Week 7: P53

Question 1

P53 protein: domain structure

Answer 1

1. Transactivation domain: TAD
2. DNA binding domain: DBD (Missense mutations are clustered here)
3. Tetramerization domain: TET
4. N terminus: where regulation occurs
5. CTD: protein stability, DNA binding, cofactor recruitment (has many positive charges)

Question 2

Evidence that p53 is a tumour suppressor gene

Answer 2

1. There is complete loss of p53 gene function through recessive genetic mechanisms such as, Missense mutations accompanied by LOH (most common), Homozygous deletions & Compound heterozygous mutations
2. p53-null mice are susceptible to tumor development
3. Germ line p53 mutations are present in families with Li-Fraumeni syndrome

Question 3

Mutant p53 as a dominant negative

Answer 3

1. In mouse knock-ins, one p53 allelle can be changed to the mutant p53, this compromises the function of the wildtype.
2. In contrast if one p53 allele is deleted, the mouse retains wildtype p53 function.

Question 4

P53 mediates the response to many stress signals

Answer 4

1. Lack of nucleotides, UV radiation, ionizing radiation etc=p53 activation=cell cycle arrest, dna repair, apoptosis etc.

Question 5

Negative feedback loop of p53

Answer 5

1. Apoptotic signal
2. Activation of p14ARF which causes P-p53 to dissociate from mdm2 (its regulator/E3 ubiquitin ligase that degrades p53)
3. Activation of P-p53 induces transcription of a number of pro-apoptotic genes, including bax and fas & cell cycle arrest genes
4. P53 levels rise so it rebinds back to mdm2 to keep it low

Question 6

P53 roles

Answer 6

1. Apoptosis, cell cycle arrest (G1/G2), etc

Question 7

Morphological changes during apoptosis

Answer 7

1. membrane blebbing
2. chromatin condensation
3. nuclear fragmentation
4. formation of apoptotic bodies
5. shrinkage of cytoplasm
6. preservation of membrane integrity

Question 8

Intrinsic and extrinsic pathways of apoptosis

Answer 8

1. DNA damage=active p53=Apoptotic signals=intrinsic or extrinsic path
2. Both pathways activate initiator caspase protease cascade
   2a. Activates CAD=cuts DNA between nucleosomes
   2b. Activates BID=pro apoptosis
   2c. Activates Noxa and puma =inhibit pro survival (Bcl2) which therefore activates pro apoptosis (Bax/Bak)

Question 9

BH3 only protein sub family

Answer 9

1. BH3 only proteins are pro-Apoptotic (inhibit the anti-apoptotic Bcl2 & Bcl-XL=activation of the pro-apoptotic proteins Bax and Bak)
2. The balance between Bcl2+Bcl-XL vs. Bax+Bak activity is what determines apoptosis through permeabilization of the mitochondrial membrane.

Question 10

Intrinsic pathway: Mitochondrial regulation

Answer 10

1. P53 activation
2. Apoptotic signals
   2a. Bad: Bad active (not bound by its inhibitor bcl2), Insertion of Bax in the OMM stimulates PT pore formation, Cytochrome C release through the PT pore activates Apaf-1, which coordinates the formation of apoptosome
   2b. Bax: pro-Apoptotic proteins (BAD/BID) inhibit anti Apoptotic (Bcl-2); Bax is relocated from cytoplasm to mitochondria

Question 11

Cytosolic and mitochondrial p53 apoptotic pathways.

Answer 11

1. Nuclear p53 induces Puma expression, which in turn releases cytosolic p53 held inactive in the cytoplasm through binding to Bcl-XL
2. p53 induces Bax oligomerization and mitochondrial translocation.
3. p53 accumulation in the cytosol is a consequence of stable monoubiquitination and is the major source for mitochondrial p53.
4. In the mitochondria, p53 induces Bax and Bak oligomerization, antagonizes the Bcl-2 and Bcl-XL antiapoptotic effect, and forms a complex with cyclophilin D.
5. These changes result in marked disruption of mitochondrial membranes and subsequent release of both soluble and insoluble apoptogenic factors

Question 12

P53 family functions

Answer 12

1. the most important role of p63 and p73 being regulation of differentiation and development
2. Role of p63 in epidermal formation
3. Regulation of developmental sympathetic neuronal survival and apoptosis by the p53 family members
   3a. The p53 protein family has been implicated in the death signaling following NGF withdrawal

Question 13

Peto’s paradox

Answer 13

1. Elephants have 20 copies of the TP53 gene, which produces the cancer-fighting p53 protein, while humans only have one copy
2. Scientists are studying how elephants’ extra copies of TP53 can apply to human cancer treatment