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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗(Shih-Chung Chang) | |
| dc.contributor.author | Chi Wei | en |
| dc.contributor.author | 魏綺 | zh_TW |
| dc.date.accessioned | 2021-06-16T13:03:41Z | - |
| dc.date.available | 2018-08-23 | |
| dc.date.copyright | 2013-08-23 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-05 | |
| dc.identifier.citation | 高翊軒 (2012) 蛋白酶體19S Rpt5 ATPase之SUMO交互作用模組功能研究，碩士論文，國立臺灣大學生命科學院生化科技學系。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61469 | - |
| dc.description.abstract | 26S蛋白酶體 (26S proteasome) 之19S regulatory particle具有辨識泛素鏈、對蛋白質進行去摺疊、開啟20S之基質通道入口等功能。19S regulatory particle ATPase 5 (Rpt5) 與其他五個Rpt次單元體共同組成19S的基座 (base)，並且與20S之alpha-ring結合，進而調控蛋白酶體之活性。
本實驗室先前的研究發現Rpt5在COS7細胞中受到SUMO2 (small ubiquitin-like modifier 2) 修飾，利用大腸桿菌SUMO修飾系統也證明Rpt5會被SUMO1與SUMO2修飾。此外也發現Rpt5具有類似SUMO-interacting motif (SIM) 的序列。將其中的SIM3突變後，Rpt5在大腸桿菌SUMO修飾系統中被SUMO修飾的情形顯著地減少了，因此我們推論Rpt5上之SIM應與其被SUMO化修飾有關。 本研究進一步以HEK293T細胞為材料，來探討SIM是否直接參與了Rpt5之SUMOylation。在HEK293T細胞中，能觀察到Rpt5有受SUMO1及SUMO2/3修飾的現象，但是突變SIM3後，Rpt5被SUMO修飾的程度不受影響，顯示SIM3可能不是Rpt5具有功能的SIM，亦或者有其他調控方式存在。而利用胞外去SUMO化 (deSUMOylation) 酵素反應，以SUMO1/sentrin specific peptidase 1 (SENP1) 處理在細胞中受SUMO修飾的Rpt3及Rpt5，可以發現其SUMOylation程度有所下降，顯示Rpt3及Rpt5受SUMO修飾的現象可以被SENP1辨識並進行截切。另外，將Rpt3及Rpt5進行胞外SUMO化酵素反應後，並沒有觀察到Rpt3及Rpt5有受SUMO修飾的現象。 | zh_TW |
| dc.description.abstract | Regulatory particle triple-A ATPase 5 (Rpt5) is one of the subunits of the 19S regulatory particle of 26S proteasome, which forms the 19S base together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. The 19S base performs several functions, such as recognizing polyubiquitin chain, unfolding substrate, gate opening of the 20S core particle, and translocation of target proteins. Our previous study has revealed that Rpt5 was modified by small ubiquitin-like modifier2 (SUMO2) in COS7 cells. In addition, Rpt5 was modified by SUMO1 and SUMO2 in the in vitro E. coli SUMOylation assay system. Furthermore, Rpt5 contains several putative SUMO interacting motifs (SIMs), and the in vitro experimental results showed that SUMOylation of Rpt5 by SUMO1 was markedly inhibited while SIM3 was mutated.
