THE JOWAL OF BIOLOGICAL C~~harsrrn B 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 16, Issue of April 22, pp. 11714-11720,1994 Printed in U.SA.

Site-directed Mutagenesis of Active Site Cysteines in Human Thioredoxin Produces Competitive Inhibitors of Human Thioredoxin Reductase and Elimination of Mitogenic Properties of Thioredoxin*

(Received for publication, August 6, 1993, and in revised form, January 14, 1994)

John E. Oblong$, Margareta Berggren, Pamela Y. Gasdaska, and Garth Powis From the Arizona Cancer Center, Tucson, Arizona 85724

Thioredoxin and thioredoxin reductase comprise a re- dox system ubiquitous in all organisms. To better under- stand the thiol chemistry of the mammalian thiore- doxin-thioredoxin reductase redox system, mutants of human thioredoxin were produced by site-directed mu- tagenesis in which the two active site cysteines were replaced by serine residues, individually (C32S and C3SS) and in combination (C32S/CSSS). CSSS and C32S/ CSSS were found to be competitive inhibitors of the re- duction of human thioredoxin by human thioredoxin re- ductase with Ki values of 1.8 and 6.7 p ~ , respectively. C32S did not inhibit thioredoxin reductase, apparently due to aggregation of the oxidized C32S species. Exami- nation of the three mutant forms of thioredoxin by cir- cular dichroism spectroscopy indicated that there were significant differences in the secondary structures when compared with thioredoxin. There were detecta- ble changes in the circular dichroism spectra when thi- oredoxin, CSSS, and C32S/CSSS were bound to thiore- doxin reductase, whereas C32S with thioredoxin reductase underwent only a small spectral change. Re- combinant human thioredoxin stimulated DNA synthe- sis and the proliferation of murine fibroblasts. The abil- ity of thioredoxin to stimulate cell proliferation could not be duplicated by either dithiothreitol or glutathi- one. C32S, CSSS, and C32S/CSSS failed to stimulate cell proliferation, showing that the redox active form of thi- oredoxin is necessary for eliciting growth stimulation.

Regulation of the intracellular redox state is critical for both cell viability and proliferation. The ability to maintain the in- tracellular redox environment is dependent on systems that utilize reducing equivalents (e.g. from NADPH) to reduce pep- tide thiols and include the ubiquitous thioredoxidthioredoxin reductase (Trx.TR)l redox system (for a review see Holmgren (1985)). Human Trx is an 11.5-kDa protein with 27% amino acid identity with Escherichia coli Trx (Gasdaska et al . , 1993). The active site of Trx is highly conserved among bacterial, plant, and mammalian forms of the protein and comprises the consensus sequence Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys, con- taining two vicinal half-cysteines that undergo reduction when

* This work was supported by National Institutes of Health Grant CA 42286 (to G. P.) and by National Institutes of Health Training Grant in Cancer Biology CA 09213 (to J. E. 0.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked sa‘aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ter, 1515 North Campbell Ave., Tucson, AZ 85724. $ To whom correspondence should be addressed: Arizona Cancer Cen-

dudase; Dm, dithiothreitol; DMEM, Dulbecco’s modified Eagle’s me- The abbreviations used are: “x, thioredoxin; TR, thioredeoxin re-

dium; PDGF, platelet-derived growth factor.

Trx is reduced by the NADPH-dependent flavoenzyme TR (EC 1.6.4.5) (Holmgren, 1985). Two half-reactions comprise the ac- tual sequence of catalytic events of Trx reduction by TR in which the reduction of the FAD prosthetic group of TR by NADPH and electron transfer to active site cysteines in TR occurs first. The second half-reaction is the reduction of bound oxidized Trx by TR. Rat liver TR (Luthman and Holmgren, 1982) and human placental TR (Oblong et al., 1993) have been purified as homodimeric complexes, comprised of 58- and 60- kDa subunits, respectively. Human TR was found to be a rela- tively stable protein with a tightly bound FAD prosthetic group (Oblong et al . , 1993).

