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Carcinogenesis Advance Access originally published online on June 15, 2006
Carcinogenesis 2006 27(12):2538-2549; doi:10.1093/carcin/bgl111
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Thioredoxin reductase is required for the inactivation of tumor suppressor p53 and for apoptosis induced by endogenous electrophiles

Pamela B. Cassidy1,2, Kornelia Edes1, Chad C. Nelson2,3, Krishna Parsawar3, F.A. Fitzpatrick1,2 and Philip J. Moos4,*

1 Huntsman Cancer Institute L.S. Skagg’s Pharmacy, Room 201, 30 S 2000 Salt Lake City, UT 84112, USA
2 The Department of Medicinal Chemistry L.S. Skagg’s Pharmacy, Room 201, 30 S 2000 Salt Lake City, UT 84112, USA
3 The Mass Spectrometry and Proteomics Core Facility L.S. Skagg’s Pharmacy, Room 201, 30 S 2000 Salt Lake City, UT 84112, USA
4 The Department of Pharmacology and Toxicology, University of Utah L.S. Skagg’s Pharmacy, Room 201, 30 S 2000 Salt Lake City, UT 84112, USA

*To whom correspondence should be addressed Email: philip.moos{at}pharm.utah.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation and apoptosis were compared and contrasted with the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone–quinone attenuates p53 function in human MCF7 cells.

Abbreviations: ACN, acetonitrile; CV, column volumes; 15-d-PGJ2, 15-deoxy-{Delta}-12,14-PGJ2; DTNB, dithiobis-5-5'-dinitrobenzoic acid; 3,4-EQ, 3,4-estronequinone; FA, formic acid; 4-HNE, 4-hydroxynonenal; LTA4, leukotriene A4; LOX, lipoxygenase; LC-MS/MS, liquid chromatography-mass spectrometry/mass spectrometry; PG, prostaglandin; PGA1-APB, prostaglandin A1-aminopentylbiotin; siRNA, small interfering RNA; TrxR, thioredoxin reductase; Trx, thioredoxin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Thioredoxin reductase (TrxR1) is a homodimeric selenoprotein that catalyses NADPH-dependent reactions (1,2). TrxR is an enzyme that can reduce multiple substrates [as reviewed in (1,3,4)] involved in the antioxidant network (5–7) and cellular proliferation (8) by providing reducing equivalents either directly or via thioredoxin (Trx) (1,3,4,9–11). The TrxR-Trx system also maintains the redox state of many transcription factors including p53, AP-1 and NF-{kappa}B (1,12–18).

Some electrophilic lipids with {alpha},ß unsaturated carbonyl substituents, derived from arachidonate metabolism, attenuate the activity of TrxR when cells are treated in a pharmacological manner or when the lipids are generated endogenously by the controlled induction of 15-LOX (19,20). By impairing TrxR, these lipids derange the protein conformation and function of the tumor suppressor p53 (20).

In the current work we characterized the interactions of electrophilic lipids with purified TrxR enzyme to clarify the kinetics and mechanism of inactivation. We compared several types of endogenous electrophiles including electrophilic eicosanoids, the 5-LOX allylic epoxide metabolite, leukotriene A4 (LTA4), the lipid peroxidation product 4-hydroxynonenal (4-HNE) and a quinone metabolite of estrogen. We used small interfering RNAs (siRNAs) to reduce cellular TrxR1 expression and compare the effects of its depletion with the effects of its inactivation by chemically reactive lipids or site-directed mutagenesis directed at the catalytic selenocysteine. We report that the C-terminal inactivated form of TrxR1 is both necessary and sufficient for the disruption of the protein conformation of wild-type p53 and that this altered form of TrxR1 is a mediator of electrophile-induced apoptosis. Our results indicate that modification of the active site selenol of TrxR is functionally distinct from loss of expression of TrxR.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Materials used included Dulbecco’s modified essential medium (DMEM), Eagle’s modified essential medium (EMEM) and supplements (GIBCO/BRL); 4-hydroxy-2-nonenal and prostaglandins (Cayman Chemicals); auranofin (ICN Biomedicals); 5,5'-dithio-bis (2-nitrobenzoic acid) (Sigma); Complete protease inhibitor mixture (Roche Molecular Biochemicals); Lipofectamine 2000 transfection reagent, Novex pre-cast polyacrylamide minigels (Invitrogen); Western Lightning enhanced chemiluminescence reagents (PerkinElmer Life Sciences); horseradish peroxidase (HRP) conjugated secondary antibodies, protein A/G PLUS-Agarose, FL393-G polyclonal antibodies against p53 (Santa Cruz Biotechnology); Neutravidin-conjugated beads (Pierce); monoclonal antibodies directed against p53 (Ab-5 wt and Ab-3 mt, Calbiochem); and antibodies against TrxR1 (custom antibody services of the Pocono Rabbit Farm & Laboratory). The University of Utah DNA/Peptide Core synthesized peptides (CIPKKLMHQAALLG and CGLSEEKAVEKFGE) from TrxR1 conserved across many species and these were conjugated to KLH and OVA as carriers, the antigens were injected, in combination, into chicken hosts and antibodies were purified from egg yokes using Eggcellent Chicken IgY purification kits (Pierce).

Cell culture
RKO colon cancer cells (gift of M.Meuth, Institute for Cancer Studies, University of Sheffield, Sheffield, U.K.) were maintained in DMEM at 37°C in a humidified incubator with 5% CO2. The medium was supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 µg/ml gentamycin and 10% (vol/vol) FBS. MCF7 breast cancer cells (ATCC) were maintained similarly in EMEM supplemented with non-essential amino acids, 1 mM sodium pyruvate, 10 µg/ml bovine insulin, 10 µg/ml gentamycin and 10% (vol/vol) FBS. The RKO–EcR cell line was generated by transfecting RKO cells with pVgRXR (Invitrogen) and selecting stable Zeocin-resistant colonies.

Immunochemical analysis (westerns)
Proteins were fractionated on 10% Tris–glycine polyacrylamide gels and then transferred to a 0.45 µm polyvinylidenedifluoride (PVDF) membrane. The membranes were blocked by incubation with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated with the appropriate primary antibody and HRP-conjugated secondary antibody or streptavidin-HRP. The membranes were treated with chemiluminescence reagents and exposed to an autoradiography film.

In other experiments, purified TrxR was incubated with 200 µM NADPH in the presence of 60 µM biotinylated prostaglandins (PGs), fractionated by SDS–PAGE, transferred to PVDF and analyzed by immunochemical detection with neutravidin-HRP to localize the site of covalently modified TrxR. In some of these experiments, auranofin (10 µM) was used as an inhibitor of TrxR to compete with the PGs. In these experiments, the amount of TrxR was assessed using the chicken anti-TrxR antibody.

Immunoprecipitation of p53
Cells were lysed in 250 mM sucrose, 50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, completeTM protease inhibitor, 2 mM NaF, 2 mM sodium orthovanadate. We sonicated the lysate twice for 5 s at 4°C. After centrifugation at 13 000x g, samples containing 200 µg of total protein were incubated for 16 h at 4°C with 1 µg of either Ab-3 or Ab-5 and 20 µl of protein A/G PLUS-Agarose in 1 ml of PBS with 0.4% Tween-20. These antibodies specifically recognize misfolded (sometimes referred to as ‘mutant’ since certain genetic changes result in misfolded protein conformations) and wild-type conformations, respectively, of p53 under non-denaturing conditions (21). The samples were centrifuged at 500x g for 5 min to isolate the immune complexes. The beads were washed twice with 1 ml of PBS/0.4% Tween-20. The samples were fractionated by SDS–PAGE as described above and the amount of conformationally misfolded or wild-type p53 in the immunoprecipitate was measured by hybridization with a separate anti-p53 polyclonal antibody (FL-393) that recognizes both the misfolded and wild-type forms of the protein.

Preparation and mass spectrometry analysis of TrxR1–lipid adducts
To a solution of 4 µg TrxR1 in 10 µl TE (50 mM Tris pH 7.4, 1 mM EDTA) was added 1 µL 2 mM NADPH. After 15 min at 25°C, 1 µl 1 mM 4-HNE in DMSO was added and the mixture was incubated for another 2 h. At this point samples for the analysis of the intact protein were desalted and analyzed. For the analysis of tryptic digests, NaBH4 (10 µl 0.02 M in 0.1 M NaOH) was added and the mixture was incubated overnight at 4°C. The next morning, excess borohydride was decomposed by the addition of 2 µl 20% acetic acid. Then 40 µl 6 M guanidine in 0.2M TE pH 8 was added. The pH was adjusted to ~7.5 by the addition of 1N NaOH (about 6 µl). The protein was denatured by heating to 60°C for 30 min. Unmodified thiol and selenol groups were alkylated by the addition of 8 µl 0.2 M iodoacetamide in TE. This mixture was incubated on ice in the dark for 2 hours. Then 1.7 µl 1M DTT was added and the solution was incubated at 37°C for 15 min. After the addition of 300 µl TE, 2 µg trypsin gold (Promega) was added and the digest was incubated at 37°C for 18 h.

All tryptic digests and intact protein samples were prepared for mass spectrometry using reverse-phase purification (C18 ZipTipTM, Milipore Corp., Billerica, MA).

For preparation for electrospray mass spectrometry (ESI/MS), ~100 pmoles of the intact native TrxR protein or trypsin digests were desalted and purified by loading the protein onto a ZipTipTM device in 20 µl of a solution containing 50 mM tris buffer, 5% acetonitrile (ACN) and 0.1% formic acid (FA). The ZipTipTM was rinsed with several 10 µl volumes of 5% ACN and 1% FA solution in nanopure water and then intact native TrxR protein or tryptic peptides were eluted in a total volume of 3–5 µl of 60% ACN and 1% formic in nanopure water.

Mass spectrometry methods
Intact TrxR protein was analyzed using positive-ion ESI/MS with a Quattro-II mass spectrometer (Micromass, Inc., Milford, MA). Samples were introduced into the instrument by direct infusion of the ZipTipTM-prepared solution at 3 µL/min. ESI was performed with 2.8 kV spray potential and a cone voltage of 50 eV. Spectra were acquired over the mass range of 800–1400 Da, and the resulting multiply charged ion series were deconvoluted into neutral molecular-mass spectra using MaxEnt software (Micromass). Typically, the resulting processed spectra are accurate within 3 Da for proteins in the range of 50–70 k Da molecular weight.

Tryptic digests of TrxR protein were analyzed using positive-ion ESI liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) using an Eksigent Nano LC-1D binary pump HPLC system (Eksigent Technologies, LLC, Dublin, CA) interfaced to a Finnigan LCQ Deca ion trap mass spectrometer (ThermoElectron Corp, CA) equipped with a Picoview Nanospray® source (New Objective, Inc., Woburn, MA). Tryptic digests were reconstituted in 5 µl of 5% ACN with 0.1 % FA, which was then manually injected using a nano injector (Valco Instrument Co, Houston, TX) onto a self-packed column (100 µm x 100 mm, 3 µm particle size; Waters Atlantis dC18). A 58 min gradient of 5–85% solvent B (A: 5% ACN/0.1% FA; B: 80% ACN /0.1% FA) was used at 400 nL/min, at 5% B for the first 3 min, followed by a linear increase to 55% B in 50 min and finally maintained at 85% B for 5 min. Spectra were acquired in automated ‘triple-play’ mode for recording of full-scan MS, Zoom scan and MS/MS data (Excalibur software, ThermoElectron). The scan range for full-scan spectra was set at m/z 400–2000. Automated analysis of peptide fragmentation or production spectra (MS/MS) was performed with MASCOT (Matrix Science, London, UK) and/or SEQUEST (ThermoElectron) computer algorithms for protein database searching and protein identification.

CDKN1A (p21WAF1) expression
Total RNA was purified from ~106 RKO–EcR cells using Qiagen RNeasy kits. One microgram of total RNA was used to synthesize first-strand cDNA using random nanomers and Superscript III (Invitrogen), diluted 1:5 and then evaluated by quantitative real-time PCR using a Chromo-4 cycler (Bio-Rad). The primers used for CDKN1A were 5'-GGCAGACCAGCATGACAGATT-3' and 5'-GCGGATTAGGGCTTCCTCTT-3' from the real-time primer database (22). We utilized ß-2 microglobulin as a house keeping control gene (primers: 5'-TCACCCCCACTGAAAAAGA-3' and 5'-GCGGCATCTTCAAACCTC-3'). We calculated the relative expression by utilizing copy number standards and then evaluating the relative expression among the different conditions.

