Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon...
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Transcript of Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon...
![Page 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.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/1.jpg)
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
![Page 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.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/2.jpg)
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
![Page 3: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/3.jpg)
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:
![Page 4: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/4.jpg)
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 (O2-)→ONOO- ( Nitration and oxidation )
NO
NO fuction: triggering of inflammation, dilating of blood vessels and penile erection No clinical drugs
![Page 5: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/5.jpg)
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 )
![Page 6: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/6.jpg)
Inflammation stimulus
NO Increased PGs production
iNOS Cox-2
L-arginine Arachidonic acid
Present reports :
Inflammation & Pain
Inflammation & cytotoxicity
Independent pathway
![Page 7: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/7.jpg)
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)
![Page 8: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/8.jpg)
Q1. To determine whether iNOS & Cox-2 interact
LPS & IFN-γRAW 264.7
(Macrophage)
Cell lysateImmunoprecipitated by Cox-2 antibody
Western blotting with against COX-2 & iNOS antibody
![Page 9: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/9.jpg)
Ans. COX-2 and iNOS bind selectively in intact cells.
![Page 10: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/10.jpg)
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
![Page 11: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/11.jpg)
Q3. To map the binding site on iNOS proteins
Glutathione S-transferase (GST) iNOS fragments COX-2 full length
Co-TransfectionHEK293T cells
Glutathione agarose beads
Western blotting with GST & COX-2 antibody
Fusion protein
Cell lysate
![Page 12: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/12.jpg)
Ans: 1 to 144 of iNOS within the oxygenase domain is required
W.B assay
GST pull-down assay
![Page 13: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/13.jpg)
Q3. To map the binding site on COX-2 proteins
Myc COX-2 fragments iNOS full length
Co-TransfectionHEK293T cells
Immunoprecipitated with Myc antibody
Western blotting with Myc & iNOS antibody
Fusion protein
Cell lysate
![Page 14: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/14.jpg)
Ans: 484 to 604 of COX-2 mediates binding
W.B assay
Immunoprecipitation assay
![Page 15: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/15.jpg)
Q4. To explore the possibility of S-nitrosylation of COX-2 by NO ( 13 cysteines )
S-nitrosylation : Cys-S-NO
Biotin switch
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GSNO: Glutathione –NO,NO donor
GSH: Glutathione
Ans: NO S-nitrosylates COX-2
NO from iNOS
![Page 17: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/17.jpg)
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
![Page 18: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/18.jpg)
Michaelis-Menten equation:
Q6. To ascertain the kinetic basis for NO activation of COX-2
( KM: Vmax /2 )
NO activates COX-2 by increasing Vmax & accelerates the release of product from COX-2.
Viscosity+-
Y= Kcat-control / kcat-viscogen
![Page 19: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/19.jpg)
Q7. To clarify the influence of NO on prostaglandin E2 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 PGE2 is about 50% in cells.
![Page 20: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/20.jpg)
Q8. To confirm iNOS & COX-2 interaction In Vivo
Macrophages from iNOS knockout mice
70% reduction
NO-induced synthesis of PGE2 is about 70% in vivo.
![Page 21: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/21.jpg)
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
Myc COX-2 ( 484-604 )or
Transfection
Myc COX-2 ( 1-483 )
![Page 22: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/22.jpg)
Ans: The truncated form COX-2 attenuates iNOS binding to COX-2 & NO-mediated activation of PGE2 production
![Page 23: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/23.jpg)
PGE2 & truncated form COX-2 were visualized with confocal microscopy
![Page 24: Inducible Nitric Oxide Synthase Binds,S-Nitrosylates,and Activates Cyclooxygenase-2 Authors: Sangwon F.K.,Daniel.A.H.,Solomon H.S. ( Johns Hopkins university.](https://reader036.fdocuments.us/reader036/viewer/2022062516/56649e605503460f94b5b143/html5/thumbnails/24.jpg)
Inflammation stimulus
NO Increased PGs production
iNOS Cox-2
L-arginineArachidonic acid
Cox-2
70% 30%
Discussion:
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