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Mammalian Thioredoxin Reductases
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[27] Mammalian Thioredoxin Reductases By TAKASHI TAMURA and THRESSA C. STADTMAN Introduction Thioredoxin reductase (TrxR) is an NADPH-dependent, FAD-containing disulfide reductase that plays an important role in cell proliferation.1 Unlike the wellcharacterized homologs from yeast and prokaryotes, the larger mammalian enzyme is a selenoprotein that contains a selenocysteine (Secys) residue2 in the sequenceCys-Secys-Gly (end) at the C terminus of each subunit.3–5 Catalysis of electron transfer from NADPH to thioredoxin, which in turn is linked to critical components of cell metabolism such as ribonucleotide reductase,6 AP-1 and NF-κB transcription factors,7–10 vitamin K epoxide reductase,11 thiol peroxidase,12 and plasma glutathione peroxidase,13 illustrates the diversity of processes that depend on this selenium-containing TrxR. The provision of reduced thioredoxin for two important cell processes, DNA synthesis and gene transcription, implicates TrxR as a key enzyme in the control of cell growth. The selenocysteine residue in mammalian thioredoxin reductase (TrxR) was first identified in the 75Se-labeled protein isolated from a human lung adenocarcinoma cell line.2 The unexpected discovery of a selenocysteine residue in a protein that proved to be mammalian TrxR originated from experiments designed to characterize a putative selenoprotein produced by nonsense mutants of a cytochrome P-450 isozyme.14 A 75Se-labeled protein was purified to apparent homogeneity from the human lung adenocarcinoma cells but its physicochemical properties did not match those of a cytochrome P-450 species. Instead of a cytochrome 1
A. Holmgren, Annu. Rev. Biochem. 54, 237 (1985). T. Tamura and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 1006 (1996). 3 V. N. Gladyshev, K.-T. Jeang, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 6146 (1996). 4 S.-Y. Liu and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 94, 6138 (1997). 5 P. Y. Gasdaska, J. R. Gasdaska, S. Cochran, and G. Powis, FEBS Lett. 373, 5 (1995). 6 L. Thelander and P. Reichard, Annu. Rev. Biochem. 48, 133 (1979). 7 G. Spyrou, M. Bjornstedt, S. Kumar, and A. Holmgren, FEBS Lett. 368, 59 (1995). 8 M. L. Handel, C. L. Watts, A. DeFazio, R. O. Day, and R. L. Sutherland, Proc. Natl. Acad. Sci. U.S.A. 92, 4497 (1995). 9 V. Makropoulos, T. Bruning, and K. Schulze-Osthoff, Arch. Toxicol. 70, 277 (1996). 10 I. Y. Kim and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 94, 12904 (1997). 11 R. B. Silverman and D. L. Nandi, Biochem. Biophys. Res. Commun. 155, 1248 (1988). 12 H. Z. Chae, S. J. Chung, and S. G. Rhee, J. Biol. Chem. 269, 27670 (1994). 13 M. Bjornstedt, J. Xue, W. Huang, B. Akesson, and A. Holmgren, J. Biol. Chem. 269, 29382 (1994). 14 S. Yamano, P. T. Nhamburo, T. Aoyama, U. A. Meyer, T. Inaba, W. Kalow, H. V. Gelboin, O. W. McBride, and F. J. Gonzalez, Biochemistry 28, 7340 (1989). 2
METHODS IN ENZYMOLOGY, VOL. 347
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chromophore the dimeric 57-kDa subunit protein contained FAD and the flavin was reduced specifically by NADPH. Unfortunately this selenocysteine-containing protein was N-blocked and N-terminal amino acid sequence information was not obtained. However, the total amino acid compositional analysis indicated similarities to the amino acid content of rat liver thioredoxin reductase.15 Indeed, the 75 Se-labeled enzyme was shown to catalyze the NADPH-dependent reduction of 5,5 -dithiobis(2-nitrobenzoate) (DTNB) and also thioredoxin as substrates, thus indicating its identity as a thioredoxin reductase. Subsequently seleniumcontaining TrxRs from other mammalian cells and tissues were identified.3,4,16 Mammalian TrxRs now appear to have three isozymes designated as TrxR1, TrxR2,17 and TrxR3.18 It seems widely accepted to designated TrxR1 as the dominant cytosolic enzyme, whereas TrxR2 is referred to as the mitochondrial type. TrxR3 was first identified in a gene sequence but its expression seems to be lower than that of TrxR1. Alternatively, TrxR3 expression may be organ specific or produced under certain physiological conditions in cells. The present article focuses on skills and techniques for studies of mammalian thioredoxin reductase and also for general selenium biochemistry. The radioisotope 75Se is a useful tool for the detection and identification of selenoproteins on sodium dodecyl sulfate (SDS)–polyacrylamide gels, and the 75Se-labeled selenocysteine residue can be chemically identified by amino acid analysis after chemical derivatization. Technical precautions in the purification of selenoproteins are also described for researchers seeking undiscovered selenoenzymes. Labeling Proteins with Selenium-75 The radioisotope selenium-75 is a powerful isotope used in selenium biochemistry. It emits γ rays with 0.265 eV (59%) and 0.280 eV (25%), and decays with a half-life time of 120.4 days. 75Se-labeled proteins can be detected by this moderately strong radioactivity, yet this half-life time gives us an opportunity to diminish the radioactivity of the biological wastes in just a few months. The presence of 75Se is readily detected with a portable Geiger–Muller counter, and the radioactivity is determined in a γ -ray counter such as the Beckman (Fullerton, CA) γ -5500 or the Wallace 1470 Wizard automatic γ counter. [75Se]Selenite can be purchased from the Research Reactor Facility, University of Missouri (Columbia, MO). The radioisotope is delivered in the form of selenious acid (H2SeO3) in a small volume of 7–30% nitric acid. Its specific activity is high enough to allow us to 15
M. Luthman and A. Holmgren, Biochemistry 21, 6628 (1982). J. Nordberg, L. Zhong, A. Holmgren, and E. S. Arner, J. Biol. Chem. 273, 10835 (1998). 17 S. R. Lee, J. R. Kim, K. S. Kwon, H. W. Yoon, R. L. Levine, A. Ginsburg, and S. G. Rhee, J. Biol. Chem. 274, 4722 (1999). 18 Q. A. Sun, Y. Wu, F. Zappacosta, K.-T. Jeang, B. J. Lee, D. L. Hatfield, and V. N. Gladyshev, J. Biol. Chem. 274, 24552 (1999). 16
