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研究生： 蔡翊琦
Tsai, Yi-Chi
論文名稱： 含雙色胺酸功能區氧化還原酶在第一型干擾素訊號傳遞路徑中的調控性角色
The regulatory role of WW domain-containing oxidoreductase in type I interferon signaling pathway
指導教授： 徐麗君
Hsu, Li-Jin
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫學檢驗生物技術學系
Department of Medical Laboratory Science and Biotechnology
論文出版年： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 78
中文關鍵詞： 腫瘤抑制因子 、訊息傳遞 、先天免疫 、發炎
外文關鍵詞： tumor suppressor, signal transduction, innate immunity, inflammation
相關次數： 點閱：114  下載：0 
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    • 在病毒感染時，被感染的細胞會釋出第一型干擾素 (interferons, IFNs) 來引起JAK-STAT訊號傳遞路徑的活化以促進抗病毒反應。當第一型IFNs與其接受器IFN-α receptor (IFNAR) 結合後，會活化下游JAK1及TYK2以吸引STAT1與STAT2聚集，進一步促使STAT1和STAT2蛋白質磷酸化。磷酸化後的STAT1和STAT2會形成異二聚體，移行入核引起干擾素刺激基因 (IFN-stimulated genes, ISGs) 的轉錄，藉以活化抗病毒反應。雖然第一型干擾素對於宿主對抗病毒感染是極有益處的，但第一型干擾素訊號傳遞路徑若過度活化，也會造成自體免疫疾病及免疫失調。為了維持良好的抗病毒功效及避免免疫疾病，細胞對第一型干擾素訊號傳遞路徑的調節是極為重要的。我們實驗室最近發現腫瘤抑制因子「含雙色胺酸功能區氧化還原酶」(WW domain-containing oxidoreductase, WWOX)會抑制腸病毒A71型和單純疱疹病毒-1的感染，但是WWOX是否調控第一型干擾素刺激細胞的訊號傳遞路徑依然是未知的。在此篇研究中，我們證實了在WWOX缺乏的細胞中，第一型干擾素介導的JAK1和STAT1蛋白質磷酸化，比起對照組，都有顯著的增加，顯示WWOX在第一型干擾素訊號傳遞路徑中扮演著調控性的角色。總結而言，我們的結果顯示WWOX在調控IFN-α訊號傳遞路徑中扮演了重要的調控角色，釐清WWOX對於第一型干擾素訊號傳遞路徑的調控將可以在瞭解WWOX於免疫系統中的功能提供全新的觀點。

    In viral infection, the infected cells release type I interferons (IFNs) to trigger Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling pathways for induction of antiviral responses. The engagement of type I IFNs with IFN-α receptor (IFNAR) activates JAK1 and tyrosine kinase 2 (TYK2) for recruitment and phosphorylation of both STAT1 and STAT2. The phosphorylated STAT1 and STAT2 form heterodimers, translocate to the nucleus and initiate the transcription of IFN-stimulated genes (ISGs) to stimulate antiviral responses. Although type I IFNs benefit host defense against virus infection, prolonged activation of type I IFN signaling has been shown to cause autoimmune diseases and immune dysregulation. To achieve proper antiviral responses and avoid immune pathogenesis, the modulation of type I IFN signaling is important. Tumor suppressor WW domain-containing oxidoreductase (WWOX) has been recently shown to suppress both enterovirus A71 and herpes simplex virus-1 infection in our laboratory. Nevertheless, whether WWOX regulates type I IFN signaling during virus infection remains unknown. In this study, we demonstrated that IFN-α-induced protein phosphorylation of JAK1 and STAT1 was increased in WWOX-knockdown cells as compared with the control cells, indicating a regulating role of WWOX in type I IFN signaling pathway. Together, our results suggest that WWOX plays an important role in regulating IFN-α-induced signaling pathway. Delineating the regulation of type I IFN signaling pathway by WWOX will provide a novel impact on the understanding of WWOX function in the immune system.

    中文摘要 I Abstract II 誌謝 III Abbreviations and Symbols IV Contents V Figure Index IX Introduction 1 Type I interferons (IFNs) during infection 1 Janus kinase (JAK) family 2 The negative regulation of JAK family proteins 2 WW domain-containing oxidoreductase (WWOX) 4 WWOX in virus infection 5 Materials and Methods 6 A. Materials 6 A-1. Cell lines 6 A-2. Plasmid and primers for construction 6 A-3. Reagents and chemicals 7 A-4. Kits 8 A-5. Antibodies 9 A-6. shRNA clones 9 A-7. PCR primers 9 A-8. Consumables 10 A-9. Instruments 10 B. Methods 13 B-1. Cell culture 13 B-2. Transfection of WWOX shRNA 13 B-3. Transfection of plasmids by electroporation 14 B-4. RNA extraction 14 B-5. Reverse transcription-polymerase chain reaction (RT-PCR) 15 B-6. Polymerase chain reaction (PCR) 16 B-7. Protein extraction 19 B-8. Protein quantification 20 B-9. Co-immunoprecipitation 21 B-10. GST pull-down assay 22 B-11. Cell fractionation 22 B-12. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting 24 B-13. Site-directed mutagenesis 27 B-14. Construction 29 B-15. Plasmid DNA purification 36 Results 38 WWOX inhibits IFN-α-induced JAK1-STAT1 signaling pathway 38 WWOX downregulates K48-linked ubiquitination of JAK1 after IFN-α stimulation. 39 WWOX neither modulates the protein expression levels of SOCS1 nor suppresses the interaction between SOCS1 and JAK1. 39 WWOX physically interacts with JAK1 and gradually dissociates from JAK1 after IFN-α treatment. 40 The N-terminal WW domain region and C-terminal SDR domain of WWOX are required for the interaction of WWOX with JAK1. 41 The kinase domain of JAK1 is critical for the association of WWOX with JAK1. 42 WWOX increases the protein levels of SHP1 but not SHP2 to suppress JAK1 phosphorylation. 42 Discussion 44 The role of WWOX in regulating protein polyubiquitination of JAK 44 The role of WWOX in inhibiting JAK1 kinase activity 44 The role of WWOX in inhibiting JAK1 phosphorylation via regulating PTPs 45 The role of WWOX in regulating other type I IFN signaling molecules 46 The role of WWOX in IFN-α-induced antiviral responses 46 The role of WWOX in other JAK-STAT signaling pathways 47 Conclusion 48 References 49 Figures 56 Appendixes 72 Appendix I. Type I IFN signaling pathway. 72 Appendix II. JAKs 73 Appendix III. WWOX 74 Appendix IV. Wwox-/- mice exhibit a higher mortality rate and body weight loss as compared with Wwox+/+ and Wwox+/- littermates after intraperitoneal (i.p.) injection of EV-A71 and HSV-1. 75 Appendix V. HA-pcDNA3 76 Appendix VI. pFLAG-CMV-2 77 Resume 78

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