L6 - Transcriptional responses to stress & infection: The p53 & HIF pathways

Question 1

What are the HIF & p53 pathways?

Answer 1

Pathways are signalling transcription factor pathways that allow the cell to respond to environmental threats or damage

Question 2

What is hypoxia?

Answer 2

Hypoxia can be defined as a lowering of the O2 concentrations compared to that at sea level ± 20.9% O2

The lowering of O2 concentrations compared to the normal levels cells are exposed to

Hypoxia is the lowering of oxygen, not the absence of oxygen

Altitude is a cause of lowering oxygen concentrations

Question 3

Why is studying hypoxia important?

Answer 3

It is involved in both physiological & pathological processes

Question 4

Physiological processes of hypoxia

Answer 4

Hypoxia is a very important regulator of embryo development

One of the main physiological responses to hypoxia is to induce the growth of new blood vessels in order to increase the supply of oxygen to the tissue concerned

This is also a feature in diseases such as cancer

Question 5

Where is the hypoxia reaction required for adaptation?

Answer 5

High altitude living

Intense muscle exercise

Question 6

Why do athletes train at high altitudes?

Answer 6

To increase their RBC production & their vasculature

Question 7

Hypoxia in cancer

Answer 7

As solid tumours grow, the centre of the tumour starts to lose oxygen because they are not properly supplied by vasculature and so the centre of the tumour becomes hypoxic

The tumours hypoxic response is to introduce more vasculature and growth of capillaries so that the tumour continues to get the oxygen it needs to grow and the nutrients through the blood supply

Targeting the growth of vasculature is a strategy for cancer treatment

Question 8

How do cells react to low oxygen levels?

Answer 8

When cells are exposed to hypoxia, the aim is to restore homeostasis (through introduction of new vasculature) to regulate the survival of the tissues

Also, can aim to introduce cell death if the conditions are too extreme

Question 9

Examples of how cells respond to low oxygen levels

Answer 9

Regulation of gene expression – some genes are turned on to help with the situation and turned off as well - can be associated with chromatin structure changes

Try to conserve energy by the blockage of the translation of mRNA so there is no energy-intensive process

DNA replication is also blocked as again it is a very energy-intensive process in the cell

There is also a microRNA response

Question 10

What does HIF stand for?

Answer 10

Hypoxia inducible factor

Question 11

What is HIF?

Answer 11

A heterodimeric transcription factor: HIF-alpha & HIF-1-beta

There are a few different alpha subunits available for HIF

Depending on the situation, one or more of the alpha subunits will become activated during hypoxia and dimerise with HIF-1-beta to give the active HIF transcription factor

Question 12

What are the 3 types of HIF-alpha?

Answer 12

HIF-1α
Ubiquitously expressed in all tissues

HIF-2α
Similar to HIF-1α, expression restricted to certain tissues

HIF-3α
Expression restricted to certain tissues and lacks C-terminus transactivation domain.
Functions as a dominant negative inhibitor for HIF-1α and HIF-2α.
More recently has also been shown to activate a different set of genes in hypoxia

Question 13

What does the C-terminal transactivation domain (CTAD) in HIF1&2-alpha do?

Answer 13

A nuclear localisation sequence to get them to the nucleus

Question 14

What does the basic helix-loop-helix domain (bHLH) in HIF1&2-alpha do?

Answer 14

Mediates the dimerisation and DNA binding of the protein

Question 15

What does the oxygen dependent degradation domain (ODD) in HIF1&2-alpha do?

Answer 15

Mediates the process by which HIF is activated in response to hypoxia

Question 16

How is HIF regulated?

Answer 16

HIF is being continually turned over in the cell until it is exposed to hypoxia

HIF is usually being made continuously in cells and it can be induced by various stimuli such as inflammation

HIF can be regulated at the translational level

HIF controls many pathways

HIF also regulates its own expression

Question 17

How is HIF regulated under normoxia conditions?

Answer 17

Under normoxia conditions, proline hydroxylases (PHDs) act on the proline residues and hydroxylate them which allows binding of the VHL protein

VHL is a ubiquitin E3 ligase meaning that it adds the ubiquitin residues to HIF, marking it for destruction by the proteasome

Question 18

How if HIF regulated under hypoxia conditions?

Answer 18

In low oxygen conditions the PHDs are no longer able to function so there is no longer hydroxylation of HIF and the protein becomes stabilised.

Turnover mechanism (hydroxylation and degradation) is switched off

The HIF-1-alpha can now dimerise with the HIF-1-beta to form the active HIF transcription factor which can then bind to its target genes, interact with transcriptional coregulators & regulate the expression of target genes

Question 19

What is p53?