Bacterial and mammalian Trxs have been shown to be in- volved in vitro in the reduction of such proteins as ribonucle- otide reductase, methionine sulfoxide reductase, and insulin (Laurent et al . , 1964; Porque et al., 1970; Luthman and Holmgren, 1982). The TrxsTR redox system also may function as a protective cellular mechanism against damaging oxidizing species (Mitsui et al., 1992). Trx catalyzes the in vitro folding of proteins by a mechanism similar to that catalyzed by protein disulfide isomerase (Lundstrom and Holmgren, 1990). Com- parison of the sequence of protein disulfide isomerase with Trx reveals two highly conserved Trx-like active site domains in protein disulfide isomerase with the consensus sequence Trp- Cys-Gly-His-Cys-Lys, the only difference being a proline to his- tidine substitution in protein disulfide isomerase (for a review see Noiva and Lennarz (1992)). Mutation of proline to histidine in the active site of E. coli Trx resulted in an increase in protein disulfide isomerase-like activity and, surprisingly, an increased affinity for bacterial and mammalian TR when compared with wild-type Trx (Krause et al . , 1991; Lundstrom et al . , 1992). Furthermore, a recombinant form of adult T-cell leukemia-de- rived factor, a mammalian protein later found to be identical to human Trx (Deiss and Kimchi, 1991; Gasdaska et al., 1994), was shown to be functional in refolding denatured RNase A (Tagaya et al., 1989).

Another function for Trx is the redox regulation of gene ex- pression through interaction with transcription factors. For example, the Trx.TR complex has been implicated in the reduc- tion of the nuclear redox factor, Ref-1, which in turn modulates the active state of the transcription factor AP-1 (Abate et al., 1990). This is supported by the findings that mutation of Cys- 154 in the Fos subunit of AP-1 results in a loss of redox regu- lation of AP-1 with a concomitant increase in specific DNA binding as well as the transforming capability of Fos (Abate et al., 1990; Okuno et al., 1993). Furthermore, addition of Trx, TR, and NADPH to nuclear extracts stimulates AP-1-specific DNA binding (Abate et al . , 1990). Similar observations of redox con- trol by Trx have been made with the transcription factors TFIIIC (Cromlish and Roeder, 1989), BZWl (Bannister et al., 1991), and NF”KB (Matthews et al., 1992), as well as with

11714

Characterization of Thioredoxin by Site-directed Mutagenesis 11715 steroid receptors (Grippo et al., 1983; Peleg et al., 1989).

Recombinant human Trx is a substrate for human TR (Ob- long et al., 1993). We now report the results of a mutagenesis study of the two active site cysteine residues in human Trx. Three mutants of Tm (C32S, C35S, and C32SIC35S) were gen- erated by site-directed mutagenesis. Both C35S and C32Sl C35S were found to be competitive inhibitors of T R , and CD spectroscopy analyses of the formation of TR complexes with Trx confirmed that Trx, C35S, and C32SlC35S interact with TR. Recombinant Tm was found to stimulate both cellular pro- liferation and DNA synthesis of murine fibroblasts. None of the thioredoxin mutants could stimulate cellular proliferation, sug- gesting that the redox active catalytic site of Tm is essential for the mitogenic effect.

EXPERIMENTAL PROCEDURES Site-directed Mutagenesis-Single-stranded DNA of the sense strand

of the human Trx cDNA (Gasdaska et al., 1993) ligated into the pBlue- script SK vector (Stratagene, La Jolla, CA) was isolated by polyethylene glycol precipitation using Arg-408 as helper phage. The isolated single- stranded DNA was used in the oligonucleotide-directed in vitro Mu- tagenesis System version 2.1 (Amersham, United Kingdom) using the oligonucleotides 5'-GCAAGGCCCAGACCACGTGGC-3',5'-GATCA"IT- T@2AAGGCCCACA-3', and 5'-GATCATlTTwAAGGCCCAGAC- CACGTGGC-3' to generate C32S, C35S, and C32S/C35S, respectively. The isolated constructs were confirmed by dideoxy sequencing of base- denatured double-stranded DNA using the Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Corp.). To facilitate cloning into the PET-3a expression vector (Studier et al., 19901, novel NdeI and EamHI sites were introduced at the 5' and 3' ends, respectively, of the Trx cDNA sequence by oligonucleotide-directed polymerase chain reaction mutagenesis. The 240-base pair NdeI-EamHI fragments containing the mutated active sites were isolated from agarose gels and ligated into NdeI-EamHI-digested PET-3a vector (Sambrook et al., 1989). Mutant Constructs were transformed into E. coli BL21 cells and confirmed by dideoxy sequencing of base-denatured double-stranded DNA as above.

Expression and Purification of Recombinant n x Variants"BL21 cells containing the pET3a::Trx variant constructs were grown to mid- log phase and then induced with 5 m~ isopropyl-l-thio-p-o-galactopy- ranoside for an additional 3 h. Cells were washed with 0.9% NaCl solution, pelleted, and resuspended in 50 m~ Tris-HCl,30 rn NaCl, 5 m~ DTT (pH 7.5). The cell suspension was sonicated and cellular debris removed by centrifugation a t 15,000 x g. The supernatant was applied to a DEAE ion exchange column (3 x 11 cm), and bound protein was eluted with a 200-ml M O O x" NaCl gradient in 50 m~ Tris-HC1,l rn EDTA (pH 7.5) (solution A). Fractions containing recombinant protein were pooled, precipitated with 85% ammonium sulfate, reduced with excess DTT, and chromatographed on a Superose 12 HR 10/30 gel fil- tration column (Pharmacia LKB Biotechnology Inc.) in solution A con- taining 200 m~ NaCl. Purification of recombinant wild-type and mutant proteins were confirmed by N-terminal sequencing of the first 6 resi- dues (Biotechnology Resource Facilities, The University of Arizona). Analysis of recombinant proteins on immunoblots with anti-human Trx antisera was performed using techniques described in Harlow and Lane (1988). Molecular weight markers for SDS-polyacrylamide gel electro- phoresis were purchased from Life Technologies, Inc.

Kinetic Analysis of DPTR Reduction-Human TR was purified from placental tissue as described previously (Oblonget al., 1993). An insulin reduction assay (Luthman and Holmgren, 1982), which monitors the oxidation of NADPH at 340 nm by TR in the presence of insulin and Trx, was used to show that purified recombinant Trx was enzymatically active. Briefly, 250 pl of 100 m~ potassium phosphate buffer (pH 7.0), 1 m~ EDTA, and 0.2 mg/ml bovine serum albumin buffer were mixed with

3 pl of NADPH (200 w), 30 pl of bovine insulin (80 w), 5 pl of TR (2.5 w), and 15 pl of Trx (2.5 p ~ ) to a final volume of 300 pl. Initial enzyme rates were monitored over a 3-min period. Specific activity was calcu- lated as described in Luthman and Holmgren (1982). For competition studies, the mutants at final concentrations of 3 and 5 p~ were added to the assay mixtures containing varying concentrations of Trx. The ki- netic constants were determined from Lineweaver-Burk double-recip- rocal plots, and the K, values were calculated from the equation,

K, = Km(l + i/Ki) (Eq. 1)

where K, represents the new K, value determined in the presence of a specific concentration of inhibitor (i) in the insulin reduction assay.

Circular Dichroism Spectroscopy-A stoppered 1-cm path length quartz cuvette was used in an Aviv model 60DS spectropolarimeter. All protein samples were dialyzed exhaustively against 5 m~ potassium phosphate (pH 7.0), and protein levels were determined using a modi- fied Lowry assay (Peterson, 1977). Scans were collected at 0.5-nm in- tervals with a bandwidth of 1.0 nm at a constant temperature of 25 "C. The dynode voltage remained below 500 V with the exception of the TR sample reduced with 20 p NADPH in which the voltage remained below 650 V. A total of three scans was signal-averaged and smoothed, from which a buffer blank scan of 5 m~ potassium phosphate (pH 7.0) was subtraded to generate the final CD spectra. The estimated per- centages of secondary structure were calculated from the CD spectra with the program PROSEC (PROtein SECondary structure estimator v2.1, Aviv) based upon the method of Chang et al. (1978).

Fluorescence Spectroscopy-Purified samples of all four Trx variants and E. coli Trx purchased from Calbiochem were dialyzed into 100 m~ potassium phosphate (pH 7.0) at concentrations of 400 pg/ml (33 p ~ ) . All fluorescence measurements were performed in a temperature-con- trolled stirred cuvette unit housed in an SLM8000C spectrofluorimeter (SLM-AMINCO, Urbana, IL) at 4 "C, using 4-nm bandpass slits and an external rhodamine standard as a reference. Following an initial scan, 1 pl of DIT was added to the 150-pl sample to give a final concentration of 60 p DIT and the sample was rescanned.

