Acyl-CoA Esters Antagonize the Effects of Ligands on Peroxisome Proliferator-activated Receptor alpha  Conformation, DNA Binding, and Interaction with Co-factors*

Morten ElholmDagger§, Inge Dam§, Claus Jørgensen§, Anne-M. Krogsdam, Dorte Holst, Irina Kratchmarova, Martin Göttlicher||**, Jan-Åke Gustafsson||, Rolf BergeDagger Dagger , Torgeir Flatmark§§, Jens Knudsen, Susanne Mandrup, and Karsten Kristiansen¶¶

From the Department of Biochemistry and Molecular Biology and Center for Experimental BioInformatics, University of Southern Denmark, DK-5320 Odense M., Denmark, the || Department of Medical Nutrition, Karolinska Institute, S-14157 Huddinge, Sweden, and the Dagger Dagger  Institute of Clinical Biochemistry and §§ Department of Biochemistry and Molecular Biology, University of Bergen, N-5021 Bergen, Norway

Received for publication, February 5, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The peroxisome proliferator-activated receptor alpha  (PPARalpha ) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha . Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha -retinoid X receptor alpha  heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha -containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Members of the nuclear receptor superfamily mediate ligand-dependent transactivation of genes controlling development, differentiation, and homeostasis in response to nutritional, metabolic, and hormonal signals (1). The peroxisome proliferator-activated receptor alpha  (PPARalpha ,1 NR1C1 (2)) belongs to the nuclear hormone receptor superfamily (3). Through heterodimerization with the retinoid X receptors (4) (NR2B1-3) and binding to DR-1 response elements, PPARalpha regulates transcription of several genes encoding enzymes involved in lipid metabolism (5, 6). Accordingly, PPARalpha is predominantly expressed in tissues with a high turnover of fatty acids (7).

Activation of nuclear receptor-mediated transcription involves an agonist-dependent release of co-repressors and recruitment of co-activators. Accumulating evidence obtained by x-ray crystallography has revealed a significant ligand-dependent conformational change involving repositioning of the conserved AF-2 helix in the ligand-binding domains of nuclear receptors (8-11). This ligand-induced conformational change has been demonstrated to be a determining event governing interactions with co-activators and co-repressors (see Refs. 12-14; reviewed in Ref. 15). The crystal structures of the PPARgamma and PPARdelta ligand-binding domains (16, 17) have revealed an overall folding pattern similar to that observed for other nuclear receptor ligand-binding domains (8-11). However, the PPAR ligand-binding pocket is substantially larger than those of other nuclear receptors, and this may in part explain the observed promiscuity in terms of ligand binding (16, 17). The interior of the ligand-binding pocket has been suggested to be solvent-accessible via a channel between helix 3 and the beta -sheet. The entrance is lined by polar side chains, and its dimension indicates that ligands may enter the cavity without affecting the overall structure of the receptor (16-18). Crystallization of a ternary complex containing the PPARgamma ligand-binding domain, the PPARgamma agonist BRL49653, and the nuclear receptor-binding domain of the steroid receptor co-activator-1 (SRC-1) has revealed that association between liganded nuclear receptors and co-activators depends on conserved residues in helix 3 and the AF-2 helix forming a charge clamp and hydrophobic interactions involving helices 3, 4, and 5 and the AF-2 helix (16). Although the three-dimensional structure of PPARalpha has yet to be reported, it has been shown that the C terminus of the ligand-binding domain is essential for the ligand-induced co-activator interaction (19-22).

A large variety of long-chain fatty acids, eicosanoids, and synthetic compounds have been shown to serve as PPARalpha ligands and activators (17, 23-25). Several natural and synthetic PPARalpha ligands are activated to the corresponding CoA esters (26-28), and these have been demonstrated to accumulate in tissues of treated rats (29, 30). Generally, the formation of CoA esters has been considered a process that merely reduces the availability of the activating PPARalpha ligands (31). In the present study, we present evidence that acyl-CoA esters directly affect PPARalpha conformation and function in a manner indicating that acyl-CoA esters may act as PPARalpha antagonists.

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INTRODUCTION
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Plasmids-- pSG5-mPPARalpha was kindly supplied by J. D. Tugwood (32). The plasmid encoding SRC-1 (pBKCMV-hSRC1) was a kind gift from B. W. O'Malley (33). pGEX-NCoR-(2239-2453) for bacterial expression of GST-NCoR (amino acids 2239-2453) was kindly provided by M. A. Lazar (34). Plasmids pTLI-mPPARDelta AB, and pTLI-mPPARDelta AB/Delta 425-468 were kindly provided by M. Leid (35). The following constructs have been described previously: rPPARalpha (p7zf-PPARBE) and rRXRalpha (prRXRT7) (4, 23), pGEX-5X-1-mPPARalpha -LBD (amino acids 166-469) (36), and pCA4-rRXRalpha (37). The plasmid pCA2-mPPARalpha was constructed by subcloning the murine PPARalpha cDNA into the BamHI/SalI site of pCA2, which is derived from pCA4 by insertion of the CUP1 promoter cassette into pRS314 (38). The murine PPARalpha fragment was obtained by polymerase chain reaction from pSG5-mPPARalpha using BamHI/SalI-tagged primers.