Purification of TrxR1 from rat liver
TrxR1 was purified from rat livers using a protocol adapted from Gromer et al. (23). Briefly, 30 rat livers were homogenized in a Waring blender containing 300 mL cold extraction buffer [10 µM FAD and 40 µM PMSF in TE (50 mM Tris, pH 7.6 and 1 mM EDTA)]. The pH was adjusted to pH 8.3 by the addition of 5 M NH4OH (~25 mL). Then 60 mL of cold (–20°C) chloroform/butanol (1:2.5 v/v) was added and the mixture was re-homogenized. The material was transferred to Teflon centrifuge bottles and centrifuged at 4000x g for 90 min. at 4°C. The supernatant was filtered through silanized glass wool and cold acetone (0.85 ml/ml supernatant) was added. The mixture was incubated for 1 h on ice then centrifuged for 15 min at 4000x g 4°C. The pellet was dissolved as completely as possible in 100 mL TE, then filtered through glass wool and dialyzed against 0.5x TE overnight at 4°C. The dialyzed solution was filtered again through glass wool and applied to a 2.5 x 56 cm DE52 column pre-equilibrated with TE at 25°C. The column was washed with 2 column volumes (CV) of TE, 1 CV 50 mM NaCl/TE, 1 CV of 100 mM NaCl/TE and 2 CV 200 mM NaCl/TE. TrxR eluted in fractions between 100 and 200 mM NaCl. The fractions were assayed for TrxR activity as described below and those with highest specific activity were concentrated to ~50 ml on a 30 kD MWCO centrifugal concentrator. The sample was then applied to a 1 x 27 cm 2'-5'-ADP-sepharose column (Pharmacia) at 4°C. The column was washed sequentially with 5 CV TE, 2 CV 500 µM NADH/TE, 2 CV 200 mM KCl/TE and 2 CV TE before elution with 1 mM NADP+/TE. Fractions were assayed for TrxR activity and then active fractions were concentrated giving protein with specific activity of 42 ± 2 U/mg, where one unit corresponds to the production of 2 µmol/min of 2-nitro-5-thiobenzoate. The protein appeared to be >95% pure by SDS–PAGE. LC-MS/MS analysis of tryptic digests confirmed the identity of the isolated protein as TrxR1 with no peptides detected from TrxR2. Some preparations appeared to be contaminated with small amounts of Trx. TrxR1 for electrospray MS experiments was further purified by chromatography on PAO-sepharose using the method of Arner (24).

Thioredoxin reductase assays
The inhibition constants for purified TrxR1 were determined in the presence of both the inhibitor and substrate using a modification of the method described by Becker et al. (25) for determination of the rate of TrxR-catalyzed reduction of the disulfide substrate dithiobis-5-5'-dinitrobenzoic acid (DTNB). To a mixture of 870 µl reaction buffer (50 mM potassium phosphate, pH 7 and 2 mM EDTA), 100 µl of 2 mM NADPH, 1 µl inhibitor in DMSO and 2 µl of enzyme (1.4 µg) was added 30 µl DTNB (0.1 M in DMSO). The rate of reduction of 3 mM DTNB in the presence of varied concentrations of electrophile was measured by monitoring the absorbance of the reaction mixtures at 412 nm. These data were used to calculate the IC50 values for each electrophile. Then using the equation Ki = IC50/(1 + [S]/KM), where [S] equals the concentration of DTNB and KM is equal to the experimentally determined value of 0.20 mM for DTNB, the Ki values listed in Table I were calculated. To evaluate the time-dependent inactivation of TrxR by electrophiles, 0.54 µg (333 nM) TrxR in 30 µL reaction buffer (50 mM potassium phosphate, pH 6.5 and 2 mM EDTA) containing 300 µM NADPH and electrophile were incubated at 25°C. At various times, 2 µl of this mixture was withdrawn and diluted 50-fold into an assay mixture containing 50 mM potassium phosphate, pH 6.5, 200 µM NADPH and 3 mM DTNB (15 x KM), for the determination of residual activity. Data were fitted using the data analysis program Origin (Microcal Software Inc.). Total TrxR activity in cell lysates treated with siRNA was measured by monitoring the oxidation of NADPH at 340 nm as described previously (20).


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  		Table I Inhibition of TrxR by endogenous electrophiles

 
siRNA and TrxR constructs
RNA oligos that are specific for TrxR1 were designed and these were synthesized in the University of Utah DNA/Peptide Synthesis Core. The siRNA directed against TrxR1 is 5'-AGACCACGUUACUUGGGCAdTdT-3' and the control is a scrambled sequence (5'-AGGCAAAUCACGGUGUCCUdTdT-3') that does not match any sequence in the GenBank human database for >16 nt (26). This siRNA is directed against the region 1034–1052 using the reference sequence NM_003330 [GenBank] for annotation. This siRNA should suppress almost all isoforms of TrxR1 since most isoforms are due to variability of transcripts at the 5' end of the gene (27) [or reviewed in (9)]. The full-length TrxR1 construct was from ATCC (#6355551) and corresponds to NM_003330 [GenBank] . The complete coding sequence was cloned into the ecdysone inducible vector, pIND(SP1)hydro (Invitrogen). This new construct does not contain the SECIS element required for selenoprotein synthesis. The C498S and U499S (from 5'-TGCTGA-3' to 5'-TCCTCA -3') mutants were prepared using QuikChange site-directed mutagenesis procedures (Strategene). This construct was made insensitive to siRNA by introducing a silent mutation (5'-CCAAGATAC-3', i.e. from CGT encoding Arg to AGA encoding Arg).

