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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 劉旻禕(Helene Minyi Liu) | |
| dc.contributor.author | Yi-Chun Peng | en |
| dc.contributor.author | 彭奕淳 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:43:06Z | - |
| dc.date.available | 2021-07-10T21:43:06Z | - |
| dc.date.copyright | 2020-09-04 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77005 | - |
| dc.description.abstract | 近期諸多研究指出，伴護蛋白質 DNAJ/HSP40 家族，除了參與病毒感染宿主細胞後複製循環的調控，也參與調控宿主細胞先天性抗病毒免疫路徑，例如第一型干擾素抗病毒反應。當 RNA 病毒感染宿主細胞，宿主細胞的模式辨認受體 (pattern recognition receptors, PRRs) 之一如 RIG-I 會分辨認病毒核酸，進而活化下游第一型干擾素生成路徑，抵抗病毒感染與複製。本篇論文首次探討編碼來源為 DNAJB4 基因的伴護蛋白質 HLJ1 在此第一型干擾素生成路徑中所扮演的角色。起初，根據未發表的小鼠實驗，同樣注射脂多醣 (Lipopolysaccharide, LPS) 之下， DNAJB4 基因剔除小鼠的存活率顯著高於野生型小鼠，表示缺少 DNAJB4 基因下，小鼠體內類鐸受體 TLR4 調控的發炎反應程度較低，亦即伴護蛋白質 HLJ1 會正調控 TLR4 下游發炎反應路徑。有鑑於先前研究已知，除了發炎訊息路徑外， TLR4 的活化也會引發第一型干擾素生成路徑，於是我們在脂多醣刺激野生型小鼠纖維母細胞 (mouse embryonic fibroblasts, MEFs) 和 DNAJB4 基因剔除的小鼠纖維母細胞後，利用 RT-PCR 檢測兩細胞第一型干擾素 IFNβ 基因表現量，發現 DNAJB4 基因剔除小鼠細胞的 IFNβ 基因表現量明顯較低，也說明了 HLJ1 蛋白質可能參與調控第一型干擾素生成路徑。除了 TLR4 下游發炎路徑外，我們利用基因體為負股單鏈 RNA 病毒的仙台病毒分別感染野生型和 DNAJB4 基因剔除小鼠纖維母細胞株，活化 RIG-I 下游之第一型干擾素生成路徑，結果發現相較於野生型，基因剔除小鼠細胞中的 IFNβ 基因表現明顯較低，且仙台病毒基因體複製程度較高，表示 DNAJB4 基因的缺失會導致 RIG-I 下游之第一型干擾素生成減少，並使得宿主細胞抗病毒能力下降；此外，從 Immunoblotting 實驗結果得知，在仙台病毒感染兩小鼠細胞下，p-IRF3 (第一型干擾素轉錄因子 IRF3 活化態)在 DNAJB4 基因剔除小鼠細胞中的相對量顯著較低，同樣顯示 DNAJB4 基因對於第一型干擾素生成路徑的重要性。除了利用小鼠細胞進行上述分析，我們也發現 Huh7 人類肝癌細胞株逐步提升 HLJ1 蛋白質外源表現並在仙台病毒感染細胞下， IFNβ 啟動子活化程度也隨之提升，再次顯示 HLJ1 蛋白質正調控第一型干擾素生成路徑。為了進一步探討 HLJ1 蛋白質調控第一型干擾素生成路徑的分子機制，我們在 Huh7 細胞株中成雙外源表現 HLJ1 和第一型干擾素生成路徑之訊息傳遞分子，下游至上游依序為 IRF3-5D (擬活化態轉錄因子)、TBK1、MAVS，結果發現，當同時外源表現 IRF3-5D 和 TBK1 活化下游第一型干擾素生成路徑下，即使逐步提升 HLJ1 蛋白質外源表現量，IFNβ 啟動子活化程度並沒有差異，然而，當外源表現 MAVS，逐步提升 HLJ1 蛋白質外源表現量，IFNβ 啟動子活化程度也隨之提升，表示 HLJ1 會正調控 TBK1 上游、 MAVS 下游的訊息傳遞分子。另外藉由細胞質/粒腺體離析實驗結合 Immunoblotting 分析得知，在仙台病毒感染下， HLJ1 蛋白質會從細胞質移動至粒腺體相關內質網膜，表示 HLJ1 蛋白質有可能參與調控 MAVS 信息小體 (signalosome)。最後，我們在外源 HLJ1 蛋白質免疫沉澱結合質譜蛋白質體學分析結果得知，在仙台病毒感染下活化第一型干擾素生成訊息傳遞路徑，可能與 HLJ1 交互作用一系列宿主蛋白質候選者身分，提供後續對於 HLJ1 蛋白質在第一型干擾素生成路徑中所扮演的調控角色更多啟發。 | zh_TW |
| dc.description.abstract | Heat shock proteins (HSPs) are a diverse group of chaperone proteins. Many of them have been proven to play key roles in response to the cellular stress conditions, such as infections or inflammations. HLJ1 encoded by the gene DNAJB4 (DnaJ Homolog, Subfamily B, Member 4) is one of the HSP40s. Previous studies showed that the depletion of DNAJB4/HLJ1 gene in mice led to the higher survival rate after lipopolysaccharide (LPS) treatment in vivo, suggesting that DNAJB4/HLJ1 could stimulate TLR4-mediated inflammatory responses. Besides inflammatory cytokines, type I interferon (IFN) is also regulated by TLR4 activation. Therefore, we monitored the relative mRNA levels of IFNβ, which belongs to the type I IFN, in the wild-type (WT) and the DNAJB4 gene-knock-out (DNAJB4 -/-) mice embryonic fibroblast cells (MEFs) respectively after LPS treatments. The results indicated that the IFNβ mRNA levels in the DNAJB4-/- MEFs were lower than those in the WT MEFs. Also, type I IFN expression triggered by Sendai virus (SenV) (ssRNA (+) virus) was also reduced in the DNAJB4-/- MEFs when compared to that in the WT MEFs. The phosphorylation of IRF3, which is the signaling active form of IRF3, was decreased in DNAJB4-/- MEFs compared with WT MEFs during SenV infection, revealing the role of DNAJB4/HLJ1 in promoting type I IFN induction signaling in response to viral infections. These results indicated that DNAJB4/HLJ1 could regulate type I IFN and/or antiviral signaling in the host cells. Furthermore, when ectopically expressed in the human hepatocyte cell line Huh7, DNAJB4/HLJ1 enhanced the IFNβ promoter activities in a protein dose-dependent manner in response to SenV infection. Our results showed that overexpression of DNAJB4 promotes the IFNβ promoter activities in the presence of MAVS but not TBK1, suggesting that DNAJB4/HLJ1 may regulate certain signaling molecules downstream of MAVS and upstream of TBK1 in type I IFN induction pathway. By cytosolic/MAM fractionation, we found that DNAJB4/HLJ1 was redistributed more to the mitochondrial/MAM fractions during SenV infection. We have identified several host proteins to interact with DNAJB4/HLJ1 in Huh7 cells in response to SenV infection by mass spectrometry analysis. The results from this analysis are anticipated to provide insights for us to further study the molecular mechanisms of how DNAJB4/HLJ1 regulates type I IFN pathway. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:43:06Z (GMT). No. of bitstreams: 1 U0001-2707202010162900.pdf: 1936102 bytes, checksum: eb55fd34c12a2964c22d6e66119ef7a4 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 中文摘要： 1 ABSTRACT: 3 TABLE OF CONTENTS 5 LIST OF FIGURES 9 CHAPTER 1: INTRODUCTION 11 1.1 Overview of the pattern recognition receptors (PRRs)-mediated innate immune responses 11 1.2 RIG-I-like (RLR) receptor family members 14 1.3 RIG-I-like (RLR)-mediated interferon induction signaling pathway and interferon response signaling pathway 15 1.4 Several chaperone proteins regulate type I IFN induction 17 1.5 The chaperone protein DNAJ/HSP40 family 18 1.6 Several DNAJ/HSP40 family members are involved in regulating viral propagation in host cells 20 1.7 Highly correlation between DNAJB1 and DNAJB4 21 1.8 Specific aim 22 CHAPTER 2: MATERIALS AND METHODS 24 2.1. Materials： 24 2.1.1. Cell line： 24 2.1.2. Competent cells 24 2.1.3 Virus 24 2.1.4 Plasmid 25 2.1.5 Reagent 25 2.1.6 Antibody 27 2.1.7 Commercial Kit 28 2.2. Methods 29 2.2.1 Cell culture 29 2.2.2 Transfection 29 2.2.3 Dual luciferase assay 30 2.2.4 Western blot 30 2.2.5 Sendai Virus cultivation 32 2.2.6 Sendai Virus infection 33 2.2.7 Mitochondria/Cytosol fractionation 33 2.2.8 RNA extraction 34 2.2.9 Real-time qPCR 35 2.2.10 Co-Immunoprecipitation (Co-IP) 36 2.2.11 Colloidal Blue Staining 37 2.2.12 V5-IP/MS 38 2.2.13 PCR amplification and Sub-Cloning: 39 CHAPTER 3: RESULTS 43 3.1. DNAJB4 was neither an interferon stimulatory gene (ISG) nor a SenV infection-inducible gene. 43 3.2. The loss of DNAJB4 gene reduced the TLR4-dependent type I IFN expression upon lipopolysaccharide (LPS) treatment. 44 3.3. DNAJB4/HLJ1 positively regulated RIG-I-mediated type I IFN induction signaling. 46 3.4. DNAJB4/HLJ1 was required for activation and phosphorylation of IRF3. 47 3.5. DNAJB4/HLJ1 enhanced the RIG-I-mediated signaling to IFNβ. 48 3.6. DNAJB4/HLJ1 positively regulated the molecules between MAVS and TBK1 in type I IFN pathway 49 3.7. DNAJB4/HLJ1 might re-localize to mitochondrial membranes or mitochondria-associated membranes (MAMs) in response to SenV infection. 50 3.8. Proteomic analysis of the DNAJB4/HLJ1 interactome network during Sendai virus infection 51 3.9. Use shRNA to silence the expression of HLJ1 at post-transcription levels. 53 CHAPTER 4: DISCUSSION 54 CHAPTER 5: REFERENCE 61 TABLE 1. PRIMER USED FOR SUB-CLONING: 80 TABLE 2. PLKO1. DNAJB4 SHRNA: 80 TABLE 3. RT-QPCR PRIMER: 81 APPENDIX 83 LIST OF FIGURES Figure 1. DNAJB4/HLJ1 was neither an ISG, nor SenV-infection-inducible gene. 69 Figure 2. The depletion of DNAJB4 gene resulted in decreased IFNβ mRNA expression during LPS treatment. 70 Figure 3. DNAJB4/HLJ1 positively regulated RIG-I-mediated type I IFN induction and IFN antiviral functions. 71 Figure 4. DNAJB4/HLJ1 is important for activation and phosphorylation of IRF3. 72 Figure 5. Ectopic Expression of DNAJB4/HLJ1 enhanced the IFNβ promoter activity during SenV infection. 73 Figure 6. DNAJB4/HLJ1 did not affect the IRF3-mediated IFNβ promoter activation 74 Figure 7. DNAJB4/HLJ1 did not affect the TBK1-mediated IFNβ promoter activation. 75 Figure 8. DNAJB4/HLJ1 enhances the IFNβ promoter activities when co-overexpressing MAVS. 76 Figure 9. DNAJB4/HLJ1 re-distributed to mitochondrial-associated membranes (MAM) in response to SenV infection. 77 Figure 10. V5-HLJ1 IP/MS proteomic analysis. 78 Figure 11. Deliver the shRNA into Huh7 cells through transfection of plasmids to silence the expression of HLJ1 at post-transcription levels. 79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 正調控 | zh_TW |
| dc.subject | 伴護蛋白質 DNAJB4/HLJ1 | zh_TW |
| dc.subject | 第一型干擾素生成路徑 | zh_TW |
| dc.subject | chaperone protein DNAJB4/HLJ1 | en |
| dc.subject | positively regulate | en |
| dc.subject | type I IFN induction pathway | en |
| dc.title | 探討伴護蛋白質 DNAJB4/HLJ1 對第一型干擾素生成路徑之調控 | zh_TW |
| dc.title | The Regulation of Type I Interferon Induction Pathway by the Chaperone Protein DNAJB4/HLJ1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.author-orcid | 0000-0002-5394-0309 | |
| dc.contributor.oralexamcommittee | 徐立中(Li-Chung Hsu),蘇剛毅(Kang-Yi Su),顏伯勳(Bo-Shiun Yan) | |
| dc.subject.keyword | 伴護蛋白質 DNAJB4/HLJ1,正調控,第一型干擾素生成路徑, | zh_TW |
| dc.subject.keyword | chaperone protein DNAJB4/HLJ1,positively regulate,type I IFN induction pathway, | en |
| dc.relation.page | 87 | |
| dc.identifier.doi | 10.6342/NTU202001891 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-07-28 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| Appears in Collections: | 生物化學暨分子生物學科研究所 | |
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