研究生: |
林叔樺 Lin, Shu-Hua |
---|---|
論文名稱: |
探討hpo-22基因在Cry5B誘發線蟲HLH-30入核及自噬作用時所扮演的角色 Characterization of hpo-22 in Cry5B 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: | 58 |
中文關鍵詞: | 自噬作用 、HLH-30/TFEB 、膜穿孔蛋白毒素 、轉譯後修飾 、mTOR訊息傳遞路徑 、秀麗隱桿線蟲 |
外文關鍵詞: | autophagy, HLH-30/TFEB, pore-forming toxin (PFT), post-translation, mTOR pathway, Caenorhabditis elegans |
相關次數: | 點閱:401 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
膜穿孔蛋白毒素已知對於許多致病菌是非常重要的毒理因子。在本研究當中,我們利用由Bacillus thurgiensis所製造的膜穿孔蛋白毒素-Cry5B,並以秀麗隱桿線蟲作為模式生物來探討在宿主受到膜穿孔蛋白毒素感染後引發的防禦機制為何。Cry5B會造成細胞膜內外離子通透的不平衡,嚴重的話甚至會導致細胞和線蟲的死亡。為了與膜穿孔蛋白毒素及細菌感染進行對抗,秀麗隱桿線蟲有多條與先天性免疫相關的路徑進行防禦,包含了自噬作用。在我們先前的研究已經發現,線蟲在受到Cry5B毒害後,其腸細胞內的轉錄因子HLH-30 (在哺乳動物為TFEB)會受到活化且進入細胞核調控自噬作用。而在本實驗中,我們的目的主要是找尋調控膜穿孔蛋白毒素誘導的HLH-30入核及自噬作用的上游因子。為此,我們利用高通量抑制基因篩選的方式找尋可能參與其中的基因,我們發現在hpo-22 (也稱作wdr-20)缺失的情況下,Cry5B所誘導的HLH-30入核的比例和細胞自噬的生物標記LGG-1的表現量皆有顯著下降的情形,缺乏hpo-22的情況下,秀麗隱桿線蟲受到Cry5B毒害的存活率也會有顯著短命的現象。過去的研究已經發現,HPO-22/WDR-20會與WDR-48以及USP-46形成複合物,以增加USP-46去泛素化的活性,且USP-46也參與在轉譯後修飾當中。同樣的,我們也證明了WDR-48與USP-46對於Cry5B所誘導的HLH-30入核與細胞自噬的的活性都是需要的。我們也證實了,HPO-22/WDR-20、WDR-48以及USP-46的缺乏會顯著的影響細胞膜孔洞修復的能力。此外,我們發現USP-46作用於mTOR/LET-363的下游以及14-3-3/FTT-2蛋白的上游,並且參與在Cry5B誘導的HLH-30入核。綜合上述結果,我們目前的研究結果表明,在秀麗隱桿線蟲受到Cry5B膜穿孔毒素感染時,HPO-22·WDR-48·USP-46複合物會藉由去泛素化的轉譯後修飾作用調控HLH-30入核並活化細胞自噬作用和內源性細胞防禦自以提供宿主免於膜穿孔毒素蛋白毒殺的保護機制。
Pore-forming toxins (PFTs) are important virulence factors for most bacterial pathogens. In this study, we used Cry5B, a PFT secreted by Bacillus thurgiensis, to investigate the host defense mechanism against PFT intoxication in Caenorhabiditis elegans. Cry5B causes the ionic imbalance between plasma membrane leading to the death of intestinal cells and even the entire organism. In order to defend PFTs and bacterial infection, C. elegans applies several innate immune mechanisms, including autophagy. In our previous studies, we found the transcription factor HLH-30, an ortholog of mammalian TFEB, can be activated and translocated into the nucleus of intestinal cells in C. elegans and mediates autophagy to fight Cry5B intoxication. Herein, we aimed to determine the upstream signal responsible for this PFT-induced HLH-30 nuclear localization and autophagy. To this end, we applied genetic suppressor screen to identify genes which are required for the Cry5B-induced HLH-30 activation. We found that the loss of the hpo-22 gene (also named wdr-20, WD repeat-containing protein 20) in C. elegans significantly reduced the Cry5B-induced HLH-30 nuclear localization and puctate signals of the autophagic biomarker, LGG-1. Knockdown and nonsense mutation of hpo-22 both confer hypersensitivity to Cry5B in the animal survival analysis. We also demonstrated that Cry5B-induced HLH-30 nuclear localization and subsequently autophagy activation require USP-46 (ubiquitin-specific protease 46), whose deubiquitinating activity is regulated by complex of WDR-20 and WDR-48. Concomitantly, loss of WDR-20, WDR-48 or USP-46 can significantly impair membrane pore-repair activity. Furthermore, we found that USP-46 acts downstream of mTOR and upstream of 14-3-3 protein, which involved in the Cry5B induced HLH-30 nuclear localization. Taken together, our current results suggested that the WDR-20/WDR-48/USP-46 complex may involve in the activation of HLH-30, autophagy, and intrinsic cellular defense against PFT intoxication plausibly through ubiquitination/deubiquitination in C. elegans.
Aroian, R. and F. G. van der Goot. "Pore-forming toxins and cellular non-immune defenses (CNIDs)." Curr Opin Microbiol 10(1): 57-61 (2007).
Bellier, A., C.-S. Chen, C.-Y. Kao, H. N. Cinar and R. V. Aroian. "Hypoxia and the hypoxic response pathway protect against pore-forming toxins in C. elegans." PLoS Pathog 5(12): e1000689 (2009).
Bischof, L. J., D. L. Huffman and R. V. Aroian. "Assays for toxicity studies in C. elegans with Bt crystal proteins." C. elegans: Methods and Applications: 139-154 (2006).
Bischof, L. J., C. Y. Kao, F. C. Los, M. R. Gonzalez, Z. Shen, S. P. Briggs, F. G. van der Goot and R. V. Aroian. "Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo." PLoS Pathog 4(10): e1000176 (2008).
Chassin, C., M. Bens, J. de Barry, R. Courjaret, J. L. Bossu, F. Cluzeaud, S. Ben Mkaddem, M. Gibert, B. Poulain, M. R. Popoff and A. Vandewalle. "Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells." Am J Physiol Renal Physiol 293(3): F927-937 (2007).
Chen, C.-S., A. Bellier, C.-Y. Kao, Y.-L. Yang, H.-D. Chen, F. C. Los and R. V. Aroian. "WWP-1 is a novel modulator of the DAF-2 insulin-like signaling network involved in pore-forming toxin cellular defenses in Caenorhabditis elegans." PloS one 5(3): e9494 (2010).
Chen, H.-D., C.-Y. Kao, B.-Y. Liu, S.-W. Huang, C.-J. Kuo, J.-W. Ruan, Y.-H. Lin, C.-R. Huang, Y.-H. Chen, H.-D. Wang, R. V. Aroian and C.-S. Chen. "HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans." Autophagy 13(2): 371-385 (2017).
Dahlberg, C. L. and P. Juo. "The WD40-repeat proteins WDR-20 and WDR-48 bind and activate the deubiquitinating enzyme USP-46 to promote the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of Caenorhabditis elegans." Journal of Biological Chemistry 289(6): 3444-3456 (2014).
Ewbank, J. J. and N. Pujol. "Local and long-range activation of innate immunity by infection and damage in C. elegans." Curr Opin Immunol 38: 1-7 (2016).
Glavis-Bloom, J., M. Muhammed and E. Mylonakis. "Of model hosts and man: using Caenorhabditis elegans, Drosophila melanogaster and Galleria mellonella as model hosts for infectious disease research." Adv Exp Med Biol 710: 11-17 (2012).
González-Juarbe, N., R. P. Gilley, C. A. Hinojosa, K. M. Bradley, A. Kamei, G. Gao, P. H. Dube, M. A. Bergman and C. J. Orihuela. "Pore-forming toxins induce macrophage necroptosis during acute bacterial pneumonia." PLoS pathogens 11(12): e1005337 (2015).
Gonzalez, M. R., M. Bischofberger, L. Pernot, F. G. van der Goot and B. Freche. "Bacterial pore-forming toxins: the (w)hole story?" Cell Mol Life Sci 65(3): 493-507 (2008).
Huffman, D. L., L. Abrami, R. Sasik, J. Corbeil, F. G. van der Goot and R. V. Aroian. "Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins." Proceedings of the National academy of Sciences of the United States of America 101(30): 10995-11000 (2004).
Joo, H.-Y., L. Zhai, C. Yang, S. Nie, H. Erdjument-Bromage, P. Tempst, C. Chang and H. Wang. "Regulation of cell cycle progression and gene expression by H2A deubiquitination." Nature 449(7165): 1068-1072 (2007).
Klionsky, D. J. and S. D. Emr. "Autophagy as a regulated pathway of cellular degradation." Science 290(5497): 1717-1721 (2000).