Caspase-3 assays
Cells were treated with electrophiles for the indicated length of time and then lysed in 25 mM Hepes, pH 7.5, 5 mM EDTA, 2 mM DTT, 0.1% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. Caspase-3 activity was measured fluorometrically using DEVD-AMC as a substrate (Peptides International) (28).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Electrophilic lipids covalently modify the C-terminus of TrxR
We investigated the kinetics and mechanisms of inactivation of isolated TrxR. We found that TrxR must be reduced with NADPH for prostaglandin A1-aminopentylbiotin (PGA1-APB) to be covalently attached (Figure 1, lane 2 versus 3). We next asked whether the C-terminal selenocysteine residue is the site of derivatization. Auranofin, a thiogold compound, is thought to act by binding to the C-terminal selenocysteine of TrxR (23). When reduced TrxR was first treated with auranofin, followed by either PGA1-APB or 15-deoxy-{Delta}-12,14-PGJ2-aminopentylbiotin (15-d-PGJ2-APB), auranofin blocked the interaction of TrxR with PGs (Figure 1, lane 3 versus 4 and lane 5 versus 6).


Figure 1
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  		Fig. 1 Electrophiles alkylate the C-terminus of TrxR. Purified TrxR was incubated with 200 µM NADPH in the presence of 60 µM 15-d-PGJ2-APB or PGA1-APB followed by fractionation by SDS–PAGE and immunochemical detection with neutravidin-HRP to localize covalently modified TrxR, containing a biotin epitope (lanes 3 and 5). In lane 2, NADPH was omitted from the mixture to illustrate the requirement for reduction of the enzyme. Auranofin (10 µM), which specifically interacts with the C-terminus of TrxR, antagonized the reaction with 15-d-PGJ2-APB or PGA1-APB (lanes 4 and 6).

 
Endogenous electrophiles are potent, time-dependent inhibitors of TrxR
We performed enzyme kinetic experiments with purified TrxR to determine the potency of the inhibition by PGs and three other chemical classes of endogenous electrophiles (Figure 2). Two of these compounds, 4-HNE and 3,4-estronequinone (3,4-EQ), are chemically similar to the PGs in that they both contain electrophilic {alpha},ß-unsaturated carbonyls. They differ from the PGs however in that 4-HNE contains an aldehyde, not a ketone, and 3,4-EQ, which is a quinone, is a potential substrate for reduction by TrxR (29). The third, LTA4, is an allylic epoxide. Inhibitory activity was measured by an assay for the reduction of the disulfide substrate DTNB (30). Table I shows the potency of inhibitors ranged from 15-deoxy-{Delta}-12,14-PGJ2 (15-d-PGJ2) with a Ki = 24 nM to LTA4 with Ki = 33 µM.


Figure 2
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  		Fig. 2 Electrophilic lipids. Arrows point to site(s) of electrophilic attack. 4-HNE and 3,4-EQ are susceptible to both 1,4- and 1,2-reactions.

 
The coincidence of a biotin label with the TrxR band in the immunochemical analysis of SDS–PAGE-fractionated reaction mixtures in Figure 1 is consistent with a covalent interaction between PGs and TrxR. Covalent modification should result in a time-dependent inactivation of TrxR when the reduced enzyme is incubated with electrophiles (10,23,25,29–31) and the results shown in Figure 3 support this hypothesis. The time-dependent inactivation of TrxR fit a first-order exponential decay model with a half-life of 11, 12 and 6 min for PGA2, 15-d-PGJ2 and LTA4-methyl ester, respectively. The inactivation of TrxR by 15-d-PGJ2 was evaluated at five different concentrations. We then calculated the apparent inactivation constant (kinact = 0.1 s–1) and binding constant (Ki = 870 µM) from the y- and x-intercepts, respectively (32), by plotting the half-life for inactivation versus the reciprocal of the inhibitor concentration (Figure 3B).


Figure 3
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  		Fig. 3 Time-dependent inactivation of TrxR by 15-d-PGJ2 and other electrophiles. (A) The rate of reduction of DTNB by TrxR was measured following incubation with PGA1, 15-d-PGJ2 or LTA4-methylester for 0–40 min. These electrophiles demonstrate first-order kinetics. (B) The half-time of inhibition of TrxR at various concentrations of 15-d-PGJ2 was plotted as a function of 1/[15-d-PGJ2]. Analysis of the resulting line gives kinact and Ki for the covalent reaction between the PG and TrxR (32). (C) 3,4-EQ and 4-HNE were evaluated as in (A). Data fit an equation described by two exponential decays, one with a half-life of 20 s and the other with a half-life of 7 min. When NADPH was left out of the pre-incubation mixture (circles and dotted line), the data fit an equation with a single exponential decay with half-life of inactivation of 20 s.

 
The kinetics of inactivation of TrxR by 4-HNE and 3,4-EQ fit a model of bi-exponential decay, with an initial half-life of 20 s and a terminal half-life of 7 min (Figure 3C). In the absence of NADPH, time-dependent inactivation fits a model of a single exponential decay, again with a half-life of ~20 s. This suggests more than one mode of inactivation of TrxR by 4-HNE and 3,4-EQ.

We tested whether the electrophiles were substrates of TrxR by monitoring the consumption of NADPH in a mixture of the electrophile, TrxR and NADPH. Only 3,4-EQ showed detectable activity, and this was very modest at 0.08 µmol NADP+/min/mg TrxR. In comparison, reduction of DTNB by TrxR occurs at the rate of 42 µmol/min/mg. Thus, these inhibitors are not mechanism-based inhibitors (suicide substrates) as defined by Ables and Maycock (33).

MS results
In our first attempts to analyze tryptic peptides from the reaction of reduced TrxR1 with the electrophiles from Table I followed by treatement with iodoacetamide, we found no lipid modified fragments and only the re-oxidized or bis-iodoacetamide-modified C-terminal selenopeptide (data not shown). We reasoned that once the protein was cleaved, the resulting fragments might undergo a retro-Michael addition and loss of the lipid modification because of the relative instability of small molecule Michael adducts in comparison with their macromolecular analogs (34). In order to prevent the loss of the lipid, we treated the adducts with sodium borohydride before tryptic digestion to reduce the lipid carbonyl moiety and thus eliminate the possibility of the retro-Michael reaction. Using this strategy we were able to observe the 4-HNE adduct to the C-terminal selenopeptide by LC-MS/MS as illustrated in Figure 4. In Figure 4, the peptide observed at mass 1359 daltons (i.e. average-isotope mass) corresponds to the C-terminal tryptic fragment of TrxR1 with one 4-HNE and one iodoacetamide modification. Figures 4A and 4B show the characteristic and definitive isotope pattern for the selenopeptide (theoretical and observed spectra are in agreement). In addition to this peptide, nine other peptides from TrxR1 were identified covering ~30% of the predicted sequence (data not shown). Neither multiple 4-HNE adducts, peptides from TrxR2 nor any other forms of the C-terminal TrxR1 peptide were observed. Unfortunately, when we used this same protocol with other electrophiles, we could no longer identify the C-terminal peptide.


