma A431 cells, which generate ROS (predominantly, hydrogen peroxide) in response to stimulation with EGF
(10). In these cells, ROS induce tyrosine phosphorylation of proteins
as a result of initial oxidation and subsequent
thioredoxin-dependent reduction of PTP1B (13). The
analogous reversible changes in the redox status of protein-disulfide
isomerase, a thioredoxin homolog and substrate for TR1, also occur in
EGF-stimulated cells (30). Exposure of A431 cells to EGF for up to 45 min resulted in a time-dependent oxidation of TR1 (Fig.
3C), indicating that oxidation of one or more thiol or
selenol groups in TR1 occurs in response to the generation of ROS in
EGF-treated cells. This is consistent with previous observation of
oxidation of the 55-kDa protein, which was suggested to be
thioredoxin reductase (30).
To identify the TR1 residues oxidized by ROS intracellularly, we
metabolically labeled A431 cells with 75Se, incubated the
labeled cell lysate with 5-IAF, and then isolated TR1. The purified
5-IAF-modified TR1 was then subjected to further alkylation with
iodoacetamide (to protect previously unmodified thiol or selenol
groups) followed by digestion with trypsin, after which the resulting
peptides were separated by reversed-phase HPLC. Analysis of fluorescein
and 75Se in the column fractions revealed the presence of
two 75Se-containing peptides but only one
fluorescein-containing peptide (Fig. 3D), the latter of
which co-eluted with the second 75Se-containing peptide.
Sequence analysis by tandem mass-spectrometry identified the
COOH-terminal selenocysteine-containing tryptic peptide in both
fractions containing 75Se. Only the second eluted
COOH-terminal tryptic peptide contained a fluorescein moiety, and the
only site of modification was the selenocysteine residue (Fig.
3E). These observations indicated that the selenol group in
TR1 is the only site of modification by 5-IAF, suggesting that
selenocysteine is the only residue of TR1 oxidized by ROS in A431 cells.
Prolonged (4 h) incubation of A431 cells with EGF resulted in an
~5-fold increase in the intracellular abundance of TR1, as revealed
by immunoblot analysis (Fig. 3F). The amount of TR1 was also
increased as a result of incubation of A431 cells with hydrogen peroxide. The TR1 present in cells after prolonged stimulation of cells
with EGF or H2O2 is most likely in a reduced
state, given that EGF- or H2O2-stimulated cells
exhibit a transient (<30 min) increase in ROS (10) and a corresponding
transient (<1 h) oxidation of PTP1B (13) and protein-disulfide
isomerase (30). The abundance of thioredoxin, the substrate of TR1, was
not affected by prolonged incubation of A431 cells with either EGF or
H2O2 (Fig. 3F).
The increase in the intracellular abundance of TR1 induced by prolonged
incubation of cells with EGF or H2O2 was also
apparent by labeling the cells with 75Se and subsequent
detection of labeled proteins by SDS-PAGE (Fig. 3G). The
latter approach (25, 31) also detected other major antioxidant
Se-containing enzymes revealing that the intracellular concentrations
of cytosolic glutathione peroxidase (GPX1) and phospholipid
hydroperoxide glutathione peroxidase (GPX4) were not substantially
affected by stimulation of cells with EGF or H2O2.
Incubation of A431 cells with DNCB mimicked the effect of EGF in that
it increased the expression of TR1 but not that of GPX1, GPX4, and the
recently discovered 15-kDa selenoprotein (32) (Fig. 3H).
DNCB specifically inhibits TR1 through irreversible modification of the
selenocysteine residue (8). Incubation of 293 cells with 20 µM DNCB for 30 min induces activation of ASK1 through
oxidation of the thioredoxin system, and TR1 inactivation by DNCB has
been implicated as a critical trigger for this process (12). Thus, ROS
and DNCB, which possess distinct chemical properties, both target
selenocysteine in TR1 and appear to induce oxidation of the thioredoxin
system and a subsequent increase in the expression of TR1. These data
are also consistent with a mechanism in which the intracellular ROS
pathway is regulated through the selenocysteine residue in TR1, rather
than through effects on thioredoxin or glutathione peroxidases.
Our results suggest a model for the role of TR in redox-regulated cell
signaling (Fig. 4). The intracellular
generation of ROS results in the direct oxidation of the selenol group
of TR (shown as a solid arrow in Fig. 4) and a
consequent transient decrease in enzyme activity. The resulting
oxidation of thioredoxin then affects thioredoxin-dependent
cellular components, including transcription factors (such as nuclear
factor
Additional characteristics of TR1 support a critical role for this
protein in redox signal transduction. Thus, TR1 is coupled with
Ca2+-phosphoinositide signaling pathways (34), and it
exhibits oncoprotein-like properties (35). Oncogene products often
regulate cell growth by simultaneously promoting cell proliferation and
sensitizing cells to apoptosis (36), both of which effects have been
demonstrated for TR1 and thioredoxin (37-42). Furthermore,
Caenorhabditis elegans contains two TR genes (Fig.
