近年來粒線體的功能的研究擴展的相當廣，其中對於調控蛋白穩態的研究正在快速發展。先前研究已得知調控粒線體外膜蛋白的穩態對於泛素—蛋白酶體系統有高度依賴性，且VCP已被證實在其中扮演相當重要的角色。粒線體相關降解（MAD）途徑在近年來的研究逐漸增多，尤其是在酵母菌的研究中已得知Doa1會與泛素和Cdc48結合，這過程對於降解粒線體外膜蛋白相當重要。本實驗著重在探討Doa1及Cdc48在哺乳動物中的同系物PLAA及VCP對於泛素化蛋白及粒線體外膜蛋白的重要性。藉由PLAA及VCP基因Knockdown，檢測相關蛋白量變化。從實驗結果得知VCP確實會影響泛素化蛋白及粒線體外膜蛋白的降解；PLAA只影響粒線體外膜蛋白的降解。

In recent years, the research on the regulation of mitochondrial protein homeostasis develops rapidly. Previous studies have known that the regulation of the homeostasis of mitochondrial outer membrane (MOM) proteins is highly dependent on the ubiquitin-proteasome system. VCP has been confirmed to play a very important role on it. In addition, research on the mitochondria-associated degradation (MAD) pathway has gradually increased, especially in yeast studies. It has been known that Doa1 binds to ubiquitin and AAA-ATPase Cdc48, which is very important for the degradation of MOM proteins. Here, we focus on the importance of PLAA and VCP, the orthologues of Doa1 and Cdc48 in mammals, respectively. By knocking down the PLAA and VCP genes, change in the amount of ubiquitinated proteins and MOM proteins were detected. From the experimental results, we knew that VCP affected the degradation of ubiquitinated proteins and MOM protein- Mcl1, Mfn1; PLAA only affected the degradation of MOM protein- Mcl1, Mfn1.

一、前文 1
中文摘要 1
ABSTRACT 2
《Acknowledgement》 3
目錄 4
二、正文 6
1. 前言介紹 6
1.1 粒線體的功能 6
1.2 粒線體外膜蛋白 7
1.3 粒線體自噬（mitophagy）與粒線體外膜蛋白之關聯性 8
1.4 粒線體相關降解途徑 Mitochondria-Associated Degradation (MAD) Pathway 10
1.5 含纈酪肽蛋白 (VCP) 及其對於MAD的重要性 12
1.6 磷脂酶A2激活蛋白 (PLAA) 及其與VCP的關係 13
2. 研究目標 15
2.1 實驗假說 15
2.2 實驗目的 15
3. 實驗器材、方法 16
3.1 細胞培養及轉染 16
3.2 小干擾核糖核酸（siRNA）基因Knockdown 16
3.3 化學處理並收成細胞 17
3.4 西方墨點法（western blotting） 18
3.5 定量和統計分析 19
4. 實驗結果 20
4.1 透過siRNA 進行VCP及PLAA基因Knockdown 20
圖1.確認VCP及PLAA之基因Knockdown 21
4.2 VCP基因Knockdown導致泛素化蛋白質的積累 23
4.3 VCP基因Knockdown導致粒線體外膜蛋白積累 23
圖2.VCP基因Knockdown對多重泛素化蛋白之影響 25
圖3.VCP基因Knockdown對粒線體外膜蛋白之影響 28
4.4 PLAA基因Knockdown對泛素化蛋白質的影響 29
4.5 PLAA基因Knockdown導致粒線體外膜蛋白積累 29
圖4.PLAA基因Knockdown對多重泛素化蛋白之影響 31
圖5.PLAA基因Knockdown對粒線體外膜蛋白之影響 33
5. 總結及討論 34
5.1 VCP和PLAA對於泛素化蛋白質和粒線體外膜蛋白降解的影響 34
5.2 VCP影響粒線體外膜蛋白量的程度大於PLAA 34
5.3 實驗遇到的困境 35
5.4 PLAA是否作為VCP的輔因子參與MAD途徑 36
6. 未來實驗方向 38
6.1 PLAA是否協助VCP調控粒線體外膜蛋白的retrotranslocation 38
6.2 觀察PLAA、VCP及粒線體外膜蛋白在空間中的位置 38
6.3 深入探討MAD相關蛋白在維持粒線體功能上扮演的角色 39
三、 參考資料 40
參考文獻 40
附錄 44
補充圖1. VCP Knockdown對PLAA的影響 44
補充圖2. PLAA Knockdown對VCP的影響 45
表格1. 實驗中用到之siRNA 序列 46

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