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• W2129338435 abstract "The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARα. Here we demonstrate thatS-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARα conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARα-retinoid X receptor α heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARα 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 PPARα to chymotrypsin digestion, and theS-hexadecyl-CoA-induced sensitivity required a functional PPARα ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARα 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 PPARα expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARα-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo. The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARα. Here we demonstrate thatS-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARα conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARα-retinoid X receptor α heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARα 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 PPARα to chymotrypsin digestion, and theS-hexadecyl-CoA-induced sensitivity required a functional PPARα ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARα 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 PPARα expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARα-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo. 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 (1Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). The peroxisome proliferator-activated receptor α (PPARα, 1The 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, glutathioneS-transferase; HNF-4α, hepatocyte nuclear factor-4α; 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. 1The 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, glutathioneS-transferase; HNF-4α, hepatocyte nuclear factor-4α; 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. NR1C1 (2Nuclear Receptors Nomenclature Committee, Cell, 97, 1999, 161, 163.Google Scholar)) belongs to the nuclear hormone receptor superfamily (3Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar). Through heterodimerization with the retinoid X receptors (4Gearing K.L. Göttlicher M. Teboul M. Widmark E. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1440-1444Crossref PubMed Scopus (343) Google Scholar) (NR2B1-3) and binding to DR-1 response elements, PPARα regulates transcription of several genes encoding enzymes involved in lipid metabolism (5Osumi T. Wen J.-K. Hashimoto T. Biochem. Biophys. Res. Commun. 1991; 175: 866-871Crossref PubMed Scopus (183) Google Scholar, 6Gulick T. Cresci S. Caira T. Moore D.D. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11012-11016Crossref PubMed Scopus (482) Google Scholar). Accordingly, PPARα is predominantly expressed in tissues with a high turnover of fatty acids (7Braissant O. Wahli W. Endocrinology. 1998; 139: 2748-2754Crossref PubMed Scopus (378) Google Scholar).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 (8Renaud J.P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1021) Google Scholar, 9Wagner R.L. Apriletti J.W. Mcgrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (805) Google Scholar, 10Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.-Å. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2916) Google Scholar, 11Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2224) Google Scholar). This ligand-induced conformational change has been demonstrated to be a determining event governing interactions with co-activators and co-repressors (see Refs. 12Zhang J. Hu X. Lazar M.A. Mol. Cell. Biol. 1999; 19: 6448-6457Crossref PubMed Scopus (91) Google Scholar, 13Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (423) Google Scholar, 14Nagy L. Kao H.-Y. Love J.D. Li C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W.R. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (344) Google Scholar; reviewed in Ref. 15Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). The crystal structures of the PPARγ and PPARδ ligand-binding domains (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar) have revealed an overall folding pattern similar to that observed for other nuclear receptor ligand-binding domains (8Renaud J.P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1021) Google Scholar, 9Wagner R.L. Apriletti J.W. Mcgrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (805) Google Scholar, 10Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.-Å. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2916) Google Scholar, 11Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2224) Google Scholar). 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 (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar). The interior of the ligand-binding pocket has been suggested to be solvent-accessible via a channel between helix 3 and the β-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 (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 18Gampe Jr., R.T. Montana V.G. Lambert M.H. Miller A.B. Bledsoe R.K. Milburn M.V. Kliewer S.A. Willson T.M. Xu H.E. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). Crystallization of a ternary complex containing the PPARγ ligand-binding domain, the PPARγ 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 (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar). Although the three-dimensional structure of PPARα 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 (19Leers J. Treuter E. Gustafsson J.