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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52823完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 湯志永 | |
| dc.contributor.author | Hsin-Yu Fang | en |
| dc.contributor.author | 方心妤 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:29:16Z | - |
| dc.date.available | 2025-12-31 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-13 | |
| dc.identifier.citation | Aguilar RC, Wendland B (2003) Ubiquitin: not just for proteasomes anymore. Current opinion in cell biology 15:184-190.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52823 | - |
| dc.description.abstract | CLC-2為一種廣泛存在於各種組織的氯離子通道，主要受到膜電位過極化、細胞體積增加、胞內氯離子濃度增加與胞外酸性微量增加等因素調控而活化；但是，CLC-2在細胞中確切的分子調控機轉則仍有待釐清。先前研究指出，cereblon (CRBN)是CLC-2的結合蛋白。近來的研究亦發現CRBN可能是cullin RING E3泛素連接酶複合體 (E3 ubiquitin ligase complex) (CRL)中的受體蛋白受器 (substrate receptor)。然而CRBN是否的確參與CLC-2的蛋白質降解作用，則尚不清楚。另一方面，本實驗室過去利用酵母菌雙雜交技術 (yeast-two hybrid screening)發現NSF是能與CLC-2交互作用的蛋白。本論文的研究方向分為二個主軸，其一是針對CLC-2的降解途徑進行探討，其二則是觀察抑制NSF對於CLC-2蛋白表現量的影響。
我們先以MG132和MLN4924這二種藥物作用的結果推論CLC-2可能是經由CRL進行蛋白酶體降解，我們接著以生化實驗發現CLC-2會與Cullin 4 (CUL4)、DDB1和CRBN存在於同一蛋白質複合體 (protein complex)。我們也證明CUL4和CRBN都會參與CLC-2的降解作用。此外，我們還以只能進行單一泛素 (monoubiquitination)作用的突變種泛素Ub-K0證明CLC-2的降解機制應該是以聚泛素鏈的形式進行泛素標定。另一方面，我們發現若對NSF進行shRNA抑制，雖然不會影響CLC-2的mRNA表現量，但卻造成CLC-2蛋白總量的增加。 | zh_TW |
| dc.description.abstract | CLC-2 is a ubiquitously expressed chloride channel, which is mainly activated by membrane potential hyperpolarization, increased cell volume, elevated intracellular chloride concentration and mild extracellular acidification; however, the precise cellular regulatory properties of CLC-2 channel still remain elusive. In recent studies, Cereblon (CRBN) has been considered to be a binding partner of CLC-2. CRBN has also been suggested to play a role as a substrate receptor of the cullin RING E3 ubiquitin ligase complex (CRL). Nevertheless, it is unclear whether CRBN is involved in the protein degradation of CLC-2. In addition, previous studies in our lab utilizing yeast-two hybrid screening of rat brain cDNA library has identified NSF as a CLC-2 interacting protein. Therefore, there are two aims in this study. One aim is to understand the degradation pathway of CLC-2, and the other aim is to observe the effect on CLC-2 protein expression level caused by NSF shRNA knockdown.
Firstly, we reason that the proteasomal degradation of CLC-2 can be mediated by CRL according to effects caused by MG132 and MLN4924 treatments. We then find out the fact that CLC-2 can coexist in the same protein complex with Cullin 4 (CUL4), DDB1 and CRBN based on our biochemical experimental results; moreover, we also prove that both CUL4 and CRBN do play a role in the degradation of CLC-2. By utilizing an ubiquitin mutant, Ub-K0, which can only undergo monoubiquitination, we further prove that CLC-2 shall be ubiquitinated through polyubiquitin chains in its degradation mechanism. Besides, we have also noted that NSF shRNA knockdown results in an increased CLC-2 total protein level. In contrast, the mRNA level of CLC-2 does not be affected by NSF shRNA knockdown. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:29:16Z (GMT). No. of bitstreams: 1 ntu-104-R01441006-1.pdf: 3556827 bytes, checksum: 4d91b71d69fd9e26b4657e805f048c1a (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 中文摘要 1
