p 53 Missense Mutations: Structural and Functional Perturbations to DNA Binding - PowerPoint PPT Presentation

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p 53 Missense Mutations: Structural and Functional Perturbations to DNA Binding

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  1. p53 Missense Mutations: Structural and Functional Perturbations to DNA Binding Bich-Chau Van Department of Biochemistry and Molecular Biology Professor Ray Luo

  2. Hudson and Abella http://www.hudsonandabella.org

  3. Presentation Outline 1. Why are we interested in p53? 2. What is p53? • Functions • Structure • Regulation • Wild type and mutants 3. Project • Goal • Methods • Results and discussion

  4. p53 is Important Because… • Acted upon by viral proteins (such as Adenovirus E1B, SV40 large T antigen, HPV E6) • Germline mutation leads to Li-Fraumeni Syndrome which predisposes the individual to many kinds of tumors (Vousden, KH, Balint, E. (2001). British Joul. Of Cancer,85(12): 1813-1823) • 50% of human cancers contain mutations in p53

  5. p53 is… • A transcription factor with tumor suppressor function • Encoded by the tumor suppressor gene TP53 • Expressed at very low levels in normal cells • Short half-life of 6-30 minutes depending upon cell or tissue type=> unstable • Optimal DNA binding sequence: RRRCWWGYYY where R is a purine, W is A or T and Y is a pyrimidine Olivier, M., et al. (2002). Human Mutation, 19: 607-614

  6. Other Functions • Activate cellular differentiation, senescence (Vousden, K. H. (2000). Cell,103: 691-694.) • Inhibit angiogenesis, maintain genetic stability through DNA repair gene induction (Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408: 307-310.) • New functions in stem cells: suppression of pluripotency and inhibition of stem cell self-renewal(Hede, S. M., et al. (2010). Journal of Oncology, 2011.)

  7. Structure of p53 • Have 393 amino acids • Active as a homotetramer

  8. Structure of p53 • Have 393 amino acids • Active as a homotetramer • 5 major, interdependent domains within each subunit: • Transcriptional activation domain (residues 1- 63) • Proline- rich domain (residues 64- 92) • Specific DNA- binding (core) domain (residues 94- 292) • Tetramerization domain (residues 326- 355) • C- terminal domain (residues 363- 393) Prives, C., Hall, P. (1999). J. Pathol, 187: 112-126.

  9. Structure of p53 http://www-p53.iarc.fr

  10. p53 Core Domain and DNA Binding Interface

  11. Viral Protein Binding Sites http://www.ncbi.nlm.nih.gov/books/NBK21551/figure/A7159

  12. Which Leads to the Activation of p53 Protein?

  13. Positive Regulation • Regulate p53 stability by inhibiting or degrading Mdm2 protein • Mdm2: an E3 ubiquitinligase

  14. Mdm2 and p53 Interaction

  15. Positive Regulation

  16. Negative Regulation

  17. p53 Network Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408: 307-310. DNA repair G1 arrest G2 arrest Mitochondria- independent Mitochondria- dependent

  18. DNA Binding Domain Mutations • 50% of human cancers contain mutations in p53, 95% of which is in the DNA- binding domain (DBD) • 75% of DBD mutations are single missense mutations. • Hot spot mutations: at residues 175, 245, 248, 249, 273, 282 Olivier, M., et al. (2000). Human Mutation, 19: 607-614

  19. Type of Cancers due to Missense Mutations Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408: 307-310.

  20. p53 Missense Mutations • Dominant negative • Mutants reside in cytoplasm Tan, Y., Luo, R. (2009). PMC Biophysics, 2:5.

  21. Side Chain Exposure Unstable sites Stable sites Tan, Y., Luo, R. (2009). PMC Biophysics, 2:5.

  22. Mechanisms for p53 Inactivation by Missense Mutations • Loss of stability and/ or • Loss of DNA contact, protein- protein contact with other domains, tetramer subunits or binding partners, or Zn contact • Loss of stability and DNA contact are main mechanisms. Tan, Y., Luo, R. (2009). PMC Biophysics, 2:5.

  23. DNA Binding Surface Mutants DNA contact Structural • R248 • R273 • Directly affect DNA binding • Recognized by PAb 1620 • R175 • G245 • R249 • R282 • Indirectly affect DNA binding • Recognized by PAb240

  24. Location of Mutations

  25. Goals of the Project • Identify the structural effects of three common missense mutations (G245S, R249S, R273H) on the p53 DNA binding interface • Effects of these mutations on DNA binding affinity of p53 • Long-term goal: to develop better pharmaceutical interventions to restore wild-type p53 functions in many mutant proteins