L5 - Transcriptional responses to stress & infection: the NF-kB pathway Flashcards by Phoebe Snow

Environmental challenges to DNA

DNA damage
Infection
Hypoxia
Physical stress

Every cell needs to be able to adapt to environmental changes

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What is NF-kB?

Nuclear Factor of the kappa Immunoglobulin light chain in B cells

NF-kappaB NOT NF-kappa-beta
B is for B cells

Protein complex that binds to small DNA sequences in the DNA enhancer region regulating kappa immunoglobulin expression

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The NF-kB transcription factor pathway

Allows the cell/organism to respond to external challenges or threats

By regulating the expression of a wide range of target genes, it helps programme the response to these, either allowing the cell/organism to survive & recover or sometimes inducing death

Role of NF-kB will vary depending on the context

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The mammalian NF-kB family

It is a family of proteins/genes – 5 members in mammals

NF-kB forms dimers to bind DNA – buttefly structure

All have an amino-terminal Rel Homology Domain (RHD) encodes the DNA binding & dimerization functions of NF-kB

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What are the 5 members of the NF-kB family?

p65 (RelA)
RelB
c-Rel
p105/p50 
p100/p52

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What do RelA, RelB & c-Rel do?

All have a transcriptional activation domain in the carboxyl terminal that enables them to act as potent regulators of transcription

These domains are all different to each other – heterogeneous & fulfil different functions

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What do p105 & p100 do?

Are precursor proteins

Synthesised as longer proteins which are then proteolytically cleaved to generate either p50 or p52

C-terminal of p105 & p100 contains inhibitory motifs that inhibit the protein – ankyrin repeats

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What are p50 & p52?

The active DNA binding forms of these proteins (p105 & p100)

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What is the shorter version of p105?

p50

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What is the shorter version of p100?

p52

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What are the non-conserved transcriptional activation domains in NF-kB?

TA1/TA2
TAD
SD1
SD2

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What is the LZ domain in NF-kB?

Leucine zipper like domain

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The Ubiquitin-Proteasome pathway (UPP)

Ubiquitination has the ability to degrade proteins

Protein gets a chain of ubiquitin molecules – an E3 ubiquitin ligase is a protein that facilitates the attachment of ubiquitin chains to a target protein

Protein targeted for destruction by the 26S proteasome – chews up the protein that is tagged by the ubiquitin chain

Ubiquitin is then recycled

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How is NF-kB found in the cell?

Held in an inactive form in the cytoplasm bound to an inhibitory protein – IkB

Stimulus can be a wide range of things

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Example of NF-kB activation with TNF-alpha

TNF binds to cell surface receptor
Leads to activation of complex signalling cascade
Results in activation of IkB kinase complex (IKK)
The complex phosphorylates IkB protein which acts as the tag for ubiquitination
Protein is then degraded by proteasome
This frees NF-kB where it can bind to its target genes in promoters & enhancers of the genes it regulates

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What is TNF-alpha?

A potent cytokine that drives the inflammatory process

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The mammalian IkB family

Consists of: 
• IkB-alpha 
• IkB-beta 
• IkB-epsilon 
• Bcl-3

Different IkB proteins can respond in different ways to different types of stimul

All have highly conserved phosphorylation sites – tag that lead to ubiquitination

Highly conserved lysine residues – ubiquitin attaches to these to start the chain

PEST domain involved in the degradation of these proteins

What is the signature motif of IkB proteins?

The ankyrin repeats (ANK) in their C termini

Ankyrin repeats mediate interaction between IkB & NFkB complex – twists & changes the conformation of the NFkB complex – prevents it binding DNA

Looks like a stacked alpha helical structure

The mammalian IkB kinase (IKK) family

Has 3 core subunits

2 catalytic ones - IKK-alpha & IKK-beta
1 regulatory subunit - NEMO

What does NEMO stand for?

NF-kB essential modifier / IKK-gamma

What does NEMO do?

Mediates signals that come from upstream that allow activation of the IKK complex

What are the 2 pathways that activate NF-kB?

Classical / Canonical pathway

Alternative / Non-Canonical pathway

Classical / Canonical pathway of NF-kB

Most commonly used pathway

IKK-beta is the kinase that phosphorylates

IKK-alpha & leads to its degradation

Most common dimer activated: p50/RelA heterodimer

Alternative / Non-Canonical pathway of NF-kB

Driven by the IKK-alpha subunit instead

Has an upstream kinase, NK, that regulates it

Most common dimer activated: p52/RelB complex

How does the cell get a rapid response?

Premade protein complexes helps to speed the process up

What is NF-kB induced by?

* Inflammatory cytokines * Bacterial products * Viral proteins & infection * DNA damage * Cell stress

What does NF-kB regulate?

* Immune responses * Stress responses * Cell survival & cell death * Cell adhesion * Proliferation This is accomplished through the regulation of 100’s of NF-kB gene targets – but not all at the same time, in the same way in all cell types

What is the central mediator of NF-kB?

IKK Allows the pathway to function

How can you see the effects of the different genes in the NF-kB pathway?

By looking at the different phenotypes displayed from doing gene knockouts in mice * Apoptosis * Immunity * Inflammation * Neurological effects

How do we regulate the response of NF-kB?

Multiple levels of regulation combine to give transcriptional specificity Each of these steps can be regulated by other signalling pathways

What does a dimer do for you?

A multigene family composed of dimers allows the formation of many different types of NF-kB, with different properties & functions Different subunits have subtly different DNA binding specificity & so can target different genes & position themselves on DNA However, many NF-kB target genes are shared between subunits Classical NF-kB binding site: P50/RelA dimer

Why is it important that NF-kB has an imperfect palindrome?

This orientates NF-kB on the DNA sequence

What can NF-kB modifications regulate?

* Nuclear import * Stability * DNA binding * Transcriptional activity * Target gene specificity * Interactions with proteins

Is NF-kB regulation ended once it enters the nucleus?

No Can then be programmed by modifications to the kB subunit to respond in a certain way Depending on the stimulation you can get different modifications – some repress & some activate

Different chromatin states at NF-kB-dependent genes

Some genes are already in an open conformation Some genes are blocked by nucleosomes

What happens if NF-kB-dependent genes are in an open conformation?

Easy for NF-kB to regulate gene expression

What happens if NF-kB-dependent genes are blocked by nucleosomes?

NF-kB cannot get to the regulatory regions – need to have chromatin remodelling enzymes to open up the chromatin to allow NF-kB activation SWI/SNF is an ATP dependent chromatin remodeller – can remove nucleosomes Allows further control of the timing of NF-kB genes

Function of beta interferon

Viral infection results in the production of alpha & beta interferon They bind to specific cell surface receptors which results in a signalling cascade leading to the activation of over 50 anti-viral genes This response needs to be tightly controlled – only want to activate a viral response when actually infected with a virus, not in response to some other immunological change

What role does the organisation of the beta interferon promoter play

If the individual transcription factor binding sites in the beta interferon enhancer are taken out of context & multidimerised they can act as a viral inducible promoter – but they have varying basal levels of activity & can respond to other inducers – not good The key therefore is in the combination & organisation of the enhancer – need the binding to happen in the correct orientation & spacing of the enhancer DNA acts as a scaffold to hold the transcription factor complex in place on the promoter of the DNA IRFs = interferon response factors

What is the role of activators on the beta interferon?

The transcription factors binding the beta interferon all interact to form the enhanceosome complex This complex forms an ‘interaction interface’ to allow the high affinity recruitment of transcriptional co-activators such as p300/CBP that activate the beta interferon gene – NF-kB cannot do this by itself

What is the role of NF-kB in beta interferon?

Its function is essential too It participates in a combination lock Only the p50/RelA(p65) complex works at the beta interferon enhancer

What can NF-kB do after chromatin remodelling?

Can directly induce transcription by helping to recruit the BTM (basal transcription machinery)

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L5 - Transcriptional responses to stress & infection: the NF-kB pathway Flashcards

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