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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 楊維元 | |
dc.contributor.author | Chun Sang | en |
dc.contributor.author | 桑淳 | zh_TW |
dc.date.accessioned | 2021-06-17T04:33:31Z | - |
dc.date.available | 2018-08-16 | |
dc.date.copyright | 2018-08-16 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-10 | |
dc.identifier.citation | 1 Abreu, S. et al. Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation. EMBO Rep 18, 765-780, doi:10.15252/embr.201643146 (2017).
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70642 | - |
dc.description.abstract | 細胞自噬為細胞內兩大降解系統的其中之一,其作用為分解受損的胞器、錯誤摺疊蛋白質的聚合體,或著是入侵細胞的病原體。細胞自噬最大的特點是利用一種具雙層膜的自噬體(autophagosome)包圍需降解的物質,最後再藉由與溶酶體的融合,利用其中的水解酶將物質降解為基本分子譬如胺基酸、脂肪酸等以便細胞的再利用,透過這個機制細胞得以對抗來自胞外或胞內的生理逆境。而在自噬體的形成過程中,一種接有脂質PE的蛋白質LC3必須參與在雙層膜的形成中,這個蛋白質與脂質形成的化學鍵可以藉由一個半胱胺酸蛋白酶Atg4來切斷,進而調控自噬體的形成。
在此篇論文中,我們透過應用光學技術FRAP來觀察細胞內Atg4B的活性,並發現其活性會在細胞飢餓的狀態下立即因為氧化自由基的作用而暫時下降。此外我們也發現氧化自由基增加的現象源自內質網大量運輸鈣離子至粒線體中,強化電子傳遞鏈的結果。粒線體中鈣離子大量增加的情況並不只反映於氧化自由基的增加,也直接導致了一個貫穿粒線體內外膜的巨型通道分子mPTP的開啟,mPTP的開啟也使得氧化自由基能夠釋放至粒線體附近的細胞質中而讓Atg4B催化位置中的半胱胺酸受到氧化,此種受到氧化的Atg4B則會失去切割LC3上PE的能力,最後在細胞自噬的下游機制裡限制自噬體的形成。 透過了解這個機制,我們可以了解細胞是如何在短時間內啟動自噬以對抗逆境,由於這個機制聚焦在膜的調控與生成上,所以細胞將之利用在調控各式細胞自噬上的可能性非常高,但仍需更進一步的支持此假說。 | zh_TW |
dc.description.abstract | Autophagy is a vital process that degrades the cytosolic components and organelles isolated by a double-membraned autophagosomes, and the substrates are finally degraded inside through the lysosomal pathway. In the autophagy mechanism, the formation of autophagosomal membrane requires the involvement of lipid-conjugated protein, LC3-PE, which phosphatidylethanolamines (PEs) could de-conjugate from LC3 by the cleavage of cysteine protease atg4, and the activity can be redox modulated. In this study, a single-cell optical technique (FRAP assay) was applied to monitor the real-time activity of atg4B, and we found that atg4B activity transiently decreased due to the oxidation of ROS immediately following starvation. As we extended the research further, a burst of mitochondrial calcium leads to the increased mitochondrial ROS level and the opening of mitochondrial transition pore (mPTP). The opened mPTP releases ROS and thus transiently decreases the activity of atg4 to induce the biogenesis of autophagosomes at the ER-mitochondria contact sites. This rapid atg4B activity change is a new finding during the early stage of autophagy induction and may act as a major regulation pathway rather than the well-known ULK1 mechanism. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T04:33:31Z (GMT). No. of bitstreams: 1 ntu-107-R05b46005-1.pdf: 3550426 bytes, checksum: e54fa9b2dd29f1f31149c9696cd14f56 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 謝誌 i
中文摘要 iii ABSTRACT iv ABBREVIATIONS v CONTENT vii FIGURE CONTENT x CHAPTER 1 INTRODUCTION 1 1.1 A brief introduction to autophagy 1 1.1.1 Mechanism of macroautophagy 1 1.2 Molecular function of Atg4-family protease and their activity regulation 7 1.3 Model in this thesis 12 CHAPTER 2 MATERIALS AND METHODS 22 2.1 Materials 22 2.1.1 Water 22 2.1.2 Reagents 22 2.2 Methods 23 2.2.1 Plasmids 23 2.2.2 Cell Culture 24 2.2.3 Live Cell Analysis of Dendra2-LC3-PE De-conjugation 25 2.2.3.1 Imaging Dendra2-LC3-PE de-conjugation (FRAP assay) 26 2.2.3.2 Measuring Nucleus : Cytoplasm (N/C) volume ratio 26 2.2.3.3 Quantification of Dendra2-LC3-PE de-conjugation rate 27 2.2.4 Western Blotting 29 2.2.5 Monitoring ROS formation 29 2.2.6 Monitoring mMPTP Activity (MPT assay) 30 2.2.7 Monitoring Mitochondrial Ca2+ level 31 2.2.8 Monitoring Autophagy Flux Through STX17/LC3 Co-localization 31 CHAPTER 3 RESULTS 33 3.1 Confirming the promising target effects of CsA and Ru360 33 3.2 Monitoring the Atg4B activity through FRAP assay 36 3.3 Monitoring the autophagy flux through the frequency of autophagosome maturation events 38 CHAPTER 4 DISCUSSION 41 CHAPTER 5 FUTURE WORKS AND DIRECTIONS 43 CHAPTER 6 REFERENCES 44 | |
dc.language.iso | zh-TW | |
dc.title | 在飢餓狀況下由內質網到粒線體之鈣離子輸送引起細胞自噬爆發 | zh_TW |
dc.title | ER to Mitochondrial Calcium Transfer Drive Autophagy Bursts During Starvation | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳光超,陳瑞華 | |
dc.subject.keyword | 細胞自噬,atg4,LC3,自噬體,FRAP,鈣離子,活性氧化物,mPTP, | zh_TW |
dc.subject.keyword | autophagy,atg4,LC3,autophagosome,FRAP,calcium,ROS,mPTP, | en |
dc.relation.page | 47 | |
dc.identifier.doi | 10.6342/NTU201802941 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-10 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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