Endocannabinoid Interaction with Human FABP1: Impact of the T94A Variant

Abstract

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.

Δ9-Tetrahydrocannabinol induces endocannabinoid accumulation in mouse hepatocytes: antagonism by Fabp1 gene ablation

“…-THC (19)(20)(21)65), AEA and 2-AG levels in WT hepatocytes (shown herein) and WT liver (shown earlier (19), are normally very low. Thus, once sufficient…”

“…in (21). While the human FABP1 T94A variant protein differs only modestly from its wild-type FABP counterpart in affinities for endocannabinoids (AEA, 2-AG) (25) as well as a variety of other ligands (26)(27)(28)(29), the conformation of FABP1 T94A variant is much less responsive to ligand-induced change (25,26) which in turn diminishes its ability to enter nuclei, interaction with, and activation of peroxisome proliferator activated receptor- (PPAR) therein (29).…”

“…Liver fatty acid binding protein (FABP1) has high affinity for the endogenous CB1 receptor agonist 2-AG (19)(20)(21). Therefore, the impact of 2-AG and/or FABP1 on hepatocyte level of AEA was examined in cultured primary hepatocytes from wild-type WT and Fabp1 gene ablated (LKO) mice.…”

Human Liver Fatty Acid Binding Protein‐1 T94A Variant, Nonalcohol Fatty Liver Disease, and Hepatic Endocannabinoid System

“…Although the mechanism(s) contributing to lipid accumulation in human livers is not yet clear, recent studies with various recombinant FABP and in FABP1 gene ablated mice suggest a role for FABP1 in binding and regulating the hepatic levels of AEA and NAE (Huang et al, ; Martin, Huang, McIntosh, Kier, & Schroeder, ). Therefore, it was important to determine the impact of the human FABP1 T94A substitution and sex on the hepatic levels of AEA and other NAE.…”

“…Recombinant murine FABP1 also binds and impacts mouse hepatic levels of 2‐AG and non‐ARA containing 2‐MAG (Huang et al, ; Martin, Huang, et al, ). Therefore, the effect of human FABP1 T94A expression and sex on hepatic levels of 2‐MAG as well as other 2‐AG was examined.…”