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ignore the original content of selenium. Because selenite is one of the more effective chemical forms in labeling the selenocysteine residue with 75Se, the sodium [75Se]selenite is directly added to the culture broth and incubated for the desired period of time. In most cases the small amount of nitric, acid added with the selenite has no effect on the growth of cells. It is strongly recommended that a small amount of “cold” or nonradioactive sodium selenite be added before use to avoid radiocolloid formation. In experiments with the human adenocarcinoma cell line NCI-H441,2 0.1 μM sodium selenite containing 75Se (368 Ci/mmol) was added to the medium and cultures were routinely incubated for 4 days. To determine the optimum time of harvest cells were collected at 6, 24, 48, 72, and 96 hr and directly analyzed by SDS–Polyacrylamide gel electrophoresis (PAGE) followed by PhosphorImager (Molecular Devices, Sunnyvale, CA) detection of radioactivity. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis and Autoradiography PhosphoImager analysis provides a convenient and sensitive method for detecting proteins labeled with 75Se after separation by SDS–PAGE and drying of gels. A cell suspension (about 5 mg wet weight in 30 μl of phosphate-buffered saline is mixed with 30 μl of SDS–PAGE sampling buffer, and boiled for 10 min. The selenocysteine residue can survive the heat treatment because of the high content of 2-mercaptoethanol. Then 5- to 20-μl portions of the heated sample are loaded on an SDS–polyacrylamide gel. Selenoproteins can be developed by SDS–PAGE without any obvious degradation unless ammonium peroxodisulfate, a radical polymerization initiator, remains in the polyacrylamide gel. Oxidizing reagents and radical species are reactive with selenocysteine residues, and 75Se may be totally eliminated from the selenoproteins during the course of electrophoresis. This can be avoided by running 10 ml of buffer containing 10% (w/v) thioglycolate through the gel before the sample is loaded, or alternatively by including 5 mM 2-mercaptoethanol or dithiothreitol (DTT) in the running buffer. Autoradiography is more conveniently and better performed by a PhosphorImager technique. Authors have noted that 3000 cpm of 75Se is sufficient for development of a clear image when the gel is exposed to an imaging plate for only 2 hr. For routine analysis, the exposure is usually performed overnight (Fig. 1). Identification of Selenocysteine Residues Identification of 75Se-labeled selenocysteine is the critical evidence for characterization of a selenocysteine-dependent selenoprotein.19,20 Some proteins can 19
R. Read, T. Bellew, J.-G. Yang, K. E. Hill, I. S. Palmer, and R. F. Burk, J. Biol. Chem. 268, 17899 (1990). 20 J. E. Cone, R. M. del Rio, J. N. Davis, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 73, 2659 (1976).
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FIG. 1. SDS–PAGE and autoradiography of 75Se-labeled proteins in the human lung adenocarcinoma cell line NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (v/v) fetal bovine serum and 680 kBq of 75Se-labeled selenite for (lane 1) 6, (lane 2) 24, (lane 3) 48, (lane 4) 72, and (lane 5) 96 hr. The cells were harvested by trypsin–EDTA treatment, boiled in SDS–PAGE sample buffer, and subjected to SDS–PAGE analysis (left; Coomassie Brilliant Blue staining). The gel was dried and subjected to autoradiography (right).
bind elemental 75Se or [75Se]selenite with high affinity, microbial samples may contain significant amounts of [75Se]selenomethionine,21 and the resulting radioactive proteins may be confused with specific selenoproteins.22,23 Selenocysteine can be identified on an amino acid analyzer but it requires chemical derivatization before the 75Se-labeled protein is subjected to acid hydrolysis. Selenocysteine can be almost completely decomposed when heated at 110◦ in 6 N HCl in the presence of trace amounts of oxygen (survival rate, 6%). Iodoacetate is frequently used for the protective derivatization of selenocysteine residues, and the resulting Se-carboxymethylselenocysteine (CM-Secys) can survive the entire procedure. A homologous derivatization can be carried out with 3-bromopropionate, which yields Se-carboxyethylselenocysteine (CE-Secys). A combination of CM-Secys and CE-Secys can illustrate that the selenium moiety of the 75Se-labeled protein is in the form of selenocysteine as described in the following procedure. Purified 75Se-labeled protein (40 μg) is washed three times with distilled water and concentrated to 40 μl with a Centricon-10 microcencentrator (Amicon, Danvers, MA). The solution is mixed with 60 μl of 100 mM NaBH4 in 20 mM NaOH aqueous solution, and incubated under argon at room temperature for 30 min. Sodium iodoacetate (or sodium 3-bromopropionate) is added to the mixture
21
M. G. M. Hartmanis and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 79, 4912 (1982). M. P. Bansal, C. J. Oborn, K. G. Danielson, and D. Medina, In Vitro 3, 167 (1989). 23 R. Sinha, M. P. Bansal, H. Ganther, and D. Medina, Carcinogenesis 14, 1895 (1993). 22
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to a final concentration of 50 μM, and the mixture is incubated under argon at room temperature for 40 min. Then, 2-mercaptoethanol is added to quench the alkylation reaction. The protein is washed three times with distilled water by ultrafiltration, taken to dryness, and then hydrolyzed in 6 M HCl at 155◦ under argon. The hydrolysate is dried, treated with small amount of NaBH4, mixed with authentic CM-Secys and CE-Secys (each at 1.3 μmol), and chromatographed on an amino acid analyzer.20,21 The eluate from the analyzer column is collected in 1-min fractrions, and the radioactivity contained in these fractions is determined with a Beckman model 5500 γ counter. On the amino acid analyzer, CM-Secys is usually eluted earlier than CE-Secys. When the labeled protein is alkylated with iodoacetate, the radioactive elution profile of the hydrolysate coincides exactly with CM-Secys (Fig. 2A). When 3-bromopropionate is used for the alkylation,
FIG. 2. Amino acid analyzer chromatogram of 75Se-labeled compounds from an acid hydrolysate of carboxymethylated (A) and carboxyethylated (B) selenoprotein. The hydrolysate was mixed with CM-Secys (I) and CE-Secys (II) before the chromatography. Solid lines represent amino acid elution and open circles represent 75Se radioactivity.
