Acyl-CoA esters antagonize the effects of ligands on peroxisome proliferator-activated receptor alpha conformation, DNA binding, and interaction with Co-factors.

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.

A large variety of long-chain fatty acids, eicosanoids, and synthetic compounds have been shown to serve as PPAR␣ ligands and activators (17,(23)(24)(25). Several natural and synthetic PPAR␣ 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 PPAR␣ ligands (31). In the present study, we present evidence that acyl-CoA esters directly affect PPAR␣ conformation and function in a manner indicating that acyl-CoA esters may act as PPAR␣ antagonists.
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 PPAR␣ was expressed in Spodoptera frugiperda Sf21 insect cells as previously described (4). The Sf21 whole cell homogenate was prepared by disrupting 4 ϫ 10 7 cells in 2.3 ml of buffer (25 mM KPO 4 , 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 g/l.
In Vitro Transcription and Translation-PPAR␣, RXR␣, and SRC-1 were synthesized in vitro by using the TNT®-coupled transcription/ translation system (Promega) with or without [ 35 S]cysteine/methionine amino acid mixture (ICN). PPAR and RXR were synthesized in the presence of 0.5-1.0 M 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 3Ј-recessive ends with [␣-32 P]dCTP (10 Ci, 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 l containing Sf21 extract, 2.4 g 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 PPAR␣ and RXR␣ were used instead of Sf21 extract, the total reaction volume was 20 l, 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-rPPAR␣ antibody (21).
Differential Protease Sensitivity Assay-2-l aliquots of the 35 S-labeled, in vitro translated protein was incubated for 20 min at 25°C in a total volume of 8 l 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 M dithioerythritol. Linoleic acid and S-hexadecyl-CoA were used in final concentrations of 120 M and 5-15 M, respectively. Chymotrypsin (Roche Molecular Biochemicals) was dissolved in 50 mM NH 4 HCO 3 . Following incubation, 2 l of chymotrypsin was added to final concentrations of 0 -120 g/ml or 0 -260 g/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, 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 mPPAR␣ and/or rRXR␣ were prepared as previously described (37). Yeast extract (7.5 l) 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 MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, and Complete TM protease inhibitor mixture (Roche Molecular Biochemicals) with 10 l of palmitoyl-CoA beads (Sigma-Aldrich) or 10 l of protein A-beads (Amersham Pharmacia Biotech). BRL49653 was added to a final concentration of 1 M, Wy14643 was added to final concentrations of 100 nM to 10 M, and 9-cis-retinoic acid was added to a final concentration of 10 M. The beads were washed twice in 150 l 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-PPAR␣ antibody was raised against the mouse PPAR␣ AB domain and affinity-purified, and anti-RXR␣ 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 l of 35 S-labeled in vitro translated mPPAR␣, 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 M Wy14643, 30 M TTA, or vehicle. 10 l 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 l of buffer A and finally once in 150 l 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).

