臺灣博碩士論文加值系統

Tmem63b 為一個11次穿膜之鈣離子通道蛋白，其功能以及對生物體的影響目前還尚未了解；利用 CRISPR/ Cas9 技術將 Tmem63b 基因剔除所建構的小鼠模式中，其肺部組織切片的型態異常，有類似肺氣腫的現象。利用Biogrid資料庫分析 Tmem63b 之交互作用蛋白，找出 Mucin 1 (Muc1) 此蛋白，其在肺部組織中有高度表現，在肺部受損時會大量表現。由於 Tmem63b基因剃除小鼠之出生率很低，無法驗證先前的發現；另外，利用免疫沉澱之方式確認Tmem63b與Muc1沒有交互作用。為了要探討Tmem63b本身之分子功能與機制，我們利用Co-IP與LC-MS/MS分析了Tmem63b之交互作用蛋白，並利用DAVID找出Tmem63b可能參與在細胞內蛋白質運輸之路徑中。 由VSVG transport assay實驗發現Tmem63b會參與在高基氏體至細胞膜之胞吐作用中。 而由免疫螢光染色發現，Tmem63b會參與在Clathrin-dependent endocytosis中，並且可能會被運送至Lysosome。 另外，Tmem63b會被ADAR2蛋白進行A-to-I之RNA編輯，導致轉譯出的氨基酸由Q轉變成R。 從蛋白質穩定性測試中發現Tmem63b編輯後之形式較不穩定，推測編輯後之蛋白較容易被降解。 利用MG132抑制蛋白酶體活性後發現並不影響蛋白質降解之能力，推測Tmem63b並不是經由蛋白酶體進行降解。 本論文發現了Tmem63b可能是參與在蛋白質運輸中一個重要的調控者。

Tmem63b is a novel transmembrane protein whose biological functions remains largely unknown. The CRISPR/ Cas9 system construct the Tmem63b gene deletion mice, show the abnormal structure in the lung tissue, which have emphysema-like phenotype. The Biogrid database show that Tmem63b is associated with Mucin1, which is as a marker when lung impaired. Because the low birth rate of Tmem63b deletion mice, which cannot repeat our preliminary results. The immunoprecipitation confirm that no interaction between Tmem63b and Muc1. In order to dissect the molecular mechanisms regulated by Tmem63b, we used co-IP and LC-MS/MS to identify the associated proteins with our target protein. Interestingly, several proteins were identified, and further Gene Ontology analysis revealed that those candidate proteins were participated in intracellular protein transport via DAVID functional annotation clustering tool. In VSVG transport assay, knockdown of Tmem63b led to the impaired of the exocytosis form Golgi-to-plasma membrane. To use immunofluorescences, we find that Tmem63b may participate in Clathrin-dependent endocytosis, which may transport to lysosome. Furthermore, Tmem63b have A-to-I RNA editing site and carried out by Adar2. In the protein stability test, we find the edited form of Tmem63b is more unstable and tend to be degraded. To use the proteasome inhibitor, MG132, show that is not affect the protein degradation between Tmem63b reference and edited form. Together, our findings demonstrated that Tmem63b may be a critical regulator in vesicles trafficking.

