1 / 24

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

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 :

kaden
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related