Thymidine Incorporation and Cell Growth Stimulation Stud&- Incorporation of radiolabeled thymidine into DNA was measured as described by Adams (1969). Briefly, Swiss murine 3T3 fibroblast cells (4 x lo4) were allowed to attach in 35-mm culture dishes with 2 ml of DMEM containing 10% fetal calf serum for 4 h, washed with DMEM, and grown in DMEM containing 0.5% fetal calf serum for 48 h. To each culture was added either D'IT (3 p ~ ) , PDGF (20 ng/ml), or varying concentrations of Trx with DIT (3 p ~ ) , and the culture was allowed to incubate for 12 h. One pCi of L3H]thymidine (specific activity, 2 Cilmmol, ICN Radiochemicals, Irvine, CA) was added, and cells were allowed to grow for an additional 12 h, aRer which cells were washed two times with phosphate-bdered saline solution and precipitated with 10% tri- chloroacetic acid. Precipitated material was scraped from culture dishes and washed with 5% trichloroacetic acid. The precipitants were pelleted by centrifugation, rewashed with 5% trichloroacetic acid, recentrifuged, and pellets were then solubilized with 10% SDS. The levels of radioac- tivity were measured by liquid scintillation using EcoLume as liquid scintillant (ICN, Costa Mesa, CA). Cell proliferation was measured by the increase in cell number relative to control cultures with time. Swiss murine 3T3 fibroblast cells (5-10 x 10') were allowed to attach in 35-mm culture dishes with 2 ml of DMEM containing 10% fetal calf serum for 4 h, washed with DMEM, and grown in DMEM containing 0.5% fetal calf serum for 24 h. To each culture was added DIT (3 p ~ ) , PDGF, or varying concentrations of Trx variants, and incubation was allowed for the indicated times. Trx and mutant Trx samples were reduced with a 3-fold excess of DTT prior to addition to the cultures for a maximal final concentration in the culture of 3 p~ DTT. Cell numbers were determined using a hemacytometer following detachment of the cells with 0.025% trypsin.

TABLE I Active site amino acid seqllenees of human thioredoxin variants

The underlined residues indicate the two active site cysteines that were mutated to serine residues in the respective mutant.

Trx C32S c35s c 3 2 s c 3 5 s

2 8 41 I I

Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-Met-Ile-Asn-Pro-Phe Ser-Ala-Thr-Trp-Ser-Gly-Pro-Cys-Lys-Met-Ile-Asn-Pro-Phe Ser-Ala-Thr-Trp-Cys-Gly-Pro-Ser-Lys-Met-Ile-Asn-Pro-Phe Ser-Ala-Thr-Trp-Ser-Glv-Pro-Ser-Lvs-Met-Ile-Asn-Pro-Phe

- - - - - -

11716 Characterization of Thioredoxin by Site-directed Mutagenesis

RESULTS

Biochemical Characterization of Variant Forms of fix-The two cysteines at positions 32 and 35 of Trx relative to the N terminus were mutated to serine residues, individually (C32S and C35S) and in combination (C32SlC35S) (Table I), and the recombinant forms of Trx were expressed in E. coli. It is im- portant to note that the residue numbers correspond to the amino acid sequence deduced from the cDNA clone and in- clude an initiator methionine residue. Expression of the re- combinant mutants did not result in any apparent decrease in either bacterial cell viability or levels of protein expression in comparison with Trx. Upon final purification, the yield of re- combinant wild-type and mutant protein averaged 3 mg of proteiditer of culture. All of the recombinant proteins gave a single 11.5-kDa band as seen by Coomassie Blue staining on a 15% SDS-polyacrylamide gel. Rabbit polyclonal antiserum raised against native recombinant human Trx (Oblong et al., 1993) recognized wild-type Trx and all three mutants with relatively equivalent cross-reactivity on immunoblots of dena- tured proteins (data not shown). Recombinant human Trx has been previously isolated as a mixture of species either con- taining or lacking the initiator methionine (Forman-Kay et al., 1989). N-terminal sequencing of the purified variant forms of recombinant human Trx revealed that the initiator methi- onines from all four variants were proteolytically removed by the bacterial host system.

