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Novel signaling paradigm regulating TOLL-like receptors in innate immune Novel signaling paradigm regulating TOLL-like receptors in innate immune

Novel signaling paradigm regulating TOLL-like receptors in innate immune - PowerPoint Presentation

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Novel signaling paradigm regulating TOLL-like receptors in innate immune - PPT Presentation

cells Samar Abdulkhalek and Myron R Szewczuk Dept Biomedical and Molecular Sciences Queens University Kingston K7L 3N6 Ontario Canada speaker A novel signaling paradigm for cell surface TLR4 and intracellular TLR7 and TLR9 ID: 655950

induced tlr neu1 activation tlr induced activation neu1 receptor cells tlr4 mmp9 raw activity blue mmp sialidase ligand lps

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Novel signaling paradigm regulating TOLL-like receptors in innate immune cellsSamar Abdulkhalek and Myron R. Szewczuk*. Dept. Biomedical and Molecular Sciences, Queen's University, Kingston, K7L 3N6 Ontario, Canada. *speaker

A novel signaling paradigm

for cell surface TLR4 and intracellular TLR-7 and TLR-9

Central to this process is that

neuromedin

B (NMBR) GPCR-Neu1-MMP9

complex is bound to

TLRs

in naive and ligand stimulated macrophage

cells.Slide2

TIR DomainEctodomainCommon Knowledge:Front-line Pattern Recognition Receptors of the innate immune system

TLRs recognize microbial pathogen-associated molecular patterns (PAMPs)

Crystal structure reveals highly

glycosylated

ectodomain

Potential 15 N-

glycosylation

sites depending on TLRStill a mystery:How TLR activation is regulated? What Is the role for TLR glycosylation in this ligand–induced TLR activation?

Toll-Like Receptors

Crystal Structure of TLR3 courtesy of Dr. David Segal, Centre for Cancer Research, NIH

TLR3Slide3

Cell surface

and intracellular

TOLL-like receptors

highly glycosylated

Sialic acid

Endoplasmic

Reticulum (ER)

Gp96

MD2

PRAT4A

(protein associated with Toll-like receptor 4)

glycosylation

MD-2:

2 N-glycosylation sites

TLR4:

9 N-glycosylation sites at the

amino terminal

ectodomain

MD2

TLR4

CD14

TLR4Slide4

 "Bruce A. Beutler - Nobel Lecture: How Mammals Sense Infection: From Endotoxin to the Toll-like Receptors". "Jules A. Hoffmann - Nobel Lecture: The Host Defense of Insects: A Paradigm for Innate Immunity". 

"Ralph M. Steinman - Nobel Lecture

:

the

Discovery of Dendritic Cells

".

The Nobel Prize in Physiology or Medicine 2011

TLR-4 receptor

TLR-4 receptorSlide5

Neu11

TLR

Ligand

Receptor

Dimerization

Hydrolyzes of

α

-2,3

sialyl

residue

PPCA

EBP

T.

cruzi

Trans-

sialidase

Strept

.

pneumoniae

neuraminidase

2

3

Enables removal of steric

hindrance

MyD88

Cellular Signalling 22 (2010) 314–324Slide6

4-MUNANA

substrate

Sialidase

activity

Neuraminidase

(

C. perfringens

)

Elastase

Control

(4-MUNANA)

Elastase

N

e

u

1

N

e

u

1

Cath

A

E

B

P

Cath

A

MMP

Elastase

Cath A-

Cathepsin A

EBP-

Elastin binding protein

MMP with

elastase

activity is required to induce NEU1 Slide7

MMP9i inhibits sialidase activity –live cell sialidase assaySlide8

MMP9i inhibits LPS-induced pNFκBSlide9

TRAF6

IRAK 1/4

IKK

I

κ

B

NF

κ

B

NF

κ

B

SEAP

P

P

MyD88

NF

κ

B-dependent SEAP

Assay – RAW-blue cells

Caffeic

acid

phenethyl

ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B.

IL-1 receptor-associated kinase-

4

LPSSlide10

MMP9-siRNA inhibits LPS-induced NFκ

B in RAW-blue macrophagesSlide11

MMP-9 forms a complex with TLR4 in naïve and LPS treated BMA macrophage cellsSlide12

MMP9 forms a Complex with NEU1Slide13
Slide14

RAW-blue

GPCR agonists induced

sialidase

activity is inhibited by

oseltamivir

phosphate

Lysophosphatidic acid (LPA)

a potent endothelium-dependent vasodilator, leading to a drop in blood

pressure

Lipid signaling

Angiotensin -vasoconstrictionSlide15

Primary WTMØ

Neu1-CathA

CathA KD

Sialidase

activity is

abscent

in Neu1 deficient primary

macrophages derived from genetically engineered mice

Bombesin

TLR3

TLR4Slide16
Slide17

TRAF6

IRAK 1/4

IKK

I

κ

B

NF

κ

B

NF

κ

B

SEAP

P

P

MyD88

Bombesin induces NFκB activation in Raw-blue cellsSlide18

Bombesin receptor neuromedin

B (NMBR)

co-IP’s with TLR4 in BMA MØ cell lysatesSlide19

Bombesin receptor NMBR co-IP’s with MMP-9 in RAW-blue MØ cell lysatesSlide20

LPS and Bombesin agonists induced SEAP activity in RAW-blue cells is blocked by MyD88 specific inhibitorSlide21

EBP

GPCR

TLR7

PPCA

Neu 1

MMP9

PPCA

Neu 1

MMP9

MMP9

MMP9

Ligand

EndosomeSlide22

NEU1 and MMP-9 form a complex with TLR7 in naïve and ligand-stimulated macrophage cellsSlide23

Tamiflu, MMP9i and BIM46174 inhibit TLR7 ligand-induced NF

κ

B activationSlide24

NEU1, MMP-9 and NMBR are essential for TLR7 activationSlide25

NMBR is involved in intracellular TLRs signalingSlide26

Tamiflu, MMP9i and BIM46174 abrogates TLR7 ligand-induced inflammatory cytokines productionSlide27

CONCLUSIONSNeu1 plays a central role in mediating nucleic acid-induced intracellular TLR activation, GPCR NMBR–MMP9–Neu1 cross-talk constitutes a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses.