, Volume 40, Issue 6, pp 559–568 | Cite as

Stability of fatty acyl-coenzyme a thioester ligands of hepatocyte nuclear factor-4α and peroxisome proliferator-activated receptor-α

  • Friedhelm Schroeder
  • Huan Huang
  • Heather A. Hostetler
  • Anca D. Petrescu
  • Rachel Hertz
  • Jacob Bar-Tana
  • Ann B. KierEmail author


Although long-chain fatty acyl-coenzyme A (LCFA-CoA) thioesters are specific high-affinity ligands for hepatocyte nuclear factor-4α (HNF-4α) and peroxisome proliferator-activated receptor-α (PPARα), X-ray crystals of the respective purified recombinant ligand-binding domains (LBD) do not contain LCFA-CoA, but instead exhibit bound LCFA or have lost all ligands during the purification process, respectively. As shown herein: (i) The acyl chain composition of LCFA bound to recombinant HNF-4α reflected that of the bacterial LCFA-CoA pool, rather than the bacterial LCFA pool. (ii) Bacteria used to produce the respective HNF-4α and PPARα contained nearly 100-fold less LCFA-CoA than LCFA. (iii) Under conditions used to crystallize LBD (at least 3 wk at room temperature in aqueous buffer), 16∶1-CoA was very unstable in buffer alone. (iv) In the presence of the respective nuclear receptor (i.e., HNF-4α and PPARα), LBD 70–75% of 16∶1-CoA was degraded after 1 d at room temperature in the crystallization buffer, whereas as much as 94–97% of 16∶1-CoA was degraded by 3 wk. (v) Cytoplasmic LCFA-CoA binding proteins such as acyl-CoA binding protein, sterol carrier protein-2, and liver-FA binding protein slowed the process of 16∶1-CoA degradation proportional to their respective affinities for this ligand. Taken together, these data for the first time indicated that the absence of LCFA-CoA in the crystallized HNF-4α and PPARα was due to the paucity of LCFA-CoA in bacteria as well as to the instability of LCFA-CoA in aqueous buffers and the conditions used for LBD crystallization. Furthermore, instead of protecting bound LCFA-CoA from autohydrolysis like several cytoplasmic LCFA-CoA binding proteins, these nuclear receptors facilitated LCFA-CoA degradation.


Nuclear Receptor Liver Fatty Acid Binding Protein Truncation Mutant Peroxisome Proliferator Activate Receptor Alpha Sterol Carrier 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



palmitoleoyl-coenzyme A


n-heptadecanoyl-coenzyme A


amino acid


acyl CoA-binding protein


coenzyme A

HNF-4α (aa 1–455), full-length hepatocyte nuclear factor 4α

HNF-4α-E (aa 132–370), N- and C-terminal truncation mutant of HNF-4α comprising aa 132–410 (i.e., ligand-binding domain E, but missing the negative regulatory domain F and the DNA-binding domain)

HNF-4α-E-F (aa 132–455)

N-terminal truncation mutant of HNF-4α comprising aa 132–455 (i.e., ligand-binding domain E and negative-regulatory domain F, but missing the DNA-binding domain)

HNF-4α-E-0.5F (aa 132–410)

N- and C-terminal truncation mutant of HNF-4α comprising aa 132–410 (i.e., ligand-binding domain E, but missing half of the negative-regulatory domain F and all of the DNA-binding domain)