Kowalski, J. R., C. L. Dahlberg and P. Juo. "The deubiquitinating enzyme USP-46 negatively regulates the degradation of glutamate receptors to control their abundance in the ventral nerve cord of Caenorhabditis elegans." Journal of Neuroscience 31(4): 1341-1354 (2011).
Lapierre, L. R., C. D. De Magalhaes Filho, P. R. McQuary, C.-C. Chu, O. Visvikis, J. T. Chang, S. Gelino, B. Ong, A. E. Davis and J. E. Irazoqui. "The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans." Nature communications 4 (2013).
Lin, Z., H. Yang, Q. Kong, J. Li, S.-M. Lee, B. Gao, H. Dong, J. Wei, J. Song and D. D. Zhang. "USP22 antagonizes p53 transcriptional activation by deubiquitinating Sirt1 to suppress cell apoptosis and is required for mouse embryonic development." Molecular cell 46(4): 484-494 (2012).
Los, F. C., T. M. Randis, R. V. Aroian and A. J. Ratner. "Role of pore-forming toxins in bacterial infectious diseases." Microbiol Mol Biol Rev 77(2): 173-207 (2013).
Luo, H., J. Xiong, Q. Zhou, L. Xia and Z. Yu. "The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans." Appl Microbiol Biotechnol 97(23): 10135-10142 (2013).
Martina, J. A., Y. Chen, M. Gucek and R. Puertollano. "MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB." Autophagy 8(6): 903-914 (2012).
Nakamura, S., Ö. Karalay, P. S. Jäger, M. Horikawa, C. Klein, K. Nakamura, C. Latza, S. E. Templer, C. Dieterich and A. Antebi. "Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals." Nature communications 7 (2016).
Ortiz, M. A., D. Noble, E. P. Sorokin and J. Kimble. "A new dataset of spermatogenic vs. oogenic transcriptomes in the nematode Caenorhabditis elegans." G3: Genes| Genomes| Genetics 4(9): 1765-1772 (2014).
Peraro, M. D. and F. G. van der Goot. "Pore-forming toxins: ancient, but never really out of fashion." Nat Rev Micro 14(2): 77-92 (2016).
Piano, F., A. J. Schetter, D. G. Morton, K. C. Gunsalus, V. Reinke, S. K. Kim and K. J. Kemphues. "Gene clustering based on RNAi phenotypes of ovary-enriched genes in C. elegans." Current Biology 12(22): 1959-1964 (2002).
Scott, R. C., O. Schuldiner and T. P. Neufeld. "Role and regulation of starvation-induced autophagy in the Drosophila fat body." Developmental cell 7(2): 167-178 (2004).
Settembre, C., R. De Cegli, G. Mansueto, P. K. Saha, F. Vetrini, O. Visvikis, T. Huynh, A. Carissimo, D. Palmer and T. J. Klisch. "TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop." Nature cell biology 15(6): 647-658 (2013).
Settembre, C., C. Di Malta, V. A. Polito, M. G. Arencibia, F. Vetrini, S. Erdin, S. U. Erdin, T. Huynh, D. Medina and P. Colella. "TFEB links autophagy to lysosomal biogenesis." Science 332(6036): 1429-1433 (2011).
Settembre, C., A. Fraldi, D. L. Medina and A. Ballabio. "Signals from the lysosome: a control centre for cellular clearance and energy metabolism." Nature reviews Molecular cell biology 14(5): 283-296 (2013).
Settembre, C., R. Zoncu, D. L. Medina, F. Vetrini, S. Erdin, S. Erdin, T. Huynh, M. Ferron, G. Karsenty and M. C. Vellard. "A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB." The EMBO journal 31(5): 1095-1108 (2012).
Spencer, W. C., R. McWhirter, T. Miller, P. Strasbourger, O. Thompson, L. W. Hillier, R. H. Waterston and D. M. Miller III. "Isolation of specific neurons from C. elegans larvae for gene expression profiling." PloS one 9(11): e112102 (2014).
Taffoni, C. and N. Pujol. "Mechanisms of innate immunity in C. elegans epidermis." Tissue Barriers 3(4): e1078432 (2015).
Taillebourg, E., I. Gregoire, P. Viargues, A.-C. Jacomin, D. Thevenon, M. Faure and M.-O. Fauvarque. "The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins." Autophagy 8(5): 767-779 (2012).
Tiller, G. R. and D. A. Garsin. "Of worms and men: HLH-30 and TFEB regulate tolerance to infection." Immunity 40(6): 857-858 (2014).
