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Mô tả: Effect of coenzymes and thyroid hormones on the dual activitiesofXenopuscytosolic thyroid-hormone-binding protein (xCTBP)with aldehyde dehydrogenase activityKiyoshi Yamauchi and Jun–ichiro NakajimaDepartment of Biology and Geoscience, Faculty of Science, Shizuoka University, Shizuoka, JapanA cytosolic thyroid-hormone-binding protein (xCTBP),predominantly responsible for the major binding activity ofT3in the cytosol of Xenopus liver, has been shown to beidentical to aldehyde dehydrogenase class 1 (ALDH1)[Yamauchi, K., Nakajima, J., Hayashi, H., Horiuchi, R. &Tata, J.R. (1999) J. Biol. Chem. 274, 8460–8469]. Within thispaper we surveyed which signaling, and other, compoundsaffect the thyroid hormone binding activity and aldehydedehydrogenase activity of recombinant Xenopus ALDH1(xCTBP/xALDH1) while examining the relationshipbetween these two activities. NAD+and NADH (each200 lM),andtwosteroids(20lM), inhibit significantly theT3-binding activity, while NADH and NADPH (each200 lM), and iodothyronines (1 lM), inhibit the ALDHactivity. Scatchard analysis and kinetic studies of xCTBP/xALDH1 indicate that NAD+and T3are noncompetitiveinhibitors of thyroid-hormone-binding and ALDH activit-ies, respectively. These results indicate the formation of aternary complex consisting of the protein, NAD+and thy-roid hormone. Although the in vitro studies indicate thatNAD+and NADH markedly decrease T3-binding toxCTBP/xALDH1 at  10)4M, a concentration equal to theNAD content in various Xenopus tissues, photoaffinity-labeling of [125I]T3using cultured Xenopus cells demonstratesxCTBP/xALDH1 bound T3within living cells. These resultsraise the possibility that an unknown factor(s) besidesNAD+and NADH may modulate the thyroid-hormone-binding activity of xCTBP/xALDH1. In comparison, thy-roid hormone, at its physiological concentration, wouldpoorly modulate the enzyme activity of xCTBP/xALDH1.Keywords: cytosolic thyroid-hormone-binding protein;aldehyde dehydrogenase; retinoic acid synthesis; Xenopuslaevis.Hydrophobic molecules that signal via nuclear receptors,such as thyroid and steroid hormones, retinoic acid andvitamin D3, predominantly exist within plasma and withinintracellular compartments bound to specific proteins. Thekinetics and the nature of the cellular responses to thesesignaling molecules are determined by these specific bindingproteins. This has been well documented for cytosolicretinoic acid and retinol binding proteins where it has beensuggested that these binding proteins may act, not only asbuffers or reservoirs of intracellular retinoids to maintainsignificant levels of free retinoids, but also as modulatorstransporting retinoids to their target sites, the retinoidresponsive genes within the nucleus and the metabolicenzymes within the cytoplasm [1–3]. Although similarfunctions have been assumed for cytosolic thyroid-hor-mone-binding proteins (CTBPs), a unified view regardingtheir function is yet to be decided due to their divergentmolecular and hormone-binding characteristics [4–8].Recently, we purified a 59-kDa CTBP from adultXenopus liver cytosol, xCTBP, which is responsible formost of the T3binding activity within the Xenopus livercytosol [9]. Sequencing of the peptide, isolated aftertreatment of xCTBP with cyanogen bromide, revealed thatxCTBP contained an amino-acid sequence similar to that ofthe mammalian and avian aldehyde dehydrogenases class 1(ALDH1) [9]. The possibility that xCTBP was XenopusALDH1 (xALDH1) was later confirmed by examining boththe 3,3¢,5-triiodo-L-thyronine (T3) binding and the ALDHactivities of the recombinant xALDH1 [10]. The concen-trations of the 59-kDa xCTBP, investigated by photoaffin-ity-labeling with [125I]T3, in the liver and the intestinalcytosol increased gradually during the