title: Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1.
abstract: We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E(2) synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.
authors.affiliation: Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Que., Canada.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Hétu
authors.forename: Marc
authors.initials: M
authors.lastname: Ouellet
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Falgueyret
authors.forename: Chidambaram
authors.initials: C
authors.lastname: Ramachandran
authors.forename: Joel
authors.initials: J
authors.lastname: Robichaud
authors.forename: Robert
authors.initials: R
authors.lastname: Zamboni
authors.forename: Denis
authors.initials: D
authors.lastname: Riendeau
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D015232:Dinoprostone / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry; D000071876:Prostaglandin-E Synthases; D011487:Protein Conformation; D014466:Ultraviolet Rays
chemical_list: D004791:Enzyme Inhibitors; D051546:Cyclooxygenase 2; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone