title: Identification of the two-phase mechanism of arachidonic acid regulating inflammatory prostaglandin E2 biosynthesis by targeting COX-2 and mPGES-1.
abstract: Through linking inducible cyclooxygenase (COX)-2 with microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), a Single-Chain Enzyme Complex (SCEC, COX-2-10aa-mPGES-1) was engineered to mimic a specific inflammatory PGE2 biosynthesis from omega-6 fatty acid, arachidonic acid (AA), by eliminating involvements of non-inducible COX-1 and other PGE2 synthases. Using the SCEC, we characterized coupling reactions between COX-2 and mPGES-1 at 1:1 ratio of inflammatory PGE2 production. AA demonstrated two phase activities to regulate inflammatory PGE2 production. In the first phase (<2 μM), AA was a COX-2 substrate and converted to increasing production of PGE2. In the second phase with a further increased AA level (2-10 μM), AA bound to mPGES-1 and inhibited the PGE2 production. The SCEC study was identical to the co-expression of COX-2 and mPGES-1. This was further confirmed by using mPGES-1 and PGH2 as a direct enzyme target and substrate, respectively. Furthermore, the carboxylic acid group of AA binding to R67 and R70 of mPGES-1 was identified by X-ray structure-based docking and mutagenesis. mPGES-1 mutants, R70A, R70K, R67A and R67K, lost 40-100% binding to [(14)C]-AA. To conclude, a cellular model, in which AA is involved in self-controlling initial initiating and later resolving inflammation by its two phase activities, was discussed.
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX 77204-5037, USA.
authors.forename: Hironari
authors.initials: H
authors.lastname: Akasaka
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX 77204-5037, USA. Electronic address: khruan@uh.edu.
authors.forename: Ke-He
authors.initials: KH
authors.lastname: Ruan
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000627:therapeutic use; D016718:Arachidonic Acid / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation; D016297:Mutagenesis, Site-Directed; D044262:Prostaglandin H2 / Q000378:metabolism*; D000071876:Prostaglandin-E Synthases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D044262:Prostaglandin H2; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone
keywords: Arachidonic acid; Cyclooxygenase-2; Microsomal prostaglandin E2 synthase; Polyunsaturated fatty acid; Prostaglandin E2