ligand-binding domain


long-chain fatty acid


long-chain fatty acyl CoA


liver fatty acid-binding protein




peroxisome proliferator-activated receptor-α


retinoic acid receptor-α


retinoid X receptor α


sterol carrier protein-2


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  1. 1.
    Hertz, R., Magenheim, J., Berman, I., and Bar-Tana, J. (1998) Fatty Acyl-CoA Thioesters Are Ligands of Hepatic Nuclear Factor-4α, Nature 392, 512–516.PubMedCrossRefGoogle Scholar
  2. 2.
    Petrescu, A.D., Hertz, R., Bar-Tana, J., Schroeder, F., and Kier, A.B. (2002) Ligand Specificity and Conformational Dependence of the Hepatic Nuclear Factor-4α (HNF-4α), J. Biol. Chem. 277, 23988–23999.PubMedCrossRefGoogle Scholar
  3. 3.
    Hertz, R., Ben-Haim, M., Petrescu, A., Kalderon, B., Berman, I., Eldad, N., Schroeder, F., and Bar-Tana, J. (2003) Rescue of MODY-1 by Agonist Ligands of HNF4α, J. Biol. Chem. 278, 22578–22585.PubMedCrossRefGoogle Scholar
  4. 4.
    Petrescu, A., Huang, H., Hertz, R., Bar-Tana, J., Schroeder, F., and Kier, A.B. (2005) Role of Regulatory F-Domain in Hepatocyte Nuclear Factor-4α Ligand Specificity, J. Biol. Chem. 280, 16714–16727.PubMedCrossRefGoogle Scholar
  5. 5.
    Elholm, M., Dam, I., Jorgenesen, C., Krogsdam, A.-M., Holst, D., Kratchamarova, I., Gottlicher, M., Gustafsson, J.A., Berge, R.K., Flatmark, T., Knudsen, J., Mandrup, S., and Kristiansen, K. (2001) Acyl CoA Esters Antagonize the Effects of Ligands on Peroxisome Proliferator Activated Receptor α Conformation, DNA Binding, and Interaction with Cofactors, J. Biol. Chem. 276, 21410–21416.PubMedCrossRefGoogle Scholar
  6. 6.
    Murakami, K., Ide, T., Nakazawa, T., Okazaki, T., Mochizuki, T., and Kadowaki, T. (2001) Fatty Acyl CoA Thioesters Inhibit Recruitment of Steroid Receptor Co-activator 1 to α and γ Isoforms of Peroxisome Proliferator Activated Receptors by Competing with Agonists, Biochem. J. 353, 231–238.PubMedCrossRefGoogle Scholar
  7. 7.
    Hostetler, H.A., Petrescu, A.D., Kier, A.B., and Schroeder, F. (2005) Peroxisome Proliferator Activated Receptor Alpha (PPARα) Interacts with High Affinity and Is Conformationally Responsive to Endogenous Ligands, J. Biol. Chem. 280, 18667–18682.PubMedCrossRefGoogle Scholar
  8. 8.
    Gossett, R.E., Frolov, A.A., Roths, J.B., Behnke, W.D., Kier, A.B., and Schroeder, F. (1996) Acyl CoA Binding Proteins: Multiplicity and Function, Lipids 31, 895–918.PubMedCrossRefGoogle Scholar
  9. 9.
    McArthur, M.J., Atshaves, B.P., Frolov, A., Foxworth, W.D., Kier, A.B., and Schroeder, F. (1999) Cellular Uptake and Intracellular Trafficking of Long Chain Fatty Acids, J. Lipid Res. 40, 1371–1383.PubMedGoogle Scholar
  10. 10.
    Knudsen, J., Jensen, M.V., Hansen, J.K., Faergeman, N.J., Neergard, T., and Gaigg, B. (1999) Role of Acyl CoA Binding Protein in Acyl CoA Transport, Metabolism, and Cell Signaling, Mol. Cell. Biochem. 192, 95–103.PubMedCrossRefGoogle Scholar
  11. 11.
    Frolov, A.A., and Schroeder, F. (1998) Acyl Coenzyme A Binding Protein: Conformational Sensitivity to Long Chain Fatty Acyl-CoA, J. Biol. Chem. 273, 11049–11055.PubMedCrossRefGoogle Scholar
  12. 12.
    Frolov, A., Cho, T.H., Billheimer, J.T., and Schroeder, F. (1996) Sterol Carrier Protein-2, a New Fatty Acyl Coenzyme A-Binding Protein, J. Biol. Chem. 271, 31878–31884.PubMedCrossRefGoogle Scholar
  13. 13.
    Frolov, A., Cho, T.H., Murphy, E.J., and Schroeder, F. (1997) Isoforms of Rat Liver Fatty Acid Binding Protein Differ in Structure and Affinity for Fatty Acids and aatty Acyl CoAs, Biochemistry 36, 6545–6555.PubMedCrossRefGoogle Scholar
  14. 14.
    Wahli, W., Devchand, P.R., Ijpenberg, A., and Desvergne, B. (1999) Fatty Acids, Eicosanoids, and Hypolipidemic Agents Regulate Gene Expression Through Direct Binding to Peroxisome Proliferator Activated Receptors, in Lipoxygenases and Their Metabolites (Nigam, S., and Pace-Asciak, C.R., eds.), pp. 199–209, Plenum Press, New York.Google Scholar
  15. 15.
    Ellinghaus, P., Wolfrum, C., Assmann, G., Spener, F., and Seedorf, U. (1999) Phytanic Acid Activates the Peroxisome Proliferator-Activated Receptor α (PPARα) in Sterol Carrier Protein-2-/Sterol Carrier Protein x-Deficient Mice, J. Biol. Chem. 274, 2766–2772.PubMedCrossRefGoogle Scholar
  16. 16.
    Sladek, F.M., Ruse, M.D., Nepomuceno, L., Huang, S.-M., and Stallcup, M.R. (1999) Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor HNF4α, Mol. Cell. Biol. 19, 6509–6522.PubMedGoogle Scholar
  17. 17.
    Cronet, P., Peterson, J.F.W., Folmer, R., Blomberg, N., Sjoblom, K., Karlsson, U., Lindstedt, E.-L., and Bamberg, K. (2001) Structure of the PPARα and γ Ligand Binding Domain in Complex with AZ 242a: Ligand Selectivity and Agonist Activation in the PPAR Family, Structure 9, 699–706.PubMedCrossRefGoogle Scholar
  18. 18.
    Wisely, G.B., Miller, A.B., Davis, R.G., Thornquest, A.D., Johnson, R., Spitzer, T., Sefler, A., Shearer, B., Moore, J.T., Miller, A.B., et al. (2002) Hepatocyte Nuclear Factor 4 Is a Transcription Factor That Constitutively Binds Fatty Acids, Structure 10, 1225–1234.PubMedCrossRefGoogle Scholar
  19. 19.
    Dhe-Paganon, S., Duda, K., Iwamoto, M., Chi, Y.-I., and Shoelson, S.E. (2002) Crystal Structure of the HNF4α Ligand Binding Domain in Complex with Endogenous Fatty Acid Ligand, J. Biol. Chem. 277, 37973–37976.PubMedCrossRefGoogle Scholar
  20. 20.
    Jorgensen, C., Krogsdam, A.-M., Kratchamarova, I., Willson, T.M., Knudsen, J., Mandrup, S., and Kristiansen, K. (2002) Opposing Effects of Fatty Acids and acyl-CoA Esters on Conformation and Cofactor Recruitment of Peroxisome Proliferator Activated Receptors, Ann. N.Y. Acad. Sci. 967, 431–439.PubMedCrossRefGoogle Scholar
  21. 21.
    Hertz, R., Sheena, V., Kalderon, B., Berman, I., and Bar-Tana, J. (2001) Suppression of Hepatocyte Nuclear Factor 4α by Acyl-CoA Thioesters of Hypolipidemic Peroxisome Proliferators, Biochem. Pharmacol. 61, 1057–1062.PubMedCrossRefGoogle Scholar
  22. 22.
    Francis, G.A., Fayard, E., Picard, F., and Auwerx, J. (2003) Nuclear Receptors and the Control of Metabolism, Annu. Rev. Physiol. 65, 261–311.PubMedCrossRefGoogle Scholar
  23. 23.
    Xu, J., Nawaz, Z., Tsai, S.Y., Tsai, M.-J., and O'Malley, B.W. (1996) The Extreme C Terminus of the Progesterone Receptor Contains a Transcriptional Repressor Domain That Functions Through a Putative Corepressor, Proc. Natl. Acad. Sci. USA 93, 12195–12199.PubMedCrossRefGoogle Scholar
  24. 24.
    Escher, P., and Wahli, W. (2000) Peroxisome Proliferator Activated Receptors: Insights into Multiple Cellular Functions, Mutat. Res. 448, 121–138.PubMedGoogle Scholar
  25. 25.
    Svensson, S., Osteberg, T., Jacobsson, M., Norstrom, C., Stefansson, K., Hallen, D., Johansson, I.C., Zachrisson, K., Ogg, D., and Jendeberg, L. (2003) Crystal Structure of the Heterodimeric Complex of LXRα and RXRβ Ligand Binding Domains in a Fully Agonistic Form, EMBO J. 22, 4625–4633.PubMedCrossRefGoogle Scholar
  26. 26.