Ligands-- Linoleic acid was purchased from Sigma-Aldrich. Wy14643 and 9-cis-retinoic acid were from Biomol. S-Hexadecyl-CoA was synthesized as described by Rosendal et al. (39), and tetradecylthioacetic acid (TTA) was synthesized as described by Berge et al. (40). BRL49653 was kindly provided by Novo Nordisk A/S.

Sf21 Whole Cell Extracts-- Rat PPARalpha was expressed in Spodoptera frugiperda Sf21 insect cells as previously described (4). The Sf21 whole cell homogenate was prepared by disrupting 4 × 107 cells in 2.3 ml of buffer (25 mM KPO4, pH 7.4, 100 mM KCl, 1 mM EDTA, 2 mM dithioerythritol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml antipain, 1 mg/ml pepstatin, 0.01 trypsin inhibitory units/ml aprotinin) with 50 strokes in an all glass Dounce homogenizer. The ionic strength was increased by slowly adding 1.2 ml of 1 M KCl, and the homogenate was incubated on ice for 30 min before centrifugation (35,000 rpm, 42 min) in a Beckman Ti70 rotor. The supernatant was stored in aliquots in liquid nitrogen. The protein concentration, as determined by spectrophotometry (41), was 3-4 µg/µl.

In Vitro Transcription and Translation-- PPARalpha , RXRalpha , and SRC-1 were synthesized in vitro by using the TNT®-coupled transcription/translation system (Promega) with or without [35S]cysteine/methionine amino acid mixture (ICN). PPAR and RXR were synthesized in the presence of 0.5-1.0 µM zinc acetate.

Electrophoretic Mobility Shift Assays-- The DNA probe was prepared by annealing 32-base pair oligonucleotides covering the PPAR/RXR binding motif in the promoter of the acyl-CoA oxidase (ACO) gene (32). The probe was labeled by filling in the 3'-recessive ends with [alpha -32P]dCTP (10 µCi, 3000 Ci/mmol; Amersham Pharmacia Biotech) using the Klenow fragment of the Escherichia coli DNA polymerase. Binding reactions were performed in a total of 25 µl containing Sf21 extract, 2.4 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 25 mM Hepes, pH 7.6, 40 mM KCl, 0.1 mM EDTA, 1 mM dithioerythritol, and 10% (v/v) glycerol. The reactions were preincubated for 20 min on ice, after which 0.5 ng of probe was added, and the reactions were incubated for 20 min at room temperature. Free DNA and DNA-protein complexes were resolved by electrophoresis (12 V/cm, 1.5 h at 4 °C) on a 4% (w/v) polyacrylamide/bisacrylamide gel (30:1) in a buffer containing 50 mM Tris-HCl, pH 8.5, 380 mM glycine, and 2 mM EDTA. Ligand-induced complex formation assays were performed as described above, except that in vitro translated PPARalpha and RXRalpha were used instead of Sf21 extract, the total reaction volume was 20 µl, and reactions contained 130 mM KCl and 5% (v/v) glycerol. Combined electrophoretic mobility assay (EMSA) and Western blotting was performed as described previously (42) using a polyclonal affinity-purified anti-rPPARalpha antibody (21).

Differential Protease Sensitivity Assay-- 2-µl aliquots of the 35S-labeled, in vitro translated protein was incubated for 20 min at 25 °C in a total volume of 8 µl of binding buffer with the addition of ligand or vehicle. The final concentrations in the binding buffer were 22 mM Tris-HCl, pH 8, 75 mM KCl, 5% (v/v) glycerol, and 2 µM dithioerythritol. Linoleic acid and S-hexadecyl-CoA were used in final concentrations of 120 µM and 5-15 µM, respectively. Chymotrypsin (Roche Molecular Biochemicals) was dissolved in 50 mM NH4HCO3. Following incubation, 2 µl of chymotrypsin was added to final concentrations of 0-120 µg/ml or 0-260 µg/ml. The digestions were carried out at 25 °C for 20 min and stopped by boiling in Tris-Tricine loading buffer (0.1 M Tris-HCl, pH 6.8, 24% (v/v) glycerol, 8% (w/v) SDS, 0.2 M dithiothreitol, 0.02% (w/v) G-250 Coomassie Brilliant Blue). The resulting peptide mixtures were separated by SDS-PAGE and visualized using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Palmitoyl-CoA-Agarose Bead Assay-- Whole cell extracts of yeast (BJ2168) expressing mPPARalpha and/or rRXRalpha were prepared as previously described (37). Yeast extract (7.5 µl) was incubated at room temperature for 30 min in 42 mM NaCl, 100 mM Tris-HCl, pH 6.7, 4% (v/v) glycerol, 1 mM dithioerythritol, 0.2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals) with 10 µl of palmitoyl-CoA beads (Sigma-Aldrich) or 10 µl of protein A-beads (Amersham Pharmacia Biotech). BRL49653 was added to a final concentration of 1 µM, Wy14643 was added to final concentrations of 100 nM to 10 µM, and 9-cis-retinoic acid was added to a final concentration of 10 µM. The beads were washed twice in 150 µl of 40 mM KCl, 100 mM Tris, pH 6.7, 4% (v/v) glycerol, 1 mM dithioerythritol, and 1 mM phenylmethylsulfonyl fluoride in the presence of ligand or vehicle. Bound proteins were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and evaluated by immunoblot analysis. The anti-PPARalpha antibody was raised against the mouse PPARalpha AB domain and affinity-purified, and anti-RXRalpha antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Enhanced chemiluminescence detection was used for visualization.

