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研究生:廖彥綸
研究生(外文):Yan Lun Liao
論文名稱:以Cdc20 Mad2-binding motif及在出芽酵母中的人類p31comet homolog為基礎的抑制胜肽和APC的結合測試
論文名稱(外文):Peptide inhibitors of APCCdc20 based on the Cdc20 Mad2-binding motif and human p31comet homolog in budding yeast can directly bind to the APC
指導教授:曲桐
指導教授(外文):S. C. Schuyler
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
論文頁數:35
中文關鍵詞:出芽酵母APC抑制胜肽
外文關鍵詞:Madz-binding motifp31comet homologbudding yeastAPC inhibitors
相關次數:
  • 被引用被引用:0
  • 點閱點閱:219
  • 評分評分:
  • 下載下載:11
  • 收藏至我的研究室書目清單書目收藏:0
在有絲分裂之中,紡錘體檢查點是一種在所有的著絲點都被附著上紡錘絲之前會延遲細胞進入分裂後期的機制,用以確保基因組穩定性。先前的研究顯示,用抗有絲分裂藥物處理腫瘤細胞會使其進行細胞滑移而繼續分裂,而當另外加入Cdc20 siRNA 時卻可以讓腫瘤細胞有顯著的死亡。這意味著APCCdc20 活性被抑制且有絲分裂拘停時間延長將可以促使癌細胞進行細胞凋亡。我們已知一個有著Mad2-binding motif 的胜肽片段DQ36 可以在in vitro 中抑制APC 的活性。在此,我建立了一套得以用來測試此類小片段胜肽與APC 的結合活性測試。對於Cdc20 是接觸在APC 的哪一個部位的問題,我也利用了這套結合性實驗並且以Apc9 次單元作為測試對象。然而在我的研究結果中,Apc9 並不是APC 和Cdc20 結合所需要的一個次單元。另一個小片段胜肽Tyc1 是來自於一個新的、功能未知的出芽酵母基因。令人意外的是,這一個胜肽也足以抑制APC 的活性。因此,我也利用了已經建立的APC 和胜肽的結合性測試來實驗這段胜肽和APC 的結合。此外,我也用了免疫共沉澱法來探討Tyc1 和Cdc20 在in vivo 中的結合。我的研究結果顯示,Tyc1 和Cd20 沒有結合現象,因此Tyc1對APC 的抑制可能是藉由結合到APC 而不是Cdc20。這些抑制性胜肽對於APC 的抑制機轉將會是我們未來的研究重心。
In mitosis, the spindle assembly checkpoint ensures genome stability by delaying the onset of anaphase until all kinetochores are properly attached to the mitotic spindle. A Previous study has shown that some proportion of tumor cells treated with anti-mitotic drugs resulted in abnormal mitotic exit, while after Cdc20 was knocked down by siRNA, it led to robust mitotic arrest followed by cell death. This report indicates that APCCdc20
inhibitor may be a potential role in development of cancer therapeutic agents. We have observed that DQ36, a peptide based on Mad2-binding motif in Cdc20, is sufficient to inhibit APCCdc20 activity in vitro. Here, I established a binding assay to verify that the peptide can directly bind to the APC in vitro. For the question that where does Cdc20 touch to the APC, we made a speculation on Apc9 subunit because of its conserved motifs. However, my result shows that Apc9 subunit is not required for APC-Cdc20 interaction. Nevertheless, we keep trying to find the answer by peptide cross-linking experiment. Another small peptide, Tyc1, is a novel tiny protein with unknown function identified from human p31comet BLAST in budding yeast genome. Amazingly, Tyc1 showed its inhibition activity in APC assay as well. Subsequently, binding tendency of Tyc1 to the APC was verified in binding assay and compared to DQ36. In addition, interaction of Tyc1 and Cdc20, a co-factor of the APC, was investigated by Co-IP. My result shows that Cdc20 is not the binding partner of Tyc1, indicating that Tyc1 peptide may disturb APCCdc20 activity by interacting with the APC. The mechanism of peptide inhibitors influencing APCCdc20 activity will be the major issue we focus on in the future.
指導教授推薦書
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誌謝 iii
中文摘要 iv
Abstract v
Table of Contents vi
Table of Figures viii
I. Introduction 1
II. Aims 6
III. Materials and Methods 7
3.1 Yeast strain 7
3.2 Cell homogenization 8
3.3 APC purification 8
3.4 Peptides binding and pulling out 9
3.5 Western blotting 9
3.6 Co-immunoprecipitation 10
IV. Results 11
V. Discussion 14
VI. Figures 18
References 25

Table of Figures
Figure 1. A model that Cdc20 flows through two pathways to regulate
the APC 18
Figure 2. Four artificial peptides used in APC binding 19
Figure 3. Binding assay process 20
Figure 4. Western blotting for DQ36 peptide binding to the APC 21
Figure 5. Western blotting for DQ36 peptide binding to APCApc9Δ 22
Figure 6. Western blotting for Tyc1 peptide binding to the APC 23
Figure 7. Western blotting for Cdc20 and Tyc1 Co-IP 24
1. H.C. Huang, et al. “Cell death when the SAC is out of
commission”, Cell Cycle 9, 11, 2049-2050, June 2010
2. Hwang, et al. “Budding yeast Cdc20: a target of the spindle
checkpoint”, Science, 279, pp. 1041-1044, February 1998.
3. Luo, et al. “The Mad2 spindle checkpoint protein has two distinct
natively folded states”, Nat. Struct. Mol. Biol., 11, pp. 338-345,
April 2004.
4. Sironi, et al. “Crystal structure of the tetrameric Mad1-Mad2
core complex: implications of a ‘safety belt’ binding mechanism
for the spindle checkpoint”, EMBO J., 21, pp. 2496-2506, May
2002.
5. Kimata, et al. “A role for the Fizzy/Cdc20 family of proteins in
activation of the APC/C distinct from substrate recruitment”. Mol.
Cell, 32, pp. 576-583, November 2008.
6. Foe, et al. “Ubiquitination of Cdc20 by the APC occurs through
an intramolecular mechanism”, Curr. Biol, 21, pp. 1870-1877,
November 2011.
7. Zhang and Lees “Identification of an overlapping binding domain
on Cdc20 for Mad2 and anaphase-promoting complex: model for
spindle checkpoint regulation”, Mol. Cell Biol, 21, pp. 5190-5199,
August 2001.
8. Chao, et al. “Structure of the mitotic checkpoint complex”,
Nature, 484, pp. 208-213, March 2012.
9. Luo, et al. “Structure of the Mad2 spindle assembly checkpoint
protein and its interaction with Cdc20”, Nat. Struct. Biol., 7, pp.
224-229, March 2000.
10. Izawa and Chen “Spindle checkpoint regulates Cdc20p stability
in Saccharomyces cerevisiae”, Genes Dev., 18, pp. 1439-1451,
June 2004.
11. Passmore, et al. “Doc1 mediates the activity of the anaphase
promoting complex by contributing to substrate recognition”,
EMBO, 22, pp. 786-796, 2003.
12. Habu, et al. “Identification of a MAD2-binding protein, CMT2,
and its role in mitosis”, EMBO J 21(23):6419-6428
13. Westhorpe, et al. “p31comet-mediated extraction of Mad2 from
the MCC promotes efficient mitotic exit”, J Cell Sci 124(Pt
22):3905-3916
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