PURIFICATION AND ANALYSIS OF HUMAN LIVER FATTY ACID BINDING PROTEIN (FABP1)
Charlene Valdez1, Ann Hertzel2, Michael Downey2, David A. Bernlohr2.
1Trinity Washington University, Washington, DC, 2University of Minnesota, Minneapolis, MN.
Obesity is a chronic metabolic disorder affecting over one-third of Americans. It is characterized by excessive accumulation of adipose tissue and is correlated with increased risk for cardiovascular disease, hepatosteatosis, and type 2 diabetes. Human liver fatty acid binding protein (FABP1) functions to metabolize long chain fatty acids in the hepatocyte and is linked to metabolic processes such as lipid storage, oxidation, and gene expression. Whole-body ablation of FABP1 in high-fat fed obese C57Bl/6J mice results in reduced hepatosteatosis and increased insulin sensitivity. This suggests that small molecule inhibitors of FABP1 may be efficacious in reducing hepatic steatosis. To facilitate FABP1 inhibitor studies, the protein was expressed in E. coli and purified using nickel affinity column chromatography followed by delipidation via lipidex column chromatography. X-ray crystallographic studies have revealed that the lipid binding domain of FABP1 is found within a large interior cavity. Lipid binding was measured using the fluorescent fatty acid analogue 1,8-ANS and revealed a binding affinity of ~1.8 μM. Moreover, fatty acids displaced 1,8-ANS from the binding cavity, thereby enabling analysis of therapeutic ligands. These studies offer the potential for identifying small molecule inhibitors of LFABP, useful in the prevention of liver steatosis. (Supported by the NIH R25 HL088728 and the University of Minnesota Life Science Summer Undergraduate Research Program.)