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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 胡孟君(Meng-Chun Hu) | |
dc.contributor.author | Shou-Hsien Huang | en |
dc.contributor.author | 黃守賢 | zh_TW |
dc.date.accessioned | 2021-06-15T06:18:05Z | - |
dc.date.available | 2015-09-09 | |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-11 | |
dc.identifier.citation | Amir R, Ciechanover A, Cohen S. 2001. [The ubiquitin-proteasome system: the relationship between protein degradation and human diseases]. Harefuah 140:1172-1176.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47779 | - |
dc.description.abstract | 肝受器同源體-1 (Liver receptor homolog-1; LRH-1) 為轉錄因子,屬於核受器 NR5A 家族的成員之一,主要表現於肝臟、小腸、胰臟、脂肪前驅細胞與卵巢。本實驗室先前發現 mLRH-1 在細胞中表現的蛋白質量不高,推測 mLRH-1 可能經由蛋白酶體降解路徑分解,而影響到 mLRH-1 蛋白質的穩定性,但是目前對於 mLRH-1 蛋白質穩定性的調控機制尚未釐清。我們將 mLRH-1 與突變型泛素 (UbK0) 共同表現於 293 細胞中,發現 mLRH-1 的蛋白質表現量會顯著增加,說明 mLRH-1 可能有泛素化反應,而影響蛋白質的穩定性。為了了解 mLRH-1 是否會與泛素鍵結,我們將帶有不同標籤的 mLRH-1 與帶有 HA 標籤的泛素 (HA-Ub) 共同表現於 293 細胞中,並利用共同免疫沉澱法與西方墨點法進行分析。結果發現 Flag-mLRH-1、Myc-mLRH-1 和 Myc-CA-mLRH-1 蛋白質皆會與泛素鏈 (poly-ubiquitin chain) 鍵結。另外,給與蛋白酶體抑制劑 MG-132 並不影響 mLRH-1 與泛素所形成的蛋白質複合體的量,我們推測 mLRH-1 與泛素形成鍵結後可能與蛋白酶體降解路徑無關。
當我們刪除 mLRH-1 蛋白質的羧基端後,mLRH-1 與泛素所形成的蛋白質複合體的量會顯著減少,說明 mLRH-1 羧基端的胺基酸序列可能為進行泛素化作用的主要區域。我們建立了持續在 293 細胞中表現 Myc-CA-mLRH-1 融合蛋白質的 stable clones,並且確定這些 stable clones 皆具有 mLRH-1 的活性。我們發現在 stable clone 中 Myc-CA-mLRH-1 蛋白質會與細胞內的內生性泛素鍵結而形成泛素鏈。綜合所有實驗結果,我們的結論是在哺乳類細胞中 mLRH-1 蛋白質會進行泛素化作用,並且可能是透過 mLRH-1 蛋白質的羧基端與泛素鏈形成鍵結。 | zh_TW |
dc.description.abstract | Liver receptor homolog-1 (LRH-1; NR5A2) is a transcriptional factor and belongs to the nuclear receptor 5A subfamily. LRH-1 is predominantly expressed in liver, intestine, exocrine pancreas, preadipocytes and ovary. Our previous studies indicated that the expression of mLRH-1 protein in transfected cells is low, suggesting that mLRH-1 could be degraded via proteasome pathway. However, the regulation of mLRH-1 protein stability is largely unknown. In the present study, overexpression of mLRH-1 and mutant type ubiquitin (UbK0) in 293 cells leaded to increased amount of mLRH-1 protein. It is suggested that mLRH-1 might be ubiquitinized, which could affect the mLRH-1 protein stability. We overexpressed mLRH-1 and HA-conjugated ubiquitin (HA-Ub) in 293 cells, co-immunoprecipitation and western blot demonstrated that Flag-mLRH-1, Myc-mLRH-1 and Myc-CA-mLRH-1 bind poly-ubiquitin chain in mammalian cells. Treatment of cells with proteasome inhibitor MG-132 had no effect on the amount of ubiquitin-conjugated mLRH-1 protein complexes, suggesting that the complexes might not related to the proteasomal degradation pathway.