1C), one of which encodes a selenocysteine-containing enzyme
that is the major, if not the only, selenoprotein in C. elegans (43), whereas the other encodes a protein with cysteine in
place of selenocysteine. In contrast, the bacterial-type TR enzymes
(also present in plants and yeast) show limited homology to animal TRs
(5, 15) and lack the COOH-terminal selenocysteine-containing extension,
suggesting a different role for TR in redox regulation in bacteria. The
well-characterized bacterial redox signaling protein OxyR, a
transcription factor, is directly activated by H2O2 and is inhibited by glutaredoxin through
the thiol-disulfide exchange process (44).
Concluding Remarks--
Our demonstration that the selenocysteine
residue of TR1 senses the presence of ROS might explain the location of
this residue at the penultimate position of the COOH terminus of the
protein. Consistent with its sensory role, selenocysteine is conserved in the newly discovered TR2 and TR3. Selenocysteine is generally more
reactive than cysteine as a result of the ionization and redox
properties of selenium (20, 21). In addition to TRs, only 11 other
mammalian selenoproteins are known (20, 21, 45). Removal of either
selenocysteine tRNA or thioredoxin genes from the mouse genome results
in embryonic death (46, 47). Finally, whereas the TR-thioredoxin system
is important in signaling by ROS, a role of the glutathione system in
signaling remains to be demonstrated. Thus, the former rather than the
latter is possibly the major cellular redox signaling system.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-counter and by immunoblot assays. Rabbit antibodies specific
for TR1 were generated against two keyhole limpet hemocyanin-conjugated
peptides corresponding to residues 383-400 and 486-499 of human TR1,
with the exception that selenocysteine 498 and cysteine 497 were
replaced with serines. Rabbit antibodies specific for TR2 were
generated in response to a keyhole limpet hemocyanin-conjugated peptide corresponding to residues 560-579 of human TR2, with the exception that selenocysteine was replaced with cysteine. Chicken antibodies specific for TR3 were generated in response to a keyhole limpet hemocyanin-conjugated peptide corresponding to residues 502-520 of
human TR3.
-counter, and
two peaks containing 75Se were detected (Fig.
3D). The fractions containing 75Se (39 and 56)
were subjected to electrospray tandem mass-spectrometric analysis,
which detected the COOH-terminal peptide in fraction 39 and the
5-IAF-labeled COOH-terminal peptide in fraction 56.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Primary structures of mammalian TR
isozymes. A, schematic representation of the domain
organization of TR1, TR2, TR3, and glutathione reductase
(GR). These four proteins possess active center disulfides,
FAD (flavin adenine dinucleotide)- and NADPH (reduced form of
nicotinamide adenine dinucleotide phosphate)-binding domains, and other
features of the pyridine nucleotide-disulfide oxidoreductase family.
TR1, TR2, and TR3 also possess a COOH-terminal extension containing a
conserved GCUG tetrapeptide sequence. TR3 contains a short
NH2-terminal extension with a putative
mitochondrial-targeting signal. TR2 contains a long
NH2-terminal extension. B, alignment of the
deduced amino acid sequences of human TR1 (hTR1) (GenBank accession no.
S79851), mouse TR1 (mTR1, partial), hTR2 (partial), mTR2 (partial),
hTR3, and mTR3 sequences. Residues conserved between corresponding
human and mouse isozymes are highlighted in light gray, and those conserved among all mammalian TR isozymes are highlighted in dark gray. Sequences of internal tryptic peptides indicated by
boxes were experimentally verified by tandem
mass-spectrometry (hTR1 and mTR3) or Edman degradation (mTR1, mTR2, and
mTR3). The putative NH2-terminal mitochondrial signal in
hTR3 and mTR3 is underlined. Residue numbers for each
protein are shown on the right. C, alignment of
the COOH-terminal sequences of mammalian and C. elegans TR
enzymes. Human glutathione reductase, which is highly homologous to TR
but lacks the COOH-terminal extension, is shown for comparison.
Selenocysteine is replaced with cysteine in one form of C. elegans TR. Residues conserved among all TRs are
shaded. GenBank accession numbers are indicated on the
right. U, selenocysteine.
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Fig. 2.
Characterization of TR1, TR2, and TR3.
