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.
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.
Hétu
Pierre-Olivier
PO
Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Que., Canada.
Ouellet
Marc
M
Falgueyret
Jean-Pierre
JP
Ramachandran
Chidambaram
C
Robichaud
Joel
J
Zamboni
Robert
R
Riendeau
Denis
D
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
D004791:Enzyme Inhibitors; D051546:Cyclooxygenase 2; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone