Lipids

, Volume 40, Issue 6, pp 559–568

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

Authors

  • Friedhelm Schroeder
    • Department of Physiology and Pharmacology, Texas A&M UniversityTVMC
  • Huan Huang
    • Department of Physiology and Pharmacology, Texas A&M UniversityTVMC
  • Heather A. Hostetler
    • Department of Physiology and Pharmacology, Texas A&M UniversityTVMC
  • Anca D. Petrescu
    • Department of Physiology and Pharmacology, Texas A&M UniversityTVMC
  • Rachel Hertz
    • Department of Human Nutrition and Metabolism, HebrewUniversity Medical School
  • Jacob Bar-Tana
    • Department of Human Nutrition and Metabolism, HebrewUniversity Medical School
    • Department of Pathobiology, Texas A&M UniversityTVMC
Articles

DOI: 10.1007/s11745-005-1416-y

Cite this article as:
Schroeder, F., Huang, H., Hostetler, H.A. et al. Lipids (2005) 40: 559. doi:10.1007/s11745-005-1416-y

Abstract

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.

Abbreviations

16∶1-CoA

palmitoleoyl-coenzyme A

17∶0-CoA

n-heptadecanoyl-coenzyme A

aa

amino acid

ACBP

acyl CoA-binding protein

CoA

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)

LBD

ligand-binding domain

LCFA

long-chain fatty acid

LCFA-CoA

long-chain fatty acyl CoA

L-FABP

liver fatty acid-binding protein

MPD

2-methyl-2,4-pentanediol

PPARα

peroxisome proliferator-activated receptor-α

RARα

retinoic acid receptor-α

RXRα

retinoid X receptor α

SCP-2

sterol carrier protein-2

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© AOCS Press 2005