p53 guardian or the genome

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p53 - guardian or the genome

• guardian of the tissuse

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p53

• product of a tumour supressor gene
• the most frequently mutated gene in human cancers
• 393 aa with 4-5 functional domains
• biological role as watch dog - vaktbikkje
• Guardian of the genome - stops the cell cycle
  upon DNA-damage
• Signalling pathway DNA-damage ? enhanced p53 ?
  activation of CDKI p21 ? G1 arrest ? activation
  of GADD45 ? stimulated DNA-repair
• Guardian of the tissue - facilitates apoptosis if
  necessary
• Signalling pathway DNA-damage ? enhanced p53 ?
  apoptosis

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p53 mutations
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p53 - protein domains
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p53 domains
C-terminal allosteric domain
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5 distinct domains in p531. TAD

• TAD N-terminal aa1-42
• aa 13-23 conserved between species
• F19, L22 and W23 necessary for transactivation in
  vivo
• F19, L22 and W23 involved in binding to TAFII70
  and TAFII31
• TAD negatively regulated through interaction with
  the MDM2 factor or the E1B-55Kd protein
• Structure of the MDM2 N-terminal domain 13-29
  peptid from p53
• MDM2 deep hydrophobic pocket
• p53 peptide amphipatic helix fitting in the
  pocket
• F19, L22 and W23 involved in binding

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Mdm2 - p53
p53-TAD
Mdm2
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p53 domains
C-terminal allosteric domain
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5 distinct domains in p532. Pro-rich domain

• Et Pro-rich region between TAD and DBD
• PxxP present 5 locations in the region 61-94
• deletion of P- rich region ? reduced
  apoptosis-response and reduced cell cycle arrest,
  but normal transcriptional response
• contains residues that become phosphorylated upon
  apoptotic response (HIPK2 phosphorylation of S46)

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p53 domains
C-terminal allosteric domain
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p53 DBD
2 ?-helical loops that contact DNA Zn
structuring CHCC-Zn Two large loops (L2
and L3) involved in minor groove contact
Scaffold ?-sandwich (two antiparalel ?-sheets)
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5 distinct domains in p533. DBD

• DBD centrally located aa102-292 folded into a
  loop-sheet-helix motif (LSH)
• protease-resistent, independent, Zn-containing
  domain CHCC-Zn
• scaffold 4- and 5-thread antiparallel ?-sheet
  structure
• 2 protruding ?-helical loops contacting DNA
  directly
• Specific base contact in major groove (K120,
  C277, R280)
• Two large loops (L2 L3) involved in minor
  groove contact (contact involve R248)
• Several H-bond contacts with sugar-phosphate-chain
  (R273)
• Two types of hotspot-mutants in human cancers
• disrupts direct DNA-interactions (R248, R273)
• disrupts the structure of DBD

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DBD mutations

• Most of the p53 mutations that cause cancer are
  found in the DNA-binding domain
• most common mutation changes arginine 248 (red),
  snaking into the minor groove of the DNA - a
  strong stabilizing interaction.
• Other key sites of mutation are shown in pink,
  including arginine residues 175, 249, 273 and
  282, and glycine 245. Some of these contact the
  DNA directly, and others are involved in
  positioning other DNA-binding amino acids.

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5 distinct domains in p533. DBD

• binds DNA as tetramer (dimer of dimer)
• DNA recognition sequence reflects this 4x RRRCW
  arranged like this

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p53 domains
C-terminal allosteric domain
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5 distinct domains in p534. Tetramerization

• Tetramerization domain aa 324-355
• 2? 2? structure
• forms tetramers
• linked with DBD via 37aa flexible linker aa
  287-323

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p53 domains
C-terminal allosteric domain
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5 distinct domains in p535. C-terminal
allosteric domain