Ligand-induced Binding of PPAR␣⅐RXR␣ 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 PPAR␣-mediated transactivation, and different protocols have been established to demonstrate that several of these activators are bona fide PPAR␣ ligands (17,(23)(24)(25). Ligandinduced 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 PPAR⅐RXR 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 PPAR␣⅐RXR␣ 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 PPAR␣ ligand, we used the fatty acid analog TTA, which has been shown to efficiently promote PPAR␣⅐RXR␣⅐ACO PPRE complex formation (24) and to induce PPAR␣-mediated transactivation.  Fig. 1, lane 4). Increasing the concentration of TTA restored PPAR␣⅐RXR␣⅐ACO 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 PPAR␣⅐RXR␣⅐ACO PPRE complex formation.
PPAR␣ Binds Palmitoyl-CoA-The ligand-induced complex formation assay described above indicated that fatty acid and acyl-CoA might compete for binding to PPAR␣ or, alternatively, that simultaneous binding of an acyl-CoA ester and an agonistic ligand to the PPAR␣⅐RXR␣ complex antagonized the effect of the PPAR␣ 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 PPAR␣ might be revealed by specific binding of PPAR␣ 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 K d 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 PPAR␣ only interacted with the free fatty acid, no specific retention of PPAR␣ would be observed.
Recombinant full-length PPAR␣ and RXR␣ 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 PPAR␣ and RXR␣ were detected by Western blotting. Fig. 2A shows that PPAR␣ preferentially was retained on the palmitoyl-CoA-agarose beads compared with the protein A-agarose beads. The addition of the PPAR␣-selective ligand Wy14643 prevented PPAR␣ binding to the palmitoyl-CoA beads in a dose-dependent manner, whereas the addition of the PPAR␥-selective ligand BRL49653 was without effect on PPAR␣ binding. In contrast, no specific interaction between RXR␣ 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 PPAR␣ ligand Wy14643 and palmitoyl-CoA compete for binding to PPAR␣.
S-Hexadecyl-CoA Increases the Sensitivity of PPAR␣ 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 PPAR␣ ligand, linoleic acid, and S-hexadecyl-CoA on PPAR␣ conformation. 35 S-Labeled PPAR␣ or RXR␣ was incubated with S-hexadecyl-CoA, linoleic acid, or vehicle (0.5% (v/v) Me 2 SO), 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 PPAR␣ 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 PPAR␣ to chymotrypsin, as indicated by the rapid disappearance of the diagnostic 26-kDa protease-resistant fragment (Fig.  3A). In comparison, incubation with 15 M S-hexadecyl-CoA did not influence the sensitivity of RXR␣ 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 PPAR␣ conformation were reversible and did not result from an irreversible denaturing action, the following experiment was performed. The 35 S-labeled PPAR␣ was incubated for 20 min with either 10 M S-hexadecyl-CoA or water, and then each mixture received either 120 M linoleic acid or vehicle (0.5% Me 2 SO). 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 PPAR␣ to chymotrypsin, whereas incubation with S-hexadecyl-CoA increased the sensitivity to chymotrypsin. It is noteworthy that the addition of linoleic acid to PPAR␣ preincubated with S-hexadecyl-CoA partially restored resistance to chymotrypsin digestion. Thus, S-hexadecyl-CoA interacted reversibly with PPAR␣ and appeared to compete with the agonist linoleic acid for binding to PPAR␣. It has previously been shown that deletion of the putative helices H10 -12 of the C-terminal region in the PPAR␣ 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 mPPAR␣⌬AB and mPPAR␣⌬AB/ ⌬425 (35) were digested in the presence of either linoleic acid or S-hexadecyl-CoA. mPPAR␣⌬AB contains the entire ligand-binding domain, whereas the putative helices 10 -12 are deleted in mPPAR␣⌬AB/⌬425 (35). As shown in Fig. 5, in the presence of linoleic acid, digestion of mPPAR␣⌬AB was decreased, whereas S-hexadecyl-CoA increased the sensitivity to chymotrypsin. However, neither linoleic acid nor S-hexadecyl-CoA affected the sensitivity of mPPAR␣⌬AB/⌬425 (Fig. 5).
Thus, it appears that helices 10 -12 are required for interaction of S-hexadecyl-CoA with PPAR␣ as well as for interaction of linoleic acid with PPAR␣.

S-Hexadecyl-CoA Abolishes Ligand-induced Interaction between SRC-1 and PPAR␣ but Enhances Interaction of NCoR with PPAR␣-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 PPAR␣ antagonist. To further address this possibility, the effects of S-hexadecyl-CoA on the interaction between PPAR␣ 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 PPAR␣ 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 PPAR␣. Thus, by these criteria, S-hexadecyl-CoA behaves like a PPAR␣ antagonist.

