臺灣博碩士論文加值系統

內質網為執行蛋白質生合成、正確摺疊與修飾的胞器。當細胞受到某種刺激後，造成內質網恆定遭到破壞，使摺疊錯誤的蛋白質累積在內質網而產生ER stress，為了消除 ER stress ，細胞會啟動UPR (unfolded protein response, UPR ) 來維持內質網的恆定。由於先前實驗室已發現腸病毒71型會調控UPR pathway。其中PERK-eIF2α pathway會被活化，但IRE1-XBP1 pathway 受到抑制，而IRE1-JNK pathway 會被活化。因此，本篇想探討腸病毒71型引發IRE1-JNK pathway對病毒複製有何影響。在 IRE1 siRNA 作用下，病毒蛋白質、RNA生合成及 plaque number 都有顯著下降且，病毒釋放至細胞外的能力也是。另外，以JNK2 siRNA 處理情況下，病毒複製與釋放受到抑制；然而先前文獻指出腸病毒會誘發apoptosis且poliovirus在感染期間會活化JNK促使Bax從細胞質進入粒線體中，產生MOMP(mitochondrial outer membrane permeabilization)，cytochrome c 釋放於細胞質，誘導apoptosis現象，達到病毒釋放；感染晚期處理JNK inhibitor (sp600125)和 QVDOPH (caspase inhibitor )，可以發現病毒複製不受影響卻會抑制病毒的釋放。因此，我們推測IRE1-JNK2 pathway 參與腸病毒71型誘發apoptosis 來幫助病毒釋放。

Enterovirus 71 (EV71) is a single-stranded RNA virus belonging to the Picornaviridae family. EV71 is known to cause hand, foot and mouth disease in young children and has been implicated in severe neurological manifestations. It has been known that many viruses in picornaviridae induce endoplasmic reticulum (ER) stress but the mechanism remains unknown. Inositol-requiring enzyme 1 (IRE1) plays an important role in unfolded protein response. In our group, we have demonstrated that EV71 could induce the increase of phosphorylated IRE1 followed by activating its downstream target, c-Jun N-terminal protein kinase (JNK). We proposed that EV71 modulates IRE1 pathway to benefit its replication and mature virus release . Thus, in this study, we tried to further investigate this regulatory mechanism. We observed that knockdown of IRE1 results in a decreased level of virus titer, which was due to the attenuated viral protein expression and viral RNA replication. Besides that, when cells were treated with JNK inhibitor in the early stage of infection or siRNA of JNK2 , virus titer and mature virus release were significantly decreased . However , addition of JNK inhibitor and QVDOPH ( caspase inhibitor ) at 5 h.p.i has no influence on virus replication but resulted in reduction of viral particle release .These results support our current hypothesis that IRE1-JNK2 pathway involved in EV71 - induced apoptosis , which in turn facilitate virus release .

論文指導教授推薦書
論文口試委員審定書
長庚大學博碩士論文著作授權書 iii
致謝 iv
縮寫表 v
中文摘要 vi
英文摘要 vii
目錄 viii
圖表目錄 x
第一章 緒論 1
一、 腸病毒之介紹 1
二、 Unfolded protein response ( UPR ) 之介紹 3
三、 IRE1 之介紹 5
四、 細胞凋亡 ( apoptosis ) 之介紹 6
五、 JNK signaling pathway 之介紹 9
第二章 研究目的 12
第三章 實驗材料與方法 13
一、 材料 13
二、 方法 15
第四章研究結果 19
一、 腸病毒 71 型感染宿主細胞對 IRE1 路徑的影響 19
二、 腸病毒71型感染複製與 IRE1 是否相關 20
三、 腸病毒71型感染與複製與 JNK 是否相關 22
四、 腸病毒 71 型之病毒釋放與 JNK 是否相關 27
五、 腸病毒 71 型之病毒釋放與JNK活化誘導 apoptosis 是否相關 29
第五章 討論 33
第六章 參考文獻 38
第七章 圖表 47
 
圖表目錄
圖一、腸病毒 71 型感染會誘導 IRE1 及JNK 活化。 47
圖二、將knockdown IRE1 之 RD 細胞感染腸病毒 71 型，病毒複製會被抑制 50
圖三、處理JNK inhibitor，會抑制病毒複製 53
圖四、JNK1 knockdown不影響病毒複製 56
圖五、JNK2參與病毒之複製 59
圖六、在感染晚期處理JNK inhibitor會抑制病毒釋放 62
圖七、處理QVDOPH，會抑制病毒釋放 65
圖八、抑制IRE1-JNK pathway 可降低腸病毒71型誘導apoptosis 70

Alter, J., Rozentzweig, D., and Bengal, E. (2008). Inhibition of myoblast differentiation by tumor necrosis factor alpha is mediated by c-Jun N-terminal kinase 1 and leukemia inhibitory factor. J Biol Chem 283, 23224-23234.
2. Autret, A., Martin-Latil, S., Mousson, L., Wirotius, A., Petit, F., Arnoult, D., Colbere-Garapin, F., Estaquier, J., and Blondel, B. (2007). Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase. J Virol 81, 7504-7516.
3. Banerjee, R., Weidman, M.K., Navarro, S., Comai, L., and Dasgupta, A. (2005). Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription. J Gen Virol 86, 2315-2322.
4. Belov, G.A., Romanova, L.I., Tolskaya, E.A., Kolesnikova, M.S., Lazebnik, Y.A., and Agol, V.I. (2003). The major apoptotic pathway activated and suppressed by poliovirus. J Virol 77, 45-56.
5. Bertolotti, A., Zhang, Y., Hendershot, L.M., Harding, H.P., and Ron, D. (2000). Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2, 326-332.
6. Bible, J.M., Pantelidis, P., Chan, P.K., and Tong, C.Y. (2007). Genetic evolution of enterovirus 71: epidemiological and pathological implications. Reviews in medical virology 17, 371-379.
7. Bogoyevitch, M.A., and Kobe, B. (2006). Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases. Microbiology and molecular biology reviews : MMBR 70, 1061-1095.
8. Calfon, M., Zeng, H., Urano, F., Till, J.H., Hubbard, S.R., Harding, H.P., Clark, S.G., and Ron, D. (2002). IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92-96.
9. Carboni, S., Antonsson, B., Gaillard, P., Gotteland, J.P., Gillon, J.Y., and Vitte, P.A. (2005). Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia. Journal of neurochemistry 92, 1054-1060.
10. Carthy, C.M., Granville, D.J., Watson, K.A., Anderson, D.R., Wilson, J.E., Yang, D., Hunt, D.W., and McManus, B.M. (1998). Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. J Virol 72, 7669-7675.
11. Chang, L., and Karin, M. (2001). Mammalian MAP kinase signalling cascades. Nature 410, 37-40.
12. Chang, S.C., Lin, J.Y., Lo, L.Y., Li, M.L., and Shih, S.R. (2004). Diverse apoptotic pathways in enterovirus 71-infected cells. J Neurovirol 10, 338-349.
13. Chen, F., Beezhold, K., and Castranova, V. (2009). JNK1, a potential therapeutic target for hepatocellular carcinoma. Biochimica et biophysica acta 1796, 242-251.
14. Clark, M.E., Lieberman, P.M., Berk, A.J., and Dasgupta, A. (1993). Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro. Mol Cell Biol 13, 1232-1237.
15. Conze, D., Krahl, T., Kennedy, N., Weiss, L., Lumsden, J., Hess, P., Flavell, R.A., Le Gros, G., Davis, R.J., and Rincon, M. (2002). c-Jun NH(2)-terminal kinase (JNK)1 and JNK2 have distinct roles in CD8(+) T cell activation. The Journal of experimental medicine 195, 811-823.
16. Danial, N.N., and Korsmeyer, S.J. (2004). Cell death: critical control points. Cell 116, 205-219.
17. Davis, R.J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103, 239-252.
18. Dong, C., Davis, R.J., and Flavell, R.A. (2002). MAP kinases in the immune response. Annual review of immunology 20, 55-72.