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75
Se in the hydrolysate coincides with that of CE-Secys (Fig. 2B). Throughout the procedure of alkylation, hydrolysis, and chromatography, the recovery of 75Se is usually 47 and 65% for CM-Secys and CE-Secys formation, respectively.
Precautions for Purifying Selenocysteine-Containing Proteins Purification can be carried out by conventional protein purification methods, but certain precautions are taken. First, selenocysteine has a lower redox potential and lower pKa than cysteine, and it requires some reducing reagent to maintain the selenol state. In the absence of such a reducing reagent, it may undergo oxidation to seleninate (–SeO2H) and decompose to a dehydroalanine residue through α,β-elimination under alkaline conditions. Dithiothreitol at 2 mM is a favorite reagent of the present authors and it is always accompanied by EDTA-Mg,K complex at 0.1 mM to prevent metal-catalyzed thiol radical formation that is also harmful to selenocysteine residues. In addition, the selenocysteine residue is also reactive toward various nucleophiles. Therefore sodium azide should be omitted from buffers used for gel-filtration column chromatography. 75Se-labeled protein can be located in the column bed if a Geiger–Muller counter is held close to the column tube during the course of purification. This physicochemical property as a radiant γ -ray emitter is particularly useful in estimating the elution of selenoproteins during gel filtration or other type of protein purification in open columns.
Separation of Thioredoxin Reductase 1 from Thioredoxin Reductase 2 in Rat Liver Homogenates A convenient and efficient method of separation of rat liver TrxR1 from TrxR2 by adjustment of pH has been described by S. G. Rhee and co-workers.17 Rat livers (1 kg) are homogenized in 4 liters of 20 mM Tris-HCl (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol, 0.05 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), pepstatin (0.5 mg/ml), and aprotinin (0.5 mg/ml). The homogenate is centrifuged at 70,000g for 30 min at 4◦ , and the resulting supernatant is adjusted to pH 5.0 with 1 M acetic acid and then centrifuged again at 70,000g for 30 min at 4◦ . The resulting pellet and supernatant are adjusted to conditions suitable for immunoblot analysis with antibodies to TrxR1 and TrxR2. TrxR1 is detected only in the supernatant, whereas TrxR2 was present mostly in the pellet. Thus, the supernatant and pellet serve as sources for purification of TrxR1 and TrxR2, respectively. For purification of TrxR1, the supernatant (40 g of protein) from the pH 5 precipitation step is adjusted to pH 7.8 with 1 M ammonium hydroxide and then applied to a DEAE-Sephacel (Pharmacia, Piscataway, NJ) column (10 × 16 cm)
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that has been equilibrated with 20 mM Tris-HCl (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol, and 0.01 mM AEBSF. The column is washed consecutively with 2.5 liters of equilibrium buffer and 2.5 liters of equilibration buffer containing 100 mM NaCl. Proteins are eluted from the column with a linear gradient of 100 to 400 mM NaCl in 5 liters of equilibration buffer, and fractions (25 ml) are collected and TrxR1 is detected by immunoblot analysis. The peak fractions (10.4 g of protein), corresponding to 300 to 380 mM NaCl on the gradient, are pooled, dialyzed overnight against 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA, 1 mM DTT, and 0.01 mM AEBSF, and then applied to a 2 ,5 ADP-agarose column (2 × 7 cm) that has been equilibrated with 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. The column is washed with 100 ml of equilibration buffer, and proteins are then eluted stepwise with 100 ml each of equilibration buffer containing 200 mM KCl, equilibration buffer containing 200 mM sodium phosphate and 200 mM KCl, and equilibration buffer containing 1 M NaCl and 200 mM KCl. TrxR1 is present almost exclusively in the fractions eluted by the buffer containing 200 mM KCl as revealed by SDS–PAGE and Coomassie blue staining and by immunoblot analysis. Peak fractions (19.8 mg of protein) are pooled and then adjusted to 1.2 M ammonium sulfate by addition of 4 M ammonium sulfate. After removal of the resulting precipitate by centrifugation, the supernatant is applied to a Phenyl-5PW high-performance liquid chromatography (HPLC) column (0.75 × 7.5 cm) that has been equilibrated with 20 mM HEPES– NaOH (pH 7.5) containing 1 mM DTT, 1 mM EDTA, and 1.2 M ammonium sulfate. The column is washed with 60 ml of equilibration buffer, and proteins are then eluted with a decreasing linear gradient of 1.2 to 0 M ammonium sulfate in 120 ml of 20 mM HEPES–NaOH (pH 7.5) containing 1 mM DTT and 1 mM EDTA. Peak fractions, corresponding to 0.8 to 0.64 M ammonium sulfate on the gradient, are pooled, concentrated, dialyzed against 20 mM HEPES–NaOH (pH 7.5) containing 1 mM DTT and 1 mM EDTA, divided into portions, and stored at −70◦ . Selective Alkylation of Selenocysteine Residue 498 of Thioredoxin Reductase 1 The essential role of selenocysteine residue 498 (Secys-498) in catalysis has been demonstrated by concomitant reduction of enzyme activity to 1% or less with selective alkylation of Secys-498.24 In the experiment, reaction of native NADPH-reduced enzyme with bromo[1-14C]acetate not only inhibited enzyme activity by 99% but also resulted in incorporation of 1.1 equivalents of alkyl group per subunit, of which >90% was present in the carboxymethyl (CM) derivative of Secys-498 and about 5% was present in the CM derivative of Cys-497. Such a highly selective alkylation can be carried out at pH 6.5 when bromoacetate instead 24