GST Pull-downs-- GST fusion proteins were captured on glutathione-Sepharose beads as described (36). 8 µl of 35S-labeled in vitro translated mPPARalpha , or SRC-1 was incubated in buffer A (50 mM NaCl, 20 mM Tris-HCl, pH 7.9, 0.1% (v/v) Nonidet P-40, 10% (v/v) glycerol, and 1% (w/v) essentially fatty acid-free milk powder) with 100 µM Wy14643, 30 µM TTA, or vehicle. 10 µl of GST fusion protein on beads was added, and interaction was allowed to proceed for 2 h at 4 °C. The beads were washed three times in 150 µl of buffer A and finally once in 150 µl of buffer A without milk powder. The bound proteins were eluted by boiling in SDS-PAGE sample buffer, resolved by electrophoresis on a 10% SDS-polyacrylamide gel, and visualized using the PhosphorImager. Quantitation of the content of discrete bands was done using the ImageQuant version 5.0 software (Molecular Dynamics).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Ligand-induced Binding of PPARalpha ·RXRalpha to the ACO PPRE Is Antagonized by the Nonhydrolyzable Palmitoyl-CoA Analog S-Hexadecyl-CoA-- It is a well established fact that a large number of fatty acids, eicosanoids, and hypolipidemic drugs activate PPARalpha -mediated transactivation, and different protocols have been established to demonstrate that several of these activators are bona fide PPARalpha ligands (17, 23-25). Ligand-induced complex formation assay provides one such simple and sensitive method for analyzing the ability of PPAR activators to bind to PPAR and induce DNA binding of a PPAR·RXR complex to a peroxisome proliferator-responsive element (PPRE) (24). We have used this well established assay to examine whether S-hexadecyl-CoA, a nonhydrolyzable analog of palmitoyl-CoA (39), was able to modulate ligand-induced binding of the PPARalpha ·RXRalpha heterodimer to the ACO PPRE. The concentration of S-hexadecyl-CoA used in these experiments was well below the critical micelle concentration (43). As a PPARalpha ligand, we used the fatty acid analog TTA, which has been shown to efficiently promote PPARalpha ·RXRalpha ·ACO PPRE complex formation (24) and to induce PPARalpha -mediated transactivation. Fig. 1 demonstrates that the addition of TTA to limiting amounts of in vitro translated PPARalpha and RXRalpha , as reported previously (24), induced a considerable increase in the binding of the PPARalpha ·RXRalpha heterodimer to the ACO PPRE probe in an electrophoretic mobility shift assay (EMSA) (Fig. 1, lanes 2 and 3), whereas no binding of the RXRalpha homodimer was observed (lane 8). Interestingly, the presence of 5 µM S-hexadecyl-CoA abrogated TTA-induced PPARalpha ·RXRalpha ·ACO PPRE complex formation (Fig. 1, lane 4). Increasing the concentration of TTA restored PPARalpha ·RXRalpha ·ACO PPRE complex formation in the presence of S-hexadecyl-CoA (Fig. 1, lanes 5-7), demonstrating that the inhibitory effect of S-hexadecyl-CoA was reversible and that competition between fatty acid and acyl-CoA appears to regulate PPARalpha ·RXRalpha ·ACO PPRE complex formation.


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Fig. 1.   S-Hexadecyl-CoA reversibly antagonizes fatty acid-induced PPARalpha ·RXRalpha ·PPRE complex formation. One µl of a mixture of in vitro transcribed/translated rPPARalpha and rRXRalpha (10:1, v/v) or rRXRalpha alone was incubated with a 32P-labeled oligonucleotide containing the PPRE of the rat ACO promoter in the presence of different concentrations of TTA and S-hexadecyl-CoA as indicated. The free and bound PPRE were separated by PAGE.

