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Micaela Roberts Pharmacology 4AA3 1 April, 2004. The NF-κB Pathway. Introduction Mechanism Activators and target genes Inhibitors. Overview. Important signalling pathway Activated by over 150 different molecules Transcribes over 150 genes Central regulator of STRESS RESPONSE

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Micaela roberts pharmacology 4aa3 1 april 2004 l.jpg

Micaela Roberts

Pharmacology 4AA3

1 April, 2004

The NF-κB Pathway


Overview l.jpg

Introduction

Mechanism

Activators and target genes

Inhibitors

Overview


Introduction l.jpg

Important signalling pathway

Activated by over 150 different molecules

Transcribes over 150 genes

Central regulator of STRESS RESPONSE

Many different steps: many different targets for interruption

Introduction


Mechanism overview l.jpg

RelA

RelA

RelA

RelA

p50

p50

p50

p50

P

P

IkB

Mechanism: Overview

IKK

IkB

IkB

IkB

ikb

kB site


The transcription factor rela nf b family l.jpg

Family of eukaryotic transcription factors

Consists of several structurally related proteins

RelA/p65, c-Rel, RelB, p50/p105, p52/p100

Form homo- or heterodimers

Two classes of Rel/NFkB proteins related by Rel homology (RH) domain - DNA binding / dimerization domain.

The transcription factor: RelA/NF-κB Family


Rela nf b cont l.jpg

Bind to 10 base pair kB sites as dimers.

NFkB commonly refers to a p50-RelA heterodimer, which is one of the most avidly forming dimers and is the major Rel complex in most cells.

Rel/NF-kB complexes in inactive state in cytoplasm bound to an inhibitor

Most common Rel/NF-kB dimer is RelA/p50

RelA/NF-κB cont.


Mechanism nf b l.jpg

RelA

p50

Mechanism: NF-κB

ikb

kB site


The inhibitor i b l.jpg

Most commonly studied is IkBa with NFkB.

IkB blocks the ability of NFkB to enter the nucleus and bind to DNA.

Covers the nuclear localization sequence of NFkB and interfere with sequences important for DNA binding.

The inhibitor: IκB


Mechanism i b l.jpg

RelA

p50

Mechanism: IκB

IkB

ikb

kB site


Degradation of i b l.jpg

Phosphorylation of 2 serines (S32, S36 in human IkBa) in the N-terminal regulatory domain of IkBs

Polyubiquitination on lysine21 and 22.

Degradation of IκB


Ubiquitination l.jpg

Ubiquitination occurs through a cascade of enzymatic reactions

E1 ubiquitin-activating enzyme forms an E1-ubiquitin thioester

E2 ubiquitin conjugating enzymes receive activated ubiquitin from E1

E3 ubiquitin-protein ligases transfer activated ubiquitin to the substrate

Ubiquitination


Ubiquitination cont l.jpg

The enzymes that catalyze the ubiquitination of phospho-IkB are constitutively active so the only regulated step in the IkB degradation pathway is the phosphorylation of the two N-terminal seines in the IkB molecule

Ubiquitination cont.


Mechanism i b degradation l.jpg

RelA are constitutively active so the only regulated step in the IkB degradation pathway is the phosphorylation of the two N-terminal seines in the IkB molecule

RelA

RelA

p50

p50

p50

P

P

IkB

Mechanism: IκB degradation

IkB

ikb

kB site


The phosphorylator ikk l.jpg

Almost all signals that lead to activation of NFkB converge in a high molecular weight complex that contains a serine-specific IkB kinase (IKK).

IKK is responsive to a number of potent NFkB activators in addition to TNFα which stimulates its activity with kinetics that match those of IkBα degradation.

Extent to which IKK is activated by a given stimulus seems to dictate the kinetics of IkB degradation.

The phosphorylator: IKK


Ikk cont l.jpg

There are three tightly associated IKK polypeptides: in a high molecular weight complex that contains a serine-specific IkB kinase (IKK).

IKKα and IKKβ are the catalytic subunits of the kinase

IKKγ is the regulatory subunit

IKK cont.


Ikk cont16 l.jpg

IKKα and IKKβ have very similar primary structures (52% overall identity)

protein kinase domains at their N-termini

leucine zippers (LZ) and helix-loop-helix (HLH) motifs in their C-terminal portions

IKKγ

no recognizable catalytic domain

three large a-helical regions, including a LZ

IKK cont.


Ikk cont17 l.jpg

The predominant form of IKK is an IKKα:IKKβ heterodimer associated with either a dimer or trimer of IKKγ

IKK activation is dependent on the presence of an intact IKKγ subunit.

Interesting results of IKKα and IKKβ knockout mice experiments

IKK cont.


Mechanism ikk l.jpg

RelA associated with either a dimer or trimer of IKKγ

RelA

RelA

p50

p50

p50

P

P

IkB

Mechanism: IKK

IKK

IkB

ikb

kB site


Dna binding l.jpg

NFkB dimers bind to kB sites in the DNA associated with either a dimer or trimer of IKKγ

consensus sequence 10 bp long

large number of different kB sites that display varying degrees of consensus

DNA binding


Dna binding cont l.jpg

Each dimer subunit contains two sets of β sheet immunoglobulin folds

N- terminal domain (NTD) contacts DNA

C- terminal domain (CTD) mediates dimerization and some DNA contacts.

NF-kB proteins use ten flexible loops extending from the secondary structure of these immunoglobulin folds to mediate DNA contacts

DNA binding cont.


Butterfly structure of nf b bound to dna l.jpg
Butterfly structure of NF-κB bound to DNA immunoglobulin folds

From Chen & Ghosh, 1999


Dna binding cont22 l.jpg

The long loop L1 reaches into the major groove of the DNA immunoglobulin folds

two arginine residues interact with conserved guanines

stabilized by hydrogen bonds from a glutamate to the cytosines

positively-charged arginine or lysine electrostatically interacts with the pyrimidines opposite of the guanine rich-strand.

DNA binding cont.


Dna binding cont23 l.jpg

Transcriptional activation is not biphasic – but it can be finely tuned based on 2 points of regulation:

Phosphorylation - phosphorylated p50 shows increased DNA binding

Variations in the primary sequence - substitutions can cause decreased binding to specific kB sites

DNA binding cont.


Mechanism dna binding l.jpg

RelA finely tuned based on 2 points of regulation:

RelA

RelA

RelA

p50

p50

p50

p50

P

P

IkB

Mechanism: DNA binding

IKK

IkB

ikb

kB site


Removal of nf b from nucleus l.jpg

Certain IkBs, such as IkBa contain both an NLS and a nuclear export sequence (NES)

Newly-synthesized IkBa enters the nucleus

Binds to NFkB dimers

Causes their exportin-mediated transport to the cytoplasm

Removal of NF-κB from nucleus


Mechanism removal of nf b l.jpg

RelA export sequence (NES)

RelA

RelA

RelA

p50

p50

p50

p50

P

P

IkB

Mechanism: Removal of NF-κB

IKK

IkB

IkB

IkB

ikb

kB site


Activators and target genes l.jpg

NFkB was initially thought to be a B-cell specific transcription factor

Activity could be induced in most cell types upon treatment with phorbol esters, the proinflammatory cytokines TNFα and IL-1, or bacterial LPS, dsRNA, certain viruses and ionizing radiation

Activators and Target Genes


Nf kb is central to the immune system l.jpg

Activated by wide variety of bacteria and bacterial products, viruses and their proteins

Many of the genes transcribed participate in the immune response (cytokines, chemokines, and receptors required for immune recognition, and for neutrophil adhesion and transmigration across blood vessel walls

NF-kB is “central to the immune system”


Nf kb induced by viruses l.jpg

Many viruses that induce NF-kB activity also have NFkB binding sites in their viral promoters

Selective advantage because as the immune system responds to the virus being present, the activation of NFkB leads to transcription of more virus.

Eg. HIV-1 has a kB site in its promoter region

NF-kB induced by viruses


Beyond the immediate immune response l.jpg

Involved in transcription of many genes whose functions extend beyond the immune response eg. acute phase proteins (angiotensinogen)

Therefore it more generally represents a regulator of stress response

Beyond the immediate immune response


Beyond immune response cont l.jpg

Induced during physiological stress extend beyond the immune response eg. acute phase proteins (angiotensinogen)

in ischemia/reperfusion, hemorrhagic shock

Physical stress

irradiation, oxidative stress

induces stress response genes such as iNOS and COX-2

Environmental stress

heavy metals, cigarette smoke, therapeutic drugs

Beyond immune response cont


Apoptosis l.jpg

Has been found that NFkB can induce both aptototic and anti-apoptotic effects in different cell types

Apoptotic: several stimuli lead both to NFkB activation and to apoptosis

this evidence is questionable because in RelA knockouts the apoptosis rate was higher

Antiapoptotic: several growth factors and mitogens stimulate or are induced by NFkB

Apoptosis


Apoptosis and stress response l.jpg

Makes sense to couple a stress responder to anti-apoptotic pathways - it responds to the stress while helping the cells survive through it.

Apoptosis and stress response


Specificity of the nf kb response l.jpg

Mostly based on which cell type is targeted pathways - it responds to the stress while helping the cells survive through it.

Combinatorial response of promoter/enhancer regions

usually need more than one transcription factor to induce transcription

Selective activation

different NFkB dimers have different preferences for different kB sites

Specificity of the NF-kB response


Inhibiting the nf kb response l.jpg

NFkB transcription factors regulate many important physiological processes

Have become sought-after molecular targets for pharmacologial intervention

Many modulators of this pathway and therefore many opportunities to disrupt it.

Inhibiting the NF-kB response


Inhibiting cont l.jpg

Inhibitors of the NFkB pathway include anti-oxidants, proteasome inhibitors, peptides, small molecules and dominant-negative or constitutively active polypeptides in the pathway

Several are general inhibitors of NFkB activation, where others inhibit specific pathways

Inhibiting cont.


Mechanism inhibitory points l.jpg

RelA proteasome inhibitors, peptides, small molecules and dominant-negative or constitutively active polypeptides in the pathway

RelA

RelA

RelA

p50

p50

p50

p50

P

P

IkB

Mechanism: Inhibitory points

IKK

IkB

IkB

IkB

ikb

kB site


Summary l.jpg

The NF-kB is an important pathway in regulating the stress response in the body

Work continues on manipulating the pathway for use in therapy

Summary


References l.jpg

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References


Suggested articles l.jpg

Boilean TW, Bray TM, Bomser JA. (2003). UV radiation modulates NF-kB activation in human lens epithelial cells. J. Biochem. Molec. Toxicol. 17(2):108-13.

Yoneda H. Miura K. Matsushima H. Sugi K. Murakami T. Ouchi K. Yamashita K. Itoh H. Nakazawa T. Suzuki M. Shirai M. 2003. Aspirin inhibits Chlamydia pneumoniae-induced NF-kappa B activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth. J. Med. Microbiol. 52(Pt 5):409-15.

Vasudevan KM, Gurumurthy S, Rangakar VM. (2004). Suppression of PTEN expression by NF-kB prevents apoptosis. Molec. Cell Biol. 24(3):1007-21.

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