19. Durbin, A.D., Hannigan, G.E., and Malkin, D. (2009a). Oncogenic ILK, tumor suppression and all that JNK. Cell Cycle 8, 4060-4066.
20. Durbin, A.D., Somers, G.R., Forrester, M., Pienkowska, M., Hannigan, G.E., and Malkin, D. (2009b). JNK1 determines the oncogenic or tumor-suppressive activity of the integrin-linked kinase in human rhabdomyosarcoma. The Journal of clinical investigation 119, 1558-1570.
21. Eferl, R., and Wagner, E.F. (2003). AP-1: a double-edged sword in tumorigenesis. Nature reviews Cancer 3, 859-868.
22. Elmore, S. (2007). Apoptosis: a review of programmed cell death. Toxicologic pathology 35, 495-516.
23. Ernest, N.J., Habela, C.W., and Sontheimer, H. (2008). Cytoplasmic condensation is both necessary and sufficient to induce apoptotic cell death. Journal of cell science 121, 290-297.
24. Galluzzi, L., Maiuri, M.C., Vitale, I., Zischka, H., Castedo, M., Zitvogel, L., and Kroemer, G. (2007). Cell death modalities: classification and pathophysiological implications. Cell Death Differ 14, 1237-1243.
25. Gingras, A.C., Svitkin, Y., Belsham, G.J., Pause, A., and Sonenberg, N. (1996). Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus. Proc Natl Acad Sci U S A 93, 5578-5583.
26. Gradi, A., Svitkin, Y.V., Imataka, H., and Sonenberg, N. (1998). Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection. Proc Natl Acad Sci U S A 95, 11089-11094.
27. Gupta, S., Barrett, T., Whitmarsh, A.J., Cavanagh, J., Sluss, H.K., Derijard, B., and Davis, R.J. (1996). Selective interaction of JNK protein kinase isoforms with transcription factors. The EMBO journal 15, 2760-2770.
28. Hambidge, S.J., and Sarnow, P. (1992). Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A. Proc Natl Acad Sci U S A 89, 10272-10276.
29. Harding, H.P., Zhang, Y., and Ron, D. (1999). Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271-274.
30. Haze, K., Okada, T., Yoshida, H., Yanagi, H., Yura, T., Negishi, M., and Mori, K. (2001). Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response. Biochem J 355, 19-28.
31. He, B. (2006). Viruses, endoplasmic reticulum stress, and interferon responses. Cell Death Differ 13, 393-403.
32. Hess, P., Pihan, G., Sawyers, C.L., Flavell, R.A., and Davis, R.J. (2002). Survival signaling mediated by c-Jun NH(2)-terminal kinase in transformed B lymphoblasts. Nature genetics 32, 201-205.
33. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993). Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev 7, 2135-2148.
34. Jheng, J.R., Lau, K.S., Tang, W.F., Wu, M.S., and Horng, J.T. (2010). Endoplasmic reticulum stress is induced and modulated by enterovirus 71. Cellular microbiology 12, 796-813.
35. Jheng, J.R., Lin, C.Y., Horng, J.T., and Lau, K.S. (2012). Inhibition of enterovirus 71 entry by transcription factor XBP1. Biochem Biophys Res Commun 420, 882-887.
36. Kaser, A., Lee, A.H., Franke, A., Glickman, J.N., Zeissig, S., Tilg, H., Nieuwenhuis, E.E., Higgins, D.E., Schreiber, S., Glimcher, L.H., et al. (2008). XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134, 743-756.
37. Kim, I., Xu, W., and Reed, J.C. (2008). Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7, 1013-1030.
38. Kim, J.H., Lee, S.C., Ro, J., Kang, H.S., Kim, H.S., and Yoon, S. (2010). Jnk signaling pathway-mediated regulation of Stat3 activation is linked to the development of doxorubicin resistance in cancer cell lines. Biochemical pharmacology 79, 373-380.
39. Klionsky, D.J., and Emr, S.D. (2000). Autophagy as a regulated pathway of cellular degradation. Science 290, 1717-1721.
