臺灣博碩士論文加值系統

在細胞受到ER stress時，C/EBP homologous protein (CHOP) 對於細胞存活與凋亡扮演重要的角色，而chop mRNA的轉譯主要受到其5’UTR上的upstream open reading frame (uORFchop) 所抑制。當細胞受到ER stress時，chop mRNA會因為uORFchop抑制功能喪失而被轉譯出來。但對於ER stress造成uORFchop喪失轉譯抑制能力的分子機制仍不清楚。本實驗室利用由CMV promoter驅動下游帶有人類uORFchop (huORFchop)及綠色螢光蛋白(green fluorescent protein,GFP) cDNA之基因構築的轉殖斑馬魚品系huORFZ當材料，因為當它受到ER stress時，便會引發huORFchop功能喪失而促使GFP表現。我們利用雷射顯微切割器(Laser microdissection) 的方式收集逆境後表現於huORFZ的GFP-(+) 細胞，接著以GFP-(-) 細胞作為比較基礎，建立microarray資料庫。當中，我們篩選到一個A值為7.2 (A值大於7表示可信度高) 及M值為2.4 (M值大於0表示強度高)的dkey基因。從全胚胎原位雜交染色(WISH)觀察斑馬魚96 hpf胚胎，發現內生性dkey mRNA在腦部與脊隨表現且經過40℃熱處理後會增量，表示dkey mRNA表現量的變化與microarray的結果吻合。為了進一步證實dkey在huORFchop擔任轉譯抑制的角色，首先在in vivo斑馬魚胚胎中注射huORFchop-luciferase及dkey DNA，發現luciferase activity會上升，表示dkey會壓制huORFchop的轉譯抑制能力。接著，為了證實chop mRNA轉譯會受dkey的影響，利用西方浸漬法得知dkey造成huORFchop轉譯抑制能力的喪失是透過eIF2α磷酸化的上升而使CHOP蛋白質的表現量增加。進一步在in vitro中同樣進行luciferase assay及西方浸漬法也得到相同的結果。接著在in vitro中證實非磷酸化eIF2α(eIF2α /S51A) 會造成dkey降低對於huORFchop轉譯抑制喪失的能力；反之持續磷酸化的eIF2α(eIF2α /S51D) 則會促進dkey對於huORFchop轉譯抑制喪失的能力，確定dkey對於huORFchop轉譯抑制喪失的能力受到eIF2α磷酸化狀態所影響。我們也透過免疫共沉澱法(Co-immunoprecipitation) 得知dkey能與eIF2α蛋白質結合。為了確定dkey對於huORFchop轉譯抑制的喪失是透過其endoribonucleases的能力，我們同時轉染dkey DNA及huORFchop-GFP DNA到HEK293T細胞株，藉由北方浸漬法的方式並且以GFP-probe偵測會造成huORFchop-GFP RNA進行RNA的切割。綜合上述實驗結果，本研究發現磷酸化eIF2α造成dkey對於huORFchop喪失轉譯抑制的能力使得CHOP蛋白質的表現量增加，另外也證實DKEY蛋白質能與eIF2α蛋白質結合。所以，我們認為dkey可能透過與eIF2α的結合而找到huORFchop的位置，然後以endoribonucleases的能力，破壞huORFchop的結構促使其轉譯抑制的喪失，而造成CHOP蛋白質的表現。

In response of endoplasmic reticulum (ER) stress, C/EBP homologous protein (CHOP) is critically involved in either cell survival or apoptosis. It has been reported that the 5’UTR of chop mRNA contains an upstream open reading frame (uORFchop) which inhibits its translation. During ER stress, the uORFchop–mediated translational inhibition is abolished, resulting in generating CHOP protein. However, underlying molecular mechanisms of uORFchop -mediated translational inhibition is not fully understood. To answer this issue, we employed the zebrafish transgenic line huORFZ, which harbors the GFP reporter fused with the human uORFchop (huORFchop) and driven by a cytomegalovirus promoter. Interestingly, GFP was expressed only when huORFZ embryos were treated with ER stresses. The number of GFP(+) cells in the brain of huORFZ embryos was dependent on the duration of heat-treatment. Taking advantage of Laser Microdissection, we collected the neuronal cells expressing GFP from the brain of heat-shocked huORFZ embryos to perform microarray analysis using GFP-negative neuronal cells as a background. Among the putative genes, dkey was selected for further study because it was one of the most up-regulated genes presented in GFP(+) cells, whose A value was 7.2 (A value greater than 7 indicates high reliability) and M value was 2.4 (M value greater than 0 indicates high intensity). Whole mount in situ hybridization demonstrated that dkey transcripts were expressed in the brain and spinal cord of embryos at 96 hpf. The expressional level of dkey was greatly increased when embryos were treated with 40℃,which was corresponding with the results obtained from microarray. To confirm whether dkey plays role on the huORFchop-mediated translational inhibition, we injected a DNA construct of huORFchop-luciferase and a DNA fragment of dkey into zebrafish embryos. Results showed that the luciferase activity of the injected embryos was increased, suggesting that overexpression of dkey did suppress the huORFchop –translational inhibition in vivo. Furthermore, Western blot analysis revealed that overexpressive dkey in embryos increased the protein levels of phosphorylated eIF2α (p-eIF2α) and CHOP. Results obtained from in vitro studies of luciferase assay and Western blot analysis were correspondent with those of in vivo study. We also demonstrated that increase of the non-phosphorylated mutant of eIF2α (eIF2α/S51A) in cells reduced the suppressive capability of dkey on the huORFchop - translational inhibition in vitro. In contrast, increase of the phosphorylated eIF2α (eIF2α/S51D) enhanced the suppressive capability of dkey on the huORFchop -mediated translational inhibition. Furthermore, co-immunoprecipitation revealed that Dkey protein was able to interact with eIF2α. Thus, we concluded that eIF2α and Dkey interact to control the huORFchop translational inhibition. Interestingly, Northern blot exhibited that Dkey could digest the huORFchop -tagged mRNA, suggesting that the endoribonucleases activity of Dkey may function in huORFchop -mediated translational inhibition. Collectively, we suggested that (1) Dkey is effectively involved in the huORFchop -mediated translational inhibition through the increase of p-eIF2α; and (2) Dkey may combine with eIF2α to locate the binding site of huORFchop, resulting in altering the huORFchop structure through its endoribonucleases ability, which in turn, the huORFchop –mediated translational inhibition is abolished, and CHOP is translated during ER stress.

中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………3
文獻回顧………………………………………………………………………………5
前言……………………………………………………………………………………18
實驗材料與方法………………………………………………………………………20
結果……………………………………………………………………………………29
討論……………………………………………………………………………………40
參考文獻………………………………………………………………………………47
圖說……………………………………………………………………………………54
附錄……………………………………………………………………………………65

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