Figure 4
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  		Fig. 4 Mass Spectrometric analysis of TrxR1-lipid adducts. (A) theoretical and (B) experimentally determined spectra from LC/MS analysis of TrxR1 C-terminal tryptic peptide containing one 4-HNE and one carboxymethyl adduct. TrxR1 in 50 mM Tris, pH 7.4, 1 mM ETDA was first reduced with 200 µM NADPH and then treated with 4-HNE (100 µM) at 25°C for 1 h. The adduct was then treated with NaBH4, and unreacted thiols and selenols were derivatized with iodoacetamide. Note the distinctive isotope pattern characteristic of selenium-containing ions. (C) MS/MS analysis of the C-terminal tryptic fragment of 4-HNE-modified TrxR1. The sequence of the C-terminal fragment is shown along with b- and y-series ions. As indicated by arrows, b- and y-type ions are also observed for losses corresponding to b-NH3, b-H2O, y-NH3 and y-H2O. Two b10-fragments are observed, one in which cysteine is modified by 4-HNE and the other having a carboxymethyl adduct (CME). (D) Inset: two major species are found in unmodified NADPH-reduced protein purified from rat livers, one at m/z = 54708 and a second at 54785. Electrospray mass spectrometric analysis of this mixture treated with 4-HNE (molecular weight 156) detects species containing 3 (55255 = 54785 + 3 x 4-HNE), 4 (55332 = 54708 + 4 x 4-HNE and 55409 = 54785 + 4 x 4-HNE) and 5 (55486 =54708 + 5 x 4-HNE and 55568 = 54785 + 5 x 4-HNE) 4-HNE molecules per TrxR1 monomer, within the error of the mass measurements (see Mass Spectrometry Methods).

 
An LC-MS/MS experiment with the 1359 dalton peptide allowed us to verify its sequence (Figure 4C). The sequence corresponded to that of the C-terminus of TrxR1 as anticipated. In addition, we were able to determine the exact amino acid residue modified with 4-HNE. Interestingly, the peak in the chromatogram corresponding to the fragment(s) with m/z of 1359 contained a mixture of two peptides, one with 4-HNE-modified cysteine and one with 4-HNE-modified selenocysteine.

We analyzed both NADPH-reduced and 4-HNE-modified intact TrxR1 by electrospray mass spectrometry (Figure 4D). Two major species of TrxR1 were found in the NADPH-reduced sample (Figure 4D inset). In the reaction of these two proteins with 4-HNE, we observed molecules with molecular weights corresponding to 3, 4 and 5 additions of 4-HNE (Figure 4D).

Electrophilic estrogen quinones disrupt the protein conformation of p53
One of the biological consequences of TrxR inhibition by PGs or 4-HNE is the derangement of the protein conformation of newly synthesized p53 in cells harboring a wild-type p53 tumor suppressor gene (20). This result, originally observed in colon cancer cells, is recapitulated in the breast cancer cell line MCF7 (Figure 5 panel A). The protein conformation of p53 is determined in an assay that takes advantage of conformation-specific antibodies to immunoprecipitate p53 either in a wild-type or misfolded conformation. We used cancer cells derived from breast tissue as this was relevant to our examination of estrogen metabolite, 3,4-EQ. We found that treatment of cells with this compound also causes the disruption of p53 conformation (Figure 5 panel B).


Figure 5
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  		Fig. 5 Endogenous electrophiles disrupt the conformation of wild-type p53. (A) MCF7 breast cancer cells were treated with PGA2 or 15-d-PGJ2 for 6 h at the concentrations specified and accumulated more p53 protein in mutant (Ab-3 epitope) conformation. Cells treated with PGs had more misfolded p53 than those treated with vehicle. (B) MCF7 cells were treated with 3,4-EQ for 5 h. Analysis of p53 conformation as in panel A demonstrated that 3,4-EQ treated cells had more misfolded p53 than did cells treated with vehicle alone. The ratio of misfolded to wild-type p53 is indicated for each treatment condition.

 
Effects of TrxR1 siRNA on PG-induced disruption of p53 conformation
Investigations on Trr1 regulation of p53 in yeast (35,36) and our prior results predicted that genetic loss of TrxR1 would have the same effect as pharmacologic inhibition by treatment with electrophiles (20,37), i.e. cells treated with TrxR1 siRNA alone or in combination with PGs should be more susceptible to conformational disruption of p53 than cells treated with PGs alone. We generated siRNA that specifically target TrxR1, lowered TrxR1 protein levels and reduced overall TrxR activity in RKO cells (Figure 6 panels A and B). In measuring the activity, our assay does not discriminate between TrxR1 and TrxR2, and this is probably the reason that the apparent reduction in activity is not as great as the reduction in TrxR1 protein levels. A scrambled siRNA control, which did not align with other sequences in the GenBank database [using the short exact match BLAST (26)], had no effect on TrxR1 protein expression. RKO cells do not display overt reductions in cell viability when TrxR1 content is reduced with siRNA. We used these siRNAs to test the effects of TrxR1 depletion on p53 conformation. We found no difference in the amount of misfolded p53 in cells treated with either TrxR1 or control siRNA (Figure 6 panel C). Figure 6 panel D shows less misfolded p53 in cells treated with TrxR1 siRNA plus PGA1 compared with cells treated with control siRNA and PGA1. The p53 in cells with reduced levels of TrxR1 protein was less susceptible to conformational derangement after PG treatment.