A, abundance of TR1, TR2, and TR3 mRNAs in various mouse
tissues. A mouse multiple-tissue Northern blot
(CLONTECH) was developed with cDNA probes
specific for mouse TR1, TR2, TR3, and actin mRNAs. The sizes of
transcripts (in kilobases) are shown on the right.
B, abundance of TR1 and TR2 proteins in various mouse
tissues. 75Se-labeled proteins in mouse tissues were
separated by SDS-PAGE and detected by PhosphorImager (Molecular
Dynamics) analysis. TR3 is less abundant than TR1 and is not apparent
in this figure. The 75Se band that appears in the liver
sample slightly above the position of testis TR2 is most likely not
TR2. The positions of glutathione peroxidases GPX1 and GPX4 and of the
15-kDa selenoprotein (which are the other major cellular
selenoproteins), in addition to those of TR1 and TR2, are indicated.
C, characterization of purified preparations of mouse liver
TR1, testis TR2, and liver TR3. The purified proteins were subjected to
SDS-PAGE and immunoblot analysis with antibodies specific for TR1
(top panel), for TR2 (second panel), or for TR3 (third panel);
75Se detection (for 75Se-labeled proteins) by
PhosphorImager analysis (fourth panel); or
Coomassie Blue staining (bottom panel). The
75Se-labeled band for TR2 is not clearly visible because
the protein was isolated from testis with a low 75Se
specific radioactivity. The positions of TR2 and of TR1 and TR3 are
indicated on the right. It is not clear why TR1 mRNA is
barely detectable in testis (see A), when the level of the
corresponding protein is high in this tissue (see B). It
should be noted that the Northern blots were carried out on a
commercial filter that had been stripped and rehybridized several times
and that the strains of mice used for mRNA and protein analysis
were different.
-helix) (Fig. 1, A and
B), suggesting that it may be a mitochondrial enzyme. The
experimentally determined NH2-terminal sequence of mouse
liver TR3 is GGQQSFDLLV, indicating the post-translational processing
of first 34 residues. This processing is consistent with the apparently
similar mobility of mouse liver TR1 and TR3 on SDS-PAGE gels.
Thioredoxin reductase has recently been isolated from mitochondria, and
the properties of the enzyme are different from those of TR1 (29). It
is possible that three mammalian TRs have related functions in
different cellular compartments.
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Fig. 3.
Role of TR1 in redox regulation of cell
signaling. A, alkylation of NADPH-reduced, but not of
oxidized, TR1 by 5-IAF. NADPH-reduced or oxidized TR1 was incubated for
10 min at 25 °C in the presence or absence of 20 µM
5-IAF, and was then subjected to immunoblot analysis with antibodies to
TR1 (upper panel) or to fluorescein
(lower panel). B, oxidation by
hydrogen peroxide of NADPH-reduced TR1 in vitro.
75Se-Labeled TR1 was reduced for 10 min with 100 µM NADPH, oxidized for 10 min with the indicated
concentration of H2O2, and then incubated for
10 min with 20 µM 5-IAF. The protein was then subjected
to SDS-PAGE and either immunoblot analysis with antibodies specific for
TR1 (top panel) or for fluorescein
(bottom panel), or to PhosphorImager analysis of
75Se (middle panel). C,
effect of stimulation of A431 cells with EGF on the redox state of TR1
in vivo. Serum-deprived A431 cells were incubated with EGF
(500 ng/ml) for the indicated times, after which cell lysates were
prepared under anaerobic conditions and incubated for 20 min with 20 µM 5-IAF. TR1 was then isolated from reacted cell lysates
using ADP-Sepharose and analyzed by immunoblot assay with antibodies to
either TR1 or fluorescein. D, co-elution of
75Se-labeled and 5-IAF-targeted tryptic peptides of TR1.
A431 cells were incubated with [75Se]selenite for 4 h, after which cell lysates were prepared under anaerobic conditions
and exposed for 20 min to 20 µM 5-IAF. TR1 was then
isolated from the lysates and digested with trypsin, and the resulting
peptides were fractionated by reversed-phase HPLC. Absorbance at 214 nm
(upper panel) and 440 nm (middle panel) was used to detect peptides and fluorescein,
respectively. The lower panel shows the
75Se radioactivity profile. E, specific
alkylation of selenocysteine in TR1 by 5-IAF. Fraction 56 from
D, containing both 75Se and fluorescein, was
subjected to mass spectrometry analysis to determine the mass of the
peptide, followed by amino acid sequencing by tandem mass spectrometry.
