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J. Biol. Chem., Vol. 278, Issue 35, 33408-33415, August 29, 2003
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From the
Department of Biochemistry,
¶Microchemical Facility, Emory University,
Atlanta, Georgia 30322 and ||Department of
Biochemistry and Molecular Biophysics, **Cancer
Center, University of Arizona, Tucson, Arizona 85721
Received for publication, October 30, 2002 , and in revised form, June 18, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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helix proximal to the active site, which formed under oxidizing
conditions. This non-active site disulfide was not a substrate for reduction
by thioredoxin reductase and delayed the reduction of the active site
disulfide by thioredoxin reductase. Within actively growing THP1 cells, most
of the active site of Trx1 was in the dithiol form, whereas the non-active
site was totally in the dithiol form. The addition of increasing
concentrations of diamide to these cells resulted in oxidation of the active
site at fairly low concentrations and oxidation of the non-active site at
higher concentrations. Taken together these results suggest that the
Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1
activity under conditions of redox signaling or oxidative stress, allowing
more time for the sensing and transmission of oxidative signals. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B, p53), controlling key aspects of cell
proliferation and survival
(1–4).
The active site of Trx1, WCGPC, is conserved among species from cyanobacteria
to humans (5). The active site
cysteines are readily accessible on the surface of the protein and become
oxidized to a disulfide upon reduction of a target protein. This disulfide is
cycled back to the dithiol by Trx reductase
(6).
Unlike Trxs from lower species, mammalian Trx1 contains additional conserved cysteine residues (at positions 62, 69, and 73 of human Trx1; See Fig. 1). Whether these non-active site Cys residues have biologic function is unknown. Cys-73 was present as an intermolecular disulfide bond (Trx1 homodimer) in x-ray crystal studies (7), suggesting a possible function for Cys-73. However, a mutant Trx1 bearing a serine at this position still appeared as a homodimer in the crystal structure, suggesting that Cys-73 was not essential for dimerization (7). More recently, S-glutathionylation of Trx1 at Cys-73 has been found during oxidative stress (8). In addition, S-nitrosylation of Cys-69 has recently been described (9).
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The midpoint potential (E0) for the active site dithiol of Trx is available for several lower species (10–14) but not for mammals. Equilibrium with NADPH in the presence of a catalytic amount of Trx reductase, where it is assumed that each mole of NADPH consumed translates into 1 mol of Trx reduced, indicated that Escherichia coli Trx had a midpoint potential of –270 mV (10, 11). An alternate approach using the equilibrium between glutathione (GSH) redox buffers and E. coli Trx yielded a similar value for E0 (15). In the latter approach, the relative amounts of reduced and oxidized Trx were determined by quantification of the number of thiols with Ellman's reagent (dithionitrobenzoic acid) because E. coli Trx contains only one redox-active pair of cysteines.
Oxidized and reduced forms of bovine Trx1 have been separated by carboxymethylation of thiols, native gel electrophoresis, and immunoblotting (16). The fully reduced (fully carboxymethylated) and fully oxidized forms of bovine Trx1 were identified, but intermediate bands on the immunoblot were only identified as "partially carboxymethylated" (16). In the present report we have used mass spectrometry to positively identify the forms of human Trx1 that are resolved by this native gel electrophoresis/Western blot (Redox Western blot) method. The results show that the resolved bands include fully reduced Trx1, a band with the active site in the disulfide form, and a band in which the protein has two disulfides, one at the active site and the other involving Cys-62 and Cys-69. The midpoint potential (E0) for the active site was found to be –230 mV. Using this value and redox Western blot analysis of THP1 cells, the steady state redox (Eh) of Trx1 was –280 mV, a value that is comparable with the Eh for GSH/GSSG in these cells (–259 mV). During oxidative stress induced by diamide, both the Trx1 active site and non-active site disulfides were observed. In vitro, the non-active site disulfide was found to inhibit the regeneration of the active form of Trx1 by thioredoxin reductase, supporting the interpretation that oxidation of this non-active site dithiol could provide a structural switch affecting Trx1 function during oxidative stress and redox signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Glutathione Redox Buffers—Redox buffers were prepared in a
100 mM potassium phosphate buffer, pH 7.0, containing 1
mM EDTA. All solutions were de-oxygenated by bubbling with
O2-free nitrogen (O2 < 0.5 ppm) for at least 2 h. The
GSH/GSSG redox potential was calculated according to the Nernst equation
(Eh = E0 + 2.3 x
RT/nF x log([GSSG]/[GSH]2), where
E0 = –240 mV at pH 7.0
(17), and n = 2 for
the 2-electron oxidation of 2GSH to GSSG). The following concentrations were
used to achieve the desired potentials (Eh, [GSH] and
[GSSG]): –150 mV, 6.5 and 43.5 mM; –180 mV, 18 and 32
mM; –210 mV, 36 and 14 mM; –240 mV, 50 and
2.5 mM; –270 mV, 50 and 0.25 mM; –300 mV, 50
and 0.025 mM. After incubation with Trx1 at room temperature for 2
h, samples were taken for the measurement of GSH and GSSG and for
determination of the redox state of Trx1. Preliminary studies showed that
equilibration periods from 15 min to 4 h yielded similar results, indicating
that equilibrium had been achieved. GSH and GSSG were quantified by HPLC as
S-carboxymethyl, N-dansyl derivatives relative to
-glutamylglutamate as an internal standard
(18).