metamorphic climaxstage [11]. In adult Xenopus, a high level of the labeledprotein was found in the cytosol from the liver and thekidney [11], although xCTBP/xALDH1 mRNA was foundpredominantly in the kidney and the intestine rather than inthe liver [10]. The restricted tissue-distribution of xCTBP/xALDH1, particularly at the metamorphosing stages, raisesthe possibility that xCTBP/xALDH1 could modulate theactions of T3in a tissue-dependent manner. By controllingthe intracellular concentrations of free T3, xCTBP/xALDH1 might play a critical role in regulating T3accessto its target sites within the nucleus and the cytoplasm [12].There have been several reports demonstrating interac-tions between mammalian ALDH1 and bioactiveCorrespondence to K. Yamauchi, Department of Biology andGeoscience, Faculty of Science, Shizuoka University, 836 Oya,Shizuoka 422-8529, Japan.Fax: + 81 54 2380986, Tel.: + 81 54 2384777,E-mail: sbkyama@ipc.shizuoka.ac.jpAbbreviations: CTBP, cytosolic thyroid-hormone-binding protein;xCTBP, Xenopus CTBP; ALDH1, aldehyde dehydrogenase class 1;xALDH1, Xenopus ALDH1; T3,3,3¢,5-triiodo-L-thyronine; T4,L-thyroxine; Triac, 3,3¢,5-triiodo-L-thyroacetic acid; MBC, maximumbinding capacity; IC50, the concentration of a chemical necessary toinhibit an activity by 50%.Enzymes: Xenopus aldehyde dehydrogenase class 1 (EC 11 February 2002, accepted 20 March 2002)Eur. J. Biochem. 269, 2257–2264 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02891.xmolecules, such as steroids [13–17], the polycyclic aromaticcompound benzo[a]pyrene [18,19], the anthracycline antibi-otic daunorubicin, which has been used as one of theeffective agents for cancer chemotherapy [20], and thesynthetic flavone flavopiridol [21]. Together with ourfindings, it would appear that ALDH1 has acquired anability to bind these molecules during the evolution ofvertebrates [22]. These observations have led us to suggestthat the above molecules might also bind to xCTBP/xALDH1 as thyroid hormones do.In this report, we examine the effects of coenzymes andseveral hydrophobic signaling molecules on T3-binding andALDH activities of xCTBP/xALDH1. We demonstratethat NAD+, NADH and two steroids inhibit theT3-binding activity of this protein, whereas NADH,NADPH and iodothyronines inhibit the ALDH activity.Detailed studies revealed that NAD+and T3each act as anoncompetitive inhibitor on the T3-binding and enzymeactivities of the protein, respectively.MATERIALS AND METHODSMaterialsT3,D-T3,L-thyroxine (T4), 3,3¢,5-triiodo-L-thyroacetic acid(Triac), all-trans-retinal, all-trans-retinoic acid, androster-one, cortisone, 11-deoxycorticosterone, dehydroisoandros-terone, 17-b estradiol, progesterone and testosterone werepurchased from Sigma. NADP+,NADPH,NAD+,NADH and disulfiram were obtained from Wako PureChemicals. Vitamin D3(cholecalciferol) was purchasedfrom Nacalai Tesque. [125I]T3(122 MBqÆlg)1; carrier free)was from NEN Life Science Products. AG 1-X8 resin wasfrom Bio-Rad. Other reagents of molecular biology gradewere purchased from either Wako Pure Chemicals, NacalaiTesque or ICN Biomedicals.All steroids and retinal were dissolved in ethanol,iodothyronines and the analogue Triac were dissolved indimethylsulfoxide, to give less < 1% (v/v) solvents. Controlassays without the above compounds were performed in thepresence of the corresponding solvent at the sameconcentration. This dilution did not affect T3-binding andALDH activities in the assays described below.Expression of recombinant xCTBP/xALDH1inEscherichia coliE. coli BL21 bearing an expression vector containingxALDH1-I (pET15b/xALDH1-I) cDNA [10] was grownand expression of the recombinant proteins was induced by0.2 mMisopropyl thio-b-D-galactoside. Purification of therecombinant proteins was performed as described previously[10]. In brief, bacteria were