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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 謝小燕(Sheau-Yann Shieh) | |
| dc.contributor.author | Zheng-Cheng Yu | en |
| dc.contributor.author | 余正晟 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:44:17Z | - |
| dc.date.available | 2021-02-24 | |
| dc.date.copyright | 2016-02-24 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-12-09 | |
| dc.identifier.citation | Ⅴ. References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51680 | - |
| dc.description.abstract | MDM2為一著名的E3泛素連接酶，而其最為著名的下游受體為抑癌蛋白p53。然而除了p53外，已經有許多MDM2的下游受體被報導，而其中有許多是參與在細胞週期調控的重要蛋白。但是關於MDM2的活性調控，尤其是本篇論文提到的遭遇氧化壓力的環境，相關的作用機制並沒有被真正的了解。在本篇論文，我們發現一著名的調控紡錘絲檢查點的激酶，hMps1，會磷酸化MDM2，進而增加MDM2對於下游受體，組蛋白子單位H2B的泛素化。透過氨基丙酸 (Ala) 突變在hMps1磷酸化的位點，會降低經由hMps1磷酸化而增加的組蛋白H2B的泛素化。在細胞內，H2B的泛素化會由於氧化壓力而增加而降低hMPs1或MDM2的表現，會降低H2B的泛素化。補回野生型 (WT) MDM2能夠回復H2B泛素化，不過如果補回的是氨基丙酸突變在Mps1磷酸化的位點的MDM2，H2B的泛素化回復的效率會變差。此實驗證明了Mps1磷酸化MDM2對於組蛋白H2B的泛素化的重要性。而本篇論文也證明了，hMps1及MDM2藉由調控H2B的泛素化，來幫助細胞在氧化壓力下的DNA損傷反應及修復。 | zh_TW |
| dc.description.abstract | MDM2 is an E3 ubiquitin ligase that targets proteins involved in cell cycle progression. Although many MDM2 substrates have been identified, how its activity is regulated is not fully understood, especially when cells are under oxidative stress. Here we show that upon oxidative stress, MDM2 can be phosphorylated by human Mps1 (hMps1)/TTK, a kinase known to function in the spindle assembly and DNA damage checkpoints. Consequently, MDM2-mediated H2B ubiquitination is enhanced, and Ala substitution at the hMps1/TTK phosphorylation sites compromises this effect. In cells, H2B ubiquitination was enhanced upon oxidative stress. Depletion of hMps1/TTK or MDM2 not only abrogated this induction but also resulted in reduced DNA repair, cell survival, and ATR signaling. Consistent with these observations, cells expressing a ubiquitination-deficient H2B mutant were also defective in DNA repair and ATR signaling. Complementation with WT MDM2 but not the phospho-deficient mutant rescued the H2B ubiquitination and DNA repair, suggesting the requirement for hMps1/TTK-mediated MDM2 phosphorylation. Taken together, we propose that hMps1/TTK phosphorylates MDM2 upon oxidative damage to promote H2B ubiquitination, which in turn facilitates oxidative DNA damage signaling and repair. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:44:17Z (GMT). No. of bitstreams: 1 ntu-104-D98445003-1.pdf: 1737577 bytes, checksum: 12e1964dcf7230f51f956c4b629ffa09 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | Contents
中文摘要…………………………………………………………………….…………け Abstract…………………………………………………………………………………げ Contents……………………………………………………………………...…………こ List of Figures……………………………………………………………..……………し Ⅰ. Introduction …………………………………………………………………………1 1.1 Functions of MDM2 beyond regulating p53………………………………...………1 1.2 Post-translational modification on MDM2………………………………………..…2 1.3 hMps1 and its canonical function in spindle assembly checkpoint (SAC) …………3 1.4 hMps1 also functions as an effector in DNA damage response…….………….……4 1.5 Histone modification－H2B ubiquitination…………………………………………5 1.6 H2B ubiquitination plays a role in DNA damage response……………………….…6 Ⅱ. Materials and Methods………………………………………………………………8 2.1 Cell lines………………………………………………………………………..……8 2.2 Plasmids and siRNA transfection……………………………………………………8 2.3 Antibodies…………………………………………………………………..………10 2.4 Phospho-specific antibodies production……………………………………………11 2.5 In vivo ubiquitination assay……………………………………………...…………11 2.6 In vitro ubiquitination………………………………………………………………12 2.7 Preparation of recombinant proteins…………………………..……………………13 2.8 GST pulldown assay…………………………………………………………..……13 2.9 Coimmunoprecipitation………………………………………………….…………14 2.10 In vitro kinase assay……………………………………………………….………15 2.11 Comet assay………………………………………………………………….……15 2.12 Immunofluorescence assay…………………………………………..……………15 2.13 Harvest of chromatin fraction………………………………………..……………16 2.14 Clonogenic cell survival assay……………………………………………………16 2.15 Protein stability assay………………………………………………………..……17 2.16 HPRT mutation frequency assay………………………………….………………17 Ⅲ. Results…………………………………………………………………………...…19 3.1 hMps1 interacts with MDM2 in vivo………………………………….……………19 3.2 Identification of the interaction domains hMps1 and MDM2…………...…………19 3.3 MDM2 can directly bind to hMps1 in vitro……………………………………...…20 3.4 MDM2 is phosphorylated by hMps1………………………………………….……20 3.5 Thr4, Thr306 and Ser307 are the major hMps1 phosphorylation sites in MDM2....21 3.6 hMps1-mediated phosphorylation promotes MDM2-dependent Histone H2B ubiquitination…………………………………………………………………...………22 3.7 H2B ubiquitination is increased by phosphomimetic mutants of MDM2……….....24 3.8 hMps1 and MDM2 are required for H2B ubiquitination after H2O2 treatment…….24 3.9 MDM2 can be phosphorylated by hMps1 upon oxidative stress…………………..25 3.10 hMps1 and MDM2 are involved in DNA repair after H2O2 treatment……………26 3.11 Impaired hMps1-MDM2 signaling leads to increased DNA mutation……………27 3.12 hMps1-MDM2 signaling modulates DNA repair through H2B ubiquitination…..28 3.13 Colocalzation of MDM2 with oxidative DNA damage repair factors…………….29 3.14 hMps1 and MDM2 are involved in DNA damage response induced by oxidative stress……………………………………………………………………………………29 3.15 Defect in H2B ubiquitination also obstructs ATR signaling under oxidative DNA damage…………………………………………………………………………………30 3.16 The hMps1-MDM2-H2B ubiquitination axis regulates the histone compaction. ..31 3.17 Recruitment of RPA and XRCC1 to damage sites was impaired in hMps1 and MDM2 downregulated cells….. ………………….……………………………………31 3.18 Physiological relevance in clinical samples………………………... ……………32 Ⅳ. Disscussion……………………. ………………………………………………..…35 4.1 The roles of hMps1 in DNA damage signaling and repair………………..……. …35 4.2 MDM2 orchestrates DNA damage signaling and repair……………………...……37 4.3 The involvement of Histone H2B ubiquitination in DNA repair………………..…38 4.4 The DNA damage signaling is regulated by chromatin compaction tuned by histone H2B ubiquitination…………………………………………………..…………………40 4.5 The clinical correlation between hMps1-MDM2 signaling axis and cancer……….42 Ⅴ. References………………………………………………………………….………44 Ⅵ.Figures………………………………………………………………………………57 List of Figures Figure 1. hMps1 interacts with MDM2…………………………………………...……57 Figure 2. Truncated form of MDM2 or hMps1 interacts with each other. ……...…..…58 Figure 3. MDM2 interacts with hMps1 directly in vitro. ………………..…….………60 Figure 4. MDM2 is phosphorylated by hMps1. ……………………………………….61 Figure 5. hMps1 phosphorylation sites on MDM2. ……………………………………62 Figure 6. Thr4, Thr306 and Ser307 at MDM2 were phosphorylated by hMps1…….…63 Figure 7. hMps1 enhances MDM2-mediated Histone H2B ubiquitination………….