S. N. Gorlotov and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 95, 8525 (1998).
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of iodoacetate is used as alkylating agent, making the reaction more selective for the fully ionized selenol group of selenocysteine. However, if the pKa of the thiol group of the adjacent cysteine residue is abnormally low, it also might be alkylated under these conditions. Amino acid analysis of the alkylated enzyme, after acid hydrolysis, showed that labeled CM-Secys accounted for at least 80% of the recovered alkyl group and that 20% or less was in CM-Cys. The amount of CM-Secys, when corrected for losses caused by the marked oxygen lability of the selenoether, corresponded to complete derivatization of one Secys-498 per subunit. In this experiment 75Se-labeled HeLa cell TrxR (11.0 nmol; 5.8 × 105 cpm) in 200 μl of 20 mM potassium phosphate (pH 7.0), 1 mM EDTA, and 10% (v/v) glycerol was reduced with 240 nmol of NADPH under argon for 20 min and then reacted with bromo[1-14C]acetic acid (175 nmol; 10 μCi) for 60 min in the dark under argon. The reaction was quenched by the addition of DTT (2 μmol) and the pH was adjusted to 8.0. Guanidine hydrochloride was added to 6 M and after 5 min the enzyme was dialyzed against 1.5 liters of 20 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA under argon for 2.5 hr. The dialyzed protein was digested with trypsin N-1-tosylamido-2-phenylethyl chloromethyl ketone, 56 μg, for 4 hr under argon and then was adjusted to pH 2.0 with HCOOH and loaded on a C18 HPLC column. Peptides were eluted with a 0–50% (v/v) linear gradient of acetonitrile in 0.05% (w/v) trifluoroacetic acid. The 75Se radioactivity of the collected fraction was detected by γ counting and a 10–20% aliquot of each fraction was analyzed for total radioactivity by liquid scintillation counting. The amount of 14C radioactivity was calculated by difference. Alkylation experiments performed at pH 8 use a similar procedure except that the initial enzyme solution is adjusted to pH 8 with potassium phosphate and the incubation time with bromoacetate is 30 min. Catalytic Role of Selenocysteine Residue Mammalian TrxR1 has two sets of redox centers, one consisting of Cys59/Cys-64 adjacent to the flavin ring of FAD and another center consisting of Cys-497/Secys-498 near the C terminus. By selective alkylation of Secys-498 it has been demonstrated that the thioredoxin-induced oxidation of Cys-59-SH/Cys64-SH is completely blocked, and that the alkylated enzyme shows negligible NADPH-disulfide oxidoreductase activity.25 Mammalian TrxR might need the redox-active C-terminal sequence for transferring the reducing equivalents from the internal dithiol group to the outer substrate, the oxidized form of thioredoxin, or some other low molecular weight compounds. Speculation as to the ternary structure of mammalian TrxR, made on the basis of mammalian glutathione reductase structure, has led to the conclusion that the redox-active C-terminal sequence 25
S. R. Lee, S. Bar-Noy, J. Kwon, R. L. Levine, T. C. Stadtman, and S. G. Rhee, Proc. Natl. Acad. Sci. U.S.A. 97, 2521 (2000).
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FIG. 3. (A) Oxidized form of N-acetyl-Ala-Gly-Cys-Secys-Gly in its most stable conformation. (B) The reduced form of the peptide in its most stable conformation. The HOMO and LUMO are designated by the red–blue cages.
appears still too far from the internal redox-active cysteines of the same subunit, but close to the active site of the other subunit.26 In a head-to-tail arrangement the C-terminal Cys-497/Secys-498 residues of one monomer might be located adjacent to the Cys-59 and Cys-64 residues of the second monomer. Accordingly, if the reductive half-reaction of TrxR is similar to that of glutathione reductase the charge transfer complex formation may be followed by exchange of the nascent Cys-59 and Cys-64 dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. X-ray crystallography is in progress for elucidating
26
L. Zong, E. S. Arner, and A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 97, 5854 (2000).
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the structure of mammalian thioredoxin reductase,27 and it should aid in deducing the reaction mechanism and the catalytic roles of Secys. Static protein structure would still require biochemical evidence to deduce the dynamic movement of the Secys-containing C-terminal tail during the course of catalytic turnover. Computation of frontier molecular orbitals has become one of the routine approaches used by organic chemists to speculate on the chemistry of novel compounds. This could also be useful in speculating on the dynamic biochemical properties of macromolecules. However, it is not realistically possible to compute the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) on the basis of whole protein structure. Instead of the entire protein molecule, we can compute molecular orbitals of the C-terminal sequence N-acetyl-Ala-Gly-Cys-Secys-Gly by the use of PC-based software available on the web.28 Figure 3A shows the oxidized form of the C-terminal sequence; a thioselenide bridge is protruding from the main chain, which takes a rather straight line at its most stable conformation. The LUMO, localized on the thioselenide bond, indicates the portion where electrons from the reductant are most likely to be accepted. Figure 3B represents the most stable conformation of the reduced form; the selenol group of Secys and thiol group of Cys are oriented in opposite directions. The HOMO, localized on the selenol group of the sequence, suggests that the selenol group of Secys would serve as the nucleophile in reducing the substrate. An interesting implication was obtained from the energy calculation of the two forms of the peptide. Calculation of the heat of formation indicated that the reduced form is more stable than the oxidized thioselenide form provided that the selenol group is deprotonated; when the selenol is protonated the C-terminal sequence is energized as highly as the oxidized form, which has the thioselenide bond between the vicinal Cys and Secys. Further biochemical evidence would be necessary to verify this computer calculation and to elucidate in detail the catalytic role of the penultimate selenocysteine residue.
