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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳瑞華(Ruey-Hwa Chen) | |
| dc.contributor.author | Yu-Tung Lin | en |
| dc.contributor.author | 林禹彤 | zh_TW |
| dc.date.accessioned | 2021-06-16T06:32:23Z | - |
| dc.date.available | 2020-07-27 | |
| dc.date.copyright | 2020-07-27 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-23 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56984 | - |
| dc.description.abstract | 蛋白質泛素化是一個複雜的轉譯後修飾，透過一些重要酵素的參與，在轉譯出的蛋白質上生成變化多端的泛素鏈而影響其命運，進而影響細胞的多個層面。泛素是一個由76個胺基酸所組成的小蛋白，其存在於絕大部分的真核生物中。泛素的胺基酸序列中含有七個離胺酸 (Lysine)，在這八個位點包含七個離胺酸以及泛素的第一個胺基酸甲硫氨酸 (Methionine) 上可以被另一個泛素修飾，以此類推，因此可以產生眾多不同組合的泛素鏈，依其不同形式可分為同型與異型鏈，其中異型鏈又可分為混鏈型以及支鏈型。在些許能夠分析泛素體的方法中，最新發展出的Ub-clipping方法利用病毒前導蛋白酶-Lbpro*結合質譜儀分析，可以更快速且容易地區分混鏈型以及支鏈型，也可以了解泛素鏈之間的連鎖。細胞自噬是一個細胞將受損老舊的胞器、錯誤摺疊的蛋白質及其他大分子等等物質送到溶酶體後進行降解，將小分子物質回收再利用的機制，其在細胞中是不可或缺的。UBE3C是一個E3泛素連接酶，它屬於HECT家族的其中一員，其已被報導會合成由Lys29以及Lys48所連接的異型泛素鏈。VPS34是第三類磷脂肌醇-3-激酶複合體中重要的一個次單元，負責產生磷脂肌醇-3-磷並幫助細胞自噬小體的生合成。先前我們實驗室已在細胞內證實UBE3C會對VPS34進行泛素化，促使它經由蛋白酶體降解。在本篇研究中，我們發現UBE3C在試管中能有效地加長VPS34上的泛素鏈，並藉由Ub-clipping的方法，證實此延長的泛素鏈是由Lys29以及Lys48所連接，更重要的是，我們發現UBE3C合成的泛素鏈屬於異型鏈中的支鏈型。而當在細胞中完全移除UBE3C的表現時，會顯著的提升VPS34及脂質化LC3。有趣的是，一個專門切除Lys29以及Lys33泛素鏈的去泛素酶-TRABID可以移除UBE3C在VPS34上合成的泛素鏈。然而，我們意外發現從細菌純化的全長UBE3C完全失去其活性，其現象同樣發生在他人的研究中，我們推測是因為蛋白質在不正常的折疊下，導致其喪失活性。我們也發現在UBE3C的HECT結構域尾端標籤上小片段胜肽會大幅地減弱其泛素化活性，說明HECT結構域的尾端在其活性上扮演重要的角色。本篇研究的最後，我們對於Lbpro*做了酵素動力的分析，發現它移除泛素鏈的方式是類似內切酶的作用。綜觀上述，我們證明UBE3C會對VPS34進行Lys29/Lys48的支鏈泛素化，使VPS34表現量下降，進而調控細胞自噬。 | zh_TW |
| dc.description.abstract | Protein ubiquitination is a complicated post-translational modification that creates a versatile connection system in cells and affects the cells in several aspects. Ubiquitin chains can be conjugated to another ubiquitin through seven lysine residues or the first methionine residue, and thus generates architecturally different types of chain including homotypic, mixed and branched chains. UBE3C, a HECT family E3 ubiquitin ligase, was reported to assembly K29- and K48-linked free ubiquitin chains. In this study, we demonstrate that UBE3C purified from baculovirus robustly elongate the polyubiquitin chain on VPS34 in vitro. However, UBE3C purified from bacteria completely lose this activity presumably due to improper folding or autoinhibition. Furthermore, the C-terminal tag conjugated UBE3C HECT domain shows a dramatic impairment in autoubiquitination activity, implying a significant role of the C-terminal residues of HECT domain in the ubiquitination activity. Importantly, using a recently developed Ub-clipping method which employs an engineered viral leader protease Lbpro* and mass spectrometry analysis to distinguish mixed from branched chain, we identify that the polyubiquitin chain on VPS34 mediated by UBE3C is a K29/K48-branched ubiquitin chain. Interestingly, a K29- and K33-specific DUB, TRABID, can potently remove the total polyubiquitin chain as well as the K48-linked ubiquitin chain on VPS34 synthesized by UBE3C in vitro. We further show that UBE3C knockout significantly upregulates VPS34 level and LC3 lipidation. Finally, our kinetic analyses revealed that Lbpro* is an endoprotease in the assembly of polyubiquitin chain. Collectively, this study demonstrates that UBE3C negatively regulates autophagy by mediating K29/K48-branched ubiquitination on VPS34, thus leading to VPS34 downregulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T06:32:23Z (GMT). No. of bitstreams: 1 U0001-2307202011475100.pdf: 3613752 bytes, checksum: 4a90c13d44d698753fa9f154ffb1e468 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | Contents 謝辭 ………………………………………………………………………………………………………………..……..…. i 摘要 …………………………………………………………………………………………………………………….…… ii Abstract ……………………………………………………………………………………………………………….……. iv Contents .......................................................................................................................... vi List of figures ................................................................................................................ viii I. Introduction ................................................................................................................... 1 1. Ubiquitination ............................................................................................................... 1 1.1. Overview ............................................................................................................. 1 1.2. Topology and biology of ubiquitin codes ............................................................ 2 1.3. Dissection of heterotypic ubiquitin chains .......................................................... 6 2. Ubiquitin protein ligase E3C (UBE3C) ....................................................................... 10 2.1. Characterization ................................................................................................. 10 2.2. Physiological and pathological function of UBE3C .......................................... 12 3. Autophagy .................................................................................................................. 14 3.1. Overview ........................................................................................................... 14 3.2. Molecular mechanism ........................................................................................ 15 3.3. VPS34 complex in autophagy ............................................................................ 16 3.4. Ubiquitination in autophagy .............................................................................. 18 II. Material and Methods ................................................................................................ 20 Plasmids ......................................................................................................................... 20 Antibodies and Reagents ................................................................................................ 20 Baculovirus expression system and purification............................................................. 21 Protein expression and purification ................................................................................ 21 In vitro ubiquitination and deubiquitination assays......................................................... 23 Lbpro* treatment of in vitro assembly reactions ............................................................... 24 Intact mass spectrometry for molecular weight determination ....................................... 24 LC-MS/MS for chain linkage analysis ............................................................................ 25 Cell culture, transient transfection, and treatment ........................................................... 27 Generation of CRISPR KO cell line ................................................................................ 27 Immunofluorescence ...................................................................................................... 28 Kinetics measurement of Lbpro* cleavage ....................................................................... 29 Western blot analysis ...................................................................................................... 29 Statistical analysis .......................................................................................................... 30 III. Results ...................................................................................................................... 31 UBE3C purified from E. coli fails to ubiquitinate VPS34 in vitro .......................... 31 UBE3C functions to elongate the polyubiquitin chain on VPS34 in vitro .............. 32 UBE3C synthesizes K29/K48-branched chains on VPS34 in vitro ......................... 33 TRABID disassembles K29/K48-linked polyubiquitin chains on VPS34 generated by UBE3C in vitro ....................................................................................................... 35 UBE3C functions as a negative regulator of autophagy via VPS34 downregulation ...................................................................................................... 35 Lbpro* acts as an “endoprotease” to execute the trimming of ubiquitin chain ........ 36 IV. Discussion ................................................................................................................ 39 V. References ................................................................................................................. 44 VI. Figures ...................................................................................................................... 52 | |
| dc.language.iso | en | |
| dc.subject | VPS34 | zh_TW |
| dc.subject | 細胞自噬 | zh_TW |
| dc.subject | 前導蛋白酶 | zh_TW |
| dc.subject | 泛素化 | zh_TW |
| dc.subject | UBE3C | zh_TW |
| dc.subject | UBE3C | en |
| dc.subject | VPS34 | en |
| dc.subject | leader protease | en |
| dc.subject | ubiquitination | en |
| dc.subject | autophagy | en |
| dc.title | UBE3C透過對VPS34進行K29/48支鏈型泛素化進而調控細胞自噬 | zh_TW |
| dc.title | UBE3C drives VPS34 K29/48 branched ubiquitination to modulate autophagy | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 吳昆峯(Kuen-Phon Wu) | |
| dc.contributor.oralexamcommittee | 王彥士(Yane-Shih Wang) | |
| dc.subject.keyword | UBE3C,VPS34,前導蛋白酶,泛素化,細胞自噬, | zh_TW |
| dc.subject.keyword | UBE3C,VPS34,leader protease,ubiquitination,autophagy, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202001767 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-07-23 | |
| 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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