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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗(Shih-Chung Chang) | |
| dc.contributor.author | Yi-Hsuan Kao | en |
| dc.contributor.author | 高翊軒 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:37:41Z | - |
| dc.date.available | 2015-08-01 | |
| dc.date.copyright | 2012-08-01 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-26 | |
| dc.identifier.citation | 劉邦宇 (2009) 哺乳類動物細胞中SUMO結合受質之蛋白質體學研究。碩士論文，國立台灣大學生命科學院微生物與生化學研究所。
劉昀瑄 (2011) 蛋白酶體19S Rpt5 ATPase受SUMO化修飾之研究。碩士論文，國立台灣大學生命科學院生化科技學系碩士班。 Baba D, Maita N, Jee JG, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M (2005) Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature 435: 979-982 Baba D, Maita N, Jee JG, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M (2006) Crystal structure of SUMO-3-modified thymine-DNA glycosylase. J Mol Biol 359: 137-147 Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345-356 Bohren KM, Nadkarni V, Song JH, Gabbay KH, Owerbach D (2004) A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65345 | - |
| dc.description.abstract | Rpt5 (19S regulatory particle triple-A ATPase 5) 是組成蛋白酶體 (26S proteasome) 的19S regulatory particle中六個 AAA ATPase的其中一個次單元體。Rpt5具有許多功能，包括對目標蛋白質進行unfolding、運送目標蛋白質進入蛋白酶體的活性中心，並藉此調控蛋白酶體的功能；另外，Rpt5也能夠以一個非蛋白酶體次單元體的角色參與在其他的反應當中，較常見的是與其他蛋白質組成複合體並調控某些轉錄的過程。
本實驗室先前的研究發現，在大腸桿菌的SUMO (small ubiquitin-like modifier) 化分析系統中，即使突變了Rpt5分子上之MKSE這個SUMO化修飾保守序列的離胺酸，Rpt5仍會受到SUMO1的修飾。 本研究經過胺基酸序列分析後發現，Rpt5上具有六個可能為SUMO interacting motif (SIM) 的序列，因此推論Rpt5或許能夠與SUMO進行非共價交互作用。我們假設SIM在Rpt5受到SUMO化修飾的過程中可能扮演著重要的角色，所以將SIM序列上的疏水性胺基酸皆突變成丙胺酸 (alanine)，並利用這些Rpt5的突變株作為受質，以大腸桿菌的SUMO化分析系統來檢測，發現當SIM3、SIM4、SIM5分別突變後，Rpt5受到SUMO1修飾的情形明顯降低；SIM3突變後同樣也使得Rpt5受到SUMO2修飾的情形明顯降低。另外，利用anti-HA binding agarose pull-down assay也發現Rpt5能夠與SUMO1進行非共價交互作用，但是其突變株與SUMO1非共價交互作用的情形，與Rpt5被SUMO1所修飾的結果並不一致；此外，本研究並無觀察到Rpt5與SUMO2之間具有非共價交互作用。 | zh_TW |
| dc.description.abstract | Regulatory particle triple-A ATPase 5 (Rpt5) is one of six AAA ATPases of 19S regulatory particle of 26S proteasome. Rpt5 performs several functions, such as unfolding target proteins and translocating these substrates to the 20S core particle, in the regulation of the proteasome catalytic activity. Furthermore, Rpt5 can also function independently from proteasome and usually participate in part of a protein complex which involved in transcription regulation.
In the previous study, we found that Rpt5 was modified by small ubiquitin-like modifier (SUMO) in an in vitro E. coli SUMOylation assay system even though its SUMOylation consensus motif MKSE was mutated. In the present study, we found that Rpt5 contains six putative SUMO interacting motifs (SIMs), herein named SIM1-6, suggesting that Rpt5 may interact with SUMO protein in a non-covalent way. Therefore, we hypothesized that the SIMs of Rpt5 may play an important role in its SUMOylation process. By using Rpt5 mutants, whose hydrophobic residues of the putative SIMs were substituted to alanine residues, as the substrates, we demonstrated that SUMOylation of Rpt5 by SUMO1 was markedly inhibited while SIM3, SIM4, or SIM5 was mutated, respectively. In addition, SUMOylation of Rpt5 by SUMO2 was also inhibited while SIM3 was mutated. Furthermore, the non-covalent interaction between Rpt5 and SUMO1 was observed by using anti-HA binding agarose pull-down assay. Mutation of SIM3 in Rpt5 also abolished its non-covalent interaction with SUMO1. However, the SIM4, SIM5, or SIM6 Rpt5 mutant displayed greater interaction with SUMO1. These findings suggest that different SIMs of Rpt5 may play various roles in regulation of its SUMOylation and non-covalent interaction with SUMO1. The non-covalent interaction between Rpt5 and SUMO2 was not detected in this study. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:37:41Z (GMT). No. of bitstreams: 1 ntu-101-R99b22029-1.pdf: 1543456 bytes, checksum: 36f24de635a65cc39580087c8998de22 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii 縮寫表 iv 第一章 緒論 1 1.1 蛋白酶體 (Proteasome) 1 1.1.1 蛋白酶體的組成 1 1.2 Regulatory Particle Triple-A ATPase 5 (Rpt5) 2 1.3 SUMO (small ubiquitin-liked modifier) 3 1.3.1 SUMO化作用 (SUMOylation) 4 1.3.2 SUMO化修飾的生理功能 6 1.4 SUMO-interacting motif (SIM) 6 1.4.1 SIM與SUMO的結合 7 1.4.2 SIM與SUMO非共價交互作用的功能 8 1.5 研究動機 9 第二章 材料與方法 10 2.1 目標基因表現載體之建構 10 2.1.1 聚合酶鏈鎖反應 10 2.1.2 限制酶切反應 10 2.1.3 DNA片段與質體DNA之純化 10 2.1.4 接合反應 10 2.1.5 質體DNA建構 10 2.2 大腸桿菌蛋白質表現系統及SUMO修飾系統 11 2.2.1 化學法製備勝任細胞 11 2.2.2 大腸桿菌細胞的轉形 11 2.2.3 重組蛋白質誘導表現 12 2.2.4 SUMO修飾系統的建立 12 2.2.5 製備含有pT-E1E2S1或pT-E1E2的勝任細胞 12 2.2.6 SUMO修飾系統及目標蛋白質的表現 13 2.3 anti-HA binding agarose pull-down assay 13 2.4 蛋白質相關基本操作方法 14 2.4.1 蛋白質定量 14 2.4.2 蛋白質SDS-PAGE膠體電泳 14 2.4.3 蛋白質轉印法 15 2.4.4 免疫染色法 15 2.4.5 免疫染色退染 15 2.5 利用Discovery StudioTM模擬Rpt5的結構 16 第三章 結果 17 3.1 確認Rpt5的SIM突變株是否會影響Rpt5受SUMO化修飾 17 3.1.1 於大腸桿菌SUMO化修飾系統中表現Rpt5及其SIM突變株 17 3.1.2 以anti-HA binding agarose pull-down assay觀察SUMO1修飾 18 3.1.3 Rpt5及其SIM突變株受SUMO2修飾的情形 18 3.2 利用anti-HA pull-down assay觀察Rpt5與SUMO的非共價交互作用 19 3.2.1 與SUMO1的非共價交互作用 19 3.2.2 與SUMO2的非共價交互作用 20 3.3 Rpt5之結構模擬 20 第四章 討論 21 4.1 Rpt5受SUMO化修飾是一種SIM-dependent的機制 21 4.2 SIM對Rpt5受SUMO1及SUMO2修飾的異同 21 4.3 Rpt5與SUMO非共價交互作用的親和性 22 4.4 Rpt5受SUMO1修飾的結果和兩者間非共價交互作用的結果並不一致 22 4.5 Rpt5模擬結構的探討 23 參考文獻 24 圖與表 31 附錄 39 | |
| dc.language.iso | zh-TW | |
| dc.subject | Rpt5 | zh_TW |
| dc.subject | SUMO | zh_TW |
| dc.subject | SIM | zh_TW |
| dc.subject | non-covalent interaction | zh_TW |
| dc.title | 蛋白酶體19S Rpt5 ATPase之SUMO交互作用模組功能研究 | zh_TW |
| dc.title | Study of the Function of the SUMO-Interacting Motif in 19S Proteasomal Rpt5 ATPase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 莊榮輝(Rong-Huay Juang),張麗冠(Li-Kwan Chang),陳威戎(Wei-Jung Chen),林翰佳(Han-Jia Lin) | |
| dc.subject.keyword | Rpt5,SUMO,SIM,non-covalent interaction, | zh_TW |
| dc.relation.page | 46 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-26 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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