PPARalpha Binds Palmitoyl-CoA-- The ligand-induced complex formation assay described above indicated that fatty acid and acyl-CoA might compete for binding to PPARalpha or, alternatively, that simultaneous binding of an acyl-CoA ester and an agonistic ligand to the PPARalpha ·RXRalpha complex antagonized the effect of the PPARalpha agonist. Due to the hydrophobic characteristics and micelle forming capacity of fatty acids and their CoA derivatives, we have been unsuccessful in performing reliable simple binding competition experiments between fatty acids and acyl-CoAs. To circumvent these problems, we reckoned that a possible direct binding of acyl-CoA to PPARalpha might be revealed by specific binding of PPARalpha to palmitoyl-CoA covalently coupled to agarose beads. Since the covalent bond between the palmitoyl-CoA and the agarose beads joins the amino group of the CoA moiety with the agarose matrix, this approach, furthermore, circumvented the inherent problem associated with assays involving acyl-CoA esters, namely the hydrolysis of the labile thioester bond. This is particularly important in the context of competition assays to determine ligand binding to PPARs, since hydrolysis of the acyl-CoA would generate a fatty acid, which would act as a regular agonist and thereby give false Kd values for the binding of acyl-CoA esters to the PPARs. In contrast, in a bead-based pull-down assay, hydrolysis of the covalently coupled palmitoyl-CoA would release the free fatty acid, and hence, if PPARalpha only interacted with the free fatty acid, no specific retention of PPARalpha would be observed.

Recombinant full-length PPARalpha and RXRalpha were expressed in yeast, and whole cell extracts were prepared as described (37). The extracts were incubated with palmitoyl-CoA agarose beads or protein A-agarose beads (control) in the absence or presence of PPAR-selective or RXR-selective ligands. After washing, bound material was recovered by boiling in SDS sample buffer, and PPARalpha and RXRalpha were detected by Western blotting. Fig. 2A shows that PPARalpha preferentially was retained on the palmitoyl-CoA-agarose beads compared with the protein A-agarose beads. The addition of the PPARalpha -selective ligand Wy14643 prevented PPARalpha binding to the palmitoyl-CoA beads in a dose-dependent manner, whereas the addition of the PPARgamma -selective ligand BRL49653 was without effect on PPARalpha binding. In contrast, no specific interaction between RXRalpha and the palmitoyl-CoA agarose beads was detected irrespective of the presence of PPAR- or RXR-selective ligands (Fig. 2B). We conclude that the established PPARalpha ligand Wy14643 and palmitoyl-CoA compete for binding to PPARalpha .


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Fig. 2.   Wy14643 and acyl-CoA compete for direct binding to PPARalpha . Immunoblot analysis of proteins retained on palmitoyl-CoA- or protein A-coupled agarose beads incubated with whole cell extracts of yeast expressing either mPPARalpha and rRXRalpha (A) or rRXRalpha alone (B). Bound proteins were recovered by boiling in SDS sample buffer and analyzed by immunoblotting. For competition, the PPARalpha -selective ligand, Wy14643, the PPARgamma -selective ligand, BRL49653, or the RXR ligand, 9-cis-retinoic acid, was added to the binding reactions as indicated.