Redox Western Blot Analysis—Separation of the redox forms of Trx1 was based upon the procedures of Holmgren and Fagerstedt (19) and Fernando et al. (16). Trx1 was carboxymethylated in guanidine-Tris solution (6 M guanidine-HCl, 50 mM Tris, pH 8.3, 3 mM EDTA, 0.5% (v/v) Triton X-100) containing 50 mM iodoacetic acid (IAA). After incubation at 37 °C for 30 min, excess IAA was removed by Sephadex chromatography (MicroSpin G-25 columns, Amersham Biosciences). Eluates were diluted in 5x sample buffer (0.1 M Tris-HCl, pH 6.8, 50% (v/v) glycerol, 0.05% (w/v) bromphenol blue) and separated on a discontinuous native polyacrylamide gel (5% stacking gel, 15% resolving gel). Gels were electroblotted to polyvinylidene difluoride membrane and probed for Trx1 using anti-Trx1 primary antibody (American Diagnostica, Greenwich, CT) and horseradish peroxidase-conjugated anti-goat immunoglobulin G secondary antibody followed by chemiluminescent detection (SuperSignal West Dura, Pierce) with x-ray film. Band intensities were determined by densitometric analysis of exposed film with Un-Scan-It software (Silk Scientific Corp.).
In-gel Digestion of Trx1—Coomassie-stained bands containing Trx1 were subjected to in-gel digestion with sequencing grade trypsin (Promega, Madison, WI), and the peptides were extracted as described (20). The extracted peptides were desalted using C18-ZipTip (Millipore, Bedford, MA) before analysis by mass spectrometry.
ESI-MS—Flow injection ESI-MS analysis of the peptides was performed on a model API3000 triple quadrupole mass spectrometer (PE-Sciex, Foster City, CA) equipped with MicroIonSpray electrospray source and operated in the positive mode of operation. Low energy collision-induced dissociation spectra of the peptides (ESI MS/MS) were performed to confirm peptide identity.
MALDI-TOF MS—Positive-ion MALDI-TOF MS analysis was
performed using model ReflexIII delayed-extraction MALDI-TOF mass spectrometer
(Bruker Daltonics, Billarica, MA) equipped with a 337-nm nitrogen laser;
-cyano-4-hydroxycinnamic acid (MALDI-Quality, Hewlett-Packard) was used
as the matrix.
Monobromobimane Labeling and Fluorescence Detection of Trx1— For some experiments samples were treated with 100 mM monobromobimane (mBBr, Molecular Probes, Eugene, OR) instead of IAA. Labeling was carried out in the guanidine-Tris solution for 30 min at 37 °C, and excess mBBr was removed with G-25 MicroSpin columns. The mBBr-labeled samples were digested with trypsin in 50 mM Tris acetate buffer, pH 8.5 (20 h at 35 °C), and then 10% aqueous trifluoroacetic acid (pH < 2) was added. The resulting peptides were separated by microbore reversed phase HPLC on an Applied Biosystems model 140A solvent delivery system using a C18 silica column (1 x 150 mm, Zorbax-SB300) with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The fluorescence of the effluent was monitored at an emission wavelength of 380 nm with an excitation wavelength of 290 nm. Eluted peaks were collected, and the identity of the peptide in each peak was established by Edman sequencing (Applied Biosystems, Foster City, CA).
