Lecture 6 (p53) Flashcards
Inherited mutation of p53 leads to what cancer
Li Fraumeni syndrome which is a familial cancer.
p53 is a nuclear protein that normally exists in the cell as a?
Homotetramer which is an assembly of 4 identical polypeptide.
What does mutant p53 do
It acts in a dominant negative manner to ‘block’ the activities of wild type (WT) p53.
What happens when there is a mutant p53 allele and WT p53 allele
The function of p53 in cell is almost gone
What happens if the tetramer has a single mutant p53 subunit
It interfere with function of the tetramer as a whole.
DNA binding:
-Is done by p53 tetramers
-Is central to p53 function
Things that affect p53 expression and activity:
- Gene transcription
- Protein stability
- Post translational modification
- Location
- Structural interactions/oligomerization
How is p53 transcription increased:
Cellular stress such as DNA damage can increase p53 transcription
P53 becomes stable in response to:
DNA damage.
Post translational modification:
For full activity, phosphorylation at key serine residues is required.
Location:
For full activity, p53 needs to be in the nucleus.
Structural interactions/oligomerization:
For full activity, p53 needs to form a tetramer
3 ways p53 expression and activity is controlled
-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.
Evidence that PTM increases p53 protein levels:
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.
DNA double strand breaks lead to:
Telomere erosion.
DNA single-strand breaks lead to:
-Stalled replication forks
-Replication errors
-UV radiation
-Chemotherapeutic drugs
Double stranded breaks activate what kinases:
ATM kinase which activates CHK2 kinase
Single stranded breaks activate what kinases:
ATR kinase which activates CHK1
p53 as a transcriptional activator:
p53 regulates transcription positively by halting the cell cycle in response to DNA damage and attempts to aid in the repair process
p53 transcriptionally activates these targets:
-p21 (CDKI)
-GADD45 (repairs DNA damage)
-BAX, Bid, APAF1(pro-apoptosis)
-Thrombospondin-1 (anti-angiogenesis)
-MDM2(p53’s negative regulator)
Mutations of p53 typically happen where?
As an amino acid substitutions in the DNA-binding domain of p53.
What does p53 do when DNA damage is detected in G1:
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.
What is PCNA
-Stands for proliferating cell nuclear antigen
-It inhibits DNA replication
-p21 competes with DNA polymerase delta for PCNA
How does p53 target Cyclin B- CDK1
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
P53 is activated by phosphorylation in response to what signals:
-Oncogenic stress
Or
-Genotoxic stress
How does oncogenic stress activate p53
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
Genotoxic stress:
Example: UV or cytotoxic drugs
Leads to ATM activating CHK2 and ATR activating CHK1kinases to phosphorylate p53, therefore activating it.
How does p53 influence the extrinsic apoptosis pathway:
p53 increases transcription of Fas and enhances Fas transport to the cell surface
How does p53 influence the intrinsic apoptosis pathway:
-Induces BAX, PUMA and NOXA (pro-apoptotic proteins)
–Induces APAF-1 (apoptosome)
–Directly binds to inhibit BCL2 and BCL2-XL (anti apoptosis)
Mutated p53 leads to:
Deregulation of apoptosis pathways and increases tumorigenesis
Events that can disable p53:
-Nucleotide depletion
-UV
-Hypoxia
-Ionizing radiation
-Exposure to nitric oxide
-Chemotherapeutic drugs
What is PRIMA-1
-Stands for P53 Reactivation and Induction of Massive Apoptosis.
-A drug that induced apoptosis
-Also known as APR-246
Mutant p53 is targeted in what cancer
In triple negative breast cancer.
APR-246 is commonly used as treatment for it.
How does PRIMA/APR-246 work?
It binds and re-folds p53 into its wild-type (pre-mutation) conformation, therefore restoring its function.
Trial on APR-246 revealed that:
-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
