臺灣博碩士論文加值系統

當紡錘體檢查點偵測到未被附著的著絲點時，可藉由調控一E3泛素連接酶─後期促進複合體(APC)的活性而抑制細胞週期的進行。細胞分裂週期蛋白20 (Cdc20)具有雙重調控功能，既可作為APC的激活因子，促使細胞進入有絲分裂後期，也可透過其Mad2-binding motif (MB-motif)和Mad2交互作用形成有絲分裂檢查點複合體(MCC)，延遲細胞周期，使細胞停留在有絲分裂期間。利用試管內的APCCdc20酵素活性定量方法，我們觀察到過量的、含有MB-motif片段的胜肽足以抑制APCCdc20活性，且利用序列經改變之胜肽，初步了解哪些胺基酸殘基影響APCCdc20的作用。除了高度保守的KILR motif，其他保守的胺基酸殘基對於抑制 APCCdc20 活性也扮演著重要角色。此外，我們觀察到MB-motif 胜肽也可抑制APCCdh1活性，顯示這些胜肽可能是透過干擾APC而非Cdc20來達到抑制APCCdc20活性的效果。藉由蛋白質體外結合測試，證實了這些胜肽確實和APC有物理性交互作用。我們推測這些含有MB-motif的胜肽可能和Cdc20上的MB-motif或C-box motif上的結合位競爭，而干擾APCCdc20活性。

The spindle checkpoint prevents cell cycle progression in response to unattached kinetochores by regulating the activity of Anaphase Promoting Complex or Cyclosome (APC), an E3-ubiquitin ligase. Cell Division Cycle 20 (Cdc20) plays a bi-functional role and can either serve as a co-activator of the APC or be targeted for mitotic arrest by interacting with one of the mitotic checkpoint components, Mitotic Arrest Deficient 2 (Mad2) via Mad2-binding motif (MB-motif). By employing an in vitro quantitative APCCdc20 enzyme assay, we have observed that excess amounts of MB-motif-based peptides were sufficient to disrupt APCCdc20 activity. Mutation analyses revealed residues important for inhibition of APCCdc20 activity. In addition to the highly-conserved KILR motif, other conserved amino acids residues were observed to play an essential role in inhibiting APCCdc20 activity. I have also observed the inhibitory effects of MB-motif derived peptides on APCCdh1 enzyme activity, indicating the peptides inhibitors might primarily targeted APC rather than Cdc20. Physical interactions between wild-type and mutant forms of peptides and the APC were demonstrated by pull-down assays. We hypothesize that Cdc20 MB-motif derived peptides may compete with MB-motif or the C-box binding sites on the APC.