27
L. Zong, K. Persson, T. Sandalova, G. Schneider, and A. Holmgren, Acta Crystallogr. D Biol. Crystallogr. 56, 1191 (2000). 28 http://www.fujitsu.co.jp/jp/soft/wimmopac/home-e.html
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[27] Mammalian Thioredoxin Reductases By TAKASHI TAMURA and THRESSA C. STADTMAN Introduction Thioredoxin reductase (TrxR) is an NADPH-dependent, FAD-containing disulfide reductase that plays an important role in cell proliferation.1 Unlike the wellcharacterized homologs from yeast and prokaryotes, the larger mammalian enzyme is a selenoprotein that contains a selenocysteine (Secys) residue2 in the sequenceCys-Secys-Gly (end) at the C terminus of each subunit.3–5 Catalysis of electron transfer from NADPH to thioredoxin, which in turn is linked to critical components of cell metabolism such as ribonucleotide reductase,6 AP-1 and NF-κB transcription factors,7–10 vitamin K epoxide reductase,11 thiol peroxidase,12 and plasma glutathione peroxidase,13 illustrates the diversity of processes that depend on this selenium-containing TrxR. The provision of reduced thioredoxin for two important cell processes, DNA synthesis and gene transcription, implicates TrxR as a key enzyme in the control of cell growth. The selenocysteine residue in mammalian thioredoxin reductase (TrxR) was first identified in the 75Se-labeled protein isolated from a human lung adenocarcinoma cell line.2 The unexpected discovery of a selenocysteine residue in a protein that proved to be mammalian TrxR originated from experiments designed to characterize a putative selenoprotein produced by nonsense mutants of a cytochrome P-450 isozyme.14 A 75Se-labeled protein was purified to apparent homogeneity from the human lung adenocarcinoma cells but its physicochemical properties did not match those of a cytochrome P-450 species. Instead of a cytochrome 1
A. Holmgren, Annu. Rev. Biochem. 54, 237 (1985). T. Tamura and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 1006 (1996). 3 V. N. Gladyshev, K.-T. Jeang, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 6146 (1996). 4 S.-Y. Liu and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 94, 6138 (1997). 5 P. Y. Gasdaska, J. R. Gasdaska, S. Cochran, and G. Powis, FEBS Lett. 373, 5 (1995). 6 L. Thelander and P. Reichard, Annu. Rev. Biochem. 48, 133 (1979). 7 G. Spyrou, M. Bjornstedt, S. Kumar, and A. Holmgren, FEBS Lett. 368, 59 (1995). 8 M. L. Handel, C. L. Watts, A. DeFazio, R. O. Day, and R. L. Sutherland, Proc. Natl. Acad. Sci. U.S.A. 92, 4497 (1995). 9 V. Makropoulos, T. Bruning, and K. Schulze-Osthoff, Arch. Toxicol. 70, 277 (1996). 10 I. Y. Kim and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 94, 12904 (1997). 11 R. B. Silverman and D. L. Nandi, Biochem. Biophys. Res. Commun. 155, 1248 (1988). 12 H. Z. Chae, S. J. Chung, and S. G. Rhee, J. Biol. Chem. 269, 27670 (1994). 13 M. Bjornstedt, J. Xue, W. Huang, B. Akesson, and A. Holmgren, J. Biol. Chem. 269, 29382 (1994). 14 S. Yamano, P. T. Nhamburo, T. Aoyama, U. A. Meyer, T. Inaba, W. Kalow, H. V. Gelboin, O. W. McBride, and F. J. Gonzalez, Biochemistry 28, 7340 (1989). 2
METHODS IN ENZYMOLOGY, VOL. 347
C 2002 by Academic Press. Copyright All rights of reproduction in any form reserved. 0076-6879/02 $35.00
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chromophore the dimeric 57-kDa subunit protein contained FAD and the flavin was reduced specifically by NADPH. Unfortunately this selenocysteine-containing protein was N-blocked and N-terminal amino acid sequence information was not obtained. However, the total amino acid compositional analysis indicated similarities to the amino acid content of rat liver thioredoxin reductase.15 Indeed, the 75 Se-labeled enzyme was shown to catalyze the NADPH-dependent reduction of 5,5 -dithiobis(2-nitrobenzoate) (DTNB) and also thioredoxin as substrates, thus indicating its identity as a thioredoxin reductase. Subsequently seleniumcontaining TrxRs from other mammalian cells and tissues were identified.3,4,16 Mammalian TrxRs now appear to have three isozymes designated as TrxR1, TrxR2,17 and TrxR3.18 It seems widely accepted to designated TrxR1 as the dominant cytosolic enzyme, whereas TrxR2 is referred to as the mitochondrial type. TrxR3 was first identified in a gene sequence but its expression seems to be lower than that of TrxR1. Alternatively, TrxR3 expression may be organ specific or produced under certain physiological conditions in cells. The present article focuses on skills and techniques for studies of mammalian thioredoxin reductase and also for general selenium biochemistry. The radioisotope 75Se is a useful tool for the detection and identification of selenoproteins on sodium dodecyl sulfate (SDS)–polyacrylamide gels, and the 75Se-labeled selenocysteine residue can be chemically identified by amino acid analysis after chemical derivatization. Technical precautions in the purification of selenoproteins are also described for researchers seeking undiscovered selenoenzymes. Labeling Proteins with Selenium-75 The radioisotope selenium-75 is a powerful isotope used in selenium biochemistry. It emits γ rays with 0.265 eV (59%) and 0.280 eV (25%), and decays with a half-life time of 120.4 days. 75Se-labeled proteins can be detected by this moderately strong radioactivity, yet this half-life time gives us an opportunity to diminish the radioactivity of the biological wastes in just a few months. The presence of 75Se is readily detected with a portable Geiger–Muller counter, and the radioactivity is determined in a γ -ray counter such as the Beckman (Fullerton, CA) γ -5500 or the Wallace 1470 Wizard automatic γ counter. [75Se]Selenite can be purchased from the Research Reactor Facility, University of Missouri (Columbia, MO). The radioisotope is delivered in the form of selenious acid (H2SeO3) in a small volume of 7–30% nitric acid. Its specific activity is high enough to allow us to 15
M. Luthman and A. Holmgren, Biochemistry 21, 6628 (1982). J. Nordberg, L. Zhong, A. Holmgren, and E. S. Arner, J. Biol. Chem. 273, 10835 (1998). 17 S. R. Lee, J. R. Kim, K. S. Kwon, H. W. Yoon, R. L. Levine, A. Ginsburg, and S. G. Rhee, J. Biol. Chem. 274, 4722 (1999). 18 Q. A. Sun, Y. Wu, F. Zappacosta, K.-T. Jeang, B. J. Lee, D. L. Hatfield, and V. N. Gladyshev, J. Biol. Chem. 274, 24552 (1999). 16