collected by centrifugation at1200 g for 30 min at4 °C. After resuspending in 0.3MNaCl,50 mMTris/HCl, pH 8.0, 10 mMimidazole, 1 mgÆmL)1lysozyme, 1 mMbenzamidine hydrochloride, 1 mMphenyl-methanesulfonyl fluoride and 50 mM2-mercaptoethanol,the cells were disrupted by sonication (UR200P type, Tomy,Japan) for 10 s repeated three times. The extract wasobtained by centrifugation at 105 000 g for 40 min at 4 °C.Recombinant proteins with a histidine tag were purified by anickel affinity column (ProBound Resin, Invitrogen, CA,USA). The purified proteins were stored in 1 mMEDTA,1mMdithiothreitol and 10% glycerol at )85 °C until furtheruse. Protein concentration was determined by the dyebinding method with bovine c-globulin as the standard [23].T3-Binding activity and photoaffinity-labelingRecombinant proteins were incubated in 250 lLof20mMTris/HCl, 1 mMdithiothreitol, pH 7.5, containing 0.1 nM[125I]T3, in the presence or the absence of 5 lMunlabeled T3for 30 min at 0 °C. [125I]T3bound to proteins was separatedfrom free [125I]T3by the Dowex method [9] and radioac-tivity levels were measured in a c-counter (Auto WellGamma System ARC-2000, Aloka, Japan). The amount of[125I]T3bound nonspecifically was obtained by measuringthe radioactivity level within the samples incubated with5 lMunlabeled T3. The nonspecific binding value wassubtracted from the amount of total bound [125I]T3to givethe values of specifically bound [125I]T3. Maximum bindingcapacity (MBC) and Kdvalues were calculated fromScatchard plots [24].Photoaffinity-labeling with underivatized [125I]T3wasperformed as described previously [9–11]. Xenopus cell linesKR and XL58, which were kindly provided by S. Iwamuro(University of Toho, Japan) and R. J. Denver (Universityof Michigan, MI, USA), respectively, were culturedaccording to the method of Smith & Tata [25]. Xenopuscytosol was incubated with 0.5 nM[125I]T3for 0.5–1.0 h at4 °C whereas the intact Xenopus cells were incubated with0.5 nM[125I]T3in 70% Leibovitz-L15 medium in theabsence of fetal bovine serum for 0.5–1.0 h at 24 °C. Thecytosol, contained within a 0.5-mL Eppendorf tube, andthe Xenopus cells, spread on a 35-mm plastic Petri dish,were placed on a UV crosslinker (CL-1000, FunakoshiCo., Japan), and exposed to UV light (254 nm, 40 W) for3min at 0°C. The resultant cytosolic proteins, andXenopus cells, detached from the Petri dish with 0.05%trypsin, were mixed separately with an equal volume of2 · SDS-sample buffer, followed by boiling for 5 min. Theproteins were resolved by SDS/PAGE. The affinity-labeledproteins were detected by autoradiography, exposed toX-ray XAR5 film (Kodak) on an intensifying screen at)85 °C for 1–3 weeks.Aldehyde dehydrogenase activityPhotometric assays were performed in triplicate in 400 lLof 50 mMTris/HCl, pH 8.0, 3.3 mMpyrazole, 100 mMKCl, 1 mMdithiothreitol, 0.33 mMNAD+and 30 lMretinal, unless otherwise stated [10]. The amount of retinoicacid formed, determined by the photometrical method, wassimilar to the result obtained from monitoring the absorb-ance at 340 nm by HPLC [26]. Kinetic constants weredetermined under initial velocity conditions, which werelinear with time and protein.Determination of NAD contentThe content of NAD (the sum of its reduced and oxidizedforms) in Xenopus tissues was determined according to themethod of Nisselbaum & Green [27]. Rat liver cytosol wasused as a control and its NAD content, determined withinthis report, was compared with those recorded in theliterature [28] to validate this method.2258 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002Statistical analysisStatistical significance between the control and the differenttreatments was determined by Student’s t-test. Differencesare considered significant at P < 0.05.RESULTSCharacterization of T3-binding activity of recombinantxALDH1 proteinWe obtained two, closely related cDNAs encoding ALDH1from a Xenopus hepatic cDNA library. Sequencing analysisof the cDNAs, xALDH1-I and xALDH1-II, revealed thatxCTBP was more likely to be xALDH1-II rather thanxALDH1-I [10]. Thus, we concentrated on binding studiesof xALDH1-II, termed xCTBP/xALDH1. [125I]T3bindingto recombinant xCTBP/xALDH1 was examined in thepresence of each compound listed in Table 1. Of threeiodothyronines and Triac, T3was the most potent compet-itor of [125I]T3binding. The resulting affinity order ofT3‡ D-T3>T4> Triac, agreed with the order of theirrelative binding affinity to xCTBP in the Xenopus cytosolfrom adult and metamorphosing tadpole liver [9,11]. AtpH 7.5, 50% inhibition of [125I]T3binding to xCTBP/xALDH1 was achieved with T3and D-T3at a concentrationof 18 nM,withT4at 450 nMand with Triac at 15 lM(Fig. 1A).ALDH1 catalyzes the formation of retinoic acid fromretinal in the presence of NAD+[29]. We thereforeexamined the effects of the substrate (retinal), product(retinoic acid), coenzymes (NAD+and NADH), relateddinucleotides (NADP+and NADPH) and a typical inhib-itor of the enzyme (disulfiram) on [125I]T3binding toxCTBP/ALDH1. NAD+and NADH, at a concentrationof 200 lM, inhibited [125I]T3binding by more than 50%while retinal, at a concentration of 12 lM, activated [125I]T3binding by 36%, although no significant difference wasobtained. The other compounds exhibited little effect on T3binding (Table 1). The effect of NAD+is shown to be dose-dependent (Fig. 1B). The concentration of NAD+neces-sary to inhibit 50% of [125I]T3binding to xCTBP/xALDH1(IC50) was 40 lM.As mammalian ALDH1 is known to bind steroids[13–17], we finally investigated the effects of seven steroidsand cholecalciferol on T3binding. Progesterone was themost potent inhibitor of T3binding for xCTBP/xALDH1(Table 1). Dose-dependence curves indicated that the IC50for progesterone was 2.6 lM(Fig. 1B).To determine how NAD+and progesterone decreasedthe specific binding of [125I]T3to xCTBP/xALDH1, westudied their effects in the presence of varying concentra-Table 1. Effects of hydrophobic signaling molecules on 3,3¢,5-triiodo-L-thyronine (T3) binding and retinoic acid formation (ALDH activity) of Xen opusclass I aldehyde dehydrogenases (xALDH1) expressed in E. coli. T3-binding activity was examined by incubating the purified xALDH1 with 0.1 nM[125I]T3for 30 min at 0 °C, as described in Materials and methods. Nonspecific binding was determined from the samples incubated in the presenceof 5 lMunlabeled T3and subtracted from the total binding. The activity of the retinoic acid formation was examined by incubating the purifiedxALDH1 with 0.33 mMNAD+and 30 lMretinal for 1–2 min at 24 °C [10]. Data are mean ± SEM from at least triplicate determina-tions.*P < 0.05; **P < 0.01; ***P < 0.001.Effector Concentration T3-binding activity ALDH activityControl 100 ± 6 100 ± 2Retinoic acid 12 lM99.3 ± 5.4 133 ± 4**NAD+200 lM22.1 ± 3.0***NADH 200 lM18.1 ± 2.7*** 38.1 ± 1.9***NADP+200 lM121 ± 10 108 ± 5NADPH 200 lM112 ± 7 20.7 ± 2.6***Control 100 ± 3 100 ± 5Retinal 12 lM136 ± 16Disulfiram 200 lM87.5 ± 3.1* 41.9 ± 4.6**L-3,3¢,5-Triiodothyronine 0.32 lM15.5 ± 1.0***1 lM34.9 ± 1.6***D-3,3¢,5-Triiodothyronine 0.32 lM18.6 ± 2.2***1 lM35.7 ± 0.6***L-Thyroxine 0.32 lM60.4 ± 4.4**1 lM36.3 ± 0.4***L-3,3¢,5-Triiodothyroacetic acid 0.32 lM95.5 ± 4.11 lM39.5 ± 1.3***Control 100 ± 2 100 ± 5Testosterone 20 lM83.7 ± 5.8 104 ± 3Androsterone 20 lM92.3 ± 3.0 96.4 ± 3.1Dehydroisoandrosterone 20 lM87.2 ± 1.9** 101 ± 1Progesterone 20 lM39.3 ± 2.3*** 96.2 ± 6.317b-Estradiol 20 lM112 ± 3* 108 ± 1Cortisone 20 lM90.0 ± 2.8* 99.4 ± 1.411-Deoxycorticosterone 20 lM61.3 ± 3.1*** 96.3 ± 4.4Cholecalciferol 200 lM117 ± 3** 136 ± 8*Ó FEBS 2002 Dual activities of xCTBP/xALDH (Eur. J. Biochem. 269) 2259tions of unlabeled T3. Scatchard plots indicated that a singleclass of binding sites existed in xCTBP/xALDH1 (Fig. 2).NAD+,ataconcentrationof200lM, significantlydecreased the MBC from 338 ± 30 pmolÆmg)1protein(n ¼ 5) to 178 ± 16 pmolÆmg)1protein (n ¼ 3), althoughthere was no significant difference in Kdvalues between theNAD+-treated and untreated samples, 66 ± 11 nM(n ¼ 3) vs. 53 ± 5 nM(n ¼ 5), respectively, as shown inFig. 2. This result indicated that the inhibitory mode ofNAD+was noncompetitive. Progesterone, at 2 lM,appeared to affect both the Kd(75 ± 2 nM, n ¼ 3)and MBC (310 ± 28 pmolÆmg)1protein, n ¼ 3) values,although no significant differences were obtained for thesevalues when compared with the Kdand MBC values for theuntreated samples.Characterization of ALDH activity of recombinantxCTBP/xALDH1Formation of retinoic acid from retinal by xCTBP/xALDH1 was examined in the presence of each compoundlisted in Table 1. The reduced forms of dinucleotides,NADH and NADPH, as well as disulfiram, were powerfulinhibitors for xCTBP/xALDH1, whereas retinoic acidslightly but significantly stimulated the enzyme activity.Iodothyronines and Triac inhibited the enzyme activity. IC50for T3was 700 nM(Fig. 3). The narrow range of theinhibitory concentration of T3indicates positive cooperati-vity. The Hill coefficient was  2.4 (Fig. 3, inset). All steroidslisted in Table 1 showed little effect on the enzyme activity ofxCTBP/ALDH1 at the concentrations investigated.Fig. 2. Scatchard plot analysis of [125I]T3binding to xCTBP/xALDH1.Purified recombinant xCTBP/xALDH1 (10 lg/250 lL) was incubatedwith 0.1 nM[125I]T3in the presence of various concentrations ofunlabeled T3with (open symbols) or without (d) the effector: 200 lMNAD+(s), 2 lMprogesterone (h), for 30 min at 0 °C. Nonspecificbinding was subtracted from total binding. Each value is the mean oftriplicate determinations. This experiment was repeated at least threetimes.Fig.3. EffectofT3on retinoic acid synthesis from retinal, catalyzed byxCTBP/xALDH1. ALDH activity was measured as the rate of retinoicacid synthesis. The reaction was performed at 24 °Cwith5lgofxCTBP/xALDH1 in the presence of various concentrations of T3.Theinset illustrates the Hill plot, log[vc/vi)1] vs. the logarithm of T3molarconcentration, the slope of which yields the Hill coefficient. vcand viarevelocities calculated in the absence and presence of various concen-trations of T3. The Hill coefficient, h,was 2.4. Each value is the mean± SEM of triplicate determinations.Fig. 1. Inhibition of [125I]T3binding to xCTBP/xALDH1 with varioushydrophobic signaling molecules. Purified recombinant xCTBP/xALDH1 (10 lg/250 lL) was incubated with 0.1 nM[125I]T3in thepresence or absence (control) of the following compounds, at variousconcentrations for 30 min at 0 °C. In (A), T3(s), D-T3(d), T4(h)orTriac (n) was added, whereas, in (B), progesterone (s)orNAD+(d)was added. Nonspecific binding was subtracted from total binding togive values for specific binding. Each value is the mean ± SEM oftriplicate determinations.2260 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002To determine how thyroid hormones interact withxCTBP/xALDH1, resulting in the decrease in the formationof retinoic acid from retinal, kinetics of the inhibition ofxCTBP/xALDH1 by T3was examined by variation ofNAD+concentration within the reaction mixture. The Kmvalue, 9 lM, was independent of the concentration of T3,but the Vmaxvalue decreased from 0.18 to 0.08 lmolÆmin)1Æmg)1with increasing concentrations of T3(Fig. 4).The Kiwas 0.28 lMand 0.31 lM,calculatedintwoindependent experiments. Next, kinetics of the inhibitionof xCTBP/xALDH1 by T3were examined when retinalconcentration was varied in the reaction mixture. As shownpreviously [10], positive cooperativity with allosteric kineticswas detected (Fig. 5). The apparent K1/2value did