The experimentally determined mass for the 5-IAF-labeled peptide in
fraction 56, [M + 2H]2+ = 779.73, matched the predicted
mass for the COOH-terminal tryptic peptide of TR1 that is modified with
a single 5-IAF group (shown as circled F) and a
single iodoacetamide group (shown as circled A),
[M + 2H]2+ = 779.48. This double-charged ion was
completely fragmented and is not seen in this figure.
yn and bn are the ions in
which amino acid residues are sequentially cleaved from the
NH2-terminal and COOH-terminal, respectively. Importantly,
the y2 ion defines the selenocysteine-glycine COOH-terminal
dipeptide modified with 5-IAF, and the b10 ion defines the
SGASILQAGC peptide containing cysteine that is not modified with 5-IAF.
Neither the corresponding y2 ion containing unmodified
selenocysteine nor b10 ion containing a 5-IAF-modified
cysteine were detected. This suggests that selenocysteine was modified,
but cysteine was not, with 5-IAF. The predicted molecular masses for
fragment ions of types b and y are shown under the sequence, and ions
observed in the spectrum are underlined.
m/z, mass/charge ratio. F, effects of
stimulation of A431 cells with EGF or H2O2 on
the expression of TR1. Cells were incubated for 4 h in the absence
(control) or presence of EGF (500 ng/ml) or 0.2 mM H2O2, after which cell lysates
were subjected to immunoblot analysis with antibodies to TR1 or to
thioredoxin (Trx). G, effects of stimulation of
A431 cells with EGF or H2O2 on the abundance of
75Se-labeled TR1, GPX1, and GPX4. Cells were labeled with
[75Se]selenite in the absence (control) or presence of
EGF (500 ng/ml) or 0.2 mM H2O2 for
4 h, after which cell lysates were subjected to SDS-PAGE and
PhosphorImager analysis of 75Se-labeled proteins. TR3 is
less abundant than TR1 and is not apparent on this figure.
H, effect of DNCB on the intracellular abundance of TR1.
A431 cells were incubated for 4 h with
[75Se]selenite and the indicated concentrations of DNCB,
after which cell lysates were subjected to SDS-PAGE and PhosphorImager
analysis of 75Se-labeled proteins.
B (NF-B)) (33), protein-tyrosine phosphatases (such as
PTP1B), and antioxidant enzymes (such as thioredoxin peroxidase). In
addition, ROS may directly target other proteins such as thioredoxin,
phosphatases, and kinases with variable efficacy (shown as
dashed arrows in Fig. 4). The system is recycled
as a result of the subsequent increased expression of TR, and possibly
through the NADPH-dependent reduction of the enzyme after
removal of the ROS by antioxidant enzymes.
View larger version (23K):
[in a new window]
Fig. 4.
Model for the role of TR in redox regulation
of cell signaling. Trx, thioredoxin; RR,
ribonucleotide reductase; Tpx, thioredoxin peroxidase;
MAPK, mitogen-activated protein kinase; PTP,
protein-tyrosine phosphatase. See text for details.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. D. Kirby and W. Lane (Harvard Microchem, Cambridge, MA) for mass spectrometric analysis of the 5-IAF-modified peptide, Dr. G. Sarath (University of Nebraska, Lincoln) for sequencing by Edman degradation, and Dr. V. Factor (NCI, National Institutes of Health, Bethesda, MD) for help in obtaining 75Se-labeled mouse tissues.
| |
Addendum |
|---|
When this manuscript was ready for submission, two
papers were published that describe the cloning and characterization of rat TrxR2 (48) and human TR
(49). Based on sequences, these proteins
are apparently the same proteins as TR3. From our study, TR1 and TR2
sequences are evolutionally more related to each other than to TR3.
Therefore, we suggest naming the three thioredoxin reductases as
presented in this paper.
| |
FOOTNOTES |
|---|
* 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) AF171053 (mTR3), AF171054 (hTR3), and AF171055 (hTR2).
Present address: SmithKline Beecham Pharmaceuticals, King of
Prussia, PA 19406.
§§ To whom correspondence should be addressed. Fax: 402-472-7842; E-mail: vgladyshev1@unl.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: ROS, reactive oxygen species; TR, thioredoxin reductase; 5-IAF, 5-iodoacetamidofluorescein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PTP1B, protein-tyrosine phosphatase 1B; ASK1, apoptosis signal-regulating kinase 1; DNCB, 1-chloro-2,4-dinitrobenzene; EGF, epidermal growth factor; HPLC, high performance liquid chromatography; GPX1, glutathione peroxidase 1; GPX4, glutathione peroxidase 4; PAGE, polyacrylamide gel electrophoresis.
| |
REFERENCES |
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|
TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
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