P53 lecture Flashcards
(37 cards)
How was P53 discovered?
- used SV40 virus - cells transformed when injected with SV40
- SV40 viral antigen is called polyoma large T
- antibodies were made for polyoma large T and RA methionine was added to fibroblasts - P53 immunoprecipitated with largeT
does P53 have synergy with Ras?
No
what were the steps that showed that P53 does not have synergy with Ras?
- when oncogenic Ras was with a deletion mutant of P53 some transformation occurred
- When oncogenic ras was with P53 cloned from tissue culture cells there was even more foci/transformation - does ras have synergy with P53?
- was later found that this form of P53 in the cells was a mutant form of P53 (Val135)
- When oncogenic Ras was with WT P53 there was no transformation of cells
- this showed that P53 is a tumour suppressor gene and does not have synergy with ras
what type of gene is P53?
tumour suppressor gene
What staining can be used to visualise SV40 antigens?
peroxidase
what gene codes for the P53 protein?
TP53
How often is P53 mutated in human cancers?
P53 is frequently mutated in human cancers - almost all cancer
Why is P53 not a typical tumours suppressor gene?
- usually when TS genes are null it results in embryonic lethality ( as TS genes control/supress cell numbers)
- But when there is a P53 null mouse it develops into an adult mouse
- doesn’t obey knudsons 2 hit hypothesis
Why does P53 not follow knudsons 2 hit hypothesis rule for tumour suppressor genes?
-primary mouse fibroblasts which were examined have 3 sources of P53 (1 exongenous from virus, and 2 endogenous)
-yet when a mutant form of P53 is introduced (from virus) there is an effect despite there being 2 WT copies still
(usually all genes must be defective for a TS gene to have an effect)
-even heterozygous P53 has an effect
what happens to mice that are null for P53 on kaplan meier plot?
mice die within a year
what is the disease caused when mutant TP53 is carried in the germline?
Li-fraumeni syndrome
What is Li-Fraumeni syndrome?
A rare disorder which increases the risk of developing cancers especially in children and young adults - caused by mutated TP53 i the germline.
what experiment looked at the mystery of the disappearing protein?
fibroblasts were labelled with radioactive methionine for 1 hour
- cells were harvested at set time points
- immunoprecipitate P53 with pab421
- pulse chase experiment - add in non RA methionine - replaces RA methionine with normal methioine
- can work out how long protein survives for
what is the half like of P53 and what does this say about it?
half - life is 20/30 minutes
- P53 goes away over time, shows this is an unstable protein as it turns over very quickly
how many kDa is P53?
53 kDa
what experiment showed the mystery of the reappearing protein?
- infect cells with SV40 large T virus
- AB pab246 cannot recognise P53 in transformed cells
- ELISA and blot with pab246 and other monoclonal ABs- found there was exactly the same amount of P53 is normal and transofmred cells
- P53 has a different structure/ is a different epitope in transformed cells (this epitope cannot be detcetd by pab 246 but can be detected by other ABs, e.g. pab 421
what is the most frequent mutation type in P53?
missense mutation (single base change/ point mutation which changes the amino acid)
what kind of facotr can P53 act as and how was this shown?
- transcription facotr
- shown in yeast using the GAL4 promoter system
- fuse piece of P53 with GAL4
- more gene product when P53 fused with GAL4 - gene transcription increases by 20 fold
can P53 repress itslef?
YES
- P53 is a TF which is capable of repressing itself
- when P53 is truncated, its expression increases
what kind of structure is P53? (e.g. monomer, dimer?)
P53 is a tetramer
how was it sown that P53 is a tetramer?
It was shown that P53 is a tetramer by sucrose density centrifugation
- protein moves through sucrose and the way it moves is due to MW
- (has marker proteins that know the MW of)
- P53 is not behaving as a monomer it behaves as a ~200kDa protein - is behaving as a tetramer
why is the tetrameric stautus of P53 important?
as it has a dominant negative effecr
- all 4 WT subunits atre required for P53 to be functional
- 16 different combinations but only 1 is all 4 WT
- explains why when mutant P53 is intoduced it has a large effect
why are ABs to P53 (pabs) unusual?
they are conformation specific
- e.g. pab246 only binds WT conformation
- pab421 binds any isoform/ conformation
what did an experiment done by Hall et al in 1993 show?
P53 becomes stabilised in human skin when exposed to high levels of UV
- transcription increases when P53 levels are stabilised