S-Hexadecyl-CoA Increases the Sensitivity of PPARalpha to Chymotrypsin-- Differential protease sensitivity assays have been widely used to examine the effect of ligand binding on receptor conformation. We applied this technique to compare the effects of a known PPARalpha ligand, linoleic acid, and S-hexadecyl-CoA on PPARalpha conformation. 35S-Labeled PPARalpha or RXRalpha was incubated with S-hexadecyl-CoA, linoleic acid, or vehicle (0.5% (v/v) Me2SO), digested with increasing concentrations of chymotrypsin for 20 min at 25 °C, and the digestion products were analyzed by SDS-PAGE. Binding of synthetic agonists to PPARalpha has been shown to decrease the sensitivity to chymotrypsin digestion, resulting in preservation of protease-resistant fragments (35). In contrast, we found that S-hexadecyl-CoA in a dose-dependent manner increased the sensitivity of PPARalpha to chymotrypsin, as indicated by the rapid disappearance of the diagnostic 26-kDa protease-resistant fragment (Fig. 3A). In comparison, incubation with 15 µM S-hexadecyl-CoA did not influence the sensitivity of RXRalpha to chymotrypsin, indicating that the effect of S-hexadecyl-CoA was receptor-dependent (Fig. 3B). To further corroborate the notion that the effects of S-hexadecyl-CoA on PPARalpha conformation were reversible and did not result from an irreversible denaturing action, the following experiment was performed. The 35S-labeled PPARalpha was incubated for 20 min with either 10 µM S-hexadecyl-CoA or water, and then each mixture received either 120 µM linoleic acid or vehicle (0.5% Me2SO). Incubation was continued for 20 min, and each of the four incubations was subjected to digestion with increasing amounts of chymotrypsin. Fig. 4 shows that incubation with linoleic acid as expected decreased the sensitivity of PPARalpha to chymotrypsin, whereas incubation with S-hexadecyl-CoA increased the sensitivity to chymotrypsin. It is noteworthy that the addition of linoleic acid to PPARalpha preincubated with S-hexadecyl-CoA partially restored resistance to chymotrypsin digestion. Thus, S-hexadecyl-CoA interacted reversibly with PPARalpha and appeared to compete with the agonist linoleic acid for binding to PPARalpha . It has previously been shown that deletion of the putative helices H10-12 of the C-terminal region in the PPARalpha ligand-binding domain abolishes agonist-induced protease protection and transactivation (35). To examine whether the presence of this region was required for S-hexadecyl-CoA-induced protease sensitivity, the truncated forms mPPARalpha Delta AB and mPPARalpha Delta AB/Delta 425 (35) were digested in the presence of either linoleic acid or S-hexadecyl-CoA. mPPARalpha Delta AB contains the entire ligand-binding domain, whereas the putative helices 10-12 are deleted in mPPARalpha Delta AB/Delta 425 (35). As shown in Fig. 5, in the presence of linoleic acid, digestion of mPPARalpha Delta AB was decreased, whereas S-hexadecyl-CoA increased the sensitivity to chymotrypsin. However, neither linoleic acid nor S-hexadecyl-CoA affected the sensitivity of mPPARalpha Delta AB/Delta 425 (Fig. 5). Thus, it appears that helices 10-12 are required for interaction of S-hexadecyl-CoA with PPARalpha as well as for interaction of linoleic acid with PPARalpha .


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Fig. 3.   S-Hexadecyl-CoA increases the sensitivity of PPARalpha to chymotrypsin digestion. In vitro transcribed/translated 35S-labeled mPPARalpha (A) or mRXRalpha (B) was incubated with increasing concentrations of S-hexadecyl-CoA or vehicle (water) as indicated for 20 min. followed by digestion with increasing concentrations of chymotrypsin for 20 min at 25 °C. The digests were analyzed by SDS-PAGE followed by PhosphorImaging.


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Fig. 4.   Opposing effects of fatty acid and acyl-CoA on the sensitivity of PPARalpha to chymotrypsin digestion. In vitro transcribed/translated 35S-labeled mPPARalpha was preincubated with either S-hexadecyl-CoA (10 µM) or vehicle for 20 min. Then each preincubation mixture was further incubated for 20 min with either linoleic acid (120 µM) or vehicle. The mixtures were subjected to limited digestion with chymotrypsin and the digests were analyzed by SDS-PAGE followed by PhosphorImaging.


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Fig. 5.   The PPARalpha C-terminal region is required for both agonist- and acyl-CoA-dependent alteration of the sensitivity to chymotrypsin digestion. In vitro transcribed/translated 35S-labeled mPPARalpha truncation mutants mPPARalpha Delta AB and mPPARalpha Delta ABDelta 425 were incubated in the presence of vehicle, 120 µM linoleic, acid or 10 µM S-hexadecyl-CoA for 20 min and then subjected to limited digestion with increasing concentrations of chymotrypsin. The digests were analyzed by SDS-PAGE followed by PhosphorImaging.

S-Hexadecyl-CoA Abolishes Ligand-induced Interaction between SRC-1 and PPARalpha but Enhances Interaction of NCoR with PPARalpha -- Nuclear receptor-mediated transactivation is controlled by a complex interplay between co-activators and co-repressors. Agonist binding enhances recruitment of co-activators, whereas the hallmarks of antagonists are decreased or abolished interaction with co-activators coupled with induced or enhanced interaction with co-repressors (44-50). The experiments presented above indicate that S-hexadecyl-CoA has the characteristics of a PPARalpha antagonist. To further address this possibility, the effects of S-hexadecyl-CoA on the interaction between PPARalpha and the co-activator SRC-1 or the co-repressor NCoR were determined using GST pull-down assays. Fig. 6A demonstrates that S-hexadecyl-CoA abolished agonist-induced recruitment of SRC-1 to PPARalpha in a dose-dependent manner and even decreased interaction below that observed with the unliganded receptor. In contrast, S-hexadecyl-CoA enhanced almost 3-fold the interaction between NCoR and PPARalpha . Thus, by these criteria, S-hexadecyl-CoA behaves like a PPARalpha antagonist.


