title: Bile Acid Recognition by NAPE-PLD.
abstract: The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide and other lipid signaling amides, including oleoylethanolamide and palmitoylethanolamide. These bioactive molecules play important roles in several physiological pathways including stress and pain response, appetite, and lifespan. Recently, we reported the crystal structure of human NAPE-PLD and discovered specific binding sites for the bile acid deoxycholic acid. In this study, we demonstrate that in the presence of this secondary bile acid, the stiffness of the protein measured by elastic neutron scattering increases, and NAPE-PLD is ∼7 times faster to catalyze the hydrolysis of the more unsaturated substrate N-arachidonyl-phosphatidylethanolamine, compared with N-palmitoyl-phosphatidylethanolamine. Chenodeoxycholic acid and glyco- or tauro-dihydroxy conjugates can also bind to NAPE-PLD and drive its activation. The only natural monohydroxy bile acid, lithocholic acid, shows an affinity of ∼20 μM and acts instead as a reversible inhibitor (IC50≈ 68 μM). Overall, these findings provide important insights into the allosteric regulation of the enzyme mediated by bile acid cofactors and reveal that NAPE-PLD responds primarily to the number and position of their hydroxyl groups.
authors: Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia , Piazza San Silvestro 12, 56127 Pisa, Italy.
authors: Eleonora
authors: E
authors: Margheritis
authors: Department of Drug Discovery-Validation, Istituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.
authors: Beatrice
authors: B
authors: Castellani
authors: Department of Drug Discovery-Validation, Istituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.
authors: Paola
authors: P
authors: Magotti
authors: Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia , Piazza San Silvestro 12, 56127 Pisa, Italy.
authors: Sara
authors: S
authors: Peruzzi
authors: Department of Drug Discovery-Validation, Istituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.
authors: Elisa
authors: E
authors: Romeo
authors: Institute Laue-Langevin (ILL) and CNR-IOM , 71 avenue des Martyrs, 38042 Grenoble, France.
authors: Francesca
authors: F
authors: Natali
authors: Department of Pharmaceutical Sciences, University of Perugia , Via del Liceo 1, 06125 Perugia, Italy.
authors: Serena
authors: S
authors: Mostarda
authors: Department of Pharmaceutical Sciences, University of Perugia , Via del Liceo 1, 06125 Perugia, Italy.
authors: Antimo
authors: A
authors: Gioiello
authors: Department of Drug Discovery-Validation, Istituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.|Department of Anatomy & Neurobiology, University of California - Irvine , Gillespie NRF 3101, Irvine, California 92697, United States.
authors: Daniele
authors: D
authors: Piomelli
authors: Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia , Piazza San Silvestro 12, 56127 Pisa, Italy.|Department of Drug Discovery-Validation, Istituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.
authors: Gianpiero
authors: G
authors: Garau
mesh_terms: D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001647:Bile Acids and Salts; D010739:Phospholipase D; C581518:NAPEPLD protein, human