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Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2

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Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K. ,Daniel.A.H. ,Solomon H.S. ( Johns Hopkins university ) Source: 23 Dec 2005 Vol 310 Science. Introduction 1. Background :

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  1. Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K. ,Daniel.A.H. ,Solomon H.S. ( Johns Hopkins university ) Source: 23 Dec 2005 Vol 310 Science

  2. Introduction 1. Background : The iNOS & Cox-2 inflammation pathway 2. Idea from reported references 3. Results a. Interaction of iNOS & COX-2 b. NO activates COX-2 via S-nitrosylation c. NO increases PGE2 formation 4. Discussion

  3. Inducible nitrite oxide synthase ( iNOS ) • one of three NOS isoforms ( eNOS:endothlial cells; nNOS: neuronal cells.) • Tissue expression: macrophages,cardiac myocytes,glial cells,vascular smooth muscle cells, endothelium , neurones. • Inducer: bacterial compounds ( LPS ) ,cytokines ( TNF-α,IL-1β,IFN-γ) • Structure:

  4. Cys-S-NO ( S-nitrosylation) NO Metal-NO ( eg: Iron,copper & zinc ,bind to heme group for activation of guanylate cyclase→cGMP ↑) NO + superoxide anion (O2-)→ONOO- ( Nitration and oxidation ) • NO formation catalyzed by NOS • NO fuction: triggering of inflammation, dilating of blood vessels and penile erection • No clinical drugs

  5. Cyclooxygenase-2 ( COX-2 ) • Known as Prostaglandin endoperoxide synthase • One of two COX isoforms ( COX-1 ) • highly induced by following Inducers: LPS,IL-1,TNF-α, IFN-γ,TGF-β,EGF,PDGF,FGF growth factors. • Structure: • Functions: synthesize Prostaglandin E2 from arachidonic acid ( Inflammation ,Fever , Control of blood pressure , Contraction & relaxation of smooth muscle ) • Clinical Cox-2 inhibitors : Vioxx ( Merck ), Celebrex ( Pfizer )

  6. Present reports : Independent pathway Inflammation stimulus iNOS Cox-2 L-arginine Arachidonic acid Increased PGs production NO Inflammation & cytotoxicity Inflammation & Pain

  7. Idea from reported references • Mediation of inflammation by encephalitogenic cells: interferon gamma induction of nitric oxide synthase and cyclooxygenase 2. ( Misko.T.P.etc..J Neuroimmunol.,1995) • Nitrite oxide activates cyclooxygenase enzymes ( Daniela S.etc.,PNAS,1993) • Regulation of prostaglandin biosynthesis by nitrite oxide is revealed by targeted deletion of iNOS ( Lawrence J.M.etc.,J. Bio Chem.2000)

  8. Q1. To determine whether iNOS & Cox-2 interact LPS & IFN-γ RAW 264.7 (Macrophage) Cell lysate Immunoprecipitated by Cox-2 antibody Western blotting with against COX-2 & iNOS antibody

  9. Ans. COX-2 and iNOS bind selectively in intact cells.

  10. Q2. To determine whether catalytic activity of the enzymes influences their binding 1400W: iNOS inhibitor SC58125: COX-2 inhibitor The binding of iNOS & COX-2 do not affected by the inhibition of catalytic activity

  11. Glutathione S-transferase (GST) iNOS fragments Q3. To map the binding site on iNOS proteins Fusion protein COX-2 full length Co-Transfection HEK293T cells Cell lysate Glutathione agarose beads Western blotting with GST & COX-2 antibody

  12. Ans: 1 to 144 of iNOS within the oxygenase domain is required W.B assay GST pull-down assay

  13. Myc COX-2 fragments Q3. To map the binding site on COX-2 proteins Fusion protein iNOS full length Co-Transfection HEK293T cells Cell lysate Immunoprecipitated with Myc antibody Western blotting with Myc & iNOS antibody

  14. Ans: 484 to 604 of COX-2 mediates binding W.B assay Immunoprecipitation assay

  15. Q4. To explore the possibility of S-nitrosylation of COX-2 by NO ( 13 cysteines ) S-nitrosylation : Cys-S-NO Biotin switch

  16. Ans: NO S-nitrosylates COX-2 GSNO: Glutathione –NO,NO donor GSH: Glutathione NO from iNOS

  17. Q5. To determine whether S-nitrosylation of COX-2 alters enzyme activity SNP: NO donor ASC: Reduce Cys-S-NO Dose-dependent 2X increase NO-induced S-nitrosylation of COX-2 increases its catalytic activity

  18. Q6. To ascertain the kinetic basis for NO activation of COX-2 Michaelis-Menten equation: ( KM: Vmax /2 ) Y= Kcat-control /kcat-viscogen Viscosity － ＋ NO activates COX-2 by increasing Vmax & accelerates the release of product from COX-2.

  19. Q7. To clarify the influence of NO on prostaglandin E2 in cells 50% inhibition LPS+ IFN-γ L-NAME: NOS inhibitor ( inhibit all NO) D-NAME: inactive isomer of L-NAME NO-induced synthesis of PGE2 is about 50% in cells.

  20. Q8. To confirm iNOS & COX-2 interaction In Vivo 70% reduction Macrophages from iNOS knockout mice NO-induced synthesis of PGE2 is about 70% in vivo.

  21. Myc Myc COX-2 ( 484-604 ) COX-2 ( 1-483) Q9. To explore the influence of of iNOS & COX-2 dissociation for new drug target Fusion protein or Transfection RAW 264.7 cells ( LPS+ IFN-γ) Immunoprecipitated with iNOS antibody Western blotting

  22. Ans: The truncated form COX-2 attenuates iNOS binding to COX-2 & NO-mediated activation of PGE2 production

  23. PGE2 & truncated form COX-2 were visualized with confocal microscopy

  24. Discussion: Inflammation stimulus × 70% 30% iNOS Cox-2 Cox-2 Arachidonic acid L-arginine NO Increased PGs production