• DNA/RNA-binding C-terminal (last 26aa)
• open protease-sensitive domain
• Basic region
• binds DNA and RNA non-specifically and can
  stimulate annealing
• binds DNA ends, internal loops or other loose
  ends from damaged duplexes
• possible function (allo)steric regulator of
  specific DNA-binding
• p53 appears to be present in a latent form
  inactive in seq.spec. DNA-binding
• Several events in the C-terminal can reactivate
  p53s central DBD
• deletion of basic C-terminal
• phosphorylation of S378 with PKC
• phosphorylation of S392 with CK2
• binding of C-terminal antibody PAb421
• small singlestranded DNA oligos

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Activation of p53- upstream inputs
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Upstream and downstream
p53 functions as sensor of upstream signals
reflecting DNA-damage /cellular stress
Upstream
activation
Downstream
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Activation of p53 - what happens?

• DNA-damage/stress ?
• 1. activation of latent p53 latent form ? active
  form
• enhanced DNA-binding activity
• probably also enhanced transactivation activity
• post-translational modifications
• 2. stabillization and a rapid increase in protein
  level? activation of response
• activation ? level increases 10-100x
• Since enhanced levels of p53 may lead to cell
  cycle-arrest and apoptosis, it is of critical
  importance that normal cells keep their p53
  levels low

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Activated by several signals

• types of activating stress
• DNA-damage (chain breaks, repair-intermediates,
  recombination-intermediates)
• Hypoxia
• protective function in tumours (tumour growth ?
  limited blood supply ? hypoxia ? p53 activation ?
  apoptosis of tumour)
• trombospondin appears to be p53 regulated, acts
  antiangiogenic, will reduce blood supply further
• NTP pool reduced
• sufficient NTP-pool for DNA-replication sensed by
  p53
• Activated oncogenes (Myc, Ras, E1A, ß-catenin)
• Foster defects

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The key to stabillization the MDM2-p53 coupling

• MDM2 associates with p53s TAD (aa 17-27)
• MDM2-binding leads to
• 1. Repression of transactivation
• 2. Destabillization of p53 since MDM2-binding
  stimulates degradation of p53
• mdm2 knock-out lethal, rescued by simultaneous
  deletion of p53

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The key MDM2-p53 coupling

• Mechanisms for stimulated degradation
• MDM2 p53-specific E3 ubiquitin protein ligase
• MDM2 cause transport of p53 from nucleus to
  cytoplasma, and export is necessary for
  degradation
• MDM2 a target gene for p53 being activated by
  p53
• Negative feedback loop - mechanism for turning
  off the p53 response
• Induced relatively late - leaves a time window
  where p53 can function
• regulation f (MDM2-p53 contact)
• Via phosphorylation
• Via associated proteins

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Several strategies to break theMDM2-p53 coupling
Before activation
Activated ? phosphorylation ? Broken binding
Activated ? phosphorylation ? inactivated E3-act
Activated ? ARF-binding ? inactivated E3-act
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Recent news

• More E3 enzymes suggesting ubiquitylation
  independent of Mdm2

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Regulation of MDM2-p53 contact through
phosphorylation of p53 TAD

• The ATM kinase
• a kinase that is the product of the ATM gene that
  is lost in pasients with ataxia-telangiectasia
• phosphorylates S15
• Weakens the p53-MDM2 interaction
• CHK2 - recently identified as a S20-kinase
• HIPK2 - recently identified as a S46-kinase
• activated as response to UV, role in apoptotic
  response
• DNA PK
• DNA-dependent protein kinase
• phosphorylates S15
• Weakens p53-MDM2 interaction

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upstream signalling pathway
Chk2 is a protein kinase that is activated in
response to DNA damage and may regulate cell
cycle arrest. Chk2-/- cells were defective for
p53 stabilization and for induction of
p53-dependent transcripts such as p21 in response
to gamma irradiation. Chk2 directly
phosphorylated p53 on serine 20, which is known
to interfere with Mdm2 binding.
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Updated p53 DNA damage
p53 functions as a molecular node in the
DNA-damage response.
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Recent HIPK2 binds and phosphorylates p53 after
UV irradiation
UV
HIPK2
leading to apoptosis
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Many covalent modifications of p53 in regulatory
N- and C-terminal

• Phosphorylation
• Acetylation
• Glycosylation
• SUMOylation
• Methylation

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Several modifications - complex regulatory
mechanisms

• The C terminus of p53 is rich in lysines, which
  are subjected to acetylation, ubiquitination and
  sumoylation.
• Acetylation of the C terminus
• has been shown to protect p53 from
  ubiquitination.
• Acetylation of p53 at K373 and K382 increases its
  DNA-binding activity and potentiates its
  interaction with other transcription factors.
• The positive effects of acetylation on p53
  activity can be reversed by deacetylation.
• p53 has also been shown to be sumoylated at K386
• although the exact role of this modification in
  the regulation of p53 is not yet clear.

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Acetylation upon p53 activation
p53 stabilization
activation
Phosphorylation followed by Acetylation
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Methylation of p53

• A novel mechanism of p53 regulation through
  lysine methylation by Set9 methyltransferase.
• Set9 specifically methylates p53 at one residue
  within the C-terminal regulatory region.
• Methylated p53 is restricted to the nucleus and
  the modification positively affects its
  stability.
• Set9 regulates the expression of p53 target genes
  in a manner dependent on the p53-methylation site.

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Turning p53 OFF - the hSir2 link

• Sir2 - silent information regulator
• conserved family identified in silencing in yeast
• function as NAD-dep deacetylase

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Deacetylation after p53 activation
p300
Mdm2
Phosphorylation followed by Acetylation
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HDAC?
hSIR2
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Model
DNA damage
p53 Stabilized Activated Acetylated
Growth arrest Apoptosis
Response ON
hSir2
Response OFF
p53 De-acetylated
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Alternative activation- the ARF input
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N-terminal control via ARF-binding
ARF (alternative reading frame) from p16INK4a The
INK4A locus (frequently mutated in cancer) ? 2
alternatively spliced transcripts ? translated
from alternative reading frames ? p16
(cdk-inhibitor) ARF binds MDM2-p53 and
inhibits the effect of MDM2s (ligase and
shuttling) ARF strongly induced by viral
oncoproteins and contributes to apoptosis of
infected cells ARF also induced by Myc
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ARF activation ? relocalization
In unstressed cells, p53 is degraded following
interaction with MDM2 and is exported to the
cytoplasm using nuclear-export signals present in
p53 and MDM2. Inhibition of MDM2-mediated
degradation occurs in response to certain stress
signals by activation of ARF expression. When ARF
binds to MDM2, the MDM2ARF complex is
relocalized to the nucleolus using
nucleolar-localization signals present in MDM2
and ARF. This leaves free, transcriptionally
active, p53 in the nucleoplasm.
Stressed cells ARF expression
Unstressed cells
p53 degradation
Stable p53
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Localization A model for PML-mediated
recruitment of p53 to NBs.

• PML (promyelocytic leukemia) org. comp. of
  nuclear bodies (NBs).
• Signal trigger
• Signals from DNA damage such as g-irradiation
  (gIR) or oncogene imbalance e.g. ras
  overexpression - trigger SUMOylation (S) and
  aggregation of PML into NBs. Factors including
  CBP, Rb, Daxx, Sp100 are also recruited to NBs.
• Modification
• Consequence phosphorylation (P) and acetylation
  (A) of p53. Result in increased and altered p53
  transcriptional activity.
• Update
• PML enhances p53 stability by sequestering Mdm2
  to the nucleolus. After DNA damage, PML and Mdm2
  accumulate in the nucleolus

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At least two main pathways
Activated p53
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Multiple pathways - diverse responses

• Multiple pathways exist to stabilize p53 in
  response to different forms of stress
• they may involve down-regulation of MDM2
  expression or regulation of the subcellular
  localization of p53 or MDM2.
• Target genes induced by gamma radiation, UV
  radiation, and the zinc-induced p53 form distinct
  sets and subsets with a few genes in common to
  all these treatments.