Deletions of the C-terminus of mLRH-1 decrease the amount of ubiquitin-conjugated mLRH-1 protein complexes. The results indicated that the ubiquitin binding site might locate at the C-terminus of mLRH-1. We established stable clones which overexpress Myc-CA-mLRH-1 protein in 293 cells. The activity of CYP11A1 promoter was significantly stimulated in these stable clones compared to the control cells. In addition, we found that mLRH-1 can bind endogenous poly-ubiquitin chain in stable clones. Our results revealed that mLRH-1 poly-ubiquitination may through the C-terminus of mLRH-1 in mammalian cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:18:05Z (GMT). No. of bitstreams: 1 ntu-99-R97441007-1.pdf: 868636 bytes, checksum: 87b7550f29473b565e493a9a1cca68f1 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 誌謝…………………………………………………………I
目錄…………………………………………………………II 圖次…………………………………………………………IV 中文摘要……………………………………………………V 英文摘要 …………………………………………………VI 第一章 序論………………………………………………1 一、LRH-1 簡介……………………………………………1 1. LRH-1 之生理功能………………………………………1 2. LRH-1 之蛋白質結構……………………………………2 3. LRH-1 之輔調控因子 …………………………………3 a. LRH-1 之輔活化子 (coactivator) …………………3 b. LRH-1 之輔抑制子 (corepressor) …………………4 4. LRH-1 之轉譯後修飾作用……………………………5 二、串連親和純化技術 (Mammalian Tandem Affinity Purification, TAP) 簡介…………………………………6 三、泛素 (Ubiquitin)–蛋白酶體 (Proteasome) 系統……………………………………………………………7 四、研究動機………………………………………………8 第二章 材料與方法 ………………………………………9 一、細胞培養………………………………………………9 二、質體 …………………………………………………9 三、暫時性轉染法 (Transient transfection)………11 四、Luciferase 活性分析………………………………12 五、Stable clone 的製備………………………………12 六、西方墨點法 (Western blot)………………………13 七、免疫沉澱法 (Immunoprecipitation)………………14 第三章 結果………………………………………………16 一、建構 TAP-mLRH-1 融合蛋白質之質體 ……………16 二、mLRH-1 可能有泛素化反應因而影響蛋白質的穩定性 ………………………………………………………………16 三、Flag-mLRH-1 和 Myc-mLRH-1 蛋白質可與泛素鏈 (poly-ubiquitin chain) 鍵結 …………………………17 四、mLRH-1 蛋白質的羧基端序列為進行泛素化作用的主要區域 ………………………………………………………………19 五、Myc-CA-mLRH-1 蛋白質可與泛素鏈鍵結 ……………19 六、選殖穩定表現 Myc-CA-mLRH-1 蛋白質的293 stable clone 細胞株 ……………………………………………………………20 七、Myc-CA-mLRH-1 蛋白質在 stable clone 中與泛素之鍵結 ………………………………………………………………21 第四章 討論…………………………………………………22 一、mLRH-1 的泛素化作用…………………………………22 二、mLRH-1 的泛素鏈接合作用……………………………22 三、mLRH-1 的泛素化接合位置……………………………23 四、泛素化作用與 mLRH-1 蛋白質的穩定性……………24 五、泛素化作用的其他功能………………………………25 參考文獻……………………………………………………26 圖……………………………………………………………34 | |
dc.language.iso | zh-TW | |
dc.title | mLRH-1 泛素化作用之探討 | zh_TW |
dc.title | Identification and characterization of mLRH-1 ubiquitination | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐立中(Li-Chung Hsu),張淑芬(Shwu-Fen Chang),李立安(Lih-Ann Li) | |
dc.subject.keyword | 肝受器同源體-1,泛素化作用,泛素鏈, | zh_TW |
dc.subject.keyword | Liver receptor homolog-1,ubiquitination,poly-ubiquitin chain, | en |
dc.relation.page | 43 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-11 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生理學研究所 | zh_TW |
顯示於系所單位: | 生理學科所 |
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ntu-99-1.pdf 目前未授權公開取用 | 848.28 kB | Adobe PDF |
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