Molecular Modeling—An energy minimization model for Trx1 with a disulfide bond between Cys-62 and Cys-69 was developed with the InsightII/Discover software package (Accelrys Inc., San Diego, CA). The starting coordinates were from the reduced human crystal structure, Protein Data Bank code 1ERT [PDB] . The model was first energy-minimized using the cvff parameter file without forming any disulfide linkages. The Cys-62–Cys-69 disulfide was then formed, and the model was further minimized. Ribbon drawings of the resulting models were prepared using MOLSCRIPT (21), BOBSCRIPT (22), and RASTER3D (23).
Site-directed Mutagenesis—Plasmid pcDNA3.1 encoding human Trx1 was a kind gift of Dr. Jiyang Cai of Emory University. Point mutations were introduced using the GeneEditor in vitro site-directed mutagenesis system of Promega. The C62S,C69S and C32S,C35S doubles mutants were generated by hybridization of the plasmid with oligonucleotides of the sequence 5'-GTGGATGACTCTCAGGATGTTGCTTCAGAGTCTGAAGTCAAATGC-3' and 5'-TTCTCAGCCACGTGGTCTGGGCCTTCCAAAATGATCAAGCC-3', respectively. Clones containing the desired mutations were selected by DNA sequencing.
Expression and Purification of Trx1 Mutants—For expression
in E. coli, Trx1 mutants were subcloned into pET-15b (Novagen) via
PCR amplification of the coding region of the pcDNA3.1/Trx1 mutants. A forward
primer encoding an NcoI restriction site and a reverse primer
encoding a 6x His tag followed by a stop codon and a BamHI
restriction site were used. Both the pET-15b vector and the PCR products were
digested with NcoI and BamHI and ligated overnight at 16
°C. Competent DL21-Gold(DE3) cells (Stratagene) were transformed with
pET/Trx1 plasmids. Expression of the mutant proteins was induced by 1
mM isopropyl-1-thio-
-D-galactopyranoside for 3 h,
and His-tagged proteins were purified under native conditions using nickel
nitrilotriacetic acid-agarose columns (Qiagen).
Reduction of Oxidized Thioredoxin by Thioredoxin Reductase and NADPH—Purified His-tagged Trx1(wt), Trx1(C32S,C35S), and Trx1(C62S,C69S) were oxidized with 1 mM H2O2 for 10 min at room temperature, then dialyzed overnight against two changes of Tris/EDTA buffer, pH 7.5 (reaction buffer). In a final volume of 0.5 ml of Tris/EDTA buffer, Trx1 mutants were reduced in the presence of 0.3 mM NADPH and 14 µM bovine thioredoxin reductase (American Diagnostica). Oxidation of NADPH was monitored by the change in absorbance at 340 nm (24).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Redox States of Trx1 after Equilibration with Redox Buffers—Purified Trx1 was equilibrated with 50 mM GSH/GSSG redox buffers over a range of potentials (–270 to –210 mV) likely to be found within cells (25, 26), carboxymethylated, and separated on a native gel as before (Fig. 4). The redox potentials of the redox buffers were verified by HPLC analysis of the GSH and GSSG content of the redox buffers at the beginning and the end of the equilibration period; these were found to be unchanged over the course of the equilibration (not shown). Experiments were also performed with GSH/GSSG redox buffers at 5 mM total concentration instead of 50 mM, and identical results were obtained. Because IAA may shift the thiol-disulfide equilibrium of proteins (27), experiments were performed to compare results obtained with 10 and 50 mM IAA. The results were not distinguishable; thus, the conditions used here (50 mM IAA) did not allow significant changes in Trx1 redox state during processing. At –270 mV, the predominant band co-migrated with the DTT-treated Trx1. MALDI-TOF analysis showed that this band (Band 3) contained only fully carboxymethylated Trx1 (see Table I). At –240 mV, Trx1 was distributed between Bands 2 and 3, and at –210 mV, Band 2 predominated. MALDI-TOF analysis showed that Band 2 contained both dithiol and disulfide forms of tryptic fragments T4 and T6, indicating that Band 2 is a mixture of two forms. One form contained a disulfide between Cys-32 and Cys-35 (active site) with Cys-62 and Cys-69 present as thiols, and the other form, which had a smaller signal, contained a disulfide between Cys-62 and Cys-69, with Cys-32 and Cys-35 present as thiols.