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At least two main pathways
Activated p53
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The outcomes of activated p53 - downstream
effects
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Upstream and downstream
p53 functions as sensor of upstream signals
reflecting DNA-damage /cellular stress
Upstream
activation
Downstream
Target genes Activated
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p53 as signal transducer - downstream response

• downstream consequences leading to repair of
  damage or apoptosis of damaged cell
• Two main types of effects of activated p53
• 1. Stop/regulation of the cell cycle
• 2. induction of apoptosis

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Downstream response

• Transcriptional effects
• Target genes inducing cell cycle arrest
• or DNA damage repair
• Target genes promoting apoptosis
• Other types of effects?

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Cytoplasmic Roles of p53 in Apoptosis

• p53 can initiate apoptosis in cells in which trx
  and translation are inhibited
• p53 polyproline domain (aa 6291) is necessary to
  cause apoptosis
• Excluding p53 from the nucleus causes apoptosis
  (gt threshold level)
• p53 affects mitochondrial apoptotic regulators
• Cytoplasmically localized p53 can either directly
  induce Bax oligomerization or liberate
  proapoptotic BH3-only proteins bound to
  Bcl2/Bcl-XL at the mitochondria. The released
  BH3-only proteins can then activate Bax
  oligomerization and thereby cause cytochrome c
  release leading to apoptosis.

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Regulation of the cell cycle via p53-Rb pathway

• Normal cell cycle regulation through four
  cooperating actors p16 - cyclin D1 - cdk4 - Rb
  which regulate the G1-S transition
• most cancers have one of these four altered
• p16 negative regulator of cyclin D1/cdk4
• Signalling pathway DNA-damage ? activated
  enhanced p53 ? activation of CDKI p21 (WAF1,
  Cip-1) ? inhibition of cdk4 ? reduced
  phosphorylation of Rb ? G1 arrest
• p21 inhibits also several of the other cdks
• p21 binds also PCNA ? stop in replication
• Signalling pathway DNA-damage ? activated
  enhanced p53 ? activation of GADD45 ? stimulated
  DNA-repair

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p53-Rb pathway
DNA damage
Cell cycle arrest
Apoptosis
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p53 as signal transducer - several downstream
responses

• p53 also a role in preventing gene amplification
• p53 also a role in G2/M checkpoint
• Induces a separate ribonucleotide reductase
  (p53R2)
• p53R2 encodes a ribonucleotide reductase that is
  directly involved in the p53 checkpoint for
  repair of damaged DNA.

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induction of apoptosis

• DNA-damage in p53/ cells ? apoptosis
• DNA-damage in p53-/- cells ? no apoptosis
• mechanisms far from fully understood
• Transcription activation necessary? only
  partially
• also TF-independent functions involved
• Bax induced by p53 - acts pro-apoptotic by
  counteracting Bcl2
• PERP is a novel effector of p53-dependent
  apoptosis
• p53AIP1 (p53-regulated Apoptosis-Inducing Protein
  1)
• upon severe DNA damage, Ser-46 on p53 is
  phosphorylated and apoptosis is induced. In
  addition, substitution of Ser-46 inhibits the
  ability of p53 to induce apoptosis and
  selectively blocks expression of p53AIP1.

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More target genes

• Genes induced by gamma radiation, UV radiation,
  and the zinc-induced p53 form distinct sets and
  subsets with a few genes in common to all these
  treatments.
• Microarray analysis of 6000 genes showed
• 107 induced and 54 repressed genes fell into
  categories of apoptosis and growth arrest,
  cytoskeletal functions, growth factors and their
  inhibitors, extracellular matrix, and adhesion
  genes.

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Signalling pathway
Latent p53
activated p53
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p53 and cancer
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p53 - in cancers

• Most frequent gene mutated in cancers
• Thousands of mutations from patients sequenced
• tumour supressor gene
• both alleles must be lost
• Common scenario 1. mutant ? 2.
  loss-of-heterozygocity (LOH)
• 90-95 develop cancer in young age
• Two types of DBD-mutations frequent in tumours
• Interferring with prot-DNA contacts
• Destabilizing the core structure of DBD