Figure 6
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  		Fig. 6 TrxR1 siRNA lowers TrxR1 protein levels and enzymatic activity and makes p53 protein less susceptible to conformational derangement. (A) RKO colon cancer cells were treated with 20 nM siRNA directed against TrxR1 or with a control, scrambled siRNA. The cells were lysed after the indicated times and protein was analyzed for TrxR1 by immunoblot. TrxR1 protein levels were reduced ~75% after 72 h incubation. (B) Enzymatic analysis of protein from cells treated with TrxR1 siRNA showed a ~70% reduction in TrxR activity compared with the control. (C) RKO cells were treated with TrxR1 and control siRNAs, and then the amount of p53 protein in misfolded and wild-type conformation was analyzed immunochemically. TrxR1 siRNA alone had no effect on p53 conformation. (D) RKO cells were treated with TrxR1 or control siRNA for 48 h and then with the indicated concentrations of PGA1 for 6 h. Analysis of misfolded p53 showed that the protein in cells pre-treated with TrxR1 siRNA was less susceptible to conformational derangement than p53 from cells treated with scrambled siRNA.

 
Expression of TrxR1 C-terminal mutant causes the disruption of p53 conformation
We considered our mechanistic model for p53 inactivation in light of our new results and those reported by Anestål and Arnér (38), who found that introduction of purified TrxR1, with C-terminal modifications, into lung cancer cells caused apoptosis, while introduction of full-length wild-type TrxR1 did not. We hypothesized that C-terminal compromised TrxR1 might be a mediator of the conformational derangement of p53. To test this hypothesis directly, we used an siRNA-resistant TrxR1 construct containing both C498S and U499S mutations that inactivate the C-terminal active site. Expression of this construct was under control of the ecdysone receptor. After first depleting wild-type TrxR1 protein by treatment with siRNA, we transfected the cells with the mutant construct, induced expression of the mutant TrxR1 and analyzed p53 conformation after 24 h. siRNA treatment lowered the expression of endogenous TrxR1 while ponasterone A treatment induced expression of the C498S and U499S mutant TrxR1 (Figure 7 panel A). Induction of mutant TrxR1 was accompanied by an increase in the amount of conformationally misfolded p53 (Figure 7, panel B). Thus C-terminal modified TrxR1 is sufficient to cause misfolded, conformationally deranged p53 protein but depletion of TrxR1 does not recapitulate this effect.


Figure 7
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  		Fig. 7 Expression of C-terminal mutant TrxR1 causes conformational disruption of p53. (A) RKO–EcR cells were transfected with siRNA directed against TrxR1 and a construct, pIND(SP1) TrxR CU-SS, that encodes a ponasterone A-inducible catalytically inactive C-terminal mutant TrxR1 and that is also resistant to siRNA. Protein from cells treated with ponasterone and vehicle was fractionated by SDS–PAGE and analyzed for TrxR1 by immunoblot. In the absence of ponasterone, TrxR1 protein was depleted. Robust expression of the mutant CU-SS TrxR1 protein was detected in ponasterone-treated cells. (B) Conformational analysis of p53 protein from cells transfected with TrxR1 siRNA and the pIND(SP1) TrxR CU-SS construct shows that expression of C-terminal mutated TrxR1 is sufficient for disruption of p53 conformation. The ratio of misfolded to wild-type p53 is indicated for each treatment condition.

 
Expression of TrxR1 C-terminal mutant causes the disruption of p53 function
We utilized the RKO inducible system in combination with siRNA to evaluate p53-mediated transactivation. Our previous studies have indicated that CDKN1A protein (p21WAF1) expression is an unreliable measure since electrophilic lipids also mediate effects on proteasome pathway functions (39,40). Therefore, we utilized quantitative real-time PCR to evaluate CDKN1A expression in response to ultraviolet light stimulation (Figure 8). Cells were stimulated with 50 J/m2 or left untreated and RNA was collected after 6 h. Cells where the C-terminal mutant TrxR1 was expressed but without p53 stimulation via UV irradiation display a modest increase in CDKN1A expression. Cells stimulated with UV irradiation display ~5- to 6-fold induction of CDKN1A; however, in cells with endogenous TrR1 suppressed by siRNA but induced to express C-terminal mutant TrxR1 do not demonstrate the p53-dependent expression of CDKN1A.


Figure 8
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  		Fig. 8 Expression of C-terminal mutant TrxR1 disrupts p53-transactivation. RKO–EcR cells were transfected with control or siRNA directed against TrxR1 and the ponasterone A-inducible C-terminal mutant TrxR1. Cells were stimulated either with 50 J/m2 UV or left untreated. UV stimulation induced CDKN1A ~5- to 6-fold; however, cells with knocked-down endogeneous TrxR1 but with induced mutant TrxR1 suppress CDKN1A expression. The relative expression was evaluated using two-way ANOVA (P < 0.01). Within group, Bonfironi comparisons indicate that UV stimulation was significantly different (P < 0.01) for all conditions except when endogenous TrxR1 was suppressed by siRNA and the C-terminal mutant TrxR1 was induced.

 
TrxR1 siRNA inhibits PG-induced apoptosis
Using TrxR1 siRNA, we investigated the role of TrxR1 in PG-induced apoptosis. Figure 9A shows the time course of effector caspase activation by 15-d-PGJ2 in RKO cells treated with TrxR1 or control siRNA. In cells treated with the control siRNA, caspase activation is maximal after 12 h of PG treatment and then begins to decline. The increase in caspase activity is much slower in cells treated with TrxR1 siRNA; activity is still increasing at 24 h, but has not reached the level observed with cells treated with control (scrambled) siRNA. Immunochemical analysis of TrxR1 protein from all treatments showed reduced levels in cells treated with TrxR1 siRNA compared with control siRNA Figure 9 panel B shows the result of caspase assays performed in TrxR1-silenced cells treated with PGA1, 4-HNE and 3,4-EQ in addition to 15-d-PGJ2. Consistent with the data for TrxR inhibition in Table I, 15-d-PGJ2 is more potent than PGA1. 4-HNE and 3,4-EQ are less efficacious than the PGs in this experiment; however 3,4-EQ is at a lower dose (32 µM for 3,4-EQ and 60 µM for the other compounds). Importantly, we observed that decreased expression of TrxR1 attenuated caspase-3 activation by all of these endogenous electrophiles.