Answer 19

The tumour suppressor gene

‘The Guardian of the Genome’

p53 is one of the major proteins that regulates cancer cell development so helps stop the development of cancer in our bodies

Question 20

Structure of p53

Answer 20

p53 has the domain structure typical for transcription factors, with distinct DNA binding & multi-dimerisation domains

• Trans – transactivation domain (TAD)
• P – proline rich domain
• NLS – nuclear localisation sequence
• Tet – tetramerization domain

Question 21

What happens when p53 is mutated?

Answer 21

It is one of the main causes of cancer

For a cancer cell to develop it needs to find a way to inactivate p53

Question 22

How is p53 found in the cell?

Answer 22

p53 is made constitutively in the cell but it is bound to its inhibitor mdm2

In response to DNA damage, hypoxia and cell cycle abnormalities, mdm2 is inactivated and p53 becomes activated

Question 23

What happens when p53 is activated?

Answer 23

Can then induce cell cycle arrest which allows DNA repair to occur and when that repair is completed, p53 gets switched off and the cell can restart

Alternatively, p53 can induce apoptosis (programmed cell death) if the damage is too extreme

A consequence of this is that p53 promotes cellular and genetic stability.

Question 24

What is Mdm2?

Answer 24

E3 ubiquitin ligase

Question 25

What is the role of Mdm2?

Answer 25

To promote the ubiquitination of p53, leading to its degradation by the proteasome

This keeps p53 levels low in undamaged cells

Question 26

What happens when the cells exposed to DNA damage?

Answer 26

p53 and mdm2 are phosphorylated, this inhibits the interaction and p53 is stabilised and it can go and regulate gene expression

There is also a negative feedback loop here as p53 actually regulates mdm2 gene expression so this can limit the extent of p53 activation

Question 27

What does the over expression of Mdm2 lead to?

Answer 27

Cancer

Functionally inactivates the p53 pathway, preventing cell cycle arrest and the induction of apoptosis in response to oncogene activation or DNA damage

Question 28

What is the role of p14-ARF?

Answer 28

Another tumour suppressor whose expression is induced in cells by oncogenes

Another major route to activating p53 that does not necessarily involve DNA damage

Question 29

How does p14-ARF work?

Answer 29

It disrupts the interaction between the p53 tumour suppressor and Mdm2

ARF binds Mdm2 and inhibits its ubiquitin ligase activity

Inactivation of Mdm2 by ARF therefore results in increased levels of transcriptionally active p53

Question 30

Inhibition of p53 activity in cancer cells

Answer 30

Viral infection - cancer-causing viruses’ express proteins that can inhibit p53, often binding directly to p53

ARF protein can be inactivated or mutated

ATM can be mutated

Mdm2 can be over expressed

p53 mutated

Question 31

Mutations in p53

Answer 31

Occur at hotspots
Tend to cluster within the DNA binding domain of p53

Mutations inactivate p53 by inactivating its ability to bind DNA and consequently inactive its ability to regulate the gene expression program that helps the cell adapt to the challenge of coping with genomic instability

p53 becomes more of a tumour promoter

Question 32

What is Li-Fraumeni syndrome

Answer 32

A hereditary genetic condition – the cancer risk can be passed from generation to generation

This condition is most commonly caused by a mutation (alteration) in a gene called TP53, which is the genetic blueprint for a protein, p53
The mutation takes away the gene’s ability to function correctly

Patients inevitably develop cancer at an early age – indication of how good of a tumour suppressor p53 usually is because cancer is usually a disease of old age and even then not every member of the population gets it

Question 33

What is the 2 hit theory of cancer causation?

Answer 33

For the normal cell with two copies of each gene, to randomly obtain a mutation in one of these genes at just the right point, given how big the genome is compared to the size of each gene, at the cellular level, this is an incredibly rare event

For a normal cell, normally one rare event has to occur followed by another rare event to occur to inactivate both copies of the gene and so it is very unlikely, but possible

With patients that have inherited a mutation in one copy of a gene, then the first rare event is not needed, only the second one

Therefore, statistically it becomes more probable for the rare event

This is a process known as a ‘loss of heterozygosity’

Question 34

Common themes of the p53, HIF & NF-kB pathways?

Answer 34

Rapid activation because the transcription factor is already being made and is poised for activation

All involve regulation of proteolysis, which contributes to ability to be rapidly activated

Question 35

Key differences in the p53, HIF & NF-kB pathways?

Answer 35

NF-κB activation involves degradation of an inhibitor (IκBα)

HIF and p53 are themselves degraded continuously in unstimulated cells. Upon exposure to an activating stimulus they become stabilised