P53 - guardian or the genome + guardian of the tissuse

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    23-Dec-2015

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  • Slide 1
  • p53 - guardian or the genome + guardian of the tissuse
  • Slide 2
  • MBV4230 Odd S. Gabrielsen 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
  • Slide 3
  • MBV4230 Odd S. Gabrielsen p53 mutations
  • Slide 4
  • p53 - protein domains
  • Slide 5
  • MBV4230 Odd S. Gabrielsen p53 domains C-terminal allosteric domain
  • Slide 6
  • MBV4230 Odd S. Gabrielsen 5 distinct domains in p53 1. 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 TAF II 70 and TAF II 31 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
  • Slide 7
  • MBV4230 Odd S. Gabrielsen Mdm2 - p53 Mdm2 p53-TAD
  • Slide 8
  • MBV4230 Odd S. Gabrielsen p53 domains C-terminal allosteric domain
  • Slide 9
  • MBV4230 Odd S. Gabrielsen 5 distinct domains in p53 2. 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)
  • Slide 10
  • MBV4230 Odd S. Gabrielsen p53 domains C-terminal allosteric domain
  • Slide 11
  • MBV4230 Odd S. Gabrielsen 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)
  • Slide 12
  • MBV4230 Odd S. Gabrielsen 5 distinct domains in p53 3. 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
  • Slide 13
  • MBV4230 Odd S. Gabrielsen 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.
  • Slide 14
  • MBV4230 Odd S. Gabrielsen 5 distinct domains in p53 3. DBD binds DNA as tetramer (dimer of dimer) DNA recognition sequence reflects this: 4x RRRCW arranged like this:
  • Slide 15
  • MBV4230 Odd S. Gabrielsen p53 domains C-terminal allosteric domain
  • Slide 16
  • MBV4230 Odd S. Gabrielsen 5 distinct domains in p53 4. Tetramerization Tetramerization domain aa 324-355 2 + 2 structure forms tetramers linked with DBD via 37aa flexible linker [aa 287-323]
  • Slide 17
  • MBV4230 Odd S. Gabrielsen p53 domains C-terminal allosteric domain
  • Slide 18
  • MBV4230 Odd S. Gabrielsen 5 distinct domains in p53 5. 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
  • Slide 19
  • Activation of p53 - upstream inputs
  • Slide 20
  • MBV4230 Odd S. Gabrielsen Upstream and downstream p53 Signal transduction pathways + +.. +.. Upstream Downstream p53 functions as sensor of upstream signals reflecting DNA-damage /cellular stress activation
  • Slide 21
  • MBV4230 Odd S. Gabrielsen 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
  • Slide 22
  • MBV4230 Odd S. Gabrielsen 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, -catenin) Foster defects
  • Slide 23
  • MBV4230 Odd S. Gabrielsen 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
  • Slide 24
  • MBV4230 Odd S. Gabrielsen 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
  • Slide 25
  • MBV4230 Odd S. Gabrielsen Several strategies to break the MDM2-p53 coupling Before activation Activated phosphorylation Broken binding Activated phosphorylation inactivated E3-act Activated ARF-binding inactivated E3-act
  • Slide 26
  • MBV4230 Odd S. Gabrielsen Recent news More E3 enzymes suggesting ubiquitylation independent of Mdm2
  • Slide 27
  • MBV4230 Odd S. Gabrielsen 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
  • Slide 28
  • MBV4230 Odd S. Gabrielsen 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.
  • Slide 29
  • MBV4230 Odd S. Gabrielsen Updated: p53 & DNA damage p53 functions as a molecular node in the DNA-damage response.
  • Slide 30
  • MBV4230 Odd S. Gabrielsen Recent : HIPK2 binds and phosphorylates p53 after UV irradiation HIPK2 UV leading to apoptosis
  • Slide 31
  • MBV4230 Odd S. Gabrielsen Many covalent modifications of p53 in regulatory N- and C-terminal Phosphorylation Acetylation Glycosylation SUMOylation Methylation 20
  • Slide 32
  • MBV4230 Odd S. Gabrielsen 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 ye