臺灣博碩士論文加值系統

細胞的有絲分裂紡綞體檢查點是一種監察機制，監視有絲分裂中期到後期時染色體的分配，並利用延遲週期的方式來避免染色體分離錯誤。有絲分裂紡綞體檢查點的目標蛋白是Cdc20，它調控細胞分裂中期到末期的轉折。其機制是利用調節Cdc20的兩個流向來調控有絲分裂紡綞體檢查點。其中一個流向會活化APC/C （細胞週期調控者之後期促進複合體）使細胞進入細胞分裂末期。另一條路徑則會與其他檢查點蛋白質結合形成細胞週期複合體，使細胞週期無法進行。但目前對於有絲分裂紡綞體檢查點的分子機制卻只有概述。為了抑制細胞週期的進行，Mad2會與APC/C競爭Cdc20上MB-Motif (Mad2-binding Motif)的KIRL序列（活化區塊）。在此篇論文中，我們發現MB-motif有新的功能，亦即活化APC/C。藉由分析單一胺基酸的功能，我們發現Cdc20的L203及Y205是活化APC/C時是必要的; 而Y205及P209則是Mad2抑制作用所必要的。這些觀察指出，Mad2的抑制與APC/C的活化兩種功能並非相互依存並且指出目前所定義的活化區塊應被擴大。在試管中審查MB-motif的胜肽時，發現一個包含整體MB-motif的胜肽「PQ-66」可能是APC/CCdc20的抑制物。總結這些觀察中，我們提出MB-motif之C端末端序列的胺基酸可能調控APC/C結合的假說，並推斷Mad2會扮演競爭型抑制物的方式與MB-motif結合，達到分離APC/C結合位點與活化位點的方式來抑制APC/C的活性。

Cells contain a surveillance mechanism called the “spindle checkpoint” that monitors spindle attachments on kinetochores, and delays the metaphase-anaphase transition to prevent chromosome mis-segregation. The target of spindle checkpoint is Cdc20, which regulates the metaphase-anaphase transition. The spindle checkpoint regulates the flow of Cdc20 into one of two pathways. One pathway leads to the activation of the APC/C (Anaphase-Promoting Complex/Cyclosome), causing the cell to go into anaphase; the other pathway leads to binding with checkpoint proteins to form the MCC (Mitotic Checkpoint Complex), leading to APC/C inactivation and cell cycle arrest. The molecular details have only been partially defined. To induce cell cycle arrest, Mad2 (Mitotic Arrest Deficient 2) competes with APC/C to bind the MB-motif (Mad2-binding motif) on Cdc20 via a KIRL motif (activator region). Here, we reported that the MB-motif has an additional novel function as an APC/C activator. By characterizing the activities of single amino acid mutants, we found L203 and Y205 in Cdc20 are necessary to activate APC/C, and Y205 and P209 are necessary for Mad2 inhibition. These observations suggest a separation of function for Mad2-inhibition and APC/C activation, and an extension of the currently defined activator region. By screening MB-motif peptides in vitro, “PQ-66”, a peptide including the full MB-motif, was identified as a potential APC/CCdc20 inhibitor. From this observation, we also hypothesize amino acids at the C-terminus of the MB-motif play a role in APC/C binding. We conclude that Mad2-mediates APC/C activity as a competitive inhibitor, which binds with MB-motif to sequester the activator region from the potential APC/C binding site.

Table of Contents
指導教授推薦書…………………………………………………………………………….
口試委員會審定書…………………………………………………………………………….
授權書……………………………………………………………………………. iii
誌謝……………………………………………………………………………. …iv
中文摘要……………………………………………………………………………. v
Abstract……………………………………………………………………………. vii
Table of Contents………………………………………………………………….. ix
Table of Figures…………………………………………………….…………… Xi
CHAPTER I Introduction……………………………………………………… 1
CHAPTER II Material and Methods…………………………………………… 6
2.1 Strain, plasmids, media, and culturing condition ……………………… 6
2.2 Plasmid purification of plasmids ………..…………………………..… 7
2.3 Preparation of 35S-Pds1 and 35S-Cdc20…………………………………. 7
2.4 Preparation of 35S-Pds1 and 35S-Cdc20 Preparation of Cdc20………….. 9
2.5 Time-course APC/C assay with Cdc20 or Cdc20-127………………….. 9
2.6 Inhibition of APC/CCdc20 activity by Mad2………………… 10
2.7 Inhibition of APC/CCdc20 activity by MB-motif peptides ………… 10
2.8 Data analysis ………………………………………………………… 11
CHAPTER III Aim………………………………….…………………………. 12
CHAPTER IV Results …………………………………….……………………. 13
4.1 Establishing a high activity APC/C enzyme assay …………………… 13
4.2 Head to head comparison of APC/CCdc20 and APC/CCdc20-127 activity . 13
4.3 Cdc20-L203A performed similar result as Cdc20-127, whereas other relative mutants do not ………………………………………………… 14
4.4 Cdc20-127 resistant to inhibition by Mad2…………………………… 15
4.5 Full MB motif peptide, PQ66, inhibits APC/C activity ……………… 16
CHAPTER V Discussion………………………………………………. 18
CHAPTER IV Figures………………………………………………… 21
REFERENCES…………………………………………………………….…… 34

Table of Figures
Figure 1. A model of how Cdc20 flow regulates APC/C activity………………. 21
Figure 2. Models of how Cdc20 activates APC/C and forms MCC……………… 22
Figure 3. Important amino acid mutant site(s) in the MB motif of Cdc20……… 23
Figure 4. Inhibition of Mad2 to APC/CCdc20 activity...………….…………………. 24
Figure 5. Measurement of IVT/T 35S-Cdc20 and 35S-Cdc20-127 in triplicate by phosphor-image density after 2 hour………………………………… 25
Figure 6. Measurement of the APC/C activity with different co-activators, Cdc20 or Cdc20-127……………………………………………………………….. 26
Figure 7. Measurement of IVT/T 35S-Cdc20-mutants and the APC/C activity with different co-activator mutants.…………………………........................... 27
Figure 8. Measurement of Mad2 inhibition of APC/CCdc20 and APC/CCdc20-127 activitie…………………………………………………………………… 28
Figure 9. Measurement of Mad2 inhibition of APC/Cco-activators activity……………. 29
Figure 10. Measurement of peptides inhibition of APC/CCdc20 activity …………… . 31
Figure 11. Measurement of PQ66 inhibition of APC/CCdc20 activity………...……... 32
Figure 12. Model of Mad2-mediated inhibition of APC/CCdc20 activity …….……... 33

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