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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃偉邦 | |
dc.contributor.author | Chih-Hung Huang | en |
dc.contributor.author | 黃植虹 | zh_TW |
dc.date.accessioned | 2021-06-15T06:18:22Z | - |
dc.date.available | 2010-08-12 | |
dc.date.copyright | 2010-08-12 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-10 | |
dc.identifier.citation | REFERENCES
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47784 | - |
dc.description.abstract | 中文摘要
細胞自噬是細胞分解大分子物質的途徑之一,在真核生物中具有高度的保守性,可以維持細胞體內的恆定。當細胞處於壓力環境,特別是營養缺乏時,細胞自噬會被誘發,將非必要的蛋白質包裹起來,再經由與溶體或者是酵母菌中的液胞癒合進而分解,而釋出的胺基酸則可以用來供應合成細胞生存所需的蛋白質。 在細胞自噬過程中所形成的雙層膜狀囊胞稱為自噬體,與其他膜狀運輸小泡不同的是,自噬體的雙層膜構造並非直接來自已經存在的膜,而是經由重新組合而成,但至今仍沒有直接證據證實單位膜的最初來源。Atg9是在目前已知參與調控細胞自噬的蛋白質裡唯一的嵌膜蛋白,對於自噬體的形成過程也是必須的,並且在自噬體形成的不同時期,Atg9會分別在自噬體前驅構造及細胞周邊胞器出現,構成一個運輸循環,因此被認為可能是把磷脂質帶到自噬體前驅構造的攜帶者。 我們假設位在自噬體前驅構造的Atg9蛋白存在某種轉譯後修飾,這樣的修飾可以傳遞訊息,讓其他下游調控細胞自噬的蛋白質移動到自噬體前驅構造以完成自噬體的形成。本篇論文研究發現,在養份缺乏所引發細胞自噬的情形下,Atg9會經過高度磷酸化修飾,此段磷酸化的區域是位於Atg9的胺基端,對於Atg9在細胞內的運輸及細胞自噬的功能相當的重要。研究利用將胺基端置換方式將可能被磷酸化的絲胺酸和蘇胺酸換成不能被磷酸化的丙胺酸模擬無法被磷酸化修飾的Atg9,結果顯示,不能被磷酸化的突變的確會造成細胞自噬活性的缺陷,也會造成Atg9在細胞體內的循環受阻;另一方面,當絲胺酸和蘇胺酸被置換成帶負電的胺基酸以模擬磷酸化現象時,此Atg9突變蛋白竟然可以自行從自噬體前驅構造離開回到細胞周邊胞器,而不需要已知參與調控此過程的Atg1蛋白的作用。此外,我們利用免疫沈降法分析發現不同程度磷酸化的Atg9對於Atg11的親和力也有所不同。綜合以上,我們認為磷酸化修飾對於Atg9在細胞體內的運輸及對於細胞自噬功能的調節扮演著重要的角色。 | zh_TW |
dc.description.abstract | Abstract
Autophagy is an evolutionally conserved degradation pathway through all eukaryotes, mediating cytosolic components turnover to maintain cellular homeostasis. It can be induced by stress signal, especially starvation condition, to make an adaptive response to environmental stimulus, by degrading cytoplasm, including entire organelles, in lysosome, or corresponding compartment in yeast, the vacuole, for necessary cell survival. Different form other secretory pathway, the formation of double-membrane vesicles, termed autophagosomes, is considered to be de novo instead of budding from a pre-existing membrane. However, the identity of the lipid membrane source in remains uncertain. Among all Atg (autophagy-related gene) proteins, Atg9 is the only integral membrane protein required for autophagosome formation. Moreover, while in the process of autophagy regulation, Atg9 cycles between peripheral sites and the PAS (pre-autophagosomal structure) in distinct steps. To be more specific, Atg9 is recruited to PAS during autophagosome formation and retrieved back to cytosolic area upon autophagosome completion. Hence, Atg9 is thought to be the candidate of membrane carrier. Here, we proposed that Atg9 may undergo some kind of modification to discriminate the PAS-localized and non-PAS-localized Atg9 in order to pass down the signal and employ different downstream proteins in definite situations. Indeed, we found that Atg9 is a phosphoprotein. Starvation treatment induces autophagy activity and causes hyper-phosphorylation of Atg9. Furthermore, the Atg9 phosphorylation sites are mapped to its N-terminus, which is important for its cellular localization and function in autophagy. Followed analyses suggested that the N-terminal alanine substitution Atg9 mutants impaired autophagy activity and blocked its retrograde transport from PAS. On the contrary, a phosphorylation-mimic