Villamil, M. A., Q. Liang and Z. Zhuang. "The WD40-repeat protein-containing deubiquitinase complex: catalysis, regulation, and potential for therapeutic intervention." Cell biochemistry and biophysics 67(1): 111-126 (2013).
Yang, Y.-p., Z.-q. Liang, Z.-l. Gu and Z.-h. Qin. "Molecular mechanism and regulation of autophagy." Acta Pharmacologica Sinica 26(12): 1421-1434 (2005).
Pena-Llopis, S., and Brugarolas, J. TFEB, a novel mTORC1 effector implicated in lysosome biogenesis, endocytosis and autophagy. Cell Cycle, 10(23), 3987-3988 (2011).
Meléndez, A., and Levine, B. Autophagy in C. elegans. Wormbook, 1-26 (2005).
Jackson, A. L. and P. S. Linsley (2010). "Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application." Nature reviews Drug discovery 9(1): 57-67.
Lin, X., X. Ruan, M. G. Anderson, J. A. McDowell, P. E. Kroeger, S. W. Fesik and Y. Shen (2005). "siRNA-mediated off-target gene silencing triggered by a 7 nt complementation." Nucleic acids research 33(14): 4527-4535.
Kao, C.-Y., F. C. Los, D. L. Huffman, S. Wachi, N. Kloft, M. Husmann, V. Karabrahimi, J.-L. Schwartz, A. Bellier and C. Ha. "Global functional analyses of cellular responses to pore-forming toxins." PLoS Pathog 7(3): e1001314 (2011).
Codogno, P. and A. Meijer. "Autophagy and signaling: their role in cell survival and cell death." Cell Death & Differentiation 12: 1509-1518 (2005).
Das, G., B. V. Shravage and E. H. Baehrecke. "Regulation and function of autophagy during cell survival and cell death." Cold Spring Harbor perspectives in biology 4(6): a008813 (2012).
Jacomin, A.-C., A. Bescond, E. Soleilhac, B. Gallet, G. Schoehn, M.-O. Fauvarque and E. Taillebourg. "The deubiquitinating enzyme UBPY is required for lysosomal biogenesis and productive autophagy in drosophila." PloS one 10(11): e0143078 (2015).
Wilkinson, K. D. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Seminars in cell & developmental biology, Elsevier (2000).
Wilkinson, K. D. "The discovery of ubiquitin-dependent proteolysis." Proceedings of the National Academy of Sciences 102(43): 15280-15282 (2005).
Medina, D. L., S. Di Paola, I. Peluso, A. Armani, D. De Stefani, R. Venditti, S. Montefusco, A. Scotto-Rosato, C. Prezioso and A. Forrester. "Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB." Nature cell biology 17(3): 288-299 (2015).
Mello, C. C., J. M. Kramer, D. Stinchcomb and V. Ambros. "Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences." The EMBO journal 10(12): 3959 (1991).
Kadandale, P., I. Chatterjee and A. Singson. "Germline transformation of Caenorhabditis elegans by injection." Microinjection: Methods and Applications: 123-133 (2009).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics, 77(1), 71-94 (1974).
Wood, W. B. The Introduction to C. elegans Biology. Cold Spring Harbor Monograph Archive, 17, 1-16 (1988).
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., and Sohrmann, M. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 421(6920), 231-237 (2003).
Bischof, L. J., D. L. Huffman and R. V. Aroian. "Assays for toxicity studies in C. elegans with Bt crystal proteins." C. elegans: Methods and Applications: 139-154 (2006).
Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., and Aguirre-Ghiso, J. A. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 8(4), 445-544 (2012).
Linares, J. F., A. Duran, T. Yajima, M. Pasparakis, J. Moscat and M. T. Diaz-Meco. "K63 polyubiquitination and activation of mTOR by the p62-TRAF6 complex in nutrient-activated cells." Molecular cell 51(3): 283-296 (2013).
Silva, G. M., D. Finley and C. Vogel. "K63 polyubiquitination is a new modulator of the oxidative stress response." Nature structural & molecular biology 22(2): 116-123 (2015).
Tenekeci, U., M. Poppe, K. Beuerlein, C. Buro, H. Müller, H. Weiser, D. Kettner-Buhrow, K. Porada, D. Newel and M. Xu. "K63-Ubiquitylation and TRAF6 Pathways Regulate Mammalian P-Body Formation and mRNA Decapping." Molecular cell 62(6): 943-957 (2016).
陳偉學. 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. (碩士), 國立成功大學 (2015).
林依穎. 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. (碩士), 國立成功大學 (2016)