Purified human TR with NADPH as a cofactor has been shown to reduce a recombinant form of human Trx that, in turn, could reduce insulin, and the K,,, values of human recom- binant Trx and E. coli Trx for human TR were 4.3 and 20 p ~ , respectively (Oblong et al., 1993). A final concentration of up to 50 of C32S, C35S, and C32SlC35S with human TR and insulin as the final electron acceptor showed no detectable oxi- dation of NADPH. These results indicate that none of the mu- tants are substrates for reduction by TR. Competition studies between Tnr and the mutant forms of Trx for reduction by TR measured by NADPH oxidation indicated that both C35S and C32SlC35S were competitive inhibitors of TR for Trx reduction, with K, values of 1.8 and 6.7 p ~ , respectively (Fig. 1). In con- trast, C32S did not inhibit Trx reduction by TR. A possible explanation for the failure of C32S to inhibit TR reduction is the aggregation of C32S. Oxidized C32S eluted as a higher molecular weight species from a gel filtration column compared with Trx, C35S, and C32SlC35S (Fig. 2 A , comparing lanes 2 and 3 with lanes 4 and 5) . However, when the same oxidized C32S sample was incubated with D m , reduced C32S eluted from the gel filtration column in the same volume as oxidized Trx, C35S, and C32SlC35S (Fig. 2B, lanes 4 and 5 ) . A reduced sample of Trx, C35S, and C32SlC35S eluted from the gel filtra- tion column with the same volume as the oxidized species (data not shown).

Circular Dichroism Spectroscopy of Variant Forms of f i x - Examination of Trx, C32S, C35S, and C32SlC35S by far-UV CD spectroscopy showed differences in the recorded spectra of all four variants (Fig. 3A). One of the more obvious spectral changes was the marked decrease in the ellipticity of C35S (scan C ) and C32SlC35S (scan D ) near 205 and 208 nm, re- spectively, compared with the CD spectra of Trx (scan A). The best estimate of the fractional components of the secondary structures of all four forms of Trx are presented in Table 11. It is relevant to note that the spectra of the oxidized and reduced form of human Trx indicated only minor secondary structural changes, as has been previously reported (Dyson et al., 1990; Forman-Kay et al., 1991). To determine if the CD spectrum of C32S was representative of the secondary structure of aggre- gated protein, oxidized C32S (Fig. 3B, scan A ) was reduced

A '1 ccb̂ d n z Y

d

B 6,

1

0.0 0.1 0.2 0.3

-1 .J FIG. 1. Competition studies of thioredoxin with thioredoxin

mutants. Purified wild-type and mutant forms of Tm were incubated with purified TR in the insulin reduction assay as described under "Experimental Procedures." Panel A, Lineweaver-Burk plots of NADPH reduction in the absence (closed circles) and presence of 3 and 5 p! C35S (open circles and squares, respectively) in the insulin reduction assay with TR and varying concentrations of T m . Panel B, Lineweaver- Burk plots of NADPH reduction in the absence (closed circles) and presence of 3 and 5 p~ C32WC35S (open circles and squares, respec- tively) in the insulin reduction assay with TR and varying concentra- tions of TR. The abscissa ( I I S ) in both panels represents the reciprocal concentrations of thioredoxin in micromolar concentrations, and the ordinate ( I /V) represents the reciprocal velocity of the insulin reduction assay calculated as micromoles of NADPH oxidized per min per reaction as described under "Experimental Procedures."

with an excess of DTT and a CD spectrum recorded (Fig. 3B, scan B ) . The difference between the scans of the oxidized and reduced forms of C32S indicated only minor changes in the protein secondary structure (Fig. 3B, scan C ) .

Changes in protein secondary structure upon substrate-re- ductase binding revealed by CD spectroscopy have been previ- ously reported for the adrenodoxin-adrenodoxin reductase re- dox system (Kimura et al., 1982). We examined changes in protein secondary structure during complex formation between Tnr and TR in the presence of NADPH by far-UV CD spectros- copy (Fig. 4A). CD spectra of C32S, C35S, and C32SlC35S with

Characterization of Thioredoxin by Site-directed Mutagenesis 11717

A Trx

C32S

c35s

C32Sl c35s

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 kDa

- 18 - 14

I

""-

- 18 - 14

- 18 - 14

- 18 - 14

15 18 21 24 27 30

(mW I

15 18 21 24 27 30

(mls) Frc. 2. Gel filtration chromatography of variant forms of hu-

man thioredoxin. Panel A, oxidized forms of purified Trx, C32S, C35S, and C32SIC35S were individually chromatographed on a Superose 12 HR 10/30 gel filtration column. Aliquots (50 pl) from alternating frac- tions (500 p1) were electrophoresed on 15% SDS-polyacrylamide gels and stained with Coomassie Blue. Panel B, a sample of oxidized C32S similar to the sample used in panel A was reduced with excess D'M' and chromatographed, and aliquots were analyzed as above. The collected volumes (mls) are indicated in the brackets below each panel, and the relative positions of molecular weight markers (kDa) are indicated to the right.

"R in the presence of NADPH were also recorded (Fig. 4, B-D, respectively). The results of these scans indicate that during complex formation of Trx, C35S, and C32S/C35S with TR, changes in protein secondary structure occurred (scan D in Fig. 4, A, C, and D , respectively). In contrast, C32S and TR elicited a much smaller CD spectral difference (Fig. 4B, scan D ) .

Fluorescence Spectroscopy of Variant Forms of IIZz"The fluo- rescence emission profile of bacterial Trx has been reported to show a 2.5-fold increased quantum yield upon reduction (Stryer et al., 1967). E. coli "rx contains 2 tryptophan residues a t positions 28 and 31 whereas human Trx contains only 1 tryp- tophan residue a t position 31. Removal of "p-31 in E. coli Trx by mutagenesis produced a mutant Trx that retained the prop- erty of increased quantum yield upon reduction (Krause and

d

195 210 22!i 240

wavelength (am)

I

195 210 225 240 wavelength (am)

ants. Panel A, purified Trx, C32S, C35S, and C32SlC35S (24 pglml) FIG. 3. Far-UV circular dichroism spectra of thioredoxin vari-'

samples collected from a final Superose 12 gel filtration column were dialyzed into 5 lll~ potassium phosphate (pH 7.0) and used to record the far-UV CD spectra a t 25 "C. (scan A, Trx; scan B, C32S; scan C, C35S; and scan D, C32SIC35S). Panel B, oxidized C32S sample was used to record the far-UV CD spectrum (scan A). The same sample was then reduced with a 3-fold excess of DTl' for 20 min a t 4 "C and a new spectrum was recorded (scan B ). The difference between scans A and B is shown as scan C . The ellipticity is given as the molar ellipticity per residue.

TABLE I1 Predicted structural content of human thioredoxin variants

Sample 0-Helix p-Sheet Turn Random coil

Trx 29.30 42.68 12.11 15.91 C32S 30.66 23.64 22.07 23.63 c35s 16.99 30.18 15.23 37.60 C32SlC35S 29.49 19.63 19.73 31.15

Holmgren, 1991). It was of interest for us to examine the fluo- rescence emission profiles of the four variants of human Trx compared with E. coli Trx. None of the forms of human Trx showed a significant change in the fluorescence quantum yield following reduction with DTT (data not shown), supporting the

11718

A "1 Characterization of Thioredoxin by Site-directed Mutagenesis

1 95 210 US wavelength (nm)

~- 240

B

195 210 US 240

wavelength (nm)

-20 4 < -20 4 195 210 225 240 195 210 U S 210

wavelength (nm) wavelength (nm)

FIG. 4. Far-W circular dichroism spectra of thioredoxin-thioredoxin reductase complex formation. Spectra of purified Trx variants

far-W CD spectra at a concentration of 200 pg/ml for scans with Trx and C32S/C35S and at 150 pg/ml for scans with C32S and C35S. Panel A: at a concentration of 6 pg/ml were recorded as in Fig. 3. Purified TR was dialyzed into 5 m potassium phosphate (pH 7.0) and used to record the

scan A, far-UV CD spectra of Trx; scan E , TR reduced with NADPH; scan C , Trx combined with TR and NADPH; and scan D, the difference of scans A and E from scan C. Panel E: scan A, far-UV CD spectra of C32S; scan E , TR reduced with NADPH; scan C, C32S combined with TR and NADPH; and scan D, the difference of scans A and E from scan C. Panel C: scan A, far-UV CD spectra of C35S; scan E , TR reduced with NADPH; scan C, C35S combined with TR and NADPH; and scan D, the difference of scans A and E from scan C. Panel D: scan A, far-W CD spectra of C32S/C35S; scan E , TR reduced with NADPH; scan C, C32S/C35S combined with TR and NADPH; and scan D, the difference of scans A and E from scan C. The ellipticity in all panels is given as the molar ellipticity per residue.

hypothesis that Trp-31 was not responsible for the change in stimulation of cellular proliferation by Trx occurred with a fluorescence emission as observed with E. coli Trx during the similar time course as that observed for PDGF (Fig. 6B). In physical fluctuations of the polypeptide between the oxidized contrast to the growth effect mediated by Trx, none of the three and reduced state. mutant forms of Trx a t concentrations up to 1 w was able to

