p53 Flashcards
What kinds of stresses might lead to the activation of p53?
- DNA damage
- oncogene activation
- hypoxia
- oxidative stress
- increased E2F
What is the TP53 gene?
- encodes p53
- one of the most frequently mutated genes in cancer
- involved in apoptosis, senesence, DNA metabolism etc
- transcription, post transcriptional and post translational roles
How does p53 work in brief?
- in response to damage, p53 is stabilised
- can promote transcription of genes involved in cell cycle arrest + repair or apoptosis
- maintains cellular and genetic stability
What is the structure of the p53 protein?
- Transactivation domain binds MDM2 and other inhibitors and also DNA
- co-activators bind at the N terminus
- sequence-specific DNA binding domain is where 95% of cancer mutations occur
- oligermirisaion domain allows tetramer formation
- carboxy-regulatory domain is modified to activate p53
Under what conditions does p53 bind the DNA?
- works best as a tetramer
- specifically binds to consensus sequence
- two copies of the sequence with a spacer region between them of 0-21 bp long
- different orientations with different affinities
What kinds of post-translational modifications can occur to p53?
- many, mostly at the c-terminus
- phosphorylation and acetylation are key
What are the other p53 family members?
- p63 and p73
- some overlap in function and domains
- p63 more important in development than apoptosis
- p73 can induce cell cycle arrest and apoptosis AND important in development
- p73 could potentially be upregulated in lieu of p53
How is p53 activity regulated in normal cells?
- present in very low amounts due to its short half life
- activation at the level of the protein rather than the gene
- allows a rapid response by stopping p53 turnover and degradation and quickly turning it back on
How does Mdm2 regulate p53 levels in normal cells?
- keeps it low in the absense of DNA damage
- works in a heterodimer with mdm4
- ubiquitin ligase that binds p53 transactivation domain, blocking its binding to DNA and causing its acetylation and ubiquitination
- shuttles it to the cytoplasm where it is degraded
What else can Mdm2 do to p53?
- can enhance p53 translation under genotoxic stress
- context-dependent role
- not fully understoof
How does p53 recognise DNA damage?
- DNA damage activates ATM/ATR kinases
- these phosphorylate p53
- the activity of these kinases is directly dependent on the presence of DNA ends/damage
What happens when kinases phosphorylate p53?
- at the N terminus in or near the Mdm2 binding region
- drives off Mdm2 and stabilises p53
- Mdm2 phosphorylation may also occur
- phosphates can undo this sometime after the damage has occurred
What can phosphorylation of the c-terminus do to p53?
- further activation
- stimulates the sequence specific DNA binding activity of p53
- enhances its transcriptional activity
How might the cell respond to huge damage?
- phosphorylation on ser46 favours transactivation of pro-apoptotic genes like Noxa and PUMA
- skips cell cycle arrest and repair and goes straight to apoptosis
How can p53 be regulated by acetylation?
- co-activators such as p300 and CBP are acetyltransferases
- chromatin remodelling enhances trnascriptional activity
- acetylation of the C-terminus end increases stability and activates the protein
How can modifications play a role in p53 target preferences?
- K120 acetylation leads to p53 accumulation at proapoptotic genes like Bax and PUMA
- K320 acetylation favours apoptosis
- ubiquitination of K320 favours growth arrest
In what two ways can p53 arrest the cell cycle?
- directly by acting as a TF and swtitching on arrest genes
- indirectly by regulating genes without a consensus sequecne
What are some examples of indirect gene regulation by p53?
- repression of transcriptional activators
- interfering with the assembly of transcriptional machinery
- recruitment of repressors such as HDACs
What kinds of genes might p53 repress?
- growth promoting or anti-apoptotic genes
- c-myc, Bcl-2, cyclin A
How does p21 inhibit the cell cycle?
- p53 binds to p21 regulatory elements and increases its expression
- p21 binds to and inhibits cyclin CDKs
- prevents cell cycle progression
- can also inhibit DNA synthesis to allow repair
Other than p21, how else can p53 cause cell cycle arrest?
- upregulates Siah-1 which degrades B-catenin and reduces cyclin D1, haulting the cell cycle
- can also arrest cells in G2 by repressing cyclin B1 and stopping mitosis
- loss of p53 - loss of G1 arrest + attenuation of G2 arrest
What role does p53 play in DNA repair?
