臺灣博碩士論文加值系統

Interferon-Stimulated Gene (ISG)是一群由Interferon (IFN) 所調控的基因，他們能夠幫助細胞對抗病毒的感染。根據先前的文獻指出ISG的調控主要是經由增加轉錄的經典路徑，然而也有猜測說可能有其他的路徑可以來協助ISG的調控。而本實驗室發現，當ISG基因接在不同的啟動子後面，再用IFN去激活，有些ISG基因能被IFN所調控。本實驗主要探討IFN調控ISG基因的非經典路徑。首先我們挑選了三個ISG基因成員，分別是IFIT1、IFIT5、PKR，將他們分別製作成能穩定表現的細胞株，再用不同濃度的IFN去激活。實驗發現，外源性的IFIT1會隨IFN的濃度而提升表現量；IFIT5則沒有顯著性的變化；而PKR則是會降低表現量。之後，我們推測IFN是藉由改變mRNA的表現量來調控蛋白質，然而，我們發現，外源性IFIT1和PKR的mRNA表現量和其蛋白質的表現量並沒有顯著性的相關。之後，我們利用處理cycloheximide來觀察蛋白質的半衰期，發現外源性IFIT1的半衰期有被延長；而PKR則是縮短。由此我們推測IFN能夠藉由改變ISG蛋白的穩定性來進行調控。之後，我們從蛋白資料庫中發現IFIT1比IFIT5多了兩個TPR domain，因此我們去掉IFIT1和PKR的一些domain來進行實驗，發現當IFIT1去除TPR5、TPR7、TPR9-10時，其蛋白質的表現量就不受IFN的調控；而PKR則是不論去除哪一段時，其蛋白質的表現量就不受IFN的調控，由此我們知道IFN是作用在ISG蛋白的特定domain上來進行調控。未來，我們將會對IFN的調控路徑做更深入的研究和探討。

Interferon-Stimulated Gene (ISG) is a group of genes regulated by Interferon (IFN). ISGs can help cells fight against viral infections. Previous studies have shown that the regulation of ISG by interferon is mainly through induction of transcription (classic pathway). However, other pathways are also believed to play a role in the regulation of ISGs by IFN. When the ISG genes are expressed under the control of an exogenous promoter, some ISG genes could also be regulated by IFN. Three ISG gene members, namely IFIT1, IFIT5, and PKR, were constructed, stably expressed in cells and treated with different doses of IFN. In this condition, expression of exogenous IFIT5 was not affected by IFN. On the other hand, exogenous IFIT1 protein amount was increased while PKR amount was decreased. Unlike the protein level, the mRNA level of exogenous IFIT1 and PKR was not significantly changed by IFN treatment. Then, cycloheximide was added and the protein half-life of IFIT1 and PKR was measured. The half-life of exogenous IFIT1 was increased while that of PKR was reduced in the presence of IFN. Therefore, IFN may regulate ISGs expression through modulation of protein stability. Comparing with IFIT5, IFIT1 has two more TPR domains (TPR5 and TPR7). Expression of exogenous IFIT1 mutants without TPR5, TPR7 or TPR9-10, unlike those mutants without TPR1, TPR2, or TPR 1-4, was not affected by IFN treatment. Neither expression of N-terminus half nor that of C-terminus half of exogenous PKR was affected by IFN treatment. Therefore, IFN may regulate the stability of some ISGs through specific domains (or sequences) in these ISGs. Further studies are required to clarify the detailed mechanisms regarding the regulation of ISGs by IFN not through the classical pathways.

中文摘要 III
Abstract IV
一、 研究背景與目的 1
1、 Interferon 1
2、 Interferon-Stimulated Gene (ISG) 1
二、 實驗材料與方法 3
1、 細胞培養 3
2、 建構質體 3
2-1、 Polymerase chain reaction (PCR) 3
2-2、 Ligation 3
2-3、 Transformation 3
2-4、 抽plasmid 4
3、 Lentiviral system 4
4、 細胞株製作 5
5、 西方墨點法 6
5-1、 蛋白萃取 6
5-2、 SDS-PAGE 6
5-3、 轉漬(transfer) 6
5-4、 Blacking 及一、二抗 6
5-5、 Enhanced chemiluminescence (ECL)呈色 7
5-6、 Alkaline phosphatase (AP)呈色 7
5-7、 ImageJ 生物影像分析軟體 7
6、 萃取RNA 7
7、 reverse transcription-PCR (RT-PCR ) 8
8、 Real-time PCR 8
三、 實驗結果 9
1、 製作出穩定的stable cell 9
2、 A549 stably with IFIT1、IFIT5、PKR treated with IFN 9
3、 The mRNA levels of IFIT1、IFIT5、PKR detected by real-time RT-PCR 9
4、 PKR蛋白分解 10
5、 IFIT1和PKR蛋白半衰期 11
6、 藉由去除不同的TPR來找出IFIT1和IFIT5的差別 11
7、 PKR去除特定domain後觀察IFN對其蛋白的影響。 12
四、 討論 13
五、 結果圖 15
六、 附錄 29
七、 參考文獻 36

