Stability of fatty acyl-coenzyme A thioester ligands of hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor-alpha.
Although long-chain fatty acyl-coenzyme A (LCFA-CoA) thioesters are specific high-affinity ligands for hepatocyte nuclear factor-4alpha (HNF-4alpha) and peroxisome proliferator-activated receptor-alpha (PPARalpha), 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-4alpha reflected that of the bacterial LCFA-CoA pool, rather than the bacterial LCFA pool. (ii) Bacteria used to produce the respective HNF-4alpha and PPARalpha 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-4alpha and PPARalpha), 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-4alpha and PPARalpha 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.
Schroeder
Friedhelm
F
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA.
Huang
Huan
H
Hostetler
Heather A
HA
Petrescu
Anca D
AD
Hertz
Rachel
R
Bar-Tana
Jacob
J
Kier
Ann B
AB
D000214:Acyl Coenzyme A / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D003460:Crystallization; D026261:Diazepam Binding Inhibitor / Q000378:metabolism; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D047493:PPAR alpha / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor; C493320:Fabp1 protein, mouse; D050556:Fatty Acid-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D008024:Ligands; D047493:PPAR alpha; D011994:Recombinant Proteins; C072087:sterol carrier proteins