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Calculation of the Midpoint Potential (E0) of the Active Site (Cys-32–Cys-35) Disulfide of Human Trx1—Because immunoblotting yielded results comparable with those obtained with Coomassie staining of the different redox states of Trx1 (e.g. compare Figs. 2 and 4), redox state analyses were performed with the redox Western blot technique. Trx1 was equilibrated with redox buffers from –300 to –150 mV, and Bands 1, 2, and 3 were quantified by densitometry. E0 was calculated by fitting the data to the Nernst equation using the intensity of Band 3 for the reduced form of the active site and the total of Bands 1 and 2 as the corresponding disulfide (Fig. 5). The line represents the best fit to the Nernst equation, yielding a midpoint potential of –230 mV for n = 2.
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To provide an independent confirmation of the E0 of the active site, Trx1 was equilibrated at different redox potentials, labeled with the thiol-specific fluorescent probe mBBr, digested with trypsin, separated by C18 liquid chromatography, and detected by fluorescence (Fig. 6). Eluates corresponding to peaks of fluorescence were collected, and the identities of the peptides were established by Edman sequencing (Table II). Because monobromobimane labels thiols, but not disulfides, the redox-dependent change in fluorescence intensity of the peaks corresponding to peptides T4, T6, and T7 was reflective of the oxidation state of the thiols in each peptide. With this analysis half of the fluorescence of the active site peptide (T4) was lost at a potential of –225 mV. These results are in good agreement with the value of –230 mV from the redox Western blot approach and show that the E0 for the active site of human Trx1 is more positive than that for E. coli Trx, i.e. in the range of –230 mV rather than –270 mV (10, 11).
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Redox Dependence of the Non-active Site Disulfide—The mass spectrometry data showed that the oxidized form of Trx1 that moved as Band 1 on the redox Western blot contained two disulfide bonds. Band 1 was detected at potentials as low as –270 mV, and the intensity of Band 1 increased at –240 mV and –210 mV. However, there was an overall loss of signal and the appearance of a fourth (minor) band at redox potentials more oxidizing than –210 mV; approximately half of the total signal (Bands 1, 2, and 3) was lost at –150 mV (Fig. 3,Fig. 3). Thus, the E0 for the non-active site is more positive than –210 mV. The nature of the changes responsible for the loss of signal in the redox Western blot at potentials more positive than –210 mV are unclear.
Redox State of Endogenous Trx1 in Cells—To determine the redox state of intracellular Trx1, extracts from control and oxidant-exposed THP1 human monocytes were analyzed by redox Western blot (Fig. 7) and compared with the redox state of Trx1 at known redox potentials (Fig. 4). About 95% of the Trx1 from proliferating cells was in the fully reduced state, with some Band 2 and no fully oxidized Trx1 (Fig. 7, lane 1). This corresponded to an Eh value of –280 + 5 mV after correction for the effect of intracellular pH (7.4). By comparison, the redox state of glutathione in these cells was –259 + 3 mV (mean ± S.D. of three separate experiments). Upon exposure of the cells to diamide, there was a rapid, dose-dependent oxidation of the endogenous Trx1. The amount of the two-disulfide form of Trx1 (Band 1) went from undetectable to a maximum of 50% of the total Trx1 after 2 min of exposure to 3 mM diamide, the highest concentration tested. At this concentration, there was a 75-mV oxidation of the active site relative to the untreated controls (i.e. from –265 to –190 mV). Even the lowest dose of diamide tested (0.1 mM) caused a 20-mV oxidation of the active site (Fig. 7).
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Modeling of the Non-active Site Disulfide in the Trx1
Structure—The crystal structure of fully reduced human Trx1 is
known but not that of the Cys-62–Cys-69-oxidized structure. In the
reduced structure, the sulfhydryls for Cys-62 and Cys-69 lie partially buried
at opposite ends of a short -helix, about 10 Å apart. In this
conformation, the Cys-62–Cys-69 disulfide bond cannot form. However,
this helix is only loosely attached to the core of the protein through two
loops, and this helix in solution could dynamically pull away from the protein
core and sufficiently unravel to allow disulfide bond formation. We modeled
this possibility starting with the crystal coordinates for the fully reduced
protein (Protein Data Bank code 1ERT
[PDB]
), connecting Cys-62 and Cys-69 and
energy-minimizing. The resulting structure
(Fig. 8) supports the
hypothesis that local unfolding would allow the disulfide to form and
substantially alter the structure in this region proximal to the active
site.