    Petrescu, A.D., Payne, H.R., Boedeker, A.L., Chao, H., Hertz, R., Bar-Tana, J., Schroeder, F., and Kier, A.B. (2003) Physical and Functional Interaction of Acyl CoA Binding Protein (ACBP) with Hepatocyte Nuclear Factor-4α (HNF4α), J. Biol. Chem. 278, 51813–51824.PubMedCrossRefGoogle Scholar
  27. 27.
    Jolly, C.A., Wilton, D.A., and Schroeder, F. (2000) Microsomal Fatty Acyl CoA Transacylation and Hydrolysis: Fatty Acyl CoA Species Dependent Modulation by Liver Fatty Acyl CoA Binding Proteins, Biochim. Biophys. Acta 1483, 185–197.PubMedGoogle Scholar
  28. 28.
    Gossett, R.E., Edmondson, R.D., Jolly, C.A., Cho, T.H., Russell, D.H., Knudsen, J., Kier, A.B., and Schroeder, F. (1998) Structure and Function of Normal and Transformed Murine Acyl CoA Binding Proteins, Arch. Biochem. Biophys. 350, 201–213.PubMedCrossRefGoogle Scholar
  29. 29.
    Chao, H., Martin, G., Russell, W.K., Waghela, S.D., Russell, D.H., Schroeder, F., and Kier, A.B. (2002) Membrane Charge and Curvature Determine Interaction with Acyl CoA Binding Protein (ACBP) and Fatty Acyl CoA Targeting, Biochemistry 41, 10540–10553.PubMedCrossRefGoogle Scholar
  30. 30.
    Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  31. 31.
    Lin, Q., Ruuska, S.E., Shaw, N.S., Dong, D., and Noy, N. (1999) Ligand Selectivity of the Peroxisome Proliferator-Activated Receptor α, Biochemistry 38, 185–190.PubMedCrossRefGoogle Scholar
  32. 32.
    Deutsch, J., Grange, E., Rapoport, S.I., and Purdon, A.D. (1994) Isolation and Quantitation of Long-Chain Acyl-Coenzyme A Esters in Brain Tissue by Solid-Phase Extraction, Anal. Biochem. 220, 321–323.PubMedCrossRefGoogle Scholar
  33. 33.
    Larson, T.R., and Graham, I.A. (2001) A Novel Technique for the Sensitive Quantification of Fatty Acyl CoA Esters from Plant Tissues, Plant J. 25, 115–125.PubMedCrossRefGoogle Scholar
  34. 34.
    Huang, H., Starodub, O., McIntosh, A., Atshaves, B.P., Woldegiorgis, G., Kier, A.B., and Schroeder, F. (2004) Liver Fatty Acid Binding Protein Colocalizes with Peroxisome Proliferator Receptor α and Enhances Ligand Distribution to Nuclei of Living Cells, Biochemistry 43, 2484–2500.PubMedCrossRefGoogle Scholar
  35. 35.
    Xu, H.E., Stanley, T.B., Montana, V.G., Lambert, M.H., Shearer, B.G., Cobb, J.E., McKee, D.D., Galardi, C.M., Plunket, K.D., Nolte, R.T., et al. (2002) Structural Basis for Antagonist-Mediated Recruitment of Nuclear Co-repressors by PPARα, Nature 415, 813–817.PubMedGoogle Scholar
  36. 36.
    Cronan, J.E., Jr. (1975) Thermal Regulation of the Membrane Lipid Composition of Escherichia coli. Evidence for the Direct Control of Fatty Acid Synthesis, J. Biol. Chem. 250, 7074–7077.PubMedGoogle Scholar
  37. 37.
    Jolly, C.A., Chao, H., Kier, A.B., Billheimer, J.T., and Schroeder, F. (2000) Sterol Carrier Protein-2 Suppresses Microsomal Acyl CoA Hydrolysis, Mol. Cell. Biochem. 205, 83–90.PubMedCrossRefGoogle Scholar
  38. 38.
    Faergeman, N.J., and Knudsen, J. (1997) Role of Long-Chain Fatty Acyl-CoA Esters in the Regulation of Metabolism and in Cell Signaling, Biochem. J. 323, 1–12.PubMedGoogle Scholar
  39. 39.
    Bogan, A.A., Dallas-Yang, Q., Ruse, M.D., Maeda, Y., Jiang, G., Nepomuceno, L., Scanlan, T.S., Cohen, F.E., and Sladek, F.M. (2000) Analysis of Protein Dimerization and Ligand Binding of Orphan Receptor HNF4α, J. Mol. Biol. 302, 831–851.PubMedCrossRefGoogle Scholar
  40. 40.
    Rajas, F., Gautier, A., Bady, I., Montano, S., and Mithieux, G. (2002) Polyunsaturated Fatty Acyl CoA Suppress the Glucose-6-phosphate Promoter Activity by Modulating the DNA Binding of Hepatocyte Nuclear Factor 4α, J. Biol. Chem. 277, 15736–15744.PubMedCrossRefGoogle Scholar