1.Andrea, D. and R. Ravera, The Interferon System: an overview. 2002.
2.Le Page, C., et al., Interferon activation and innate immunity. Rev Immunogenet, 2000. 2(3): p. 374-86.
3.Schneider, W.M., M.D. Chevillotte, and C.M. Rice, Interferon-stimulated genes: a complex web of host defenses. Annu Rev Immunol, 2014. 32: p. 513-45.
4.Au-Yeung, N., R. Mandhana, and C.M. Horvath, Transcriptional regulation by STAT1 and STAT2 in the interferon JAK-STAT pathway. JAKSTAT, 2013. 2(3): p. e23931.
5.Kadota, S. and K. Nagata, Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I. Nucleic Acids Res, 2014. 42(12): p. 7642-53.
6.Ooi, E.L., et al., Novel antiviral host factor, TNK1, regulates IFN signaling through serine phosphorylation of STAT1. Proc Natl Acad Sci U S A, 2014. 111(5): p. 1909-14.
7.Schoggins, J.W., Interferon-Stimulated Genes: What Do They All Do? Annu Rev Virol, 2019. 6(1): p. 567-584.
8.Wang, W., et al., Transcriptional Regulation of Antiviral Interferon-Stimulated Genes. Trends Microbiol, 2017. 25(7): p. 573-584.
10.De Andrea, M., et al., The interferon system: an overview. Eur J Paediatr Neurol, 2002. 6 Suppl A: p. A41-6; discussion A55-8.
11.Fensterl, V. and G.C. Sen, The ISG56/IFIT1 gene family. J Interferon Cytokine Res, 2011. 31(1): p. 71-8.
12.Garcia, M.A., et al., Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev, 2006. 70(4): p. 1032-60.
13.Platanias, L.C., Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol, 2005. 5(5): p. 375-86.
14.Polyak, S.J., et al., The P58 cellular inhibitor complexes with the interferon-induced, double-stranded RNA-dependent protein kinase, PKR, to regulate its autophosphorylation and activity. J Biol Chem, 1996. 271(3): p. 1702-7.
15.Schroder, K., et al., Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol, 2004. 75(2): p. 163-89.
16.Stark, G.R. and J.E. Darnell, Jr., The JAK-STAT pathway at twenty. Immunity, 2012. 36(4): p. 503-14.
17.Entrez Gene: EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase
18.Platanias L.C. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol.2005;5:375–386.
19.Gal-Ben-Ari S, Barrera I, Ehrlich M, Rosenblum K. PKR: A Kinase to Remember. Front Mol Neurosci. 2019 Jan 9;11:480. doi: 10.3389/fnmol.2018.00480. PMID: 30686999; PMCID: PMC6333748.
20.Kotenko S.V. IFN-lambdas.Curr. Opin. Immunol.2011;23:583–590.
21.Galani I.E. Type III interferons (IFNs): emerging master regulators of immunity.Adv. Exp. Med. Biol.2015;850:1–15.
22.Fenner J.E. Suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type I interferon activity.Nat. Immunol.2006;7:33–39.
23.Farrar M.A., Schreiber R.D. The molecular cell biology of interferon-gamma and its receptor.Annu. Rev. Immunol.1993;11:571–611.
24.Babon J.J. The molecular regulation of Janus kinase (JAK) activation.Biochem. J.2014;462:1–13.
25.Steen H.C., Gamero A.M. STAT2 phosphorylation and signaling JAKSTAT .2013;2:e25790.
26.Pestka, S., Langer, J. A., Zoon, K. C. & Samuel, C. E. Interferons and their actions.Annu. Rev. Biochem.56, 727–777 (1987).
27.Pestka, S., Krause, C. D. & Walter, M. R. Interferons, interferon-like cytokines, and their receptors.Immunol. Rev.202, 8–32 (2004).
28.Chen, J., Baig, E. & Fish, E. N. Diversity and relatedness among the type I interferons.J. Interfon Cytokine Res.24, 687–698 (2004).
29.Stephens, L.M.; Varga, S.M. Function and Modulation of Type I Interferons during Respiratory Syncytial Virus Infection. Vaccines 2020, 8, 177. https://doi.org/10.3390/vaccines8020177
30.Pidugu VK, Pidugu HB, Wu MM, Liu CJ, Lee TC. Emerging Functions of Human IFIT Proteins in Cancer. Front Mol Biosci. 2019 Dec 19;6:148. doi: 10.3389/fmolb.2019.00148. PMID: 31921891; PMCID: PMC6930875.
31.Qiu L, Wang T, Tang Q, Li G, Wu P, Chen K. Long Non-coding RNAs: Regulators of Viral Infection and the Interferon Antiviral Response. Front Microbiol. 2018 Jul 19;9:1621. doi: 10.3389/fmicb.2018.01621. PMID: 30072977; PMCID: PMC6060254.
32.Michalska A, Blaszczyk K, Wesoly J, Bluyssen HAR. A Positive Feedback Amplifier Circuit That Regulates Interferon (IFN)-Stimulated Gene Expression and Controls Type I and Type II IFN Responses. Front Immunol. 2018 May 28;9:1135. doi: 10.3389/fimmu.2018.01135. PMID: 29892288; PMCID: PMC5985295.