title: Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.
abstract: Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.
authors: Department of Structural Biology, The State University of New York at Buffalo, Buffalo, NY 14203, USA.
authors: Benjamin J
authors: BJ
authors: Orlando
authors: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, IL 61802, USA; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61802, USA; Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61802, USA.
authors: Daniel R
authors: DR
authors: McDougle
authors: Department of Structural Biology, The State University of New York at Buffalo, Buffalo, NY 14203, USA.
authors: Michael J
authors: MJ
authors: Lucido
authors: New York Structural Biology Center, New York, NY 10027, USA.
authors: Edward T
authors: ET
authors: Eng
authors: Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University Medical School, Nashville, TN 37232, USA.
authors: Leigh Ann
authors: LA
authors: Graham
authors: Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University Medical School, Nashville, TN 37232, USA.
authors: Claus
authors: C
authors: Schneider
authors: New York Structural Biology Center, New York, NY 10027, USA.
authors: David L
authors: DL
authors: Stokes
authors: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, IL 61802, USA; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61802, USA; Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61802, USA.
authors: Aditi
authors: A
authors: Das
authors: Department of Structural Biology, The State University of New York at Buffalo, Buffalo, NY 14203, USA; Hauptman-Woodward Medical Research Institute, Buffalo, NY 14203, USA. Electronic address: malkowski@hwi.buffalo.edu.
authors: Michael G
authors: MG
authors: Malkowski
mesh_terms: D000818:Animals; D055162:Biocatalysis; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000378:metabolism*; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D036103:Nanotechnology / Q000379:methods*; D019308:Palmitic Acid / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D008051:Lipid Bilayers; D010743:Phospholipids; D019308:Palmitic Acid; D051546:Cyclooxygenase 2
keywords: Arachidonic acid; Aspirin; Cyclooxygenase; Nanodisc; Nonsteroidal anti-inflammatory drugs; Phospholipid