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.
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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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Schroeder, F., Huang, H., Hostetler, H.A. et al. Stability of fatty acyl-coenzyme a thioester ligands of hepatocyte nuclear factor-4α and peroxisome proliferator-activated receptor-α. Lipids 40, 559–568 (2005). https://doi.org/10.1007/s11745-005-1416-y
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DOI: https://doi.org/10.1007/s11745-005-1416-y