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研究生： 張欣慈
Chang, Hsin-Tzu
論文名稱： 探討usp-46基因在細菌穿孔蛋白誘發線蟲HLH-30/TFEB入核時所扮演的角色
The function of usp-46 in the bacterial pore-forming toxin induced HLH-30/TFEB nuclear localization 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： 2019
畢業學年度： 107
語文別： 英文
論文頁數： 50
中文關鍵詞： 秀麗隱桿線蟲 、膜穿孔蛋白 、TFEB/HLH-30轉錄因子 、自嗜作用
外文關鍵詞： Caenorhabditis elegans, pore-forming toxin (PFT), transcription factor TFEB/HLH-30, autophagy
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    • 膜穿孔蛋白毒素是病原細菌重要的毒力因子，當細菌感染宿主後，宿主為了抵抗細菌所分泌的膜穿孔蛋白，會產生許多機制來防禦膜穿孔蛋白的毒害，其中包含細胞自嗜作用。在過去實驗室的研究中發現當秀麗隱桿線蟲受到膜穿孔蛋白感染時，線蟲腸道細胞內的HLH-30轉錄因子(在哺乳類動物當中為TFEB)會入核調控細胞自嗜作用基因來抵禦膜穿孔蛋白Cry5B所造成的毒害，而在其他文獻中也有提到mTOR會將TFEB/HLH-30磷酸化，然後抑制TFEB/HLH-30入核。然而對於HLH-30入核的機制目前仍有許多未解之謎。為了找到調控HLH-30入核的新因子，我們利用高通量篩選找到wdr-20基因可能是線蟲受到Cry5B毒害時調控HLH-30入核的重要基因。在我們的研究中證實線蟲WDR-20, WDR-48, USP-46所形成的複合物可以調控HLH-30入核並促進細胞自嗜作用來抵抗Cry5B的毒害，並且在受到Cry5B毒害時USP-46會在細胞質對HLH-30去除其泛素化修飾，進而促進HLH-30入核，不僅如此USP-46還會與HLH-30一同入核停留在細胞核中，而且USP-46也參與在mTOR調控路徑當中。總結我們的發現，受到膜穿孔蛋白感染時，USP-46是一個重要的因子調控HLH-30入核以及停留在核內，藉此促進自嗜作用抵抗膜穿孔蛋白的毒害。本篇研究也提供了對於穿孔蛋白感染時產生的機制，並且為未來藥物治療提出貢獻。

    Pore forming toxins (PFTs) are important virulence factors of many bacterial pathogens. To defense PFT intoxication, host have evolved some conserved mechanisms, one of which is autophagy. In our previous study, we demonstrated that the transcription factor HLH-30, the ortholog of mammalian TFEB, can be activated and translocated into nucleus of intestinal cells in C. elegans and mediates autophagy activation to defend PFT intoxication. Furthermore, it has been reported that phosphorylation of TFEB/HLH-30 by mTOR inhibits its nuclear localization. However, the detailed mechanisms involved in the PFT-induced HLH-30 nuclear localization remained largely unknown. In our study, we aim to find novel host factors regulate HLH-30 nuclear localization by genetic suppressor screen. From the screen, we identified wdr-20 as an essential gene for the Cry5B-PFT induced HLH-30 nuclear localization. Our data demonstrated that the WDR-20, WDR-48 and USP-46 may form a complex to regulate the nuclear localization of HLH-30 and to induce autophagy to defend Cry5B intoxication in C. elegans. USP-46 induced HLH-30 nuclear localization by mediating deubiquitination in cytosol, it also colocalized with HLH-30 and kept in nucleus in Cry5B intoxication. Moreover, USP-46 may regulate HLH-30 translocate into nucleus and autophagy activation by a mTOR-dependent manner. In conclusion, we find that USP-46 plays an important role in the PFT-induced HLH-30 nuclear localization and promotes autophagy to defend PFT intoxication. Thus we provide mechanistic insights into the pathogenesis of PFT, as well as potential therapeutic treatment.

    致謝 i 中文摘要 ii Abstract iii Content iv Introduction 1 Pore-forming toxins 1 Autophagy 2 HLH-30/TFEB 3 Ubiquitin-specific proteases 46 (USP-46) 4 Materials and methods 6 C. elegans strains 6 Bacterial strains 7 Media and chemicals 7 DNA microinjection 7 RNA interference (RNAi) 8 Fluorescent microscopy image 8 C. elegans autophagy analysis 9 Pore-repair assay 9 Lifespan assays 10 Worm lysis, Immunoblotting, and Immunoprecipitation 10 Data analysis 11 Results 12 The complex of WDR-20, WDR-48 and USP-46 regulated the nuclear localization of HLH-30 and induced autophagy and pore- repair activity to defend Cry5B-PFT intoxication in C. elegans 12 USP-46 plays an important role in regulating the cellular localization of HLH-30 by deubiquitinating 14 USP-46 induced HLH-30 nuclear localization in cytosol, moreover it also kept HLH-30 in nucleus in Cry5B intoxication 15 LET-363/mTOR signaling pathway also involved in HLH-30 nuclear localization and induced autophagy and pore-repair activity in Cry5B intoxication 17 USP-46 might involve in LET-363/mTOR signaling pathway to induce HLH-30 nuclear localization 18 Discussion 20 Figures 23 Fig. 1 Cry5B induced HLH-30::GFP translocate into nucleus of intestinal cells in C. elegans 23 Fig. 2 HLH-30 nuclear localization in the wdr-20, wdr-48, usp-46 mutant animals 24 Fig. 3 The complex of wdr-20, wdr-48, usp-46 regulated HLH-30 nuclear localization 25 Fig. 4 Cry5B induced autophagy activity in C. elegans 26 Fig. 5 Autophagy activity in the wdr-20, wdr-48, usp-46 mutant animals 27 Fig. 6 Pore-repaired assay in the Cry5B intoxicated worms 28 Fig. 7 wdr-20, wdr-48 and usp-46 contribute to membrane pore-repair in Cry5B intoxication 29 Fig. 8 wdr-20, wdr-48, usp-46 against Cry5B intoxication 31 Fig. 9 usp-46 plays an important role in regulating the cellular localization of HLH-30 32 Fig. 10 The ubiquitination of HLH-30 was reduced in Cry5B intoxication 33 Fig. 11 usp-46 induced HLH‐30 nuclear localization in cytosol in Cry5B intoxication 34 Fig. 12 usp-46 kept HLH-30 in nucleus by Cry5B intoxication 35 Fig. 13 usp-46 kept HLH-30 in nucleus by Cry5B intoxication 36 Fig. 14 USP-46 colocalized with HLH-30 in nucleus in Cry5B intoxication 38 Fig. 15 Analyzing confocal image of Fig. 14 39 Fig. 16 Let-363/mTOR signaling pathway involved in HLH-30 nuclear localization and induced autophagy and pore- repair activity in Cry5B intoxication 41 Fig. 17 usp-46 is epistatic to let-363 in Cry5B induced-HLH-30 nuclear localization 43 Fig. 18 ftt-2 is epistatic to usp-46 in Cry5B induced-HLH-30 nuclear localization 44 Fig. 19 Model of regulating HLH-30/TFEB nuclear localization by WDR-20/WDR-48/USP-46 complex to against Cry5B 45 Fig. 20 The blast result of HLH-30 and TFEB 46 Reference 47

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