Abstract 2 附圖目錄 8 第一章 9 導論 9 1.1氯離子通道 (Chloride channels) 9 1.2 CLC-2所參與之生理功能 10 1.3 CLC-2的結構與特性 12 1.4 與CLC-2可能相關的疾病 16 1.5 可能影響CLC-2蛋白表現量或通道功能的蛋白分子 17 1.5.1 影響CLC-2蛋白表現量的其他蛋白分子 17 1.5.2 影響CLC-2通道功能表現的其他蛋白分子 19 1.5.3 其他與CLC-2具交互作用但影響未明的蛋白分子 20 1.6 蛋白質的降解 (Protein degradation) 21 1.7 泛素與蛋白酶體系統 (Ubiquitin and proteasome system) 21 1.8 E3泛素連接酶 (E3 ubiquitin ligase) 23 1.9 研究目的 24 第二章 25 材料與方法 25 2.1 DNA構築質體 (DNA constructs) 25 2.2 細胞培養 (cell culture) 26 2.3 DNA磷酸鈣轉染 (DNA calcium phosphate transfection) 26 2.4 蛋白樣品製備與西方墨點法 (Preparation of protein samples and western blot) 27 2.5 共同免疫沉澱 (Co-immunoprecepitation) 29 2.6 細胞表面生物素標定 (Biotinylation) 30 2.7 Cycloheximide細胞處理 (Cycloheximide treatment) 30 2.8 MLN4924細胞處理 (MLN4924 treatment) 31 2.9 17-AAG細胞處理 (17-AAG treatment) 31 2.10 慢病毒製備與感染 (Lentivirus production and infection) 32 2.11 CLC-2 stable cell line製備 (CLC-2 stable cell line production) 33 2.12 RNA萃取與逆轉錄PCR (RNA extraction and RT-PCR) 33 2.13資料統計分析 (Data analysis) 36 第三章 37 結果 37 3.1 CLC-2的降解途徑探討 37 3.1.1 MG132、HA-Ub-K0與MLN4924使CLC-2蛋白表現總量增加 37 3.1.2 CUL4可能參與CLC-2之蛋白酶體降解之調控 38 3.1.3 DDB1與CLC-2具交互作用 40 3.1.5 CRBN可能扮演CLC-2之substrate receptor 42 3.1.6 DN-CUL4A與DN-CUL4B並未影響CLC-2之代謝半衰期 42 3.1.7 HA-Ub-K0、Flag-DN-CUL4A、Flag-DN-CUL4B使CLC-2在細胞膜上表現量增加 43 3.1.8 HA-CRBN使CLC-2在細胞膜上表現量減少 44 3.1.9 17-AAG使CLC-2蛋白總量增加 44 3.1.10 Caco-2細胞株具內生性CLC-2蛋白表現 45 3.2 抑制NSF對於CLC-2的影響 46 3.2.1 CLC-2與NSF具交互作用 46 3.2.2 大量表現NSF對CLC-2蛋白總量與其在細胞膜上表現量均無影響 (含前人研究) 46 3.2.3 以shRNA壓制NSF對CLC-2 mRNA與蛋白總量表現的影響 47 第四章 50 討論 50 4.1 蛋白酶體降解途徑調控CLC-2之蛋白表現量 50 4.2 CUL4A與CUL4B參與CLC-2之蛋白酶體降解途徑 51 4.3 DDB1扮演CLC-2蛋白酶體降解途徑之adaptor protein 57 4.4 CRBN扮演CLC-2蛋白酶體降解途徑之substrate receptor 57 4.5 Hsp90 抑制劑17-AAG提升CLC-2之蛋白質表現量 59 4.6 抑制NSF對於CLC-2的影響 60 4.7 待釐清之問題以及後續的實驗設計 61 4.7.1 缺少ubiquitination實驗故無法直接證明CUL4A/ 4B與CRBN可參與CLC-2之泛素化 61 4.7.2 缺少壓制CUL4A/ 4B、CRBN的實驗數據 61 4.7.3 CLC-2的聚泛素鏈鍵結型式尚待驗證 62 4.7.4 需在stable cell line中壓制NSF並重複CLC-2之RT-PCR實驗 62 4.7.5 進行shRNA壓制實驗時需控制各系統中的CLC-2表達效率差異 62 結論 63 圖表 64 附圖 92 參考資料 98 | |
| dc.language.iso | zh-TW | |
| dc.title | E3泛素連接酶與NSF蛋白對第二型氯離子通道的調節機制 | zh_TW |
| dc.title | Modulation of CLC-2 chloride channel by E3 ubiquitin ligase and NSF | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭瓊娟,卓貴美 | |
| dc.subject.keyword | CLC-2氯離子孔道,蛋白質降解,E3泛素連接?,NSF, | zh_TW |
| dc.subject.keyword | CLC-2 chloride channel,protein degradation,E3 ubiquitin ligase,NSF, | en |
| dc.relation.page | 115 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-13 | |
| 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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