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Reduction of Trx1 Mutants by Thioredoxin Reductase—Earlier
studies showed that fully oxidized mammalian Trx1 was much more slowly reduced
by thioredoxin reductase than was Trx1 that was oxidized only at the active
site (24,
28,
29). These results imply that
oxidation of the non-active site thiols could alter the rate of reduction by
thioredoxin reductase and thereby provide a redox mechanism for control of
Trx1 function. To determine whether the Cys-62–Cys-69 disulfide was
responsible for the altered reactivity with thioredoxin reductase, a mutant of
Trx1 bearing Cys 
Ser mutations at positions 62 and 69 was generated and
purified (C62S,C69S). Fig. 9
shows the reduction of wild type and mutant Trx1 by thioredoxin reductase and
NADPH according to the reaction NADPH + H+ + oxidized Trx1
NADP+ + reduced Trx1. As reported previously
(24,
28,
29), wild type Trx1 exhibited
an early lag phase during which it was slowly reduced followed by a period of
more rapid reduction. In contrast, C62S,C69S did not exhibit the lag period;
the rate of reduction was greatest initially and slowed only as the amount of
substrate (oxidized C62S,C69S) became limiting. The total amount of NADPH
oxidized was consistent with the interpretation that the mutant form contained
one less disulfide than the wild type Trx1.
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An oxidized mutant of Trx1 in which the active site was mutated (C32S,C35S)
was not a substrate for thioredoxin reductase
(Fig. 9). However, upon the
addition of a small amount of wild type Trx1
(
of the amount of mutant
Trx1 on a molar basis), 1 mol of NADPH was reduced per mol of the active site
mutant. These results show that there was one disulfide in this mutant without
active site thiols and that this non-active site disulfide was reduced by wild
type Trx1. Thus, the non-active site disulfide is a substrate for reduction by
the active site of Trx1, and the active site is, in turn, reduced by
thioredoxin reductase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 helix, and Cys-35 is in the linker
region between the 2 and 3 helices
(30). The
E0 of the active site dithiol of Trx1 is –270 mV in
E. coli (10,
11), –240 mV in yeast
(12), and –230 mV in
bacteriophage T4 (13). The
present data show that the active site of human Trx1 has an
E0 of –230 mV, i.e. at the higher end of
the range of known values. The redox state of endogenous E. coli Trx
has been estimated to be 30–60% reduced
(19,
31), although a more recent
report has suggested that E. coli Trx1 is up to 90% reduced
(32). In mammalian systems,
bovine Trx1 was completely reduced in endothelial cells
(16), and human Trx1 was 90%
reduced in MCF-7 cells (33)
and in HaCaT keratinocytes
(34). Thus, our results show
that human Trx1, like bovine Trx1, is maintained in a more reduced state
within cells than is E. coli Trx1 and that the redox Western blot
yields an estimate of the redox state of cellular Trx1 that agrees well with
previous estimates.
In addition to the active site cysteines, mammalian Trx1 contains three
additional cysteines that are not found in thioredoxins from other species or
in mammalian mitochondrial Trx2. Cys-62 and Cys-69 are within the 3
helix, and Cys-73 is on a hydrophobic patch on the surface of the protein (see
Fig. 8). The data presented
here show that a disulfide bond can form between Cys-62 and Cys-69. In
vitro analysis of purified proteins demonstrated that oxidized forms of
human (35), bovine
(24), and rat
(28) Trx1 contained no
detectable thiols as measured by reduction of dithionitrobenzoic acid
(24,
35), supporting the
interpretation that the non-active site thiols of mammalian Trx1 are subject
to oxidation. However, oxidation of Cys-62 and Cys-69 was not seen in the
crystal structure of oxidized human Trx1
(7). This could have resulted
from the method of sample preparation because the crystals of oxidized Trx1
were obtained by air oxidation of the reduced form. The physical constraints
within the crystallized reduced protein could have prevented the formation of
the second disulfide upon air oxidation
(7). Solution structures of
oxidized and reduced human Trx1 are available
(36) but do not provide
information on the non-active site disulfide because a mutant of Trx1 was used
in which all three non-active site cysteines were replaced with alanines.
Thus, the present study provides the novel and potentially important finding
that non-active site cysteines form a disulfide upon mildly oxidizing
conditions.
