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. Kier*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

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-4cx 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.

Original languageEnglish
Pages (from-to)559-568
Number of pages10
JournalLipids
Volume40
Issue number6
DOIs
StatePublished - Jun 2005

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