Lecture 6 (p53)

Question 1

Inherited mutation of p53 leads to what cancer

Answer 1

Li Fraumeni syndrome which is a familial cancer.

Question 2

p53 is a nuclear protein that normally exists in the cell as a?

Answer 2

Homotetramer which is an assembly of 4 identical polypeptide.

Question 3

What does mutant p53 do

Answer 3

It acts in a dominant negative manner to ‘block’ the activities of wild type (WT) p53.

Question 4

What happens when there is a mutant p53 allele and WT p53 allele

Answer 4

The function of p53 in cell is almost gone

Question 5

What happens if the tetramer has a single mutant p53 subunit

Answer 5

It interfere with function of the tetramer as a whole.

Question 6

DNA binding:

Answer 6

-Is done by p53 tetramers
-Is central to p53 function

Question 7

Things that affect p53 expression and activity:

Answer 7

1. Gene transcription
2. Protein stability
3. Post translational modification
4. Location
5. Structural interactions/oligomerization

Question 8

How is p53 transcription increased:

Answer 8

Cellular stress such as DNA damage can increase p53 transcription

Question 9

P53 becomes stable in response to:

Answer 9

DNA damage.

Question 10

Post translational modification:

Answer 10

For full activity, phosphorylation at key serine residues is required.

Question 11

Location:

Answer 11

For full activity, p53 needs to be in the nucleus.

Question 12

Structural interactions/oligomerization:

Answer 12

For full activity, p53 needs to form a tetramer

Question 13

3 ways p53 expression and activity is controlled

Answer 13

-DNA damage causes levels of p53 protein to increase.
-The ubiquitin ligase MDM2 inactivates p53 when bound to it.
-Post translational modifications stabilizes p53 protein.

Question 14

Evidence that PTM increases p53 protein levels:

Answer 14

A 8hr post-radiation Western blot was done. It revealed that PTM events meditated by several kinases stabilizes p53 and inhibits MDM2 binding to p53. These kinases become activated following DNA damage.

Question 15

DNA double strand breaks lead to:

Answer 15

Telomere erosion.

Question 16

DNA single-strand breaks lead to:

Answer 16

-Stalled replication forks
-Replication errors
-UV radiation
-Chemotherapeutic drugs

Question 17

Double stranded breaks activate what kinases:

Answer 17

ATM kinase which activates CHK2 kinase

Question 18

Single stranded breaks activate what kinases:

Answer 18

ATR kinase which activates CHK1

Question 19

p53 as a transcriptional activator:

Answer 19

p53 regulates transcription positively by halting the cell cycle in response to DNA damage and attempts to aid in the repair process

Question 20

p53 transcriptionally activates these targets:

Answer 20

-p21 (CDKI)
-GADD45 (repairs DNA damage)
-BAX, Bid, APAF1(pro-apoptosis)
-Thrombospondin-1 (anti-angiogenesis)
-MDM2(p53’s negative regulator)

Question 21

Mutations of p53 typically happen where?

Answer 21

As an amino acid substitutions in the DNA-binding domain of p53.

Question 22

What does p53 do when DNA damage is detected in G1:

Answer 22

p53 will halt cell cycle progression by increasing CDKI p21 levels. p21 inhibits CDK2-cyclin E activity which results in RB hypophosphorylation and E2F will not be released. So no S-phase genes will be made.

Question 23

What is PCNA

Answer 23

-Stands for proliferating cell nuclear antigen
-It inhibits DNA replication
-p21 competes with DNA polymerase delta for PCNA

Question 24

How does p53 target Cyclin B- CDK1

Answer 24

3 ways:
1. It can target the 14-3-3 sigma protein which sequesters cyclinB-CDK1 complex in the cytoplasm, prevents it from moving into nucleus which prevents Mitosis until DNA repair has been successful.
2. Targets GADD45 which binds to and dissociates cyclin B-CDK1 kinases
3. Increases p21 which inhibits B/CDK1

Question 25

P53 is activated by phosphorylation in response to what signals:

Answer 25

-Oncogenic stress
Or
-Genotoxic stress

Question 26

How does oncogenic stress activate p53

Answer 26

Example of oncogenic stress: mutated Ras or Myc
-Leads to increase in Arf which inhibits binding of MDM2 to p53. Causes p53 to be active

Question 27

Genotoxic stress:

Answer 27

Example: UV or cytotoxic drugs
Leads to ATM activating CHK2 and ATR activating CHK1kinases to phosphorylate p53, therefore activating it.

Question 28

How does p53 influence the extrinsic apoptosis pathway:

Answer 28

p53 increases transcription of Fas and enhances Fas transport to the cell surface

Question 29

How does p53 influence the intrinsic apoptosis pathway:

Answer 29

-Induces BAX, PUMA and NOXA (pro-apoptotic proteins)
–Induces APAF-1 (apoptosome)
–Directly binds to inhibit BCL2 and BCL2-XL (anti apoptosis)

Question 30

Mutated p53 leads to:

Answer 30

Deregulation of apoptosis pathways and increases tumorigenesis

Question 31

Events that can disable p53:

Answer 31

-Nucleotide depletion
-UV
-Hypoxia
-Ionizing radiation
-Exposure to nitric oxide
-Chemotherapeutic drugs

Question 32

What is PRIMA-1

Answer 32

-Stands for P53 Reactivation and Induction of Massive Apoptosis.
-A drug that induced apoptosis
-Also known as APR-246

Question 33

Mutant p53 is targeted in what cancer

Answer 33

In triple negative breast cancer.
APR-246 is commonly used as treatment for it.

Question 33

How does PRIMA/APR-246 work?

Answer 33

It binds and re-folds p53 into its wild-type (pre-mutation) conformation, therefore restoring its function.

Question 34

Trial on APR-246 revealed that:

Answer 34

-Its anti-proliferative activity was more potent in p53 mutants then WT
-It inhibited cell migration in mutated cells
-p53 proteins levels can be a biomarker for response to APR-246