notchange in the incubations with and without T3(2.8 ± 0.3vs. 2.6 ± 0.1 lM, n ¼ 6), but the Vmaxvalue decreased by64% when 5 lMT3was added to the reaction mixture. TheHill coefficient did not change significantly in incubationswith and without 5 lMT3, 2.3 ± 0.1 vs. 2.2 ± 0.1 (Fig. 5,inset). These results indicated that T3acts as a noncompet-itive inhibitor against both NAD+and retinal upon theenzyme activity of xCTBP/xALDH1.T3binding to xCTBP/xALDH1 in intactXenopuscellsThe present studies on the dual activities of xCTBP/xALDH1 have indicated that NAD+is required atconcentrations of 10-5)10-4Mfor expression of ALDHactivity, whereas  10)4Mof NAD+or NADH pro-foundly inhibits the T3-binding activity. However, we haveno information regarding NAD+,NADHorNAD(thesum of NAD+and NADH) content within Xenopus tissues,although NAD content in rat liver is known to be0.7–0.9 lmolÆ(g fresh weight))1[27,28]. As both NAD+and NADH showed similar inhibitory effects on T3-bindingto xCTBP/xALDH1 (Table 1), we assumed that the sumof NAD+and NADH is important for evaluating theinhibitory effect. NAD content within rat liver was756 ± 49 lmolÆ(kg fresh weight))1(n ¼ 3), which agreedwith values reported previously [27,28]. On the other hand,Xenopus liver had a low NAD content, 201 ± 23 lmolÆ(kgfresh weight))1(n ¼ 6), less than one third of that in rat liver(Table 2). There were no significant differences in NADcontents among various Xenopus tissues. Next, T3-bindingactivity of xCTBP/xALDH1 was directly examined byphotoaffinity-labeling using intact Xenopus cells. Analysesof the cytosol obtained from the cell lines (KR and XL58)and the adult liver revealed the presence of single labeled59-kDa xCTBP (lanes 1–3 in Fig. 6). Photoaffinity-labelingof [125I]T3using intact KR and XL58 cells revealed, viaautoradiography, a labeled protein band of the same size(lanes 4 and 5 in Fig. 6), demonstrating that xCTBP/xALDH1 is capable of binding T3within the Xenopus cells.Fig. 4. Kinetics of the inhibition of xCTBP/xALDH1 by T3whenNAD+concentration was varied within the reaction mixture. Thereaction was performed at 24 °Cwith5lg of xCTBP/xALDH1. Theconcentration of retinal was 30 lMand the concentrations of T3were 0(d), 0.4 (e), 0.6 (n), 0.8 (h)and1lM(s).The buffer used was 50 mMTris/HCl, pH 8.0. Each value is the mean of triplicate determinations.This experiment was repeated twice, each with similar results.Fig. 5. Kinetics of the inhibition of xCTBP/xALDH1 by T3when ret-inal concentration was varied within the reaction mixture. The reactionwas performed at 24 °Cwith5lg of xCTBP/xALDH1. The concen-tration of NAD+was 0.33 mMand the concentrations of T3was 0(s), or 5 lM(d). The buffer used was 50 mMTris/HCl,pH8.0.Theinset depicts the Hill plots. Each value is the mean of triplicate deter-minations. SEMs, which were less than the size of symbols, are notshown. This experiment was repeated six times, each with similarresults.Table 2. Contents of NAD in rat liver and various Xenopus tissues. Dataare expressed as the mean ± SEM (number of samples). NAD contentis the sum of the oxidizaed and reduced forms.Species/tissue NAD (lmolÆ kg wet weight)1)RatLiver 756 ± 49 (3)XenopusLiver 201 ± 23 (6)Kidney 234 ± 83 (5)Stomach 232 ± 7 (3)Intestine 291 ± 69 (3)Ovary 294 ± 94 (4)Heart 177 ± 29 (3)Skeletal muscle 199 ± 37 (3)Ó FEBS 2002 Dual activities of xCTBP/xALDH (Eur. J. Biochem. 