-Å. Mol. Cell. Biol. 1998; 18: 6001-6013Crossref PubMed Scopus (92) Google Scholar, 20Dowel P. Ishmael J.E. Avram D. Peterson V.J. Nevrivy D.J. Leid M. J. Biol. Chem. 1997; 272: 33435-33443Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 21Treuter E. Albrektsen T. Johansson L. Leers J. Gustafsson J.-Å. Mol. Endocrinol. 1998; 12: 864-881Crossref PubMed Scopus (0) Google Scholar, 22Dowel P. Ishmael J.E. Avram D. Peterson V.J. Nevrivy D.J. Leid M. J. Biol. Chem. 1999; 274: 15901-15907Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).A large variety of long-chain fatty acids, eicosanoids, and synthetic compounds have been shown to serve as PPARα ligands and activators (17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 23Göttlicher M Widmark E. Li Q. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (796) Google Scholar, 24Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1850) Google Scholar, 25Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisley G.B. Kolbe C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1870) Google Scholar). Several natural and synthetic PPARα ligands are activated to the corresponding CoA esters (26Bronfman M. Amingo L. Morales M.N. Biochem. J. 1986; 239: 781-784Crossref PubMed Scopus (85) Google Scholar, 27Lygre T. Aarsaether N. Stensland E. Aarsland A. Berge R.K. J. Chromatography. 1986; 38: 95-105Crossref Scopus (31) Google Scholar, 28Aarsland A. Berge R.K. Biochem. Pharmacol. 1991; 41: 53-61Crossref PubMed Scopus (60) Google Scholar), and these have been demonstrated to accumulate in tissues of treated rats (29Nilsson A. Thomassen M.S. Christiansen E. Lipids. 1984; 19: 187-194Crossref PubMed Scopus (46) Google Scholar, 30Berge R.K. Aarsland A. Biochim. Biophys. Acta. 1985; 837: 141-151Crossref PubMed Scopus (63) Google Scholar). Generally, the formation of CoA esters has been considered a process that merely reduces the availability of the activating PPARα ligands (31Hertz R. Berman I. Bar-Tana J. Eur. J. Biochem. 1994; 221: 611-615Crossref PubMed Scopus (40) Google Scholar). In the present study, we present evidence that acyl-CoA esters directly affect PPARα conformation and function in a manner indicating that acyl-CoA esters may act as PPARα antagonists.DISCUSSIONIn the present work, we present evidence suggesting that acyl-CoA esters directly affect the conformation and function of PPARα. Using a variety of in vitro approaches, we show that the nonhydrolyzable acyl-CoA analogue, S-hexadecyl-CoA, antagonizes ligand-induced formation of a PPARα·RXRα·ACO PPRE complex. We were able to demonstrate specific binding of PPARα to immobilized palmitoyl-CoA, and furthermore, we show thatS-hexadecyl-CoA increases the sensitivity of PPARα 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 PPARα-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 (56Elholm M. Garras A. Neve S. Tornehave D. Lund T.B. Skorve J. Flatmark T. Kristiansen K. Berge R. J. Lipid Res. 2000; 41: 538-545Abstract Full Text Full Text PDF PubMed Google Scholar), are compatible with the notion that acyl-CoA esters also in vivo might be involved in the regulation of PPARα activity. Our results are furthermore supported by recent data showing interaction between acyl-CoA esters and PPARα and PPARγ in competition binding experiments with the labeled synthetic dual agonist, KRP-297 (57Murakami K. Ide T Nakazawa T. Okazaki T. Mochizuki T. Kadowaki T. Biochem. J. 2001; 353: 231-238Crossref PubMed Scopus (38) Google Scholar).Long-chain acyl-CoA esters have been estimated to have a van der Waals volume of not less than 850 Å3 (58Bogan A.A. Dallas-Yang Q. Ruse Jr, M.D. Maeda Y. Jiang G. Nepomuceno L. Scanlan T.S. Cohen F.E. Sladek F.M. J. Mol. Biol. 2000; 302: 831-851Crossref PubMed Scopus (96) Google Scholar). 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 (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar). It was recently reported that docosahexaenoic acid is a ligand for RXRα, raising the question of whether acyl-CoA esters might also influence the PPARα·RXRα heterodimer via RXRα. However, as mentioned above, the size of the ligand-binding pocket of RXRα is not compatible with specific binding of acyl-CoA esters, and accordingly, we detected no alteration in the sensitivity to chymotrypsin digestion when RXRα was incubated withS-hexadecyl-CoA, and similarly, we observed no binding of RXRα 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 (13Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (423) Google Scholar, 14Nagy L. Kao H.-Y. Love J.D. Li C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W.R. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (344) Google Scholar, 15Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 59Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (520) Google Scholar). 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 PPARγ (13Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (423) Google Scholar, 14Nagy L. Kao H.-Y. Love J.D. Li C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W.R. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (344) Google Scholar, 15Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 59Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (520) Google Scholar). 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 (13Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (423) Google Scholar, 15Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 59Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (520) Google Scholar). Interestingly, the crystal structure of PPARγ 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 (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 18Gampe Jr., R.T. Montana V.G. Lambert M.H. Miller A.B. Bledsoe R.K. Milburn M.V. Kliewer S.A. Willson T.M. Xu H.E. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 60Uppenberg J. Svensson C. Jaki M. Bertilsson G. Jendeberg L. Berkenstam A. J. Biol. Chem. 1998; 273: 31108-31112Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), 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 (10Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.-Å. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2916) Google Scholar). Our finding thatS-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 PPARα to chymotrypsin digestion upon binding of S-hexadecyl-CoA is also indicative of a less compact conformation.Examination of the crystal structure of PPARγ and PPARδ (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar) led to the suggestion that ligands might enter the ligand-binding pocket via a channel between helix 3 and the β-sheet. In addition, the crystal structure of liganded PPARγ and PPARδ revealed prominent interactions between the polar head group of the different agonists and the AF-2 helix (16Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1659) Google Scholar, 17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Lehmann J.M. Wisely G.B. Wilson T.M. Kliewer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-406Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 18Gampe Jr., R.T. Montana V.G. Lambert M.H. Miller A.B. Bledsoe R.K. Milburn M.V. Kliewer S.A. Willson T.M. Xu H.E. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). In contrast, co-crystallization of the partial agonist GW0072 with the ligand-binding domain of PPARγ 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 (61Oberfield J.L. Collins J.L. Holmes C.P. Goreham D.M. Cooper J.P. Cobb J.E. Lenhard J.M. Hull-Ryde E.A. Mohr C.P. Blanchard S.G. Parks D.J. Moore L.B. Lehmann J.M. Plunket K. Miller A.B. Milburn M.V. Kliewer S.A. Willson T.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6102-6106Crossref PubMed Scopus (313) Google Scholar). In this context, it is intriguing that we observe specific binding of PPARα 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 β-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 PPARα with immobilized PPARα agonists would clearly be of interest to examine this possibility.Several genes are transcriptionally regulated by antagonistic cross-talk between PPAR and HNF-4α through a shared DNA binding motif (62Hertz R. Bishara-Shieban J. Bar-Tana J. J. Biol. Chem. 1995; 270: 13470-13475Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 63Hertz R. Seckbach M. Zakin M.M. Bar-Tana J. J. Biol. Chem. 1996; 271: 218-224Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 64Rodriguez J.C. Ortiz J.A. Hegardt F.G. Haro D. Biochem. Biophys. Res. Commun. 1998; 242: 692-696Crossref PubMed Scopus (29) Google Scholar). It is well established that PPARα is activated by polyunsaturated fatty acids (23Göttlicher M Widmark E. Li Q. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (796) Google Scholar, 24Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1850) Google Scholar), and recently myristoyl-CoA and palmitoyl-CoA were reported to bind to HNF-4α and activate HNF-4α-mediated transactivation, whereas ω-3 and ω-6 polyunsaturated acyl-CoA esters and stearoyl-CoA were shown to antagonize HNF-4α-mediated transactivation (65Hertz R. Magenheim J. Berman I. Bar-Tana J. Nature. 1998; 392: 512-516Crossref PubMed Scopus (452) Google Scholar). 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 PPARα and HNF-4α (65Hertz R. Magenheim J. Berman I. Bar-Tana J. Nature. 1998; 392: 512-516Crossref PubMed Scopus (452) Google Scholar). However, it should be noted that recent data based on molecular modeling of HNF-4α and protease protection experiments have questioned the role of acyl-CoA esters in the regulation of HNF-4α activity (58Bogan A.A. Dallas-Yang Q. Ruse Jr, M.D. Maeda Y. Jiang G. Nepomuceno L. Scanlan T.S. Cohen F.E. Sladek F.M. J. Mol. Biol. 2000; 302: 831-851Crossref PubMed Scopus (96) Google Scholar). Thus, it remains to be established conclusively whether HNF-4α is a target for acyl-CoA-dependent regulation. If so, our findings add another level to the interplay between PPARα and HNF-4α, indicating that acyl-CoA esters, apart from activating HNF-4α, down-regulate PPARα-mediated transactivation via direct binding to PPARα, thereby imparting a conformation that reduces co-activator interaction and enhances recruitment of co-repressors (Fig.8). 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 (1Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). The peroxisome proliferator-activated receptor α (PPARα, 1The 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, glutathioneS-transferase; HNF-4α, hepatocyte nuclear factor-4α; 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. 1The 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, glutathioneS-transferase; HNF-4α, hepatocyte nuclear factor-4α; 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. NR1C1 (2Nuclear Receptors Nomenclature Committee, Cell, 97, 1999, 161, 163.Google Scholar)) belongs to the nuclear hormone receptor superfamily (3Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar). Through heterodimerization with the retinoid X receptors (4Gearing K.L. Göttlicher M. Teboul M. Widmark E. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1440-1444Crossref PubMed Scopus (343) Google Scholar) (NR2B1-3) and binding to DR-1 response elements, PPARα regulates" @default.
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• W2129338435 title "Acyl-CoA Esters Antagonize the Effects of Ligands on Peroxisome Proliferator-activated Receptor α Conformation, DNA Binding, and Interaction with Co-factors" @default.
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