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 )

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

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 )

Inflammation stimulus

NO Increased PGs production

iNOS Cox-2

L-arginine Arachidonic acid

Present reports :

Inflammation & Pain

Inflammation & cytotoxicity

Independent pathway

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-γRAW 264.7

(Macrophage)

Cell lysateImmunoprecipitated 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

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

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

Myc COX-2 fragments iNOS full length

Co-TransfectionHEK293T cells

Immunoprecipitated with Myc antibody

Western blotting with Myc & iNOS antibody

Fusion protein

Cell lysate

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

GSNO: Glutathione –NO,NO donor

GSH: Glutathione

Ans: NO S-nitrosylates COX-2

NO from iNOS

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

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

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.

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.

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 )

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

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

Inflammation stimulus

NO Increased PGs production

iNOS Cox-2

L-arginineArachidonic acid

Cox-2

70% 30%

Discussion:

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