title: Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs.
abstract: Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I(2), thromboxane A(2) and prostaglandin F(2alpha). The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H(2) production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.
authors.affiliation: School of Biological Sciences, University of Bristol, Bristol, UK.
authors.forename: Maged E
authors.initials: ME
authors.lastname: Mohamed
authors.forename: Colin M
authors.initials: CM
authors.lastname: Lazarus
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D053898:Biosynthetic Pathways; D051545:Cyclooxygenase 1 / Q000096:biosynthesis / Q000235:genetics; D051546:Cyclooxygenase 2 / Q000096:biosynthesis / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D015237:Dinoprost / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D011464:Epoprostenol / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008954:Models, Biological; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013928:Thromboxane A2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D013930:Thromboxane-A Synthase / Q000096:biosynthesis / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000235:genetics
chemical_list: D000893:Anti-Inflammatory Agents; D011994:Recombinant Proteins; D013928:Thromboxane A2; D003577:Cytochrome P-450 Enzyme System; D015237:Dinoprost; D011464:Epoprostenol; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; D013930:Thromboxane-A Synthase