Fatty acid-binding protein 7 triggers α-synuclein oligomerization in glial cells and oligodendrocytes associated with oxidative stress.

We previously show that fatty acid-binding protein 3 (FABP3) triggers α-synuclein (Syn) accumulation and induces dopamine neuronal cell death in Parkinson disease mouse model. But the role of fatty acid-binding protein 7 (FABP7) in the brain remains unclear. In this study we investigated whether FABP7 was involved in synucleinopathies. We showed that FABP7 was co-localized and formed a complex with Syn in Syn-transfected U251 human glioblastoma cells, and treatment with arachidonic acid (100 M) significantly promoted FABP7-induced Syn aggregation, which was associated with cell death. We demonstrated that synthetic FABP7 ligand 6 displayed a high affinity against FABP7 with Kd value of 209 nM assessed in 8-anilinonaphthalene-1-sulfonic acid (ANS) assay; ligand 6 improved U251 cell survival via disrupting the FABP7-Syn interaction. We showed that activation of phospholipase A2 (PLA2) by psychosine (10 M) triggered oligomerization of endogenous Syn and FABP7, and induced cell death in both KG-1C human oligodendroglia cells and oligodendrocyte precursor cells (OPCs). FABP7 ligand 6 (1 M) significantly decreased Syn oligomerization and aggregation thereby prevented KG-1C and OPC cell death. This study demonstrates that FABP7 triggers α-synuclein oligomerization through oxidative stress, while FABP7 ligand 6 can inhibit FABP7-induced Syn oligomerization and aggregation, thereby rescuing glial cells and oligodendrocytes from cell death.

Analysis of binding affinity and docking of novel fatty acid-binding protein (FABP) ligands.

Fatty acid-binding proteins (FABPs) belong to a family of proteins that transports fatty acids in the cytosol and regulates cellular functions like membrane phospholipid synthesis, lipid metabolism, and mitochondrial ß oxidation. In this study, we synthesized ten novel derivatives from BMS309403, a biphenyl azole compound specific for FABP4, and analyzed their affinity and specificity for FABP3, FABP4, and FABP5, which possess 60% of homology in amino acid sequence. Here, we used 1-anilinonaphthalene 8-sulfonic acid (ANS) displacement assay and found that Ligand 1 has highest affinity for FABP3, with comparable affinity for FABP4 and FABP5. The apparent dissociation constant of BMS309403 was identical to that of arachidonic acid and docosahexaenoic acid. Docking studies with X-ray structural data showed that these novel derivatives obtained by the substitution of phenoxyacetic acid in BMS309403 but not BMS309403 have high or moderate affinity for FABP3. We further found that substitution of a phenyl group and alkyl group caused steric hindrance between 16F, the portal loop and 115L, 117L, respectively, leading to decrease in their affinity for FABPs. In conclusion, our study provides a novel strategy for development of specific ligand for each FABP.

Development of FABP3 ligands that inhibit arachidonic acid-induced α-synuclein oligomerization.

In Parkinson's disease (PD), α-synuclein (αSyn) accumulation and inclusion triggers dopamine neuronal death and synapse dysfunction in vivo. We previously reported that fatty acid-binding protein 3 (FABP3) is highly expressed in the brain and accelerates αSyn oligomerization when cells are exposed to 1-Methyl-1,2,3,6-tetrahydropiridine (MPTP). Here, we demonstrate that αSyn oligomerization was markedly enhanced by co-overexpressing FABP3 in neuro-2A cells when cells were treated with arachidonic acid (AA). We developed FABP3 ligands, which bind to the fatty acid binding domain of FABP3, using an 8-Anilinonaphthalene-1-sulfonic acid (ANS) assay with a recombinant FABP3 protein. The prototype for the FABP4 ligand, BMS309403, has no affinity for FABP3. We developed more FABP3-specific ligands derived from the chemical structure of BMS309403. Like AA, ligands 1, 7, and 8 had a relatively high affinity for FAPB3 in the ANS assay. Then, we evaluated the inhibition of αSyn oligomerization in neuro-2A cells co-overexpressing FABP3 and αSyn. Importantly, AA treatments markedly enhanced αSyn oligomerization in the co-expressing cells. Ligands 1, 7, and 8 significantly reduced AA-induced αSyn oligomerization in neuro-2A cells. Taken together, our results indicate that FABP3 ligands that target FABP3 may be used as potential therapeutics that inhibit αSyn aggregation in vivo.

In silico and in vitro approaches to elucidate the thermal stability of human UDP-glucuronosyltransferase (UGT) 1A9.

UDP-Glucuronosyltransferases (UGTs) are predominant drug metabolizing enzymes in the liver and extrahepatic tissues. Human UGT1A9 is uniquely stable against heat treatment. To understand the unique properties of UGT1A9, the three-dimensional structure was constructed by homology modeling using a crystal structure of TDP-epi-vancosaminyltransferase as template. Sequence alignment analysis revealed that 13 amino acid residues (Arg42, Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Asn152, Leu173, Leu219, His221, Arg222, and Glu241) are unique to UGT1A9 as compared with UGT1A7, UGT1A8 and UGT1A10. To examine the roles of these residues in the conformational stability of UGT1A9, molecular dynamics simulation of the structures was carried out at 310 K and 360 K in aqueous solution for 3.0 nanoseconds. Root mean square deviation analyses revealed that Arg42, Leu173, Leu219, His221 and Arg222 were responsible for the thermal stability. Root mean square fluctuation analyses and a dynamical cross correlation map revealed that Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Leu219, His221, Arg222 and Glu241 were responsible for the thermal stability. In vitro study using mutants of these residues demonstrated that all these amino acids may be collectively involved in the thermal stability of UGT1A9. The results presented here provide a molecular basis for the thermal stability of human UGT1A9.

Electronic and geometric structures of the blue copper site of azurin investigated by QM/MM hybrid calculations.

The electronic and geometric structures of the copper-binding site in a fully solvated azurin were investigated using quantum mechanics (QM) and molecular mechanics (MM) hybrid calculations. Two types of computational models were applied to evaluate the effects of the environment surrounding the active site. In model I, long-distance electrostatic interactions between QM region atoms and partial point charges of the surrounding protein moieties and solvent water were calculated in a QM Hamiltonian, for which the spin-unrestricted Hartree-Fock (UHF)/density functional theory (DFT) hybrid all-electron calculation with the B3LYP functional was adopted. In model II, the QM Hamiltonian was not allowed to be polarized by those partial point charges. Models I and II provided different descriptions of the copper coordination structure, particularly for the coordinative bonds including a large dipole. In fact, the Cu-O(Gly45) and Cu-S(Cys112) bonds are sensitive to the treatment of long-distance electrostatic interactions in the QM Hamiltonian. This suggests that biological processes occurring in the active site are regulated by the surrounding structures of protein and solvent, and therefore the effects of long-range electrostatic interactions involved in the QM Hamiltonian are crucial for accurate descriptions of electronic structures of the copper active site of metalloenzymes.