指導教授推薦書
口試委員會審定書
致謝 iii
中文摘要 iv
Abstract vi
目錄 viii
圖表目錄 x
第一章 緒論 - 1 -
第一部分：探討Tmem63b基因在肺部組織中之功能 - 1 -
1.1.1 TMEM63B 簡介 - 1 -
1.1.2 Mucin 1 簡介 - 3 -
1.1.3 肺氣腫 (Emphysema) 簡介 - 5 -
1.1.4 研究動機 - 5 -
1.1.5 實驗設計 - 6 -
第二部分：探討Tmem63b基因之功能與分子機制 - 9 -
1.2.1 ADAR 簡介 - 9 -
1.2.2 胞吐作用 (Exocytosis) 簡介 - 11 -
1.2.3 內吞作用 (Endocytosis) 簡介 - 12 -
1.2.4 研究動機 - 13 -
1.2.5實驗設計 - 13 -
第二章 材料與方法 - 15 -
2.1 細胞培養 (Cell culture) - 15 -
2.2 細胞轉染 (Cell transfection) - 16 -
2.3 RNA 萃取 (RNA extraction) - 16 -
2.4 反轉錄聚合酶鏈鎖反應 (Reverse Transcription Polymerase Chain Reaction, RT-PCR) - 17 -
2.5 即時定量聚合酶鏈鎖反應 (Quantitative Real-Time PCR) - 17 -
2.6 蛋白質分析 (Protein analysis) - 18 -
2.7 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) - 19 -
2.8 表現載體之建構 (Construction of expression vector) - 19 -
2.9 Tmem63b 抗體製備 (Tmem63b antibody preparation) - 20 -
2.10 免疫沉澱 (Immunoprecipitation) - 21 -
2.11 免疫螢光染色 (Immunofluorescence) - 22 -
2.12 水泡性口炎病毒蛋白G運輸方法(Vesicular stomatitis virus protein G, VSVG Transport Assay) - 22 -
2.13 銀染法 (Silver staining) - 23 -
第三章 實驗結果 - 24 -
第一部分：探討Tmem63b基因在肺部組織中之功能 - 24 -
3.1.1 Tmem63b 專一性抗體測試 - 24 -
3.1.2 Mucin 1 抗體測試 - 25 -
3.1.3 建構 Muc1 HA tag 表現載體 - 26 -
3.1.4 分析小鼠肺組織中Tmem63b與Muc1基因之關係 - 27 -
3.1.5 分析N2A與A549細胞中Tmem63b與Muc1基因之關係 - 27 -
3.1.6 利用慢病毒載體專一性抑制Tmem63b基因 - 28 -
3.1.7 確認TMEM63B與MUC1蛋白間之交互作用 - 28 -
第二部分：探討Tmem63b基因之功能與分子機制 - 29 -
3.2.1 分析TMEM63B在細胞中所扮演之分子功能與機制 - 29 -
3.2.2 探討TMEM63B是否參與在高基氏體至細胞膜(Golgi-to-Plasma membrane)之運輸 - 30 -
3.2.3 探討TMEM63B是否會參與在內吞作用中並分析其路徑 - 31 -
3.2.4 利用Lysotracker 測試TMEM63B是否會運送至Lysosome - 32 -
3.2.5 蛋白質穩定性測試 - 32 -
3.2.6 分析TMEM63B編輯前後蛋白質泛素化之程度 - 33 -
第四章 結果討論 - 35 -
第一部分：探討Tmem63b基因在肺部組織中之功能 - 35 -
第二部分：探討Tmem63b基因之功能與分子機制 - 35 -
4.2.1 TMEM63B參與在細胞膜運輸中的角色 - 35 -
4.2.2 TMEM63B編輯前後蛋白質穩定性有差異 - 37 -
4.2.3 TMEM63B與神經傳導之關聯 - 38 -
4.2.4 TMEM63B在不同組織間表現量不同以及專一性地在腦區被ADAR2編輯對於生物體之生理意義 - 39 -
第五章 文獻 - 40 -
第六章 實驗圖表 - 44 -

 
圖目錄
圖 一、Tmem63b 專一性抗體測試與Tmem63b mRNA表現量分析 - 45 -
圖 二、Mucin 1抗體測試 - 48 -
圖 三、Muc1 HA tag表現載體測試 - 50 -
圖 四、小鼠肺組織中 Tmem63b 與 Muc1 基因間的表現量 - 51 -
圖 五、Tmem63b與Muc1基因在N2A細胞中的表現量 - 52 -
圖 六、TMEM63B與MUC1基因在A549細胞中的表現量 - 53 -
圖 七、利用慢病毒載體在細胞中進行Knockdown之測試 - 54 -
圖 八、確認TMEM63B與MUC1蛋白間之交互作用 - 55 -
圖 九、分析TMEM63B在細胞中所扮演之分子功能 - 57 -
圖 十、TMEM63B與AP2A1蛋白有交互作用 - 59 -
圖 十一、TMEM63B會參與在高基氏體至細胞膜之運輸 - 63 -
圖 十二、TMEM63B會經由clathrin-dependent endocytosis進入細胞 - 67 -
圖 十三、TMEM63B會被運送至Lysosome - 69 -
圖 十四、TMEM63B蛋白質穩定性測試 - 71 -
圖 十五、TMEM63B編輯前後泛素化程度之分析 - 73 -
圖 十六、TMEM63B參與在蛋白質運輸之路徑模式圖 - 74 -

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