Stability of fatty acyl-coenzyme A thioester ligands of hepatocyte nuclear factor-4α and peroxisome proliferator-activated receptor-α
Stability of fatty acyl-coenzyme A thioester ligands of hepatocyte nuclear factor-4α and peroxisome proliferator-activated receptor-α
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DOI：
10.1007/s11745-005-1416-y
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发表时间：
2005-06-01
期刊：
影响因子：
1.9
通讯作者：
Kier, AB
中科院分区：
文献类型：
--
作者：
Schroeder, F;Huang, H;Kier, AB
虽然长链脂肪酰基辅酶A （LCFA- coa）硫酯是肝细胞核因子-4 α (HNF-4 α)和过氧化物酶体增殖物激活受体- A （PPAR α）的特异性高亲和力配体，但纯化后的重组配体结合域（LBD）的x射线晶体不含ILCFA-CoA，而是表现出结合LCFA或在纯化过程中失去了所有配体。如图所示：(i) LCFA结合重组HNF-4 α的酰基链组成反映了细菌LCFA- coa库的组成，而不是细菌LCFA库的组成。（ii）用于产生HNF-4 α和PPAR α的细菌所含的LCFA- coa比LCFA少近100倍。（iii）在用于LBD结晶的条件下（在室温水溶液缓冲液中至少3周），16:1-CoA在单独的缓冲液中非常不稳定。（iv）在各自的核受体（即HNF-4 α和PPAR α）存在的情况下，室温下结晶缓冲液1天后，16:1-CoA的70-75%的ILBD被降解，而16:1-CoA的94-97%在3周内被降解。(v)胞质LCFA-CoA结合蛋白，如酰基- coa结合蛋白、甾醇载体蛋白-2和肝- fa结合蛋白，与其各自对该配体的亲和力成正比，减缓了16:1-CoA降解过程。综上所述，这些数据首次表明，结晶的HNF-4 α和PPAR α中LCFA-CoA的缺失是由于细菌中ILCFA-CoA的缺乏，以及LCFA-CoA在水缓冲液中的不稳定性和LBD结晶所用的条件。此外，这些核受体不像其他细胞质LCFA-CoA结合蛋白那样保护结合的LCFA-CoA免受自水解，而是促进LCFA-CoA的降解。
Although long-chain fatty acyl-coenzyme A (LCFA-CoA) thioesters are specific high-affinity ligands for hepatocyte nuclear factor-4 alpha (HNF-4 alpha) and peroxisome proliferator-activated receptor-a (PPAR alpha), X-ray crystals of the respective purified recombinant ligand-binding domains (LBD) do not contain ILCFA-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-4 alpha reflected that of the bacterial LCFA-CoA pool, rather than the bacterial LCFA pool. (ii) Bacteria used to produce the respective HNF-4 alpha and PPAR alpha 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-4 alpha and PPAR alpha), ILBD 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-4 alpha and PPAR alpha was due to the paucity of ILCFA-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.