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ignore the original content of selenium. Because selenite is one of the more effective chemical forms in labeling the selenocysteine residue with 75Se, the sodium [75Se]selenite is directly added to the culture broth and incubated for the desired period of time. In most cases the small amount of nitric, acid added with the selenite has no effect on the growth of cells. It is strongly recommended that a small amount of “cold” or nonradioactive sodium selenite be added before use to avoid radiocolloid formation. In experiments with the human adenocarcinoma cell line NCI-H441,2 0.1 μM sodium selenite containing 75Se (368 Ci/mmol) was added to the medium and cultures were routinely incubated for 4 days. To determine the optimum time of harvest cells were collected at 6, 24, 48, 72, and 96 hr and directly analyzed by SDS–Polyacrylamide gel electrophoresis (PAGE) followed by PhosphorImager (Molecular Devices, Sunnyvale, CA) detection of radioactivity. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis and Autoradiography PhosphoImager analysis provides a convenient and sensitive method for detecting proteins labeled with 75Se after separation by SDS–PAGE and drying of gels. A cell suspension (about 5 mg wet weight in 30 μl of phosphate-buffered saline is mixed with 30 μl of SDS–PAGE sampling buffer, and boiled for 10 min. The selenocysteine residue can survive the heat treatment because of the high content of 2-mercaptoethanol. Then 5- to 20-μl portions of the heated sample are loaded on an SDS–polyacrylamide gel. Selenoproteins can be developed by SDS–PAGE without any obvious degradation unless ammonium peroxodisulfate, a radical polymerization initiator, remains in the polyacrylamide gel. Oxidizing reagents and radical species are reactive with selenocysteine residues, and 75Se may be totally eliminated from the selenoproteins during the course of electrophoresis. This can be avoided by running 10 ml of buffer containing 10% (w/v) thioglycolate through the gel before the sample is loaded, or alternatively by including 5 mM 2-mercaptoethanol or dithiothreitol (DTT) in the running buffer. Autoradiography is more conveniently and better performed by a PhosphorImager technique. Authors have noted that 3000 cpm of 75Se is sufficient for development of a clear image when the gel is exposed to an imaging plate for only 2 hr. For routine analysis, the exposure is usually performed overnight (Fig. 1). Identification of Selenocysteine Residues Identification of 75Se-labeled selenocysteine is the critical evidence for characterization of a selenocysteine-dependent selenoprotein.19,20 Some proteins can 19
R. Read, T. Bellew, J.-G. Yang, K. E. Hill, I. S. Palmer, and R. F. Burk, J. Biol. Chem. 268, 17899 (1990). 20 J. E. Cone, R. M. del Rio, J. N. Davis, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 73, 2659 (1976).
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FIG. 1. SDS–PAGE and autoradiography of 75Se-labeled proteins in the human lung adenocarcinoma cell line NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (v/v) fetal bovine serum and 680 kBq of 75Se-labeled selenite for (lane 1) 6, (lane 2) 24, (lane 3) 48, (lane 4) 72, and (lane 5) 96 hr. The cells were harvested by trypsin–EDTA treatment, boiled in SDS–PAGE sample buffer, and subjected to SDS–PAGE analysis (left; Coomassie Brilliant Blue staining). The gel was dried and subjected to autoradiography (right).
bind elemental 75Se or [75Se]selenite with high affinity, microbial samples may contain significant amounts of [75Se]selenomethionine,21 and the resulting radioactive proteins may be confused with specific selenoproteins.22,23 Selenocysteine can be identified on an amino acid analyzer but it requires chemical derivatization before the 75Se-labeled protein is subjected to acid hydrolysis. Selenocysteine can be almost completely decomposed when heated at 110◦ in 6 N HCl in the presence of trace amounts of oxygen (survival rate, 6%). Iodoacetate is frequently used for the protective derivatization of selenocysteine residues, and the resulting Se-carboxymethylselenocysteine (CM-Secys) can survive the entire procedure. A homologous derivatization can be carried out with 3-bromopropionate, which yields Se-carboxyethylselenocysteine (CE-Secys). A combination of CM-Secys and CE-Secys can illustrate that the selenium moiety of the 75Se-labeled protein is in the form of selenocysteine as described in the following procedure. Purified 75Se-labeled protein (40 μg) is washed three times with distilled water and concentrated to 40 μl with a Centricon-10 microcencentrator (Amicon, Danvers, MA). The solution is mixed with 60 μl of 100 mM NaBH4 in 20 mM NaOH aqueous solution, and incubated under argon at room temperature for 30 min. Sodium iodoacetate (or sodium 3-bromopropionate) is added to the mixture
21
M. G. M. Hartmanis and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 79, 4912 (1982). M. P. Bansal, C. J. Oborn, K. G. Danielson, and D. Medina, In Vitro 3, 167 (1989). 23 R. Sinha, M. P. Bansal, H. Ganther, and D. Medina, Carcinogenesis 14, 1895 (1993). 22
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to a final concentration of 50 μM, and the mixture is incubated under argon at room temperature for 40 min. Then, 2-mercaptoethanol is added to quench the alkylation reaction. The protein is washed three times with distilled water by ultrafiltration, taken to dryness, and then hydrolyzed in 6 M HCl at 155◦ under argon. The hydrolysate is dried, treated with small amount of NaBH4, mixed with authentic CM-Secys and CE-Secys (each at 1.3 μmol), and chromatographed on an amino acid analyzer.20,21 The eluate from the analyzer column is collected in 1-min fractrions, and the radioactivity contained in these fractions is determined with a Beckman model 5500 γ counter. On the amino acid analyzer, CM-Secys is usually eluted earlier than CE-Secys. When the labeled protein is alkylated with iodoacetate, the radioactive elution profile of the hydrolysate coincides exactly with CM-Secys (Fig. 2A). When 3-bromopropionate is used for the alkylation,
FIG. 2. Amino acid analyzer chromatogram of 75Se-labeled compounds from an acid hydrolysate of carboxymethylated (A) and carboxyethylated (B) selenoprotein. The hydrolysate was mixed with CM-Secys (I) and CE-Secys (II) before the chromatography. Solid lines represent amino acid elution and open circles represent 75Se radioactivity.