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Fig. 6.   S-Hexadecyl-CoA antagonizes ligand-induced interaction between PPARalpha and SRC-1, but enhances PPARalpha -NCoR interaction. A, bacterially expressed GST-PPARalpha (ligand binding domain) immobilized on glutathione-Sepharose was incubated with in vitro transcribed/translated 35S-labeled SRC-1 in the presence of vehicle, Wy14643, or S-hexadecyl-CoA. B, bacterially expressed GST-mNCoR (amino acids 2239-2453) immobilized on glutathione-Sepharose was incubated with in vitro transcribed/translated 35S-labeled mPPARalpha in the presence of vehicle, Wy14643, or S-hexadecyl-CoA. Bound proteins were recovered by boiling in SDS sample buffer, separated by SDS-PAGE, and quantified by PhosphorImaging.

S-Hexadecyl-CoA Exerts Its Action on PPARalpha Even When Complexed to Its Natural Carrier Protein, the Acyl-CoA-binding Protein-- In the cell, the concentrations of free acyl-CoA esters and fatty acids are kept in the low or medium nanomolar range by the buffering action of the acyl-CoA-binding protein (ACBP) and fatty acid-binding proteins, respectively (for a review, see Ref. 51). Thus, in order to evaluate the possible biological significance of acyl-CoA esters or for that matter fatty acids in PPAR-mediated signaling, it is imperative to establish assay conditions that mimic or at least approach in vivo conditions. These requirements have only been met partially in one report analyzing the interaction between fatty acids and PPARalpha using a fluorescence-based assay (52). During our initial EMSA experiments using saturating or nearly saturating amounts of in vitro translated PPARalpha and RXRalpha , we noted that the mobility of the PPARalpha ·RXRalpha ·ACO PPRE complex in the presence of S-hexadecyl-CoA or TTA-CoA was marginally increased (data not shown). Subsequently, we discovered that this effect was much more pronounced in whole cell extracts of Sf21 insect cells expressing recombinant rat PPARalpha , suggesting that this might form the basis for a highly sensitive assay for S-hexadecyl-CoA interaction with PPARalpha . No binding was observed in extract prepared from mock-infected cells (Fig. 7A). The increase in electrophoretic mobility was not observed using millimolar concentrations of the detergent lauryl-sarcosine, underscoring the specific action of S-hexadecyl-CoA (results not shown). Sf21 cells contain an RXR analog, in this work referred to as Sf "RXR" (related to ultraspiracle (NR2B4 (2)) in Drosophila (53)), with which PPARalpha is able to heterodimerize and subsequently bind to the ACO PPRE (4, 54). Accordingly, combining in vitro translated PPAR with extract of mock-infected cells allowed efficient binding to the ACO PPRE in EMSA experiments (results not shown). Using this assay, we next asked whether a preformed complex between S-hexadecyl-CoA and ACBP was able to modulate the mobility of the rPPARalpha ·Sf"RXR"·ACO PPRE complex. Fig. 7B demonstrates that even in the presence of a molar surplus of ACBP, S-hexadecyl-CoA was able to increase the mobility of the rPPARalpha ·Sf"RXR"·ACO PPRE complex. By combining EMSA with Western blotting (42) we demonstrated that the shifted band indeed contained PPARalpha (Fig. 7C). Using the established Kd for binding of long-chain acyl-CoA to ACBP, the concentration of free S-hexadecyl-CoA in a solution containing 10 µM S-hexadecyl-CoA and 15 µM ACBP was calculated as 0.2 nM. Thus, using conditions that mimic the in vivo conditions with respect to acyl-CoA availability, S-hexadecyl-CoA imparted an increase in the electrophoretic mobility of the rPPARalpha ·Sf"RXR"·ACO PPRE complex.


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Fig. 7.   The electrophoretic mobility of the rPPARalpha ·Sf"RXR"·PPRE complex is increased by S-hexadecyl-CoA as well as by S-hexadecyl-CoA complexed with ACBP. Whole cell extracts of Sf21 cells infected with an expression vector without (Sf21) or with (Sf21(PPARalpha )) the rPPARalpha cDNA were incubated with a 32P-labeled oligonucleotide containing the PPRE of the rat ACO promoter in the presence of vehicle or S-hexadecyl-CoA (A). B, S-hexadecyl-CoA was added as a preformed complex with ACBP as indicated. C, combined EMSA and Western blotting revealing the shifted position of PPARalpha in the presence of S-hexadecyl-CoA.

