1. MDM2 inhibits p300-mediated p53 acetylation and activation by forming a ternary complex with the two protein 生科系 04 級 賴保諺 891635 Eric Kobet, Xiaoya Zeng, Yong Zhu, David Keller, and Hua Lu PNAS | November 7, 2000 | vol. 97 | no. 23 | 12547-12552

2. p53 : a tumor suppressor gene which can induce cell growth arrest and apoptosis a transcription factor MDM2 is transcriptionally activated by p53

3. MDM2 : possess ubiquitin-transferase activity p53 MDM2 ubiquitin p53 ubiquitin ubiquitination degradation

4. p53 DNA transcription MDM2 p53 ubiquitination p53 degradation translation negative feedback loop

5. p300 : histone acetyl-transferase (HAT) p300 ace p53 acetyl - CoA p53 ace

6. p53 posttranslation modification  phosphorylation by DNA-dependent protein kinase may contribute to transactivation and stability  acetylation by p300 leading to activation of sequence-specific DNA binding ability

7. MDM2 could form a complex with p300 and p53 in vitro. known

8. HeLa Nuclear Extract and marker chromatography western

9. western marker 100 90 70 50 40 KD add antibody

10. MDM2 can form a ternary complex with p300 and p53 in the nucleus. p300 ~ 400kDa p53 ~ 212kDa (homotetramer) MDM2 ~ 90kDa total ~ 700kDa

11. Whether the purified protein complex could acetylate p53 ?

12. The purified p300 was able to acetylate p53-dependent on acetyl CoA in a dose-dependent fashion. western by using antiacetylated Lys antibody

13. MDM2 、 p53 、 p300 complex p300, once complexed to MDM2, may not be able to target the p53 Lys residues. isotope-labeled acetyl CoA (autoradiography)

14. MDM2 was preincubated with p300 before adding to the reaction mixture ( MDM2 interacted with the CH1 domain of p300) MDM2 can inhibit p53 acetylation by p300 probably through direct association.

15. Whether the inhibition requires the association of MDM2 with p53 compare wild-type MDM2 with its N-terminally deleted form lacking amino acid 58-89 (p53 binding domain) This suggest that for MDM2 to inhibit p300-mediated p53 acetylation, it must form a ternary complex with p53 and p300.

16. Whether MDM2 could influence the p300 acetylase activity on different substrates that do not interact with MDM2. Futher investigation

17. p73 C-teminal domain (could be acetylated by p300 in vitro)

18. Association of MDM2 with p53 is required for its inhibitory effect on p53 acetylation.

19. What’s the functional consequence of the inhibition of p300-mediated p53 acetylation by MDM2 ? We examine the effect of MDM2 on p300-stimulated p53 sequence-specific DNA-binding activity.

20. Electrophoretic Mobility-Shift Assay ( EMSA ) add DNA-binding protein probe (DNA) shift add protein antibody super shift p53 binding sequence p53 p53 antibody

21. MDM2 eliminates the ability of p300 to stimulate sequence-specific DNA-binding activity of p53 in vitro.

22. Finally, we examine whether MDM2 inhibits p53 acetylation by p300 in vivo. transfect human lung carcinoma cells with plasmids coding p53, MDM2, p300

24. Test whether inhibition of p53 acetylatoin by MDM2 affects sequence–specific DNA-binding activity of p53 in vivo. p53 antibody super shift These results indicate that MDM2 also inhibits sequence- specific DNA-binding activity of p53 in vivo.

25. MDM2 inhibits p53 acetylation and activation mediated by p300 in vivo + MDM2 can form a ternary complex with p300 and p53 in the nucleus MDM2 inhibits p300-mediated p53 acetylation and activation by forming a ternary complex with the two protein.

26. UV and γ irradiation differentially regulate MDM2 expression and p53 acetylation.

28. MDM2 expression was repressed by UV (not by (not by γ irradiation). DNA binding ability of p53 was enhanced by UV ( not by ). ( not by γ irradiation). DNA was damaged and p53 was activated and cells may undergo apoptosis.