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75
Se in the hydrolysate coincides with that of CE-Secys (Fig. 2B). Throughout the procedure of alkylation, hydrolysis, and chromatography, the recovery of 75Se is usually 47 and 65% for CM-Secys and CE-Secys formation, respectively.
Precautions for Purifying Selenocysteine-Containing Proteins Purification can be carried out by conventional protein purification methods, but certain precautions are taken. First, selenocysteine has a lower redox potential and lower pKa than cysteine, and it requires some reducing reagent to maintain the selenol state. In the absence of such a reducing reagent, it may undergo oxidation to seleninate (–SeO2H) and decompose to a dehydroalanine residue through α,β-elimination under alkaline conditions. Dithiothreitol at 2 mM is a favorite reagent of the present authors and it is always accompanied by EDTA-Mg,K complex at 0.1 mM to prevent metal-catalyzed thiol radical formation that is also harmful to selenocysteine residues. In addition, the selenocysteine residue is also reactive toward various nucleophiles. Therefore sodium azide should be omitted from buffers used for gel-filtration column chromatography. 75Se-labeled protein can be located in the column bed if a Geiger–Muller counter is held close to the column tube during the course of purification. This physicochemical property as a radiant γ -ray emitter is particularly useful in estimating the elution of selenoproteins during gel filtration or other type of protein purification in open columns.
Separation of Thioredoxin Reductase 1 from Thioredoxin Reductase 2 in Rat Liver Homogenates A convenient and efficient method of separation of rat liver TrxR1 from TrxR2 by adjustment of pH has been described by S. G. Rhee and co-workers.17 Rat livers (1 kg) are homogenized in 4 liters of 20 mM Tris-HCl (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol, 0.05 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), pepstatin (0.5 mg/ml), and aprotinin (0.5 mg/ml). The homogenate is centrifuged at 70,000g for 30 min at 4◦ , and the resulting supernatant is adjusted to pH 5.0 with 1 M acetic acid and then centrifuged again at 70,000g for 30 min at 4◦ . The resulting pellet and supernatant are adjusted to conditions suitable for immunoblot analysis with antibodies to TrxR1 and TrxR2. TrxR1 is detected only in the supernatant, whereas TrxR2 was present mostly in the pellet. Thus, the supernatant and pellet serve as sources for purification of TrxR1 and TrxR2, respectively. For purification of TrxR1, the supernatant (40 g of protein) from the pH 5 precipitation step is adjusted to pH 7.8 with 1 M ammonium hydroxide and then applied to a DEAE-Sephacel (Pharmacia, Piscataway, NJ) column (10 × 16 cm)
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that has been equilibrated with 20 mM Tris-HCl (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol, and 0.01 mM AEBSF. The column is washed consecutively with 2.5 liters of equilibrium buffer and 2.5 liters of equilibration buffer containing 100 mM NaCl. Proteins are eluted from the column with a linear gradient of 100 to 400 mM NaCl in 5 liters of equilibration buffer, and fractions (25 ml) are collected and TrxR1 is detected by immunoblot analysis. The peak fractions (10.4 g of protein), corresponding to 300 to 380 mM NaCl on the gradient, are pooled, dialyzed overnight against 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA, 1 mM DTT, and 0.01 mM AEBSF, and then applied to a 2 ,5 ADP-agarose column (2 × 7 cm) that has been equilibrated with 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. The column is washed with 100 ml of equilibration buffer, and proteins are then eluted stepwise with 100 ml each of equilibration buffer containing 200 mM KCl, equilibration buffer containing 200 mM sodium phosphate and 200 mM KCl, and equilibration buffer containing 1 M NaCl and 200 mM KCl. TrxR1 is present almost exclusively in the fractions eluted by the buffer containing 200 mM KCl as revealed by SDS–PAGE and Coomassie blue staining and by immunoblot analysis. Peak fractions (19.8 mg of protein) are pooled and then adjusted to 1.2 M ammonium sulfate by addition of 4 M ammonium sulfate. After removal of the resulting precipitate by centrifugation, the supernatant is applied to a Phenyl-5PW high-performance liquid chromatography (HPLC) column (0.75 × 7.5 cm) that has been equilibrated with 20 mM HEPES– NaOH (pH 7.5) containing 1 mM DTT, 1 mM EDTA, and 1.2 M ammonium sulfate. The column is washed with 60 ml of equilibration buffer, and proteins are then eluted with a decreasing linear gradient of 1.2 to 0 M ammonium sulfate in 120 ml of 20 mM HEPES–NaOH (pH 7.5) containing 1 mM DTT and 1 mM EDTA. Peak fractions, corresponding to 0.8 to 0.64 M ammonium sulfate on the gradient, are pooled, concentrated, dialyzed against 20 mM HEPES–NaOH (pH 7.5) containing 1 mM DTT and 1 mM EDTA, divided into portions, and stored at −70◦ . Selective Alkylation of Selenocysteine Residue 498 of Thioredoxin Reductase 1 The essential role of selenocysteine residue 498 (Secys-498) in catalysis has been demonstrated by concomitant reduction of enzyme activity to 1% or less with selective alkylation of Secys-498.24 In the experiment, reaction of native NADPH-reduced enzyme with bromo[1-14C]acetate not only inhibited enzyme activity by 99% but also resulted in incorporation of 1.1 equivalents of alkyl group per subunit, of which >90% was present in the carboxymethyl (CM) derivative of Secys-498 and about 5% was present in the CM derivative of Cys-497. Such a highly selective alkylation can be carried out at pH 6.5 when bromoacetate instead 24
