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研究生:陳緯學
研究生(外文):Wei-HsuehChen
論文名稱:分析膜穿孔毒素誘發線蟲轉錄因子HLH-30之轉譯後修飾作用
論文名稱(外文):Analysis of the Posttranslational Modification of the Transcription Factor HLH-30 after Pore Forming Toxin Intoxication in C. elegans
指導教授:陳昌熙
指導教授(外文):Chang-Shi Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學暨分子生物學研究所
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:2015
畢業學年度:103
語文別:英文
論文頁數:57
中文關鍵詞:秀麗隱桿線蟲轉譯後修飾作用精氨酸甲基轉移酶
外文關鍵詞:C. elegansHLH-30post-translational modificationprotein arginine methyltransferase
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膜穿孔蛋白毒素是由細菌所分泌製造的,其主要作用於宿主的細胞膜上,宿主為了抵抗膜穿孔毒素,我們發現細胞自噬的機制能夠調控宿主細胞對此毒素的忍受度。過去的研究指出,在哺乳動物中細胞自噬是由轉錄因子TFEB所調控,而在線蟲中則是其同源基因HLH-30所控制。我們發現給予線蟲Cry5B膜穿孔毒素後,其會刺激腸細胞的HLH-30由細胞質進入細胞核的現象,為了瞭解此現象所參與的訊息傳遞路徑,我們利用高通量抑制基因篩選的方式發現抑制蛋白質精胺酸甲基轉移脢(PRMT)此種參與在轉譯後修飾作用的酵素能夠降低由Cry5B所引發HLH-30入核的現象,我們後續以RNA干擾實驗測試了線蟲所有的PRMT並確認其對於HLH-30入核的影響。此外我們也發現prmt突變株同樣可以觀察到抑制HLH-30的入核,而其中prmt-7突變株若曝處於Cry5B時,壽命有明顯的縮短,因此,我們接著觀察此突變株體內細胞自噬的現象是否有所改變,發現細胞自噬的生物標記LGG-1的表現量相較於野生種N2線蟲有顯著下降的情況。綜合以上結果,我們證實了當線蟲受到Cry5B膜穿孔毒素攻擊時,prmt-7會藉由轉譯後修飾作用調控HLH-30入核並啟動細胞自噬以提供宿主免於膜穿孔毒素毒殺的保護機制。
Pore-forming toxins (PFTs) secreted by bacteria can damage the plasma membrane of their target host cells. In order to defend PFT, autophagy plays an essential role in controlling the tolarance of host cells. According to recent research, autophagy in mammals is regulated by the transcription factor EB (TFEB) and the C. elegans orthologue HLH-30 is also required for auphagy regulation. Our previous study demonstrated that HLH-30 translocates from the cytosol into the nucleus of the intestinal cells of C. elegans after Cry5B-PFT intoxication. In this study, we performed a high-throughput genetic suppressor screen to identify novel signaling pathways that control HLH-30 nuclear localization induced by Cry5B-PFT. We found that prmt (protein arginine methyltransferases) RNAi significantly abolished the Cry5B-induced HLH-30 nuclear localization. Thus, our data suggested that the PRMT protein family may participate in the regulation of HLH-30 to activate autophagy. In order to reconfirm whether and which PRMT is involved in HLH-30 nuclear localization, we tested RNAi of all five CePRMTs in C. elegans in the liquid and agar-based assays. Even though there were different efficiency in the inhibiton of nuclear localization, all five CePRMTs can regulate HLH-30 nuclearlization. Furthermore, the phenomena are also comfirmed in the five prmt mutant animals. We found that HLH-30 nuclear localization and the expression of LGG-1, a downstream gene of HLH-30, were significantly decreased in the prmt-7 mutant animal. Taken all together, these results demonstrated that prmt-7 could regulate the activity of HLH-30 by posttranslational modification, i.e.-arginine methylation to control autophagy in response to pore-forming toxin intoxification in C. elegans.
中文摘要 II
Abstract III
誌謝 V
Introduction 1
Materials and Methods 5
Nematode strains 5
Maintenance of C. elegans strains 5
Bacterial strains 6
Bacterial killing assays 6
C. elegans autophagy and HLH-30 nuclear localization analysis and microscopy 7
Genetic suppressor screen and analysis 8
Generation of hlh-30::gfp; prmt and lgg-1::gfp; prmt worms 9
Real-time quantitative RT-PCR 10
Data analysis 11
Results 12
Pore-forming toxin Cry5B actives autophagy through transcriptional regulation 12
Transcription factor HLH-30 regulates autophagy-related genes to support defense of C. elegans against Cry5B 13
Transcription factor HLH-30 may be regulated by posttranscriptional modification 15
Protein arginine methyltransferases regulate HLH-30 nuclear localization and were involved in Cry5B-acvivated defense 17
PRMT-7 regulates the Cry5B-induced HLH-30 nuclear localization and autophagy to defense against Cry5B intoxication 19
Discussion 21
Reference 25
Figures 31
Figure 1 31
Figure 2 32
Figure 3 34
Figure 4 36
Figure 5 37
Figure 6 38
Figure 7 39
Figure 8 41
Figure 9 43
Figure 10 44
Figure 11 45
Figure 12 47
Figure 13 49
Figure 14 51
Figure 15 53
Figure 16 55
Figure 17 56
Figure 18 57

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