A model for the Cys-62–Cys-69 linkage
(Fig. 8) displays local
unfolding of the intervening helix. A likely mechanism for this disulfide
formation would involve local unfolding before bond formation. The helix is
attached to the rest of the protein through two loops, consistent with the
possibility of helix dynamics. The temperature factors for the helix in the
x-ray structure, which is a measure of its disorder in the crystal, are about
average for the structure (14.7 Å2 for the main-chain atoms
versus 14 Å2 overall). However, movement of the
helix is blocked in the dimer found in the crystal and so does not reflect its
unrestricted dynamics. The loops connecting the helix are not restricted in
the crystal and have higher temperature factors. Therefore, the possibility of
disulfide bond formation between the Cys residues of the 3 helix is
supported by the crystal structure.
The formation of a disulfide between Cys-62 and Cys-69 is predicted to have
a profound effect by disrupting the helical structure of the 3 helix of
Trx1 (see Fig. 8). In
co-crystallization experiments, amino acids within this helix were shown to
come in contact with Ref-1 and NF-B peptides
(37,
38), two substrates for Trx1.
Thioredoxin reductase most likely contacts Trx1 in this region as well; when
Trx1 was in the homodimer form, stabilized by an intermolecular
Cys-73–Cys-73' disulfide bond in close proximity to the
3-helix, the active site was inaccessible to thioredoxin reductase
(29). The biphasic reduction
of the two-disulfide form of Trx1 (see
Fig. 9) provides further
support for the interpretation that the interaction between Trx1 and
thioredoxin reductase is sensitive to the presence of a non-active site
disulfide in the 3-helix. A mutant of Trx1 (C62S,C69S) that was unable
to form the Cys-62–Cys-69 disulfide was not inactivated as a substrate
for thioredoxin reductase by extensive oxidation, highlighting the importance
of these two residues. It should be noted that this mutant still contained
Cys-73 and was, thus, still capable of forming disulfide-bonded homodimers
(29,
35). Therefore, dimerization
was probably not responsible for the early lag phase in the kinetics of
reduction by thioredoxin reductase under the conditions used here.
The presence of the non-active site disulfide decreased the rate at which the active site of Trx1 was regenerated by thioredoxin reductase. Because the midpoint potential of the non-active site dithiol is more negative than that of the active site, formation of this non-active site disulfide is more likely to occur under conditions in which the active site is also oxidized (and therefore inactive). Thus, the non-active site disulfide may provide a mechanism by which Trx1 can be temporarily inactivated and made resistant to regeneration by thioredoxin reductase. This would allow time for redox-dependent signaling processes to occur. Signaling processes that would benefit from the inactivation of thioredoxin include activation of the transcription factor Nrf2 via dissociation from Keap1 in the cytoplasm (39) and loss of inhibition of the pro-apoptotic kinase apoptosis signal-regulating kinase 1 (40).
In conclusion, E0 for the active site of human Trx1 is
–230 mV, among the highest of Trx proteins characterized to date. In
addition to oxidation at the active site, human Trx1 also forms a second
disulfide under relatively mild oxidizing conditions. Although the redox state
of the active site is regulated by thioredoxin reductase, both Trx1 disulfides
are at redox potentials within the range of the intracellular GSH/GSSG redox
buffer. The location of the non-active site disulfide within the protein
suggests a number of possible functions, such as substrate recognition
(NF-B), the regulation of other proteins through redox-sensitive
binding to Trx1 (apoptosis signal-regulating kinase 1), and regulation of
activity through interactions with thioredoxin reductase, as demonstrated in
the current report. Thus, reversible oxidation of the conserved non-active
site Cys residues of mammalian Trx1 may play an important regulatory role in
the function of Trx1.
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FOOTNOTES |
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To whom correspondence should be addressed: Emory University School of
Medicine, Dept. of Biochemistry, Rollins Research Center, 1510 Clifton Rd.,
Atlanta, GA 30322. Tel.: 404-727-5984; Fax: 404-727-3231; E-mail:
wwatson{at}emory.edu.
1 The abbreviations used are: Trx1, human thioredoxin-1; GSH, reduced
glutathione; GSSG, oxidized glutathione; DTT, dithiothreitol; HPLC, high
pressure liquid chromatography; ESI-MS, electrospray ionization triple
quadrupole mass spectrometry; MALDI-TOF MS, matrix assisted laser desorption
ionization time-of-flight mass spectrometry; IAA, iodoacetic acid; mBBr,
monobromobimane.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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