Martin Gregory G, Huang Huan, McIntosh Avery L, Kier Ann B, Schroeder Friedhelm
Department of Physiology and Pharmacology, Texas A&M University , College Station, Texas 77843-4466, United States.
Department of Pathobiology, Texas A&M University , College Station, Texas 77843-4467, United States.
Biochemistry. 2017 Sep 26;56(38):5147-5159. doi: 10.1021/acs.biochem.7b00647. Epub 2017 Sep 11.
Using recombinant human wild-type fatty acid binding protein 1 (WT FABP1 T94T) and a variant (FABP1 T94A) protein, fluorescence binding assays, and circular dichroism, it was shown for the first time that WT FABP1 and the T94A variant each have a single, relatively hydrophobic site for binding fluorescent NBD-labeled analogues of N-arachidonoylethanolamide and 2-arachidonoylglycerol with high affinity. Most native N-acylethanolamides (NAEs) but only one 2-monoacylglycerol [i.e., 2-arachidonoylglycerol (2-AG)] displaced WT FABP1-bound fluorescently labeled endocannabinoids (ECs). While the T94A variant did not differ in affinity for AEA and most other NAEs, it exhibited a modestly higher affinity for OEA, as well as a higher affinity for 2-AG. Binding of AEA and 2-AG altered WT FABP1's secondary structure more extensively than any other previously examined ligand did. The T94A variant without a ligand was more susceptible to temperature-induced unfolding. While the T94A variant was much less sensitive to ligand (i.e., AEA or 2-AG)-induced conformational change, nevertheless binding of AEA and 2-AG significantly stabilized the T94A structure to thermal unfolding. These data provide the first evidence that ECs not only bind to but also alter the secondary structure of the human FABP1, with the latter markedly impacted by the T94A substitution, a variant strongly associated with hepatic accumulation of lipids and non-alcoholic fatty liver disease (NAFLD). Importantly, NAFLD has been associated with elevated hepatic levels of ECs and FABP1.
通过使用重组人野生型脂肪酸结合蛋白1（WT FABP1 T94T）和一种变体（FABP1 T94A）蛋白、荧光结合测定法和圆二色性，首次表明WT FABP1和T94A变体各自具有一个单一的、相对疏水的位点，用于以高亲和力结合N-花生四烯酰乙醇胺和2-花生四烯酰甘油的荧光NBD标记类似物。大多数天然N-酰基乙醇胺（NAE），但只有一种2-单酰甘油[即2-花生四烯酰甘油（2-AG）]能够取代与WT FABP1结合的荧光标记内源性大麻素（EC）。虽然T94A变体对AEA和大多数其他NAE的亲和力没有差异，但它对OEA表现出适度更高的亲和力，以及对2-AG更高的亲和力。与任何其他先前检测的配体相比，AEA和2-AG的结合更广泛地改变了WT FABP1的二级结构。没有配体的T94A变体更容易受到温度诱导的解折叠影响。虽然T94A变体对配体（即AEA或2-AG）诱导的构象变化不太敏感，但AEA和2-AG的结合显著稳定了T94A结构以抵抗热解折叠。这些数据提供了首个证据，表明EC不仅与人FABP1结合，还会改变其二级结构，后者受到T94A取代的显著影响，T94A是一种与肝脏脂质积累和非酒精性脂肪性肝病（NAFLD）密切相关的变体。重要的是，NAFLD与肝脏中EC和FABP1水平升高有关。