As shown above, the presence of S-hexadecyl-CoA differentially altered the ability of PPARalpha to interact with co-activators and co-repressors. Formally, it was therefore possible that the increased electrophoretic mobility reflected an altered molecular mass of the rPPARalpha ·Sf"RXR"·ACO PPRE complex. However, a Ferguson analysis (55) revealed that the presence of 10 µM S-hexadecyl-CoA, 10 µM ACBP, or a 10 µM ACBP plus 10 µM S-hexadecyl-CoA complex did not alter the molecular mass of the rPPARalpha ·Sf"RXR"·ACO PPRE complex (results not shown). Thus, the increased electrophoretic mobility of the rPPARalpha ·Sf"RXR"·ACO PPRE complex in the presence of S-hexadecyl-CoA is not due to an altered molecular mass of the bound heterodimer but rather reflects an altered conformation or change in charge of the heterodimer.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

In the present work, we present evidence suggesting that acyl-CoA esters directly affect the conformation and function of PPARalpha . Using a variety of in vitro approaches, we show that the nonhydrolyzable acyl-CoA analogue, S-hexadecyl-CoA, antagonizes ligand-induced formation of a PPARalpha ·RXRalpha ·ACO PPRE complex. We were able to demonstrate specific binding of PPARalpha to immobilized palmitoyl-CoA, and furthermore, we show that S-hexadecyl-CoA increases the sensitivity of PPARalpha to chymotrypsin digestion in a manner that depended on the integrity of the ligand-binding pocket. We show that S-hexadecyl-CoA, like well established antagonists for other receptors, abolishes ligand-induced interaction with a co-activator, SRC-1, and conversely increases recruitment of a co-repressor, NCoR. Importantly, we show that S-hexadecyl-CoA is able to affect a PPARalpha -containing complex in the presence of a molar excess of the natural cellular acyl-CoA carrier, ACBP. These observations, taken together with our recent finding that ACBP and acyl-CoA esters are present in the nuclei of rat hepatocytes (56), are compatible with the notion that acyl-CoA esters also in vivo might be involved in the regulation of PPARalpha activity. Our results are furthermore supported by recent data showing interaction between acyl-CoA esters and PPARalpha and PPARgamma in competition binding experiments with the labeled synthetic dual agonist, KRP-297 (57).

Long-chain acyl-CoA esters have been estimated to have a van der Waals volume of not less than 850 Å3 (58). This size would exclude acyl-CoA esters from the ligand-binding pocket of most nuclear receptors except for the PPARs with ligand-binding pockets of ~1300 Å3 (16, 17). It was recently reported that docosahexaenoic acid is a ligand for RXRalpha , raising the question of whether acyl-CoA esters might also influence the PPARalpha ·RXRalpha heterodimer via RXRalpha . However, as mentioned above, the size of the ligand-binding pocket of RXRalpha is not compatible with specific binding of acyl-CoA esters, and accordingly, we detected no alteration in the sensitivity to chymotrypsin digestion when RXRalpha was incubated with S-hexadecyl-CoA, and similarly, we observed no binding of RXRalpha to palmitoyl-CoA.

Biochemical and structural studies have revealed a unifying principle determining the interaction of nuclear receptors with co-activators and co-repressors involving an at least partially overlapping binding site (13-15, 59). The hydrophobic face of helical regions in the receptor interacting domains of co-activators or co-repressors harboring an LXXLL core motif or a related CoRNR motif, respectively, interacts with a hydrophobic pocket formed by helices 3-5 and the AF-2 helix in PPARgamma (13-16, 59). Ligand-dependent positioning of the AF-2 helix and differences in the regions flanking the LXXLL and CoRNR motifs are critically involved in the differential interaction of co-activators and co-repressors with liganded and unliganded nuclear receptors, respectively (13, 15, 59). Interestingly, the crystal structure of PPARgamma shows that the AF-2 helix even in the unliganded receptor may fold back against the body of the receptor, assuming a conformation similar to the conformation stabilized by interactions between the polar head group of ligands and the AF-2 helix (16, 18, 60), and as a consequence, interaction with co-activators and co-repressors may be less stringently regulated by ligands in the PPAR subfamily in comparison with other nuclear receptor subfamilies.

From the analysis of the structure of the estrogen receptor bound to agonists or antagonists, it is evident that subtle distortions in the placement of the AF-2 helix may have a profound effect on the interaction with co-activators or co-repressors (10). Our finding that S-hexadecyl-CoA decreases interaction with SRC-1 and increases recruitment of NCoR indicates that the bulky CoA head influences directly or indirectly the positioning of the AF-2 helix. Thus, the bulky CoA head group of S-hexadecyl-CoA may prevent the AF-2 helix from folding back, forcing the AF-2 helix to adopt an extended conformation contrasting with the unliganded conformation that allows the AF-2 helix to fold back. The increased sensitivity of PPARalpha to chymotrypsin digestion upon binding of S-hexadecyl-CoA is also indicative of a less compact conformation.