S-Hexadecyl-CoA Exerts Its Action on PPAR␣ 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 PPAR␣ using a fluorescence-based assay (52). During our initial EMSA experiments using saturating or nearly saturating amounts of in vitro translated PPAR␣ and RXR␣, we noted that the mobility of the PPAR␣⅐RXR␣⅐ACO 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 PPAR␣, suggesting that this might form the basis for a highly sensitive assay for S-hexadecyl-CoA interaction with PPAR␣. 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 PPAR␣ is able to heterodimerize and subsequently bind to the ACO PPRE (4,54). Accordingly, combining in vitro translated PPAR with extract of mockinfected 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 rPPAR␣⅐Sf"RXR"⅐ACO PPRE complex. Fig. 7B demonstrates that even in the presence of a molar surplus of ACBP, Shexadecyl-CoA was able to increase the mobility of the rPPAR␣⅐Sf"RXR"⅐ACO PPRE complex. By combining EMSA with Western blotting (42) we demonstrated that the shifted band indeed contained PPAR␣ (Fig. 7C). Using the established K d for binding of long-chain acyl-CoA to ACBP, the concentration of free S-hexadecyl-CoA in a solution containing 10 M S-hexadecyl-CoA and 15 M 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 rPPAR␣⅐Sf"RXR"⅐ACO PPRE complex.
As shown above, the presence of S-hexadecyl-CoA differentially altered the ability of PPAR␣ 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 rPPAR␣⅐Sf"RXR"⅐ACO PPRE complex. However, a Ferguson analysis (55) revealed that the presence of 10 M S-hexadecyl-CoA, 10 M ACBP, or a 10 M ACBP plus 10 M S-hexadecyl-CoA complex did not alter the molecular mass of the rPPAR␣⅐Sf"RXR"⅐ACO PPRE complex (results not shown). Thus, the increased electrophoretic mobility of the rPPAR␣⅐Sf"RXR"⅐ACO 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 In the present work, we present evidence suggesting that acyl-CoA esters directly affect the conformation and function of PPAR␣. Using a variety of in vitro approaches, we show that the nonhydrolyzable acyl-CoA analogue, S-hexadecyl-CoA, antagonizes ligand-induced formation of a PPAR␣⅐RXR␣⅐ACO PPRE complex. We were able to demonstrate specific binding of PPAR␣ to immobilized palmitoyl-CoA, and furthermore, we show that S-hexadecyl-CoA increases the sensitivity of PPAR␣ 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 PPAR␣-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 PPAR␣ activity. Our results are furthermore supported by recent data showing interaction between acyl-CoA esters and PPAR␣ and PPAR␥ 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 Å 3 (58). This size would exclude acyl-CoA esters from the ligand-binding pocket of most nuclear receptors except for the PPARs with ligandbinding pockets of ϳ1300 Å 3 (16,17). It was recently reported that docosahexaenoic acid is a ligand for RXR␣, raising the question of whether acyl-CoA esters might also influence the PPAR␣⅐RXR␣ heterodimer via RXR␣. However, as mentioned above, the size of the ligand-binding pocket of RXR␣ is not compatible with specific binding of acyl-CoA esters, and accordingly, we detected no alteration in the sensitivity to chymotrypsin digestion when RXR␣ was incubated with S-hexadecyl-CoA, and similarly, we observed no binding of RXR␣ 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)(14)(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 PPAR␥ (13)(14)(15)(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 PPAR␥ 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 corepressors (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 PPAR␣ to chymotrypsin digestion upon binding of S-hexadecyl-CoA is also indicative of a less compact conformation.
Examination of the crystal structure of PPAR␥ and PPAR␦ (16,17) led to the suggestion that ligands might enter the ligand-binding pocket via a channel between helix 3 and the ␤-sheet. In addition, the crystal structure of liganded PPAR␥ and PPAR␦ 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 PPAR␥ revealed a mode of binding in which the carboxylic group of GW0072 was oriented toward the loop region between helices 2Ј and 3 with no contacts to the AF-2 helix (61). In this context, it is intriguing that we observe specific binding of PPAR␣ 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 ␤-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 PPAR␣ with immobilized PPAR␣ agonists would clearly be of interest to examine this possibility.
Several genes are transcriptionally regulated by antagonistic cross-talk between PPAR and HNF-4␣ through a shared DNA binding motif (62)(63)(64). It is well established that PPAR␣ 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.
is activated by polyunsaturated fatty acids (23,24), and recently myristoyl-CoA and palmitoyl-CoA were reported to bind to HNF-4␣ and activate HNF-4␣-mediated transactivation, whereas -3 and -6 polyunsaturated acyl-CoA esters and stearoyl-CoA were shown to antagonize HNF-4␣-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 PPAR␣ and HNF-4␣ (65). However, it should be noted that recent data based on molecular modeling of HNF-4␣ and protease protection experiments have questioned the role of acyl-CoA esters in the regulation of HNF-4␣ activity (58). Thus, it remains to be established conclusively whether HNF-4␣ is a target for acyl-CoA-dependent regulation. If so, our findings add another level to the interplay between PPAR␣ and HNF-4␣, indicating that acyl-CoA esters, apart from activating HNF-4␣, down-regulate PPAR␣-mediated transactivation via direct binding to PPAR␣, thereby imparting a conformation that reduces co-activator interaction and enhances recruitment of co-repressors (Fig. 8).