  41. 41.
    DiRusso, C.C., Heimert, T.L., and Metzger, A.K. (1992) Characterization of FadR, a Global Transcriptional Regulator of Fatty Acid Metabolism in Escherichia coli. Interaction with the fadB Promoter Is Prevented by Long Chain Fatty Acyl Coenzyme A, J. Biol. Chem. 267, 8685–8691 [published erratum appears in J. Biol. Chem. 267, 22693 (1992)].PubMedGoogle Scholar
  42. 42.
    van Aalten, D.M.F., Milne, K.G., Zou, J.Y., Kleywegt, G.J., Bergfors, T., Ferguson, M.A.J., Knudsen, J., and Jones, T.A. (2001) Binding Site Differences Revealed by Crystal Structures of Plasmodium falciparum and Bovine Acyl-CoA Binding Protein, J. Mol. Biol. 309, 181–192.PubMedCrossRefGoogle Scholar
  43. 43.
    Berge, R.K. (1980) Physiochemical Properties of the Long Chain Acyl CoA Hydrolase from Rat Liver Microsomes, Eur. J. Biochem. 111, 67–72.PubMedCrossRefGoogle Scholar
  44. 44.
    Mentlein, R., Suttorp, M., and Heymann, E. (1984) Specificity of Purified Monoacylglycerol Lipase, Palmitoyl-CoA Hydrolase, Palmitoyl-Carnitine Hydrolase, and Nonspecific Carboxylesterase from Rat Liver Microsomes, Arch. Biochem. Biophys. 228, 230–246.PubMedCrossRefGoogle Scholar
  45. 45.
    Yamada, J., Suga, K., Furihata, T., Kitahara, M., Watanabe, T., Hosokawa, M., Satoh, T., and Suga, T. (1998) cDNA Cloning and Genomic Organization of Peroxisome Proliferator-Inducible Long Chain Acyl CoA Hydrolase from Rat Liver Cytosol, Biochem. Biophys. Res. Commun. 248, 608–612.PubMedCrossRefGoogle Scholar
  46. 46.
    Broustas, C.G., and Hajra, A.K. (1995) Purification, Properties, and Specificity of Rat Brain Cytosolic Fatty Acyl CoA Hydrolase, J. Neurochem. 64, 2345–2353.PubMedCrossRefGoogle Scholar
  47. 47.
    Svensson, L.T., Enberg, S.T., Aoyama, T., Usada, N., Alexson, S.E.H., and Hashimoto, T. (1998) Molecular Cloning and Characterization of a Mitochondrial Peroxisome Proliferator Induced Acyl CoA Thioesterase from Rat Liver, Biochem. J. 329, 608.Google Scholar
  48. 48.
    Westin, M.A.K., Alexson, S.E.H., and Hunt, M.C. (2004) Molecular Cloning and Characterization of Two Mouse Peroxisomal Proliferator Activated Receptor α (PPARα)-Regulated Peroxisomal Acyl CoA Thioesterases, J. Biol. Chem. 279, 21841–21848.PubMedCrossRefGoogle Scholar
  49. 49.
    Duda, K., Chi, Y.-I., and Shoelson, S.E. (2004) Structural Basis for HNF4α Activation by Ligand and Coactivator Binding, J. Biol. Chem. 279, 23311–23316.PubMedCrossRefGoogle Scholar
  50. 50.
    Brenner, S. (1988) The Molecular Evolution of Genes and Proteins: A Tale of Two Serines, Nature 343, 528–530.CrossRefGoogle Scholar
  51. 51.
    Kursula, P., Ojala, J., Lambier, A.M., and Wieringa, R.K. (2002) The Catalytic Cycle of Biosynthetic Thiolase: A Conformational Journey of an Acetyl Group Through Four Binding Modes and Two Oxyanion Holes, Biochemistry 41, 15543–15556.PubMedCrossRefGoogle Scholar

Copyright information

© AOCS Press 2005

Authors and Affiliations

  • Friedhelm Schroeder
    • 1
  • Huan Huang
    • 1
  • Heather A. Hostetler
    • 1
  • Anca D. Petrescu
    • 1
  • Rachel Hertz
    • 2
  • Jacob Bar-Tana
    • 2
  • Ann B. Kier
    • 3
    Email author
  1. 1.Department of Physiology and Pharmacology, Texas A&M UniversityTVMCCollege Station
  2. 2.Department of Human Nutrition and Metabolism, HebrewUniversity Medical SchoolJerusalemIsrael
  3. 3.Department of Pathobiology, Texas A&M UniversityTVMCCollege Station

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