Department of Physiology and Pharmacology, Texas A&M University , TVMC, College Station, Texas 77843-4466, United States.
Biochemistry. 2013 Dec 23;52(51):9347-57. doi: 10.1021/bi401014k. Epub 2013 Dec 10.
Although the human liver fatty acid binding protein (L-FABP) T94A variant arises from the most commonly occurring single-nucleotide polymorphism in the entire FABP family, there is a complete lack of understanding regarding the role of this polymorphism in human disease. It has been hypothesized that the T94A substitution results in the complete loss of ligand binding ability and function analogous to that seen with L-FABP gene ablation. This possibility was addressed using the recombinant human wild-type (WT) T94T and T94A variant L-FABP and cultured primary human hepatocytes. Nonconservative replacement of the medium-sized, polar, uncharged T residue with a smaller, nonpolar, aliphatic A residue at position 94 of the human L-FABP significantly increased the L-FABP α-helical structure content at the expense of β-sheet content and concomitantly decreased the thermal stability. T94A did not alter the binding affinities for peroxisome proliferator-activated receptor α (PPARα) agonist ligands (phytanic acid, fenofibrate, and fenofibric acid). While T94A did not alter the impact of phytanic acid and only slightly altered that of fenofibrate on the human L-FABP secondary structure, the active metabolite fenofibric acid altered the T94A secondary structure much more than that of the WT T94T L-FABP. Finally, in cultured primary human hepatocytes, the T94A variant exhibited a significantly reduced extent of fibrate-mediated induction of PPARα-regulated proteins such as L-FABP, FATP5, and PPARα itself. Thus, while the T94A substitution did not alter the affinity of the human L-FABP for PPARα agonist ligands, it significantly altered the human L-FABP structure, stability, and conformational and functional response to fibrate.
尽管人类肝脂肪酸结合蛋白 (L-FABP) T94A 变体源自整个 FABP 家族中最常见的单核苷酸多态性，但人们对这种多态性在人类疾病中的作用仍知之甚少。有人假设 T94A 取代导致配体结合能力完全丧失，功能类似于 L-FABP 基因缺失。使用重组人野生型 (WT) T94T 和 T94A 变体 L-FABP 以及培养的原代人肝细胞来解决这个可能性。在人类 L-FABP 的第 94 位用较小的非极性脂肪族 A 残基取代中到大的、极性、不带电荷的 T 残基，会显著增加 L-FABP 的α-螺旋结构含量，而降低β-折叠结构含量，同时降低热稳定性。T94A 不改变 L-FABP 对过氧化物酶体增殖物激活受体 α (PPARα) 激动剂配体（植烷酸、非诺贝特和非诺贝特酸）的结合亲和力。虽然 T94A 不改变植烷酸的影响，仅略微改变非诺贝特对人 L-FABP 二级结构的影响，但活性代谢物非诺贝特酸对 T94A 二级结构的影响比对 WT T94T L-FABP 的影响大得多。最后，在培养的原代人肝细胞中，T94A 变体显示出对纤维酸介导的 PPARα 调节蛋白（如 L-FABP、FATP5 和 PPARα 本身）诱导的程度显著降低。因此，尽管 T94A 取代不改变人 L-FABP 对 PPARα 激动剂配体的亲和力，但它显著改变了人 L-FABP 的结构、稳定性以及对纤维酸的构象和功能反应。
