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| 研究生： |
林依穎 Lin, I-Yin |
|---|---|
| 論文名稱： |
探討 prmt-7 基因在膜穿孔毒素誘發線蟲HLH-30入核及活化自噬作用時所扮演的角色 Characterization of the Role of prmt-7 in Pore-Forming Toxin Induced HLH-30 Nuclear Localization and Autophagy Activation in C. elegans |
| 指導教授： |
陳昌熙
Chen, Chang-Shi |
| 學位類別： |
碩士 Master |
| 系所名稱： |
醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology |
| 論文出版年： | 2017 |
| 畢業學年度： | 105 |
| 語文別： | 英文 |
| 論文頁數： | 74 |
| 中文關鍵詞： | 自噬作用 、秀麗隱桿線蟲 、膜穿孔蛋白毒素 、轉譯後修飾 |
| 外文關鍵詞： | autophagy, Caenorhabditis elegans, HLH-30/TFEB, pore-forming toxin (PFT), post-translation modification(PRMT), mTOR pathway |
| 相關次數： | 點閱：153 下載：7 |
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膜穿孔蛋白毒素其主要作用於宿主的細胞膜上竟而造成宿主死亡，在許多致病菌中都有其毒理因子。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，我們也證實給予線蟲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，進而活化自噬作用並抵抗膜穿孔毒素，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)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，我們後續實驗測試了線蟲所有的PRMT並確認其對於HLH-30入核的影響，而其中prmt-7突變株若曝處於Cry5B時，HLH-30入核的現象表現下降。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。在先前的文獻也已知HLH-30會調控自噬反應流、孔洞修復及對Cry5B的防禦，結果證實prmt-7確實會影響。此外，我們也發現對Cry5B敏感並不是因生理特性改變造成。同時我們觀察帶有prmt-7螢光轉綠的線蟲給予膜穿孔蛋白毒素Cry5B後其螢光表現增加並表現在腸道，在反轉錄PCR實驗中也證實prmt-7 參與調控自噬作用。最後，我們也找到了prmt-7是參與在mTOR這條傳遞路徑的下游來調控HLH-30入核的重要角色。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。
The pore-forming toxins (PFTs) that damage the integrity of the plasma membrane of target cells are essential for the pathogenesis of numerous disease-causing bacteria. In order to defend PFT, autophagy plays essential roles in controlling the tolerance of host cells. According to recent research, the C. elegans transcription factor HLH-30, an ortholog of the mammalian transcription factor EB (TFEB), can regulate this PFT-induced autophagy. The Cry5B-PFT can induce the nuclear translocation of the HLH-30, and this event is required for the cellular defense against Cry5B intoxication. Our previous study demonstrated that HLH-30 translocated into the nucleus of the intestinal cells of C. elegans and autophagy activation after Cry5B-PFT intoxication. In this study, we performed a genetic suppression screen to identify novel factors that control HLH-30 nuclear localization induced by Cry5B-PFT. We found Cry5B-induced HLH-30 nuclear localization was significantly abolished in prmt-7 (protein arginine methyltransferase 7) mutant strains, the suggested that PRMT-7 may participate in the regulation of HLH-30 to activate autophagy. PRMTs functioned to transfer methyl groups to arginine residues of their substrate proteins. Additionally, the prmt-7 loss-of-function mutation significantly abolished HLH-30 nuclear localization and cellular LGG-1 multi-puncta formation, a biomarker for autophagy activation. Additionally, we demonstrate prmt-7 can regulated autophagy flux, pore repair and Cry5B defense that was regulated by HLH-30. And we found prmt-7 signals were increased when treated Cry5B in prmt-7 transcription reporter strains and expression on intestines. In the RT-PCR analysis, we suggested prmt-7 was required to activate autophagy when treading Cry5B. And the wild-type N2 treated Cry5B the expression of prmt-7 have the significant increase. Furthermore, we found that prmt-7 acted downstream to mTOR pathway and involved in the Cry5B induced HLH-30 nuclear. Taken all together, our current data suggested that prmt-7 arginine methylation, a posttranslational modification, of HLH-30 may involve in HLH-30 cellular localization and autophagy activation in C. elegans.
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