269) 2261DISCUSSIONThe present work was undertaken with the aim of deter-mining which signaling molecules, and other molecules,affected the T3-binding and ALDH activities of xCTBP/xALDH1. We have obtained evidence that the[125I]T3-binding activity of xCTBP/xALDH1 was markedlyinhibited by NAD+, NADH, progesterone and 11-deoxy-corticosterone, as well as iodothyronines and Triac, but notby NADP+, NADPH, disulfiram and retinal. On the otherhand, the ALDH activity was inhibited by NADH,NADPH, disulfiram, iodothyronines and Triac, but notby any of the steroids tested. We initially expected xCTBP/xALDH1 to be one of the target sites for endocrinedisrupting chemicals, because amphibian malformationsfound in field studies were very similar to those found inindividuals experimentally treated with retinoids [30].However, treatment with bisphenol A, nonylphenol, octyl-phenol, and benzo[a]pyrene had little effect on ALDHactivity of xCTBP/xALDH1 (data not shown). NADH wasthe only compound to affect both the thyroid hormonebinding and enzymatic activities of xCTBP/xALDH1,suggesting that the binding of a compound to xCTBP/xALDH1 will not necessarily inhibit both activities. Asimilar result was observed for flavopiridol [21]. Its bindingto human ALDH1 did not affect the enzyme activity ofALDH1. Study of the interaction of ALDH1 with bioactivemolecules revealed that the mammalian enzymes have asignificant affinity for thyroid hormone [31], progesterone,deoxycorticosterone, diethylstilbestrol, dehydroepiandros-terone [13,14,32], dihydroandrosterone, 17,b-estradiol,hydrocortisone [15–17] and benzo[a]pyrene [18,19]. As thebinding of the first three compounds to xALDH1 was alsowitnessed in the present study (Table 1), the ability ofALDH1 to bind the compounds appears to have occurredat an early step during vertebrate evolution.Detailed studies revealed that NAD+noncompetitivelyinhibited the T3-binding activity of xCTBP/ALDH1whereas T3inhibited the ALDH activity in a noncompet-itive fashion against both NAD+and retinal. These resultssuggested the formation of a ternary complex consisting ofxCTBP/xALDH1, NAD+and T3. For human mitochon-drial and cytoplasmic ALDHs, T3and Triac were compet-itive inhibitors against NAD+and uncompetitive inhibitorsagainst propionaldehyde [31]. These distinct inhibitorymodes might reflect the differences of the iodothyroninebinding pocket within xALDH1 and mammalian ALDHs.The inhibitory interactions of NAD+upon T3binding toxCTBP/xALDH1 and of T3upon its enzyme activity mustoccur in a more complex fashion. Binding studies demon-strated that xCTBP/xALDH1 had a high affinity for T3,with a Kdof 53 nM(Fig. 2), whereas the Kivalue for T3against NAD+on ALDH activity was 0.3 lM(Fig. 4). Wecan not precisely determine why there was a differencebetween the calculated Kdand Kivalues. It may be possiblethat xCTBP/ALDH1 forms different conformations whenbound to NAD+and/or T3, This possibility is considereddue to the presence of positive cooperativity upon ALDHactivity (the Hill coefficient, h ¼ 2.2) when the concentra-tion of retinal was varied (Fig. 5) and the presence ofpositive cooperativity upon the inhibition of ALDH activity(h ¼ 2.4) when the concentration of T3was varied (Fig. 3).T3may be a selective, allosteric inhibitor of the xALDH1enzyme. Such an allosteric conformational change wasproposed for human alcohol dehydrogenase when bound totestosterone, where testosterone acts as a noncompetitiveinhibitor with respect to ethanol and NAD+[33]. Alter-natively, it is possible that thyroid hormone alters theequilibrium between the tetramer and dimer conformationsor between the dimer and monomer conformations ofxCTBP/ALDH1, as found in glutamate dehydrogenase,where T4and T3induce dissociation [34]. To explore thesecond possibility, the hepatic xCTBP/xALDH1, in thepresence or absence of 5 lMT3, were subjected to centrif-ugation in a glycerol density gradient. However, tetramericxCTBP/xALDH1 was not found to dissociate into its dimeror monomer forms (data not shown). Thus, the secondpossibility is unlikely to occur in xCTBP/xALDH1.There are many reports of the inhibitory effects of thyroidhormones upon the activity of several dehydrogenases: pigheart malic dehydrogenase [34], beef liver glutamic dehy-drogenase [34–36], pig heart malate dehydrogenase [37],horse and human alcohol dehydrogenases [38–40] andhuman aldehyde dehydrogenases [31]. These observationsraise the possibility of the presence of a dehydrogenase-specific binding site for thyroid hormone. In ALDH1, thebinding sites for NAD+/NADH and retinal reside in theN-terminal region, termed the NAD-binding domain, andin the C-terminal region, termed the catalytic domain,respectively [41]. We found previously that the thyroid-hormone-binding site is located in the NAD-bindingdomain of xCTBP/xALDH1 [10]. Zhou & Weiner [31]reached the same result by eluting human ALDHs bound toAMP-affinity column with T3or Triac. These resultssupport the possibility of a dehydrogenase-specific bindingsite for thyroid hormone as the coenzyme-binding domainswithin dehydrogenases have a relatively conserved ternarystructure [42] when compared to their catalytic domains.However, Kivalues for thyroid hormone binding to alldehydrogenases, including those calculated for xCTBP/xALDH1, were in the 10-7)10-4Mrange. These are highconcentrations, even if the local distribution or accumula-tion of intracellular thyroid hormones was considered.The present studies demonstrate that xCTBP/xALDH1can bind T3in intact cells (Fig. 6). However, the NADcontent corresponding to 0.2 mMconcentration wouldrestrict T3-binding activity of xCTBP/xALDH1 within theXenopus cells compared to the binding activity witnessedin vitro. It should be noted that retinal, at a concentration of12 lM, activated the T3-binding activity by 36%, althoughno significant difference was obtained. In the previousstudies, the affinity-labeled xCTBP/xALDH1 was found atFig. 6. Photoaffinity-labeling of xCTBP/xALDH1 in Xenopus cells.Xenopus cytosol from KR cells (lane 1), XL58 cells (lane 2) and adultliver (lane 3), and the intact KR (lane 4) and XL58 (lane 5) cells werephotoaffinity-labeled with 0.5 nM[125I]T3. The resultant proteins wereanalysed on a 10% SDS/PAGE, followed by autoradiography.2262 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002a higher level in the liver cytosol than in the kidney cytosol[11], whereas xCTBP/xALDH1 mRNA was found morepredominantly in the kidney than in the liver [10]. Therefore,it is possible that T3binding to xCTBP/xALDH1 mightbe under the control of an unknown factor(s) besidescoenzymes within the cells, while poorly influencing itsALDH activity.ACKNOWLEDGEMENTSWe would like to thank Mr Takashi Honda for the preparation ofrecombinant xCTBP/xALDH1. We also wish to thank Drs S. Iwamuroand R. J. Denver for providing the Xenopus cell lines. This work wassupported by Grant-in-Aid for Scientific Research (B) from the JapanSociety for the promotion of Science (no. 13559001).REFERENCES1. Chanbon, P. (1995) The molecular and genetic dissection ofthe retinoid signaling pathway. Recent. Prog. Horm. Res. 50,317–332.2. Napoli, J.L., Posch, K.C., Fiorella, P.D., Boerman, M.H.E.M.,Salerno, G.J. & Burns, R.D. 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(1974) Chemical andbiological evolution of a nucleotide-binding protein. Nature 250,194–199.2264 K. Yamauchi and J. Nakajima (Eur. J. Biochem. 269) Ó FEBS 2002 . Effect of coenzymes and thyroid hormones on the dual activities of Xenopus cytosolic thyroid- hormone-binding protein (xCTBP) with aldehyde dehydrogenase activity Kiyoshi Yamauchi and Jun–ichiro. concentration, would poorly modulate the enzyme activity of xCTBP/xALDH1. Keywords: cytosolic thyroid- hormone-binding protein; aldehyde dehydrogenase; retinoic acid synthesis; Xenopus laevis. Hydrophobic. xCTBP/xALDH1. There are many reports of the inhibitory effects of thyroid hormones upon the activity of several dehydrogenases: pig heart malic dehydrogenase [34], beef liver glutamic dehy- drogenase

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