Mitogenic Properties of %"I'm and adult T-cell leukemia- significantly stimulate cell proliferation (Fig. 7). Furthermore, derived factor, an autocrine growth factor for lymphoid cells D l T (3 w) and glutathione (10 p) had no effect on cellular (Tagaya et al., 1989), have been shown to be identical species proliferation. The values presented in Figs. 5-7 were deter- (Deiss and Kimchi, 1991; Gasdaska et al., 1993). We therefore mined from three separate repeated experiments and three examined the ability of Trx to stimulate DNA synthesis by individual cultures for each averaged data point. serum-deprived Swiss 3T3 murine fibroblasts in the presence of radiolabeled thymidine and increasing concentrations of Trx DISCUSSION up to 1 (Fig. 5). Trx showed a dose-dependent stimulation of We have conducted a site-directed mutagenesis study of the DNA synthesis, maximal at 0.5 1.1~ Trx, to a level approximately active site cysteines in human Trx. Three mutant forms of Trx 90% of that seen with PDGF used as a separate positive control. were generated in which the two active site cysteines were D" alone at 3 final concentration had no detectable effect mutated to serine residues individually ((232s and C35S) or in on the levels of DNA synthesis. combination (C32S/C35S). Biochemical characterization of the

The effect of Trx on cellular proliferation was examined by mutant forms showed that C35S and C32SIC35S were both measuring the increase in cell number of serum-deprived mu- strong competitive inhibitors of TR for Trx reduction, indicat- rine fibroblasts over time. Trx stimulated fibroblast prolifera- ing that TR recognized both mutants with nearly equivalent tion in a dose-dependent manner with a maximal effect ob- affinity as to Trx. The single mutant C35S was more potent served at 0.25 w Trx, a t a level approximately 85% of that seen than C32S/C35S at inhibiting the reduction of Trx by TR with with PDGF used as a separate positive control (Fig. 6A). The a K, value of 1.8 w compared with 6.7 VM for C32S/C35S. The

Characterization of Thioredoxin by Site-directed Mutagenesis 11719

0'51 0.0

0.00 0.25 0.50 0.75 1.00

thioredoxin (p3l I

FIG. 5. Effects of human recombinant thioredoxin on DNAsyn- thesis. Swiss 3T3 murine fibroblast cells were stimulated with Trx in the presence of [3Hlthymidine as described under "Experimental Pro- cedures." The amount of radioactivity incorporated into trichloroacetic acid-precipitated material was then determined and compared with the levels of control cultures (value of 3.95 x lo4 dpm). The maximal level of radioactivity in precipitable material when cells were stimulated with PDGF (20 ng/ml) is indicated by the dotted line (value of 6.54 x lo4 dpm). The cultures in all panels contained 3 p~ DTI'. A total of three separate cultures was used to obtain the average values with the error bars indicating the calculated standard deviations.

inability of C32S to inhibit Trx reduction by TR may be due to the aggregated state of this recombinant protein, mediated pre- sumably by intermolecular disulfide bond formation. The active site of mammalian Trx has been determined to protrude from the protein surface as opposed to being located in an active site cleft (Forman-Kay et al., 1991), which could facilitate intermo- lecular disulfide bond formation between C32S monomers. We have not seen any evidence suggesting that oxidized human Trx is present as an aggregate nor was there detectable inac- tivation of oxidized Trx over time when incubated in the insulin reduction assay as has been previously reported for rat liver Trx (Luthman and Holmgren, 1982; Holmgren, 1989). The fact that C35S does not aggregate may be explained by the obser- vation that the Cys-32 thiolate anion is stabilized by hydrogen bonding in human Trx and, presumably, prevents formation of an intermolecular disulfide bond (Forman-Kay et al., 1991, 1992). Removal of Cys-32 may be conducive for Cys-35 to un- dergo intermolecular disulfide bond formation, leading to ag- gregation of C32S monomers. However, since the disulfide bond arrangement of oxidized Trx is unknown, aggregation through the non-active site cysteines cannot be ruled out. The CD spec- tra comparing oxidized and reduced C32S indicated that ag- gregation of the monomer does not result in significant changes in the secondary structure.