S. N. Gorlotov and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 95, 8525 (1998).
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of iodoacetate is used as alkylating agent, making the reaction more selective for the fully ionized selenol group of selenocysteine. However, if the pKa of the thiol group of the adjacent cysteine residue is abnormally low, it also might be alkylated under these conditions. Amino acid analysis of the alkylated enzyme, after acid hydrolysis, showed that labeled CM-Secys accounted for at least 80% of the recovered alkyl group and that 20% or less was in CM-Cys. The amount of CM-Secys, when corrected for losses caused by the marked oxygen lability of the selenoether, corresponded to complete derivatization of one Secys-498 per subunit. In this experiment 75Se-labeled HeLa cell TrxR (11.0 nmol; 5.8 × 105 cpm) in 200 μl of 20 mM potassium phosphate (pH 7.0), 1 mM EDTA, and 10% (v/v) glycerol was reduced with 240 nmol of NADPH under argon for 20 min and then reacted with bromo[1-14C]acetic acid (175 nmol; 10 μCi) for 60 min in the dark under argon. The reaction was quenched by the addition of DTT (2 μmol) and the pH was adjusted to 8.0. Guanidine hydrochloride was added to 6 M and after 5 min the enzyme was dialyzed against 1.5 liters of 20 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA under argon for 2.5 hr. The dialyzed protein was digested with trypsin N-1-tosylamido-2-phenylethyl chloromethyl ketone, 56 μg, for 4 hr under argon and then was adjusted to pH 2.0 with HCOOH and loaded on a C18 HPLC column. Peptides were eluted with a 0–50% (v/v) linear gradient of acetonitrile in 0.05% (w/v) trifluoroacetic acid. The 75Se radioactivity of the collected fraction was detected by γ counting and a 10–20% aliquot of each fraction was analyzed for total radioactivity by liquid scintillation counting. The amount of 14C radioactivity was calculated by difference. Alkylation experiments performed at pH 8 use a similar procedure except that the initial enzyme solution is adjusted to pH 8 with potassium phosphate and the incubation time with bromoacetate is 30 min. Catalytic Role of Selenocysteine Residue Mammalian TrxR1 has two sets of redox centers, one consisting of Cys59/Cys-64 adjacent to the flavin ring of FAD and another center consisting of Cys-497/Secys-498 near the C terminus. By selective alkylation of Secys-498 it has been demonstrated that the thioredoxin-induced oxidation of Cys-59-SH/Cys64-SH is completely blocked, and that the alkylated enzyme shows negligible NADPH-disulfide oxidoreductase activity.25 Mammalian TrxR might need the redox-active C-terminal sequence for transferring the reducing equivalents from the internal dithiol group to the outer substrate, the oxidized form of thioredoxin, or some other low molecular weight compounds. Speculation as to the ternary structure of mammalian TrxR, made on the basis of mammalian glutathione reductase structure, has led to the conclusion that the redox-active C-terminal sequence 25
S. R. Lee, S. Bar-Noy, J. Kwon, R. L. Levine, T. C. Stadtman, and S. G. Rhee, Proc. Natl. Acad. Sci. U.S.A. 97, 2521 (2000).
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FIG. 3. (A) Oxidized form of N-acetyl-Ala-Gly-Cys-Secys-Gly in its most stable conformation. (B) The reduced form of the peptide in its most stable conformation. The HOMO and LUMO are designated by the red–blue cages.
appears still too far from the internal redox-active cysteines of the same subunit, but close to the active site of the other subunit.26 In a head-to-tail arrangement the C-terminal Cys-497/Secys-498 residues of one monomer might be located adjacent to the Cys-59 and Cys-64 residues of the second monomer. Accordingly, if the reductive half-reaction of TrxR is similar to that of glutathione reductase the charge transfer complex formation may be followed by exchange of the nascent Cys-59 and Cys-64 dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. X-ray crystallography is in progress for elucidating
26
L. Zong, E. S. Arner, and A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 97, 5854 (2000).
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the structure of mammalian thioredoxin reductase,27 and it should aid in deducing the reaction mechanism and the catalytic roles of Secys. Static protein structure would still require biochemical evidence to deduce the dynamic movement of the Secys-containing C-terminal tail during the course of catalytic turnover. Computation of frontier molecular orbitals has become one of the routine approaches used by organic chemists to speculate on the chemistry of novel compounds. This could also be useful in speculating on the dynamic biochemical properties of macromolecules. However, it is not realistically possible to compute the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) on the basis of whole protein structure. Instead of the entire protein molecule, we can compute molecular orbitals of the C-terminal sequence N-acetyl-Ala-Gly-Cys-Secys-Gly by the use of PC-based software available on the web.28 Figure 3A shows the oxidized form of the C-terminal sequence; a thioselenide bridge is protruding from the main chain, which takes a rather straight line at its most stable conformation. The LUMO, localized on the thioselenide bond, indicates the portion where electrons from the reductant are most likely to be accepted. Figure 3B represents the most stable conformation of the reduced form; the selenol group of Secys and thiol group of Cys are oriented in opposite directions. The HOMO, localized on the selenol group of the sequence, suggests that the selenol group of Secys would serve as the nucleophile in reducing the substrate. An interesting implication was obtained from the energy calculation of the two forms of the peptide. Calculation of the heat of formation indicated that the reduced form is more stable than the oxidized thioselenide form provided that the selenol group is deprotonated; when the selenol is protonated the C-terminal sequence is energized as highly as the oxidized form, which has the thioselenide bond between the vicinal Cys and Secys. Further biochemical evidence would be necessary to verify this computer calculation and to elucidate in detail the catalytic role of the penultimate selenocysteine residue.
27
L. Zong, K. Persson, T. Sandalova, G. Schneider, and A. Holmgren, Acta Crystallogr. D Biol. Crystallogr. 56, 1191 (2000). 28 http://www.fujitsu.co.jp/jp/soft/wimmopac/home-e.html