40. Kuan, C.Y., Yang, D.D., Samanta Roy, D.R., Davis, R.J., Rakic, P., and Flavell, R.A. (1999). The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron 22, 667-676.
41. Kuo, R.L., Kung, S.H., Hsu, Y.Y., and Liu, W.T. (2002). Infection with enterovirus 71 or expression of its 2A protease induces apoptotic cell death. J Gen Virol 83, 1367-1376.
42. Li, M.L., Hsu, T.A., Chen, T.C., Chang, S.C., Lee, J.C., Chen, C.C., Stollar, V., and Shih, S.R. (2002). The 3C protease activity of enterovirus 71 induces human neural cell apoptosis. Virology 293, 386-395.
43. Lipson, K.L., Ghosh, R., and Urano, F. (2008). The role of IRE1alpha in the degradation of insulin mRNA in pancreatic beta-cells. PLoS One 3, e1648.
44. Liu, J., and Lin, A. (2005). Role of JNK activation in apoptosis: a double-edged sword. Cell research 15, 36-42.
45. Liu, J., and Lin, A. (2007). Wiring the cell signaling circuitry by the NF-kappa B and JNK1 crosstalk and its applications in human diseases. Oncogene 26, 3267-3278.
46. Liu, Y., Adachi, M., Zhao, S., Hareyama, M., Koong, A.C., Luo, D., Rando, T.A., Imai, K., and Shinomura, Y. (2009). Preventing oxidative stress: a new role for XBP1. Cell Death Differ 16, 847-857.
47. Metzstein, M.M., Stanfield, G.M., and Horvitz, H.R. (1998). Genetics of programmed cell death in C. elegans: past, present and future. Trends in genetics : TIG 14, 410-416.
48. Minor, P.D. (2004). Polio eradication, cessation of vaccination and re-emergence of disease. Nat Rev Microbiol 2, 473-482.
49. Morishima, Y., Gotoh, Y., Zieg, J., Barrett, T., Takano, H., Flavell, R., Davis, R.J., Shirasaki, Y., and Greenberg, M.E. (2001). Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of Fas ligand. The Journal of neuroscience : the official journal of the Society for Neuroscience 21, 7551-7560.
50. Nasrin, N., Kaushik, V.K., Fortier, E., Wall, D., Pearson, K.J., de Cabo, R., and Bordone, L. (2009). JNK1 phosphorylates SIRT1 and promotes its enzymatic activity. PLoS One 4, e8414.
51. Ng, C.P., and Bonavida, B. (2002). X-linked inhibitor of apoptosis (XIAP) blocks Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of prostate cancer cells in the presence of mitochondrial activation: sensitization by overexpression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pl (Smac/DIABLO). Molecular cancer therapeutics 1, 1051-1058.
52. Ng, D.T., Watowich, S.S., and Lamb, R.A. (1992). Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene. Mol Biol Cell 3, 143-155.
53. Nishitoh, H., Matsuzawa, A., Tobiume, K., Saegusa, K., Takeda, K., Inoue, K., Hori, S., Kakizuka, A., and Ichijo, H. (2002). ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16, 1345-1355.
54. Oikawa, D., Tokuda, M., and Iwawaki, T. (2007). Site-specific cleavage of CD59 mRNA by endoplasmic reticulum-localized ribonuclease, IRE1. Biochem Biophys Res Commun 360, 122-127.
55. Palacios, G., and Oberste, M.S. (2005). Enteroviruses as agents of emerging infectious diseases. J Neurovirol 11, 424-433.
56. Pan, J., Zhao, Y.X., Wang, Z.Q., Jin, L., Sun, Z.K., and Chen, S.D. (2007). Expression of FasL and its interaction with Fas are mediated by c-Jun N-terminal kinase (JNK) pathway in 6-OHDA-induced rat model of Parkinson disease. Neuroscience letters 428, 82-87.
57. Rao, R.V., Ellerby, H.M., and Bredesen, D.E. (2004). Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ 11, 372-380.
58. Reddien, P.W., Cameron, S., and Horvitz, H.R. (2001). Phagocytosis promotes programmed cell death in C. elegans. Nature 412, 198-202.
59. Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8, 519-529.
60. Sabapathy, K., Jochum, W., Hochedlinger, K., Chang, L., Karin, M., and Wagner, E.F. (1999). Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2. Mechanisms of development 89, 115-124.
61. Schmidt, N.J., Lennette, E.H., and Ho, H.H. (1974). An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 129, 304-309.
62. Schulze-Osthoff, K., Bakker, A.C., Vanhaesebroeck, B., Beyaert, R., Jacob, W.A., and Fiers, W. (1992). Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem 267, 5317-5323.
63. Shao, Z., Bhattacharya, K., Hsich, E., Park, L., Walters, B., Germann, U., Wang, Y.M., Kyriakis, J., Mohanlal, R., Kuida, K., et al. (2006). c-Jun N-terminal kinases mediate reactivation of Akt and cardiomyocyte survival after hypoxic injury in vitro and in vivo. Circulation research 98, 111-118.
64. Tang, W.F., Yang, S.Y., Wu, B.W., Jheng, J.R., Chen, Y.L., Shih, C.H., Lin, K.H., Lai, H.C., Tang, P., and Horng, J.T. (2007). Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication. J Biol Chem 282, 5888-5898.
65. Tirasophon, W., Lee, K., Callaghan, B., Welihinda, A., and Kaufman, R.J. (2000). The endoribonuclease activity of mammalian IRE1 autoregulates its mRNA and is required for the unfolded protein response. Genes Dev 14, 2725-2736.
66. Tirasophon, W., Welihinda, A.A., and Kaufman, R.J. (1998). A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev 12, 1812-1824.
67. Tolskaya, E.A., Romanova, L.I., Kolesnikova, M.S., Ivannikova, T.A., Smirnova, E.A., Raikhlin, N.T., and Agol, V.I. (1995). Apoptosis-inducing and apoptosis-preventing functions of poliovirus. J Virol 69, 1181-1189.
68. Tournier, C., Hess, P., Yang, D.D., Xu, J., Turner, T.K., Nimnual, A., Bar-Sagi, D., Jones, S.N., Flavell, R.A., and Davis, R.J. (2000). Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 288, 870-874.
69. Tournier, C., Whitmarsh, A.J., Cavanagh, J., Barrett, T., and Davis, R.J. (1997). Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc Natl Acad Sci U S A 94, 7337-7342.
70. Ventura, J.J., Hubner, A., Zhang, C., Flavell, R.A., Shokat, K.M., and Davis, R.J. (2006). Chemical genetic analysis of the time course of signal transduction by JNK. Molecular cell 21, 701-710.
71. Voncken, J.W., Niessen, H., Neufeld, B., Rennefahrt, U., Dahlmans, V., Kubben, N., Holzer, B., Ludwig, S., and Rapp, U.R. (2005). MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1. J Biol Chem 280, 5178-5187.
72. Wang, X., Destrument, A., and Tournier, C. (2007). Physiological roles of MKK4 and MKK7: insights from animal models. Biochimica et biophysica acta 1773, 1349-1357.
73. Wang, Y., Singh, R., Lefkowitch, J.H., Rigoli, R.M., and Czaja, M.J. (2006). Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 281, 15258-15267.
74. Weston, C.R., and Davis, R.J. (2007). The JNK signal transduction pathway. Current opinion in cell biology 19, 142-149.
75. Yoneda, T., Imaizumi, K., Oono, K., Yui, D., Gomi, F., Katayama, T., and Tohyama, M. (2001). Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress. J Biol Chem 276, 13935-13940.
76. Yoshida, H., Matsui, T., Hosokawa, N., Kaufman, R.J., Nagata, K., and Mori, K. (2003). A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 4, 265-271.
77. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001). XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881-891.
78. Zhong, S., Zhang, Y., Jansen, C., Goto, H., Inagaki, M., and Dong, Z. (2001). MAP kinases mediate UVB-induced phosphorylation of histone H3 at serine 28. J Biol Chem 276, 12932-12937.