Table of Contents
指導教授推薦書…………………………………………………
口試委員會審定書………………………………………………
致謝…………………………………………………………… iii
Chinese Abstract………………………………………………… iv
English Abstract………………………………………………… v
Table of Contents………………………………………………… vi
List of Figures……………………………………………………… ix
List of Tables……………………………………………………… x
Chapter 1 Introduction……………………………………………
1.1 SAC ensure accurate chromosome segregation………… 1
1.2 SAC is activated at kinetochore that lack microtubule attachment or tension………………………………………………………………………………………………… 2
1.3 APC is the downstream target of SAC…………………… 3
1.4 SAC regulates the APC activity by targeting its coactivator,
Cdc20……………………………………………………… 4
1.5 Study rational……………………………………………… 6
Chapter 2 Aims …………………………………………………… 9
Chapter 3 Materials and Methods …………………………………
3.1 Yeast strains, plasmids, media and culture conditions…… 10
3.2 Preparation of yeast extract……………………………… 10
3.3 Isolation of plasmid pSCSB96, pSCSB176, pSCSB183… 12
3.4 Preparation of radioactive-labeled substrate, 35S-Pds1…… 12
3.5 Preparation of co-activator Cdc20 and Cdh1…………… 14
3.6 purification of APC………………………………………… 14
3.7 In vitro enzyme assays for APCCdc20 and APCCdh1 activity… 14
3.8 In vitro enzyme assay for inhibition of APCCdc20 and APCCdh1
activity by MB-motif peptides…………………………… 15
3.9 Data analysis……………………………………………… 16
3.10 APC binding and competition assays………………… 16
Chapter 4 Results
4.1 Investigation of potential peptide inhibitors of APCCdc20 activity
and their ability to bind APC…………………………… 17
4.2 Identification of important amino acid residues for APCCdc20
activity by mutant peptides………………………………… 17
4.2.1 Both mutant forms of DQ36 could disrupt APCCdc20
inhibition……………………………………………… 18
4.2.2 DQ36-Mut2 weakly bound to APC…………………… 18
4.2.3 PQ65-Mut1 decreases APCCdc20 inhibition activity whereas
PQ65-Mut2 does not…………………………………… 19
4.2.4 SQ19-Mut lost ability to inhibit APCCdc20 and bind APC… 19
4.3 DQ36 could compete with DQ36-Mut1 for binding APC… 20
4.4 Inhibitory effects of MB-motif-based peptides are not specific to APCCdc20 and also appear against APCCdh1……………………
4.4.1 DQ36 and PQ65 can disrupt APCCdh1………………… 20
4.4.2 Both DQ36-Mut1 and DQ36-Mut2 could interfere with
APCCdh1 inhibition……………………………………… 20
Chapter 5 Discussion
5.1 MB-motif peptides are able to bind APC and serve as APCCdc20 inhibitors…………………………………………………… 22
5.2 Identification of important amino acid residues for APCCdc20 activity by mutant peptides…………………………………. 22
5.2.1 KILR motif is not required for binding APC but is required for inhibiting APC activity………………………………… 23
5.2.2 Untouched conserved sequences among fungi and yeast contribute to both APC binding and activation………… 23
5.3 Inhibitory effects of MB-motif-based peptides are not specific to
APCCdc20, and also appear against APCCdh1…………………　24
5.4 Unsolved questions and future directions………………… 25
Chapter 6 Figures………………………………………………… 27
Chapter 7 Tables…………………………………………………… 42
References………………………………………………………… 43


List of figures
Figure 1. Analysis of Cdc20 bypass mutant alleles……………… 27
Figure 2. DK27 interact with APC and excess amount of DK27 inhibit APCCdc20 activity………………………………………… 28
Figure 3. DQ36 interact with APC and excess amount of DQ36 inhibit APCCdc20 activity ……………………………………… 29
Figure 4. PQ65 interact with APC and excess amount of PQ65 inhibit APCCdc20 activity ……………………………………… 30
Figure 5. PS46 interact with APC and excess amount of PS46 inhibit APCCdc20 activity ……………………………………… 31
Figure 6. SQ19 interact with APC and excess amount of SQ19 inhibit APCCdc20 activity ……………………………………… 32
Figure 7. Both DQ36-Mut1 and DQ36-Mut2 could disrupt APCCdc20 inhibition. DQ36-Mut2 weakly bound to APC………… 33
Figure 8. PQ65-Mut1 can disrupt APCCdc20 inhibition whereas PQ65-Mut2 cannot ……………………………………………… 35
Figure 9. SQ19-Mut lost ability to inhibit APCCdc20 and bind APC…………… 36
Figure 10. DQ36 could compete with DQ36-Mut1 for binding APC…………… 37
Figure 11. DQ36 and PQ65 can disrupt APCCdh1. …………………… 38
Figure 12. Both DQ36-Mut1 and DQ36-Mut2 could interfere with APCCdh1 inhibition. …………………………………… 39
Figure 13. Summary of the important regions on MB-motif……… 40
Figure 14. Similar sequences shared between MB-motif and C-box motif on Cdc20………………………………………………………… 41


List of tables
Table 1. Sequences of MB-motif-derived peptides………………… 42

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