Figure 9
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  		Fig. 9 siRNA directed against TrxR1 affects electrophile-induced caspase activation. (A) RKO cells were transfected with TrxR1 siRNA or control siRNA. After 24 h, cells were treated for the indicated time with 60 µM 15-d-PGJ2. Cells were then harvested and analyzed for caspase 3. The relative caspase activities were analyzed by ANOVA and were found to be significantly different (P < 0.03) at all time points except 16 and 20 h. (B) RKO cells were transfected with TrxR1 siRNA or control siRNA for 24 h and then treated with 15-d-PGJ2 and PGA1 (60 µM) and 3,4-EQ (32 µM) for 20 h. In all cases, caspase 3 activity was lower in TrxR1 siRNA-treated cells compared with those treated with control siRNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We examined two important biological consequences of the interaction of electrophiles with TrxR1, the conformational disruption of p53 and the induction of apoptosis. The apoptotic effect is somewhat counterintuitive considering the disruption of p53 function; however, the electrophilic lipids that we utilized in these studies have demonstrated p53-independent promotion of apoptosis (41). We also show that electrophiles disrupt the conformation of p53 in breast cancer cells in a manner similar to that observed for colon cancer cells. Thus, the process appears to be general from a chemical and cellular perspective.

Mammalian TrxRs are flavoenzymes which contain a C-terminal cysteine-selenocysteine active site that is responsible for the reduction of their cognate substrate Trx (1,2,42). The C-terminal selenocysteine is especially vulnerable to electrophilic and oxidative modification (43). The high reactivity of this residue is a result of the low pKa (~5.5) of the selenol group, which is fully ionized at physiological pH. Data shown in Figure 1 supports the conclusion that the reduced selenol form of TrxR is required for the reaction of the enzyme with electrophilic PGs.

Inflammation likely exposes cells to a mixture of electrophiles in quantities sufficient to affect TrxR as well as other nucleophilic cellular targets. Here we have expanded the scope of endogenous electrophiles examined to include LTA4, another arachidonic acid metabolite produced by 5-lipoxygenase, and 3,4-EQ, a metabolite produced by the hydroxylation and subsequent oxidatation of estrogen by cytochrome P450 (44). Recently, Rogan and colleagues showed that levels of 4-catechol estrogens and their quinone conjugates were highly significant predictors of breast and other cancers (45,46). We report that electrophilic estrogens, and the arachidonic acid metabolites, are potent, time-dependent irreversible inhibitors of TrxR in vitro. Our kinetic data suggest that all of these lipid electrophiles share a common mechanism for modification of TrxR, which requires its C-terminal active site in a reduced form. 3,4-EQ and 4-HNE have a second, separate effect. This second mode of inhibition has a very short half-life of inactivation, which does not involve the reduced enzyme but still results in the irreversible disruption of the active site, perhaps by Schiff-base formation with histidine or lysine. Our mass spectrometric analyses are entirely consistent with this model in that tryptic digests of the 4-HNE adduct provide evidence that the C-terminus is indeed covalently modified, and analysis of the intact protein indicates that as many as four other residues are also subject to modification. Isolation of tryptic peptides containing lipid adducts required reduction of the complex with sodium borohydride prior to digestion, providing evidence for adduct reversibility. Also supportive of this contention is the fact that both cysteine and selenocysteine adducts were observed. This observation can be rationalized if one considers that the kinetically favored product (the selenocysteine adduct formed by reaction with the highly nucleophilic selenocysteine anion) might give rise to the thermodynamically more stable cysteine adduct if the system were to approach equilibrium. While this work was in revision, Fang and Holmgren reported that 4-HNE was an inhibitor of Trx and TrxR in vivo and in vitro (47). We have previously reported the inhibition of TrxR activity by 4-HNE in colon cancer cells (20), but here we performed a more thorough enzymatic characterization. Fang found that the time-dependent inactivation of TrxR1 was consistent with first-order exponential decay of enzyme activity. We speculate that the difference between their observations and our own could be due to the fact that they worked with recombinant rat TrxR1 and we isolated the protein from rat liver. Although they did mass spectrometric characterization of the covalent adducts of 4-HNE with Trx, they did not report any of this type of data for TrxR1.

We could not reconcile the experimentally determined molecular weights of TrxR1 purified from rat liver with any combination of published sequences, modifications of those sequences (such as N to R in TrEMBL #O89849 at residue 52, which we observed in the LC-MS/MS experiments) or any possible post-translational modifications (Biolynx software, Micromass Inc.). Gladyshev et al. (48) similarly determined a molecular weight irreconcilable with predicted sequences in their analysis of human TrxR1. As to the presence of two major species, we reasoned that since these proteins were isolated from the livers of ~30 different animals, we could be observing the protein readout of a single nucleotide polymorphism. At the second position of either of two different serine codons, a C exchanged for A would result in a change of serine to tyrosine in the resulting protein with a corresponding mass difference of 76 daltons, very close to the 77 daltons that we measured experimentally. The mass accuracy of the measured mass of an intact protein at 54k Da is expected to be within 3 Da (see Mass Spectrometry Methods).

Inflammatory exudate contains a blend of electrophiles typified by {alpha},ß-unsaturated aldehydes derived from eicosanoid biosynthesis or lipid peroxidation (4-HNE), {alpha},ß-unsaturated ketones derived from eicosanoid metabolism (15-keto-PGF2{alpha}, 15-keto-PGE2, 5-, 12-, and 15-oxo-ETE), and {alpha},ß-unsaturated ketones derived from albumin-catalyzed dehydration of PGE2 and PGD2 (PGA2, {Delta}12-PGJ2 and 15-d-PGJ2) (49–51). Indeed, the level of endogenous 4-HNE alone in tissues ranges from 0.1 to 3.0 µM and increases to ~10 µM in conditions of oxidative stress (52). However, there is considerable controversy regarding the amount of electrophilic metabolites of PGJ2 (in particular 15-d-PGJ2) formed in vivo. One study indicates that 15-d-PGJ2 is influential in the resolution phase of inflammation (53). In another study, that also evaluated 15-d-PGJ2 as a potential PPAR{gamma} ligand, investigators used a sensitive mass spectrometry-based technique to show that the concentration of free 15-d-PGJ2 in inflamed tissues and pre-adipocytes was extremely low (2 pM) (54). However their assertion that low levels of free 15-d-PGJ2 found in their study eliminates the possibility that this class of electrophilic PG has any physiological significance fails to take into account biological and chemical studies of the reactivity of this class of molecules with biological thiols. Narumiya et al. (55) found that (3H){Delta}-12,14-prostaglandin J2 (a cross-conjugated dienone precursor of 15-d-PGJ2) accumulates in the nucleus of cells in culture in a form that is not extractable with acidic ethyl acetate, as would be expected for the free PG, but is released by dilute alkali or protease treatment. This property is consistent with the rapid, physiologically irreversible interaction of {Delta}-12,14-PGJ2 with protein thiols. Suzuki et al. (34) performed a series of chemical studies of the reactions of {Delta}-7-prostaglandin A1 (a cross-conjugated dienone regio-isomer of 15-d-PGJ2 with chemical reactivity toward thiols, which is directly analogous to 15-d-PGJ2). They found that in reactions with low molecular weight thiols such as glutathione, {Delta}-7-prostaglandin A1 rapidly formed reversible 1,4-adducts at the endocyclic cyclopentenone double bond. However in reactions with a synthetic high molecular weight polymer-bound thiol designed to mimick protein thiols, they found that the PG was rapidly and completely bound to the thiol in a form that was reversed only at pH > 9.5. This difference in the reversibility of small thiol- versus large thiol-PG adducts is explained by the increased molecular motion of the small adduct in comparison with the large adduct, which allows the former increased access to the transition state of the uncatalyzed retro-Michael reaction. Finally, a recent in vivo study using a model of acute inflammation in PGD2 synthase null mice strongly supports the hypothesis that 15-d-PGJ2 has biological relevance (PGD2 is a precursor to 15-d-PGJ2) (56). Mice in the present study displayed a severe inflammatory response that failed to resolve. This effect was rescued by 15-d-PGJ2 but not PGD2. Thus there is evidence that electrophilic PGJ2 metabolites have a physiologically significant role despite the low levels of the free PG found in some tissues.

When TrxR is treated with the electrophilic compound 2,4-dinitrochlorobenzene, the selenocysteine residue is modified and the resulting C-terminal nitroarylated enzyme produces superoxide in the presence of NADPH and oxygen (31). We were able to reproduce this result, but when we treated TrxR with PGs, 4-HNE or 3,4-estrogen quinone, we found no evidence of superoxide production in vitro (data not shown). Consequently, we do not believe that TrxR1 is directly involved in PG-induced production of ROS (57,58). Consistent with our contention is the work of Nordberg et al. (4), who showed that while many covalent modifications of the C-terminal selenocysteine of TrxR1 inhibited normal enzyme activity, only modifications containing an aromatic nitro group were able to produce superoxide. They speculated that the peculiar redox properties of the nitro group allowed this chemical moiety to shuttle single electrons from the N-terminal thiol-flavin charge transfer complex of TrxR to oxygen in solution resulting in the production of superoxide. Recently curcumin has been added to the list of electrophiles capable of inhibiting normal TrxR activity and causing the production of superoxide (59).

Anestål and Arnér determined that C-terminal inactivated TrxR1 proteins have distinct activity compared with the authentic wild-type species containing unmodified selenocysteine (38). Using BioPORTER to introduce proteins directly into lung cancer cells, they found that there was an increase in apoptosis with selenium-compromised TrxR1s and that the susceptibility to apoptosis was increased markedly compared with cells treated with wild-type TrxR1. We have presented data from experiments that exploit the genetic depletion of TrxR1 to investigate the role of this selenoprotein in the inactivation of the tumor suppressor p53 and the induction of apoptosis by endogenous electrophiles. Our initial hypothesis that depletion of TrxR1 would potentiate the conformational derangement of redox-sensitive p53 in electrophile-treated cells due to depletion of reduced Trx was based on the study by Merwin et al. (37). They showed that reporter gene activity of human p53 was compromised in Trr1 null yeast. We found that reduction of TrxR1 levels protected p53 conformation in colon cancer cells treated with endogenous electrophiles and also antagonized electrophile-induced apoptosis. Our data are consistent with the observations of Anestål and Arnér (38) and support a model in which C-terminal modified TrxR1 is the species through which these two important biological activities of endogenous electrophiles are manifested. A model of this gain-of-function role for modified TrxR is illustrated in Figure 10. The newly identified functions of chemically and mutationally modified TrxR are currently under investigation. TrxR synthesis and activity are directly related to selenium levels (60,61). Thus, the observation that selenium sufficiency both increases TrxR activity and spares p53 function in cells treated with electrophilic lipids (20) provides an explicit mechanistic framework in which to understand how dietary selenium confers protection against cancer.


Figure 10
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  		Fig. 10 Gain-of-function model for the biological activity of C-terminal-modified TrxR1. Unmodified TrxR1 is a homodimer. The N-terminal active site contains two cysteines that are reduced by NADPH through an FAD cofactor. The N-terminal active site of one subunit then transfers reducing equivalents to the C-terminal cysteine-selenocysteine redox-pair of the other subunit. This reduced form of the unmodified enzyme is able to reduce the substrate Trx and is susceptible to covalent modification by cyclopentenone PGs or other electrophiles. C-terminal modified TrxR1 is unable to reduce Trx, but has new functions. Modified TrxR1 mediates the conformational disruption of p53 and PG-induced apoptosis via activation of caspase 3. Similar models have been proposed by others (9,38).

 


   Acknowledgments
 
The authors would like to thank Dr Diana Stafforini for her valuable editorial suggestions and Kelly Doyle for critical reading of this manuscript. This work was supported by the Huntsman Cancer Foundation; United States Public Health Services Grant AI26730 and CA073992 (to F.A.F.), CA115616 (to P.J.M); and by the Congressionally Directed Medical Research Program xgrant DAMD17-03-1-0649 (to P.B.C).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 3, 2005; revised May 26, 2006; accepted May 29, 2006.


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