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研究生:張惠宥
研究生(外文):Wen Chun Chang
論文名稱:探討 RTN3 蛋白分子在 mTOR 訊號傳遞中扮演的角色
論文名稱(外文):Biological Functions of RTN3 in mTOR Signaling Transduction
指導教授:洪錦堂洪錦堂引用關係
指導教授(外文):Jim Tong Horng
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:84
中文關鍵詞:RTN3蛋白分子AKT蛋白分子細胞自噬轉譯細胞增生
外文關鍵詞:Reticlon 3AKTAutophagyTranslationProliferation
相關次數:
  • 被引用被引用:0
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  • 下載下載:2
  • 收藏至我的研究室書目清單書目收藏:0
Reticulons (RTNs),是一種只存於真核細胞生物中、龐大、有多樣性且會與內質網 (endoplasmic reticulum, ER) 結合並影響其構形的蛋白質。所有的RTNs 在其C-terminal 都有一段具高度保留性的reticulon homology domain (RHD),此段區域對於RTNs 的分布以及多樣性的功能扮演重要的角色。目前已知RTNs 與細胞內物質的運輸、細胞凋亡、細胞自噬 (autophagy) 和阿茲海默症有關。近來也有文獻指出,RTN4A (Nogo-A) 中的Nogo 區域可以透過降低AKT(S473) 磷酸化 而抑制神經軸突的生長。
本篇研究發現,同樣具Nogo 區域的Reticulons 3 (RTN3) 確實會抑制AKT (S473) 以及4EBP1(T37/46) 的磷酸化,最後抑制細胞內的轉譯以及增生。此外,我們也發現當RTN3 蛋白減少時,會促進autophagy 形成相關的ULK1 (S555) 磷酸化上升,藉由增進Vps34與Beclin 1 蛋白的交互作用,加強autophagy nucleation,最後促使autophagy 產生。在此,我們證實RTN3 對於細胞內的轉錄及autophagy 的形成中扮演重要的角色。
The reticulons (RTNs) are a family of morphogenic, endoplasmic reticulum (ER) membrane-shaping proteins that stabilize highly curved ER membrane tubules. They are all characterized by the conserved reticulon homology domain (RHD) that is located at the C-terminal and is highly crucial for localization and function of the protein. RTNs are
related to membrane trafficking, neuron regeneration, apoptosis and autophagy. The Nogo domaino of RHD in RTN4A (Nogo-A) can inhibit nurite outgrowth by reducing the phosphorylation of AKT at Ser473.
Here we demonstrated that the Nogo region of RTN3 inhibited phosphorylation of AKT at Ser473 and suppressed mammalian target of rapamycin complex 1 (mTORC1) downstream substrate 4EBP1 (T37/46) phosphorylation that might suppress translation in mammalian cells. In addition, we investigate whether RTN3 also involves autophagy process.
The results indicated knockdown of RTN3 could increase autophagy through phosphorylation of uncoordinated-51 like kinase 1 (ULK1) at Ser555 and then enhanced autophagy nucleation that might induce Vps34 complex activity directly. These results suggested that RTN3 plays an important role in cellular translation and autophagy process.
指導教授推薦書
口試委員審定書
誌謝 iii
縮寫表 iv
中文摘要 vii
Abstract viii
目 錄 ix
圖目錄 xii
第一章 前言 - 1 -
1.1 Reticulon family - 1 -
1.2 Mammalian target of rapamycin (mTOR) pathway - 3 -
1.3 Autophagy - 8 -
1.4 ULK - 13 -
1.5 Cell cycle - 16 -
第二章 研究目的 - 18 -
第三章 材料與方法 - 19 -
3.1 溶液配製 - 19 -
3.2 細胞培養 - 20 -
3.3 利用shRNA建立RTN3蛋白表現量低的肝臟細胞株 - 20 -
3.4 質體與抗體 - 21 -
3.5 西方點墨法 (Western blot) - 22 -
3.6 免疫螢光染色 - 23 -
3.7 Turnover of LC3-II - 23 -
3.8 免疫共同沉澱 - 24 -
3.9 定量反轉錄聚合酶連鎖反應 - 25 -
3.10 Reporter assay - 26 -
3.11 細胞週期同步化 (Synchronization of cell cycle) - 26 -
3.12 流式細胞技術 - 27 -
第四章 結果 - 28 -
4.1 RTN3 Nogo區域可抑制AKT S473的磷酸化 - 28 -
4.2 RTN3在mTOR pathway訊號傳遞中所扮演的角色 - 28 -
4.3 RTN3 Nogo區域透過4EBP1 (T37/46) 影響轉譯及增生 - 30 -
4.4 大量表現RTN3蛋白使細胞週期停滯於G1 phase - 31 -
4.5 降低RTN3蛋白表現量可活化ULK1 (S555) 磷酸化促進autophagy進行- 32 -
4.6 在RTN3蛋白表現低時促進autophagy nucleation透過加強Vps34、Atg14和Beclin 1蛋白之間的交互作用 - 35 -
第五章 討論 - 36 -
RTN3蛋白的Nogo區域可抑制AKT (S473) 的磷酸化 - 36 -
RTN3在mTOR pathway訊號傳遞中所扮演的角色 - 37 -
RTN3 Nogo區域透過4EBP1 (T37/46) 影響細胞轉譯及增生 - 37 -
大量表現RTN3蛋白使細胞週期停滯於G1 phase - 38 -
降低RTN3蛋白表現量可活化ULK1 (S555) 磷酸化促進autophagy進行 - 39 -
在RTN3蛋白表現低時促進autophagy nucleation透過加強Vps34、Atg14和Beclin 1蛋白之間的交互作用 - 41 -
第六章 圖表 - 43 -
附錄1 - 53 -
附錄2 - 54 -
附錄3 - 55 -
附錄4 - 56 -
參考文獻 - 57 -

圖目錄
Fig. 1 AKT (S473) 磷酸化受 RTN3 Nogo 區域抑制.................... -43-
Fig. 2 RTN3 蛋白對 mTOR pathway 訊號傳遞的影響................ -44-
Fig. 3 細胞轉譯和增生受到 RTN3 Nogo 區域的抑制.................. -45-
Fig. 4 大量表現 RTN3 蛋白使細胞週期停滯於 G1 phase............ -47-
Fig. 5 RTN3 蛋白可透過 ULK1 影響 autophagy進行 ................ -49
Fig. 6 RTN3 調控 autophagy nucleation藉由 Vps34、Atg14及 Beclin 1 之間的交互作用.....-52-
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