…65 Figure 8. hMps1 phosphorylation of MDM2 did not alter its E3 ligase specifity….…..66 Figure 9. hMps1 phosphorylates MDM2 at Thr4,Thr306 and Ser307 to enhance H2B ubiquitination in part through the elevated stability………………………………....…67 Figure 10. The 3A mutation of MDM2 did not alter the intrinsic E3 ligase activity…..68 Figure 11. Phosphomimetic mutation at hMps1 phosphorylation sites of MDM2 increased the E3 ligase activity……………………………………………………...…69 Figure 12. Depletion of hMps1 or Mdm2 results in decrease in H2B Ubiquitination…70 Figure 13. Depletion of hMps1 and MDM2 compromises the H2B ubiquitination...….71 Figure 14. Complementation with MDM2 WT but not 3A mutant restored H2B ubiquitination. ………………………………………………………….………………73 Figure 15. Specificity analysis of anti-phospho Thr4 and Thr306 MDM2 antibodies…75 Figure 16. MDM2 phosphorylation at Thr4 and Thr306 were induced upon oxidative stress.……………………………………………………...……………………………76 Figure 17. Downregulation of hMps1 or MDM2 impaired DNA repair and cell survival upon oxidative stress. ………………………………………………………….………77 Figure 18. Complementation with MDM2 WT but not 3A mutant rescued DNA repair upon oxidative stress. …………………………………………………………….……79 Figure 19. Downregulation of hMps1 or MDM2 delayed repair of 8-oxoguanine under oxidative stress. …………………………………………………………………..……81 Figure 20. Increase of WT MDM2 impaired mutation frequency from oxidative stress but 3A mutant compromised this ability. ………………………………………………82 Figure 21. Defect in histone H2B ubiquitination dampened DNA repair upon oxidative stress. ………………………………………………………………………………..…83 Figure 22. MDM2 co-localized with effectors for base excision repair….…………….85 Figure 23. Depletion of hMps1 or MDM2 diminishes ATR signaling after H2O2 treatment………………………………………………………………………..………87 Figure 24. Expression of 2KR mutant histone H2B dampened the ATR signaling…….89 Figure 25. H2O2-induced eviction of histones from chromatin was impaired by knockdown of hMps1 or MDM2………………………………………………….……90 Figure 26. Foci formation of RPA or XRCC1 was dampened upon oxidative stress in hMps1 or MDM2-depleted cells. ………………………………………………………91 Figure 27. Expression or Variation of copy numbers of hMps1 or MDM2 in human sarcoma. …………………………………………………………………………..……92 | |
| dc.language.iso | en | |
| dc.subject | hMps1激? | zh_TW |
| dc.subject | MDM2泛素連接? | zh_TW |
| dc.subject | 組蛋白H2B泛素化 | zh_TW |
| dc.subject | 氧化壓力 | zh_TW |
| dc.subject | DNA損傷反應 | zh_TW |
| dc.subject | DNA damage response | en |
| dc.subject | hMps1 | en |
| dc.subject | MDM2 | en |
| dc.subject | Histone H2B ubiquitination | en |
| dc.subject | oxidative stress | en |
| dc.title | 激酶TTK/hMps1藉由磷酸化MDM2調控組織蛋白H2B泛素化以及氧化壓力下的DNA損傷及修復反應 | zh_TW |
| dc.title | MDM2 Phosphorylation by TTK/hMps1 Promotes Oxidative DNA Damage Response and Repair through Histone H2B Ubiquitination | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 李財坤(Tsai-Kun Li),陳小梨(Show-Lin Chen),沈志陽(Chen-Yang Shen),張智芬(Zee-Fen Chang) | |
| dc.subject.keyword | MDM2泛素連接?,hMps1激?,組蛋白H2B泛素化,氧化壓力,DNA損傷反應, | zh_TW |
| dc.subject.keyword | MDM2,hMps1,Histone H2B ubiquitination,oxidative stress,DNA damage response, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-12-09 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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