- increases DNA polymerase B activtiy
- cells without p53 are unable to efficiently repair DNA lesions
Which function of p53 appears to be more important in tumour suppression?
- mice with mutated p53 that can’t arrest the cell cycle but can induce apoptosis are abble to suppress cancers
- pro-apoptotic role more important?
What is the role of p53 in cellular senescense?
- induces senescence and prevents lesions from developing into malignant tumours
- inactivation of p53 prevents ras-induces senescence
- sustained p53 + p16 expression can lead to senescence
What is senescense?
permanent withdrawl from the cell cycle
How do cells make the choice between repair or die? (3)
- post-transcriptional modifications of p53 favour specific promoter binding
- different affinities to different response elements
- low p53 levels favour growth arrest and high or persisting levels override this and cause apoptosis
WHat is the ASPP family of proteins?
- apoptosis stimulating proteins of p53
- direct p53 towards proapoptotic genes
- upregulation of E2F or DNA damage activate ASPP 1+2
How are ASPP proteins regulated?
- turned on by DNA damage or E2F upregulation
- iASPP prevents ASPPs from binding to p53 and allow growth and proliferation
- ASPP 1+2 can also be silenced by methyatlion
How might p53 be inactivated in cancer? (4)
- missense p53 mutations seen in over hald of cancers
- mutations in upstream activators such as ATM
- increased levels of inhibitors such as MDM2 (sarcomas)
- interaction with oncogenic viral proteins that bind and inactivate p53
How might mutant p53 act? (3)
- not binding its consensus sequence
- not activating indirectly by promoters
- not suppressing promoter activity
What makes p53 mutations different to other oncogenes?
most oncogene mutations are frameshifts but not p53 are missense mutations
What kinds of mutations occur in the DNA binding domain of p53?
- contact mutations that prevent protein-DNA interaction
- conformation mutations that destabilise the core structure (more common)
Why does p53 usually have mutations that destabilise the structure?
- has a selective advantage
- only 1 copy needs to be mutated out of the 4 proteins in the tetramer to drive them all into mutant conformation and stop it binding DNA
What are gain of function p53 mutations?
- tumours may benefit from mutant p53 as they can do more
- mutations can regulate transcription on promoters without p53 binding sites
- tetramer may be locked in a mutant conformation
What is Li Fraumeni syntdrome?
weakly penetrant p53 mutations are inherited and lead to increased cancer predisposition
How does p14ARF stabilise p53 in response to stress?
- binds Mdm2 and blocks its ubiquitin ligase activity
- leads to stabilisation of p53
- E2F can activate p14ARF
- loss of ARF promoters or overexpression of repressors are associated with cancer development
How do E2F and p53 work together?
- E2F upregulation constitutes oncogenic stress
- upregulates expression fo proteins that stabilise/activate p53 such as ATM, ARF and ASPPs
How does PI3K signalling suppress p53 function?
- Akt phosphorylates Mdm2 and triggers its translocation to the nucleus where it binds p53
- PTEN inhibits PI3K signalling and restricts Mdm2 to the cytoplasm where it cant bind p53
How can viral oncoproteins interact with p53?
- have similar effects as mutations
- complex with the WT protein in tetramers and inactivate them
- adenovirus, HPV E6+E7
- viral oncoproteins bind both Rb and p53 to block their pathways to allow viral infected cells to continue proliferating
How are Rb and p53 important together in cancer?
- loss of Rb must be combined with apoptosis resistance to promote tumour development
- selective pressure to reduce the otherwise apoptotic consequences of regulated E2F
What are the main goals in targeting p53 for cancer therapy?
- reactivating WT functions
- stabilising p53
How might mutant WT functions be reactivated in p53?
- small molecule drugs
- can restore the WT conformation and sequence-specific binding
- can restore protein folding of mutated p53 into a formation more similar to WT
What is the target for increasing p53 stabilisation?
- targeting Mdm2
- Nutilin binds to Mdm2 and prevents its interaction with p53
- can activate p53 pathways in cancer cells
- small molecules can also inhibit Md2m
- has side effects in normal tissue
Why might it be appealing to inhibit p53 in cancer therapy?
- most cancer therapy side effects are cause by p53-mediated apoptosis
- could protect from lethal side effects
- could allow the use of higher doses in those less responsive to treatment
What is p53 gene therapy?
- retroviral vector containing WT-p53 gene and an actin promoter
- injected directly into tumours of lung cancer patients
- not yet approved
