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 :
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
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:
 NO formation catalyzed by NOS
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 fuction:
triggering of inflammation, dilating of blood vessels and penile erection
 No clinical drugs
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 )
Present reports :
Independent pathway
Inflammation stimulus
iNOS
L-arginine
Cox-2
Arachidonic acid
NO
Inflammation
& cytotoxicity
Increased PGs
production
Inflammation
& Pain
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)
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
Ans. COX-2 and iNOS bind selectively in intact cells.
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
Q3. To map the binding site on iNOS proteins
Fusion protein
Glutathione S-transferase (GST)
iNOS fragments
COX-2 full length
Co-Transfection
HEK293T cells
Cell lysate
Glutathione agarose beads
Western blotting with GST & COX-2 antibody
Ans: 1 to 144 of iNOS within the oxygenase domain is required
W.B assay
GST pull-down
assay
Q3. To map the binding site on COX-2 proteins
Fusion protein
Myc
COX-2 fragments
iNOS full length
Co-Transfection
HEK293T cells
Cell lysate
Immunoprecipitated with Myc antibody
Western blotting with Myc & iNOS antibody
Ans: 484 to 604 of COX-2 mediates binding
W.B assay
Immunoprecipitation
assay
Q4. To explore the possibility of S-nitrosylation of COX-2 by NO ( 13 cysteines )
S-nitrosylation :
Cys-S-NO
Biotin switch
Ans: NO S-nitrosylates COX-2
GSNO: Glutathione –NO,NO donor
GSH: Glutathione
NO from iNOS
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
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.
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.
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.
Q9. To explore the influence of of iNOS & COX-2 dissociation for new drug target
Fusion protein
Myc
COX-2 ( 1-483 )
or
Myc
COX-2 ( 484-604 )
Transfection
RAW 264.7 cells ( LPS+ IFN-γ)
Immunoprecipitated with iNOS antibody
Western blotting
Ans: The truncated form COX-2 attenuates iNOS binding to COX-2 & NOmediated activation of PGE2 production
PGE2 & truncated form COX-2 were visualized with confocal microscopy
Discussion:
Inflammation stimulus
iNOS
×
70%
Cox-2
30%
Cox-2
Arachidonic
acid
L-arginine
NO
Increased PGs production