In this study, HEK293T cells were utilized to investigate the function of SIM3 in Rpt5. The results showed that Rpt5 was modified by SUMO1 and SUMO2 in HEK293T cells; however, the SUMOylation level of Rpt5 was not reduced when SIM3 was mutated, which suggesting SIM3 is probably not involved in the SUMOylation of Rpt5. The in vitro deSUMOylation assay was conducted to examine whether the SUMO conjugates of Rpt3 and Rpt5 obtained from HEK293T cells can be removed by SUMO1/sentrin specific peptidase 1 (SENP1). The data showed that the SUMO conjugates of Rpt3 and Rpt5 were efficiently cleaved by SENP1, suggesting that the formation and de-conjugation of SUMO conjugates on Rpt3 and Rpt5 were reversible. The in vitro SUMOylation assay showed that Rpt3 and Rpt5 from HEK293T cells were not modified by SUMO2. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T13:03:41Z (GMT). No. of bitstreams: 1 ntu-102-R00b22042-1.pdf: 2589541 bytes, checksum: fe06ecef328a88661bb7b7b810fdb9cc (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii 縮寫表 ii 第一章 緒論 1 1.1 泛素-蛋白酶體系統 (ubiquitin-proteasome system) 1 1.1.1 泛素與泛素化作用 1 1.1.2 蛋白酶體之組成 2 1.1.2.1 20S Core Particle (CP) 2 1.1.2.2 19S Regulatory Particle (RP) 2 1.2 Regulatory Particle Triple-A ATPase 5 (Rpt5) 3 1.3 SUMO修飾系統 4 1.3.1 SUMO protein 4 1.3.2 SUMO化作用 (SUMOylation) 5 1.3.3 SUMO修飾的保守性序列 7 1.3.4 去SUMO化作用 8 1.3.5 SUMO化修飾的生理功能 8 1.3.6 SUMO-Interacting Motif (SIM) 9 1.4 研究動機 11 第二章 材料與方法 12 2.1 實驗材料 12 2.1.1 真核細胞株 12 2.1.2 大腸桿菌 12 2.1.3 真核表現系統載體 12 2.1.4 原核表現系統載體 12 2.2 目標基因表現質體之建構 13 2.2.1限制酶切反應 13 2.2.2 核酸純化 13 2.2.3 接合反應 13 2.2.4 小量質體DNA製備 13 2.3 大腸桿菌蛋白質表現系統 14 2.3.1 化學法製備勝任細胞 14 2.3.2 大腸桿菌細胞的轉形 14 2.3.3重組蛋白質誘導表現 15 2.4 重組蛋白質之純化方法 15 2.4.1 6xHis重組蛋白質親和性層析法 16 2.4.2 GST重組蛋白質親和性層析法 16 2.5 真核細胞表現系統 16 2.5.1真核細胞轉染 16 2.6 蛋白質相關基本操作方法 16 2.6.1 蛋白質定量 16 2.6.2 蛋白質SDS-PAGE膠體電泳 17 2.6.3 蛋白質轉印法 17 2.6.4 免疫染色法 18 2.6.5 免疫染色退染 18 2.6.6 免疫沈澱 18 2.7 去SUMO化酵素反應 19 2.8 胞外SUMO化修飾反應 19 2.9 模擬Rpt5之結構 19 第三章 結果 20 3.1 Rpt5在真核細胞中之SUMO化修飾 20 3.1.1 於HEK293T細胞中表現Rpt5並以免疫沈澱分析其SUMO化修飾的情況 20 3.1.2 於HEK293T細胞中表現Rpt5 SIM3 mutant並分析其SUMO化修飾的情況 21 3.2 分析Rpt3與Rpt5的SUMOylation現象是否可被SENP1降解 21 3.2.1去SUMO化酵素重組蛋白質表現與純化 21 3.2.2 SENP1對Rpt3及Rpt5的基質專一性辨識 22 3.3利用胞外SUMO修飾系統進行Rpt3及Rpt5 SUMOylation的研究 23 3.3.1 E1、E2、SUMO1及SUMO2重組蛋白表現與純化 23 3.3.2 Rpt3及Rpt5的胞外SUMO修飾反應 23 第四章 討論 25 4.1 Rpt5受SUMO1及SUMO2/3修飾 25 4.2 突變SIM3不影響Rpt5受SUMO2/3化修飾的程度 25 4.3 SENP1能辨識Rpt3與Rpt5的SUMO化修飾 26 4.4無法藉由in vitro SUMOylation assay觀察來自細胞的Rpt3與Rpt5之SUMOylation 26 參考文獻 28 圖與表 37 附錄 49 | |
| dc.language.iso | zh-TW | |
| dc.subject | Rpt5 | zh_TW |
| dc.subject | SUMO | zh_TW |
| dc.subject | SIM | zh_TW |
| dc.subject | SUMOylation | zh_TW |
| dc.subject | SENP1 | zh_TW |
| dc.title | 蛋白酶體19S Rpt5 ATPase受SUMO化修飾之調控機制研究 | zh_TW |
| dc.title | Study of the regulation mechanism of the SUMOylation of proteasome 19S Rpt5 ATPase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| 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,SUMOylation,SENP1, | zh_TW |
| dc.relation.page | 51 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-05 | |
| 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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