Examination of the crystal structure of PPARgamma and PPARdelta (16, 17) led to the suggestion that ligands might enter the ligand-binding pocket via a channel between helix 3 and the beta -sheet. In addition, the crystal structure of liganded PPARgamma and PPARdelta revealed prominent interactions between the polar head group of the different agonists and the AF-2 helix (16-18). In contrast, co-crystallization of the partial agonist GW0072 with the ligand-binding domain of PPARgamma revealed a mode of binding in which the carboxylic group of GW0072 was oriented toward the loop region between helices 2' and 3 with no contacts to the AF-2 helix (61). In this context, it is intriguing that we observe specific binding of PPARalpha to palmitoyl-CoA immobilized via the CoA head group. If the palmitoyl-CoA entered the ligand-binding pocket via the channel between helix 3 and the beta -sheet, this suggests that the orientation of palmitoyl-CoA mimicked that of GW0072. Alternatively, positioning of the palmitoyl-CoA molecule with the acyl chain in the characteristic tail-down configuration would imply that the acyl-CoA ligand entered the ligand-binding pocket via the AF-2 side. Interaction of PPARalpha with immobilized PPARalpha agonists would clearly be of interest to examine this possibility.

Several genes are transcriptionally regulated by antagonistic cross-talk between PPAR and HNF-4alpha through a shared DNA binding motif (62-64). It is well established that PPARalpha is activated by polyunsaturated fatty acids (23, 24), and recently myristoyl-CoA and palmitoyl-CoA were reported to bind to HNF-4alpha and activate HNF-4alpha -mediated transactivation, whereas omega -3 and omega -6 polyunsaturated acyl-CoA esters and stearoyl-CoA were shown to antagonize HNF-4alpha -mediated transactivation (65). Based on this finding, it was proposed that the ratio of fatty acids to acyl-CoA esters and the composition of acyl-CoA esters might regulate cross-talk between PPARalpha and HNF-4alpha (65). However, it should be noted that recent data based on molecular modeling of HNF-4alpha and protease protection experiments have questioned the role of acyl-CoA esters in the regulation of HNF-4alpha activity (58). Thus, it remains to be established conclusively whether HNF-4alpha is a target for acyl-CoA-dependent regulation. If so, our findings add another level to the interplay between PPARalpha and HNF-4alpha , indicating that acyl-CoA esters, apart from activating HNF-4alpha , down-regulate PPARalpha -mediated transactivation via direct binding to PPARalpha , thereby imparting a conformation that reduces co-activator interaction and enhances recruitment of co-repressors (Fig. 8).


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Fig. 8.   Model of the opposing effects of transcriptional agonists and acyl-CoA esters on PPARalpha . A, ligands and acyl-CoA compete for binding to PPARalpha , resulting in acyl-CoA-dependent suppression of the ligand-induced formation of a PPARalpha (P)·RXR (R)·PPRE complex. This phenomenon is observed when the concentrations of PPARalpha and RXRalpha are limiting. B, when the concentrations of PPARalpha and RXRalpha are high, formation of the PPARalpha ·RXRalpha ·PPRE complex is unaffected by ligands. The transcriptional activity of the DNA-bound PPARalpha ·RXRalpha complex is determined by the recruitment of cofactors. Acyl-CoA antagonizes the ligand-enhanced recruitment of the co-activator SRC-1 but imparts an increased affinity for the co-repressor NCoR. Arrows in gray denote DR-1 half-sites with the consensus sequence AGGTCA.


    ACKNOWLEDGEMENTS

We thank Drs. J. D. Tugwood, B. W. O'Malley, M. A. Lazar, and Dr. M. Leid for the generous gift of plasmids and Novo Nordisk A/S for supplying ligands.

    FOOTNOTES

* This work was supported in part by grants from the Danish Biotechnology Program, the Danish Natural Science Research Council, and the Novo Nordisk Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a grant from the Nordic Research Academy. Present address: Dept. of Biochemistry and Molecular Biology, University of Bergen, N-5009 Bergen, Norway.

§ These authors contributed equally to this work.

** Present address: Institut für Genetik, Kernforschungszentrum, D-7602 Karlsruhe, Germany.

¶¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5320 Odense M., Denmark. Tel.: 45 6550 2408; Fax: 45 6550 2467; E-mail: kak@bmb.sdu.dk.

Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M101073200

1 The abbreviations and trivial names used are: PPAR, peroxisome proliferator-activated receptor; ACBP, acyl-CoA-binding protein; ACO, acyl-CoA oxidase; BRL49653, (±)-5-([4-[2-methyl-2-(pyridylamino)-ethoxy]phenyl]methyl) 2,4-thiazolidinedione; DR-1, direct repeat separated by one nucleotide; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; HNF-4alpha , hepatocyte nuclear factor-4alpha ; NCoR, nuclear receptor co-repressor; PAGE, polyacrylamide gel electrophoresis; mPPAR, mouse peroxisome proliferator-activated receptor; rPPAR, rat peroxisome proliferator-activated receptor; RXR, retinoid X receptor; rRXR, rat RXR; PAGE, polyacrylamide gel electrophoresis; SRC-1, steroid receptor co-activator-1; TTA, tetradecylthioacetic acid; Wy14643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid; PPRE, peroxisome proliferator-responsive element; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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