lecture 5

Question 1

What is the purpose of a sensor in a signalling pathway?

Answer 1

to detect any DNA damage (p53), infection (NF-κB), hypoxia (HIF), physical stress

Question 2

Give examples of effects in response to a signalling pathway?

Answer 2

gene expression, repair, programmed death, immune response

Question 3

What is NF-κB?

Answer 3

• NF-κB is a nuclear factor of the kappa immunoglobulin light chain in B cells
• A transcription factor which is activated in stress, infection, radiation, etc.
• It increases transcription of genes involved in inflammation and immune response

Question 4

What is the purpose of the NK-κB pathway/

Answer 4

By regulating the expression of many target genes it helps programme the response to these either allowing the cell/organism to survive or induce death

Question 5

What controls the functions of NF-κB?

Answer 5

The Rel Homology Domain encodes the DNA binding and dimerisation functions of NF-κB

Question 6

What are the different types of NF-κB?

Answer 6

• p50 and p52 processed from p105 and p100
• TA1/TA2, TAD, SB1, SDII - non-transcriptional activation domains

Question 7

Describe the structure and function of p100 and p105

Answer 7

they contain ankyrin repeats in their C-termini that allow them to function and IκB-like inhibitors

Question 8

What did NF-κB evolve from?

Answer 8

primitive eukaryotes

Question 9

What is E3 Ubiquitin ligase?

Answer 9

a protein that facilitates the attachment of ubiquitin chains to a target protein to detect and degrade proteins

Question 10

Describe the ubiquitin-proteasome pathway

Answer 10

1) ATP dependent proteins degrade intracellular proteins bound to Ub - a chain of 5 Ub molecules attached to the protein substrate is enough to be recognised by the 26S proteasome
2) Ubiquitin acts as a tag and attaches to protein (via ubiquitin ligases) destining them for degradation by proteasome to form amino acids

Question 11

What is NF-κB induced by?

Answer 11

inflammatory cytokines, bacterial products, viral proteins & infection, DNA damage, cell stress

Question 12

What does NF-κB regulate?

Answer 12

the immune and inflammatory response, stress responses, cell proliferation survival, cell death, cell adhesion, tumour promotion and metastasis, angiogenesis

Question 13

Describe the process of NF-κB activation

Answer 13

1) Adapter proteins bind to the ligand bound receptor complex (eg: TNFα) which recruits and activates IKK Kinase
2) IκB is phosphorylated by the IκB kinase complex (IKK), which leads to ubiquitin-mediated degradation of IκB by the 26S proteasome
3) NF-κB is released from the cytoplasmic inhibitory complex, further activated by post-translational modifications (PTMs)
4) It translocates into the nucleus where it binds as a dimer to κB sites at target gene and induces transcription through the recruitment of co-activators and co-repressors

Question 14

What are the three core subunits of the IκB kinase (IKK) complex?

Answer 14

NEMO (also known as IKKγ), IKKα, IKKβ

Question 15

What is the structure of the IKK complex?

Answer 15

a long coiled dimer of NEMO bound by a molecule each of IKKα and IKKβ

Question 16

Which stimuli is IκB-α most common in?

Answer 16

inflammatory

Question 17

What are the inhibitors of the NF-κB family?

Answer 17

IκB-α, IκB-β, IκB-ε and Bcl-3

Question 18

What makes up the IκB proteins?

Answer 18

anykrin repeat motifs (ANK) and their C-termini, PEST, domain rich in proline, glutamate, serine and threonine

Question 19

What is the non-canonical (alternative) pathway of NF-κB activation?

Answer 19

TNFR activates NIK4 inducing receptors
NF-κB-inducing kinase (NIK) activates IKK-α, which phosphorylates NFKB2
This results in proteasomal removal of an inhibitory C-terminal IkB-δ domain, generating the p52 subunit, which leads to accumulation of p52/RelB heterodimers in the nucleus.

Question 20

What makes up the majority of a diseased NF-κB cell?

Answer 20

nucleus

Question 21

How is NF-κB regulated?

Answer 21

DNA binding and gaining access to the promotor/enhances

Question 22

What is Swi/Snf?

Answer 22

an ATP dependent chromatin remodeller that can move/remove nucleosomes

Question 23

What are the different chromatin states at stim/unstim NF-κB-dependent genes?

Answer 23

unstimulated cells - wound or unwound histones
stimulated cells - chromatin remodellers (HATs, Swi, Snf) recruited

Question 24

How did NF-κB regulated genes react differently to COVID-19 to normal respiratory viruses?

Answer 24

inflammation, immune cell rectruitmeny by COVID = CXCLs, IL-6, IL-1 and Normal = chemokines
Covid = low, ISGs, limited antiviral state, IFNβ, IFNλ
Normal = high ISGs, potent antiviral state, IFNβ, IFNλ

Question 25

What is the function of IFNβ?

Answer 25

Produced in response to viral infections Binds to specific cell surface receptors to trigger signalling cascade leaving to activation of antiviral genes

Question 26

Why does IFNβ need to be tightly controlled?

Answer 26

so a viral response is activated only when infected not for other immunological challenges

Question 27

What role does the IFNβ promotor play?

Answer 27

if transcription factor binding sites of IFNβ enhancer are multimerised they can act as a viral inducible promotor - but they have varying basal levels of activity and can respond to other inducers which isn't needed

Question 28

What is the coactivator interaction interface?

Answer 28

the transcription factors binding the IFNβ enhancer interact to form this complex Allows high affinity recruitment of transcriptional activators such as p300/CBP which allow binding of more than one transcription factor at a time

Question 29

What NF-κB complex works at the IFN-β enhancer?

Answer 29

p50/ReIA(p65)