Atg9 mutant with aspartate/glutamate substitution revealed no typical PAS-localized punctate structures in cells with deletion of ATG1, which is essential for recycling Atg9 from PAS back to the peripheral sites. Moreover, the co-immunoprecipitation assay demonstrated that the Atg9N1A exhibited stronger affinity to its interacting protein, Atg11, than wild type Atg9 and Atg9N1D. Together, we concluded that the phosphorylation modification of Atg9 plays a key role in its trafficking and autophagy regulation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:18:22Z (GMT). No. of bitstreams: 1 ntu-99-R97b41019-1.pdf: 1906448 bytes, checksum: 2bbcfb2e05dbce28d6d52f3d7e516a01 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | CONTENTS
致謝 I 中文摘要 II ABSTRACT III CHAPTER 1: INTRODUCTION 1 1.1 OVERVIEW OF AUTOPHAGY 1 1.2 ROLE OF AUTOPHAGY IN CELLS 3 1.3 SELECTIVITY OF AUTOPHAGY 5 1.4 THE MOLECULAR MECHANISM OF AUTOPHAGY 8 1.5 THE ROLE OF ATG9 IN AUTOPHAGY IN THE BUDDING YEAST SACCHAROMYCES CEREVISIAE 12 CHAPTER 2: MATERIALS AND METHODS 15 2.1 STAINS AND MEDIA 15 2.2 PLASMID CONSTRUCTS 16 2.3 IMMUNOBLOT ANALYSIS 17 2.4 YEAST-TWO-HYBRID ASSAY 18 2.5 GFP CLEAVAGE ASSAY 18 2.6 PULL-DOWN ASSAY 19 2.7 IN VITRO ALKALINE PHOSPHATASE TREATMENT 19 2.8 FLUORESCENCE MICROSCOPY 20 CHAPTER 3: RESULTS 21 3.1 ATG9 IS A PHOSPHOPROTEIN 21 3.2 OVEREXPRESSION OF PPH21 EXHIBITS ATG9 PHOSPHORYLATION AND AUTOPHAGIC FUNCTIONAL DEFECTS 22 3.3 ATG9Y6A SHOWS NO AUTOPHAGIC DEFECT 25 3.4 THE PHOSPHORYLATION SITES OF ATG9 ARE MAPPED TO ITS N-TERMINUS 25 3.5 THE N-TERMINUS OF ATG9 IS IMPORTANT FOR ITS PHOSPHORYLATION AND AUTOPHAGY REGULATION 26 3.6 ATG9 MUTANTS WITH N-TERMINAL MULTIPLE ALANINE SUBSTITUTIONS TEND TO FORM PUNCTATE STRUCTURES AND ACCUMULATED AT THE PAS 28 3.7 THE PHOSPHORYLATION-MIMIC ATG9 MUTANTS DO NOT AFFECT AUTOPHAGIC PROCESS 29 3.8 ATG9N1D DOES NOT ACCUMULATE AT THE PAS IN THE ABSENCE OF RESPECTIVE ATG1, ATG2, AND ATG18 30 3.9 SELF-INTERACTION OF ATG9 IS IMPORTANT FOR ITS TRAFFICKING 32 3.10 THE PHOSPHORYLATION STATUS OF ATG9 MAY INFLUENCE ITS AFFINITY TO ITS INTERACTING PROTEIN, ATG11 33 CHAPTER 4: DISCUSSION 36 4.1 ATG9 IS A PHOSPHOPROTEIN WHICH IS HYPER-PHOSPHORYLATED UNDER NUTRIENT DEPLETION CONDITIONS 36 4.2 OVEREXPRESSION OF PHOSPHATASE PPH21 RESULTED IN PARTIAL ATG9 PHOSPHORYLATION AND AUTOPHAGIC DEFECTS 37 4.3 THE NON-PHOSPHORYLATED ATG9 DAMAGED AUTOPHAGIC PROCESS AND BLOCKED ITS TRAFFICKING 38 4.4 ATG9N1D COMPLETE RETROGRADE TRANSPORTATION IN SEPARATE ATG1Δ, ATG2Δ, AND ATG18Δ BACKGROUNDS 39 4.5 SELF-INTERACTION OF ATG9 REGULATED ITS CYCLING 41 4.6 PHOSPHORYLATION STATUS OF ATG9 INFLUENCED ITS AFFINITY TO ITS INTERACTING PROTEIN, ATG11 42 REFERENCES 44 TABLES 52 FIGURES 54 | |
dc.language.iso | en | |
dc.title | Atg9磷酸化修飾對其細胞內運輸及細胞自噬之影響 | zh_TW |
dc.title | Atg9 phosphorylation status regulates its cycling and function in autophagy | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李心予,陳俊宏,朱家瑩 | |
dc.subject.keyword | 細胞自噬, | zh_TW |
dc.subject.keyword | Atg9,autophagy, | en |
dc.relation.page | 74 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-11 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 動物學研究所 | zh_TW |
顯示於系所單位: | 動物學研究所 |
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