The substitution of the serine residues for the active site cysteine residues resulted in changes in the secondary struc- ture in all three mutants of compared with the wild-type protein. These results were somewhat surprising since a prop- erty of E. coli Trx is the ability to introduce amino acid substi- tutions in the hydrophobic core without undergoing major structural alterations (Hellinga et al., 1992). Furthermore, a mutant of E. coli Trx termed C32S/C35S/L78C has been shown to have native-like structure and denaturation profiles (Wynn and Richards, 1993). The CD studies supported the competition studies, showing that TR was capable of interacting with C35S and C32SlC35S. C35S and C32SlC35S thus retained enough structural information to be recognized by TR. Again, the in-

A -F _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - _ -

100 0.00 0.25 0.50 0.7s 1.00

B lm]

thioredoxin (pM)

1

A

0 _ , . , 1 1 i

0 10 20 30 40 50 60 70

time (hours)

proliferation of Swiss 3T3 murine fibroblasts. Panel A, the dose- FIG. 6. Effects of human recombinant thioredoxin on cellular

response curve for the effect of various concentrations of Trx on cell

Cell number was determined 48 h after the addition of Trx and ex- proliferation performed as described under "Experimental Procedures."

pressed as a percentage relative to the cell number in control cultures containing 3 p~ DTI'. The growth stimulation in the presence of PDGF (20 ng/ml) is indicated by the dotted line. All of the sample cultures also contained 3 p~ DIT. Panel B , a separate experiment showing the time course of growth stimulation with 3 p~ DTT (closed circles) and in the presence of 1.0 p~ Trx (closed squares), and 20 ng/ml PDGF (open squares). Cell number was determined 48 h after the addition of each factor and is expressed as a percentage relative to the number of cells originally in control cultures. A total of three separate cultures was used to obtain the average values with the error bars indicating the calcu- lated standard deviations.

ability of C32S to interact with TR may be due to aggregation of C32S monomers.

We examined the mitogenic properties of Trx since the Ihr.TR system may exert a key regulatory function for eukary- otic cell growth. I t was also of interest for us to understand the role of the Trx.TR system in mitogenesis since it has been shown that TR is a target for inhibition by some quinoid anti- cancer drugs (Mau and Powis, 1992). Adult T-cell leukemia- derived factor, which is identical to Trx (Deiss and Kimchi, 1991; Gasdaska et al., 19931, has been shown to be an autocrine growth factor that can stimulate proliferation of leukemic cell lines (Tagaya et al., 1989). Furthermore, some human tumors

Characterization of Thioredoxin by Site-directed Mutagenesis

amining the signaling pathway(s) utilized by Trx in stimulat- ing cellular proliferation.

Acknowledgments-We thank both Edmundo Chantler and Polly T Kintzel for technical assistance with the preparation of placental ex-

D l T C32S C35S C32SI Trx PDCF c35s

doxin on cellular proliferation of Swiss ST3 fibroblasts. The ef- FIG. 7. Effects of mutant forms of human recombinant thiore-

fect of the mutant forms of Trx on cell proliferation was performed as described under “Experimental Procedures.” Cell number was deter- mined 48 h after the addition of 1.0 1.1~ Trx, C32S, C35S, and C32SK35S and is expressed as a percentage relative to the number of cells in

ng/ml) was used as a positive control. All cultures contained 3 p~ Dl”. control cultures. The growth stimulation in the presence of PDGF (20

A total of three separate cultures was used to obtain the average values

asterisk (*) denotes that the stimulation by Trx was statistically signifi- with the error bars indicating the calculated standard deviations. The

cant above the control levels with 3 p~ DTT alone ( p < 0.05).

show substantially elevated levels of Tm mRNA (Gasdaska et al., 1994). In our study, recombinant human Trx was found to be capable of stimulating both cellular proliferation and DNA synthesis of murine fibroblasts. The time course of growth stimulation by Trx was identical to that observed with PDGF. C32S, C35S, and C32SfC35S were unable to stimulate cell growth. This indicates that the mitogenic effect of Trx is de- pendent upon a catalytically active redox site or other struc- tural features of Trx imparted by the active site cysteines, since none of the mutants were able to be reduced by TR. The effect of reduced Trx on cell proliferation appears to be due to a site-specific redox reaction because higher levels of D’M’ and glutathione than those of Trx could not duplicate the effect of Trx on cell proliferation. The mechanism(s) by which Trx func- tions to stimulate cellular proliferation and DNA synthesis is not known at the present. Current efforts are focusing on ex-

tracts and thymidine incorporation studies; respectively. We also thank Raul Martinez and Bob Gillies for assistance with fluorescence spec- troscopy.

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