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 PGE 2 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.  NO formation catalyzed by NOS Cys-S-NO ( S-nitrosylation ) Metal-NO ( eg: Iron,copper & zinc,bind to heme group for activation of guanylate cyclase→cGMP ↑) NO + superoxide anion (O 2 - )→ONOO - ( Nitration and oxidation ) NO  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. Inflammation stimulus NO Increased PGs production iNOSCox-2 L-arginineArachidonic acid Present reports : Inflammation & Pain Inflammation & cytotoxicity Independent pathway

7. Idea from reported references 1.Mediation of inflammation by encephalitogenic cells: interferon gamma induction of nitric oxide synthase and cyclooxygenase 2. ( Misko.T.P.etc..J Neuroimmunol.,1995) 2. Nitrite oxide activates cyclooxygenase enzymes ( Daniela S.etc.,PNAS,1993) 3. 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. Q3. To map the binding site on iNOS proteins Glutathione S-transferase (GST)iNOS fragments COX-2 full length Co-Transfection HEK293T cells Glutathione agarose beads Western blotting with GST & COX-2 antibody Fusion protein Cell lysate

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

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

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

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

18. Michaelis-Menten equation: Q6. To ascertain the kinetic basis for NO activation of COX-2 ( K M : V max /2 ) NO activates COX-2 by increasing V max & accelerates the release of product from COX-2. Viscosity ＋－ Y= K cat -control / k cat -viscogen

19. Q7. To clarify the influence of NO on prostaglandin E 2 in cells LPS+ IFN-γ 50% inhibition L-NAME: NOS inhibitor ( inhibit all NO) D-NAME: inactive isomer of L-NAME NO-induced synthesis of PGE 2 is about 50% in cells.

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

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

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

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

24. Inflammation stimulus NOIncreased PGs production iNOSCox-2 L-arginine Arachidonic acid Cox-2 70%30% Discussion: ×