L5/6 NF-kB, P53 and HIF pathways

Question 1

What do the NF-kB, p53 and HIF pathways all do for the cell?

Answer 1

They allow the cell to respond to environmental stress

Question 2

The NF-kB Family is made up of 5 family members. They all have a Rel Homology Domain (RHD). What does the RHD encode?

Answer 2

DNA binding and dimerisation

Question 2

What are the 5 family members of NF-kB?

Answer 2

• RelA (p65)
• RelB
• c-Rel
• p105 which gets cleaved to p50
• p100 which gets cleaved to p52

Question 3

What do p105 and p100 (NF-kB) both contain which allow them to function as IkB-like inhibitors? Also, what else involved in the NF-kB signalling contains this?

Answer 3

• Ankyrin
• IkB inhibitors also contain ankyrin

Question 4

What is NF-kB induced by?

Answer 4

Inflammatory cytokines
Bacterial products
viral proteins and infections
DNA-damage
Cell stress

Question 5

Describe the pathway of NF-kB activation.

Answer 5

1. Ligand binds to receptor eg TNF-a which indicates cell stress.
2. This activates IkB kinase (IKK) which phosphorylates the inhibitor protein IkB.
3. Phosphorylation results in ubiquitination of IkB and subsequent degradation.
4. NF-kB components form homo or heterodimers then it translocates to nucleus

Question 6

IkB is the inhibitor protein which keeps NF-kB in its inactive form. IKK phosphorylates IkB, causing NF-kB to become free in times of cell stress. What makes up the IkB kinase (IKK) complex?

Answer 6

NEMO - IKK gamma.
IKKa and IKKb - catalytic subunits - these contain the kinase domain.

Question 7

Canonical and non-canonical are two pathways for NF-kB. What does each pathway involve?

Answer 7

1. Canonical - Activated by TNF, IL-1 and LPS, this pathway involves mainly the IKKb component which mediates the activation of p50 and RelA.
2. Non-canonical - activated by LPS, CD40, lymphotoxin receptors. This pathway involves mainly IKKa component. Mediates phosphorylation of p100 to p52.

Question 8

Different dimers of NF-kB components can influence which genes are regulated. What is an example of a dimer?

Answer 8

p50/RelA heterodimer binds DNA with a distinct orientation.

Question 9

Once NF-kB is in the nucleus, certain things can happen to it to alter gene expression. What can happen?

Answer 9

Chromatin remodelling - HATs and Swi-snf

Example - Phosphorylation of RelA/p50 allows it to bind to p300/CBP. This is a transcriptional coactivator and is a histone acetyl transferase which causes transcriptional activation.

Question 10

What is the beta interferon enhancer and enhancesome?

Answer 10

Beta interferon enhancer contains precisely arranged transcription factor binding sites, one for NF-kB. When the transcription factors are activated eg by cell stress or viral infection, beta interferon expression occurs. When the Transcription factors interact with the enhancer the enhancesome is created. This acts as a landing pad for transcriptional regulators (eg p300/CBP which is a transcriptional activator). As a result of this binding, beta interferon gene expression occurs, which results in the direct recruitment of basal transcription machinery.

Question 11

Hypoxia is the lowering of O2 levels. What is the normal oxygen % at sea level?

Answer 11

20.9%

Question 12

HIF is hypoxia inducible factor. When O2 levels decrease (hypoxia), HIF which is a transcription factor is activated and gene expression changes. What happens to HIF1-alpha when O2 levels are normal, and when they drop?

Answer 12

HIF alphas have an O2 dependent degradation domain. When O2 levels are normal, Proline hydroxylases (PHDs) and FIH (Factor inhibiting HIF - an asparagine hydroxylase) use O2 to hydryoxylate residues within the HIF-alpha subunit. Hydroxylation of ODD signals for VHL (Von hippel lindau - a ubiquitin E3 ligase) which leads to proteosomal degradation of the HIF.

When o2 levels drop, PHDs and FIHs are inhibited, meaning no hydroxylation occurs. This means HIF1-alpha is stabilised. it then binds with HIF2-alpha and target gene expression is activated.

Question 13

When O2 levels are normal, the O2 dependent degradation domain gets hydroxylated. What amino acid specifically gets hydroxylated here?

Answer 13

Proline to hydroxyproline

Question 14

p53 responds to things like DNA damage, Cell cycle abnormalities and hypoxia by inducing either cell cycle arrest to allow the cell to repair and survive, or apoptosis. What happens to p53 when a negative stimulus occurs?

Answer 14

In a normal cell, p53 is bound to its negative inhibitor Mdm2, which inactivates p53.

When there are cell stresses, p53 dissociates from Mdm2 and becomes activated and can now do its job.

Question 15

What is the role of Mdm2?

Answer 15

Acts as a E3 ubiquitin ligase which causes ubiquitination p53 by the proteasome in a normal cell. This keeps the levels of p53 low in undamaged cells.

In response to DNA damage, p53 and Mdm2 are phosphorylated at serine 15 by ATM or ATR kinases. The phosphorlaytion causes p53 to split from Mdm2.

Question 16

What can happen if Mdm2 is over expressed?

Answer 16

p53 is always inactivated which prevents cell cycle arrest and apoptosis, which leads to cancer.

Question 17

p14ARF is another tumour suppressor that is stimulated by oncogenes (cancer genes). What is its role?

Answer 17

It disrupts the interaction of p53 with Mdm2, and binds to Mdm2, inhibiting it E3 ubiquitin ligase activity, which would normally degrade p53. This means that p53 can become active and leading to tumour suppression.

Question 18

What is Li-Fraumeni Syndrome?

Answer 18

hereditary genetic condition where there is a mutation in the TP53 gene which is linked to p53. The mutation takes away the proper function of the gene, which leads to cancer.

Question 19

What are the key differences with degradation in NF-kB, p53 and HIF pathways?

Answer 19

NF-kB involves degradation of the inhibitor IkB to activate it.

P53 and HIF are continuously degraded in unstimulated cells but are not degraded in damaged cells.

They all involve E3 ubiquitin ligase.