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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉旻禕(Helene Minyi Liu) | |
| dc.contributor.author | Jhih-Pu Lin | en |
| dc.contributor.author | 林志璞 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:33:21Z | - |
| dc.date.available | 2021-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-02 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50233 | - |
| dc.description.abstract | 宿主在受到RNA病毒感染時，宿主細胞內之RIG-I相似受器家族如RIG-I或者MDA5具有辨識細胞質內病毒遺傳物質RNA，並進一步活化下游訊息傳遞路徑，以產生第一型干擾素抵抗病毒感染與複製的功能。RIG-I相似受器家族通常被認為具有不同的雙股RNA辨認親和性，RIG-I被認為會優先辨認長度小於200個鹼基對的雙股RNA，而MDA5則通常被認為會辨認長度大於2000個鹼基對的雙股RNA。根據先前的研究指出，當病毒感染細胞時，RIG-I必須藉由蛋白質14-3-3ε的幫助使RIG-I能夠由細胞質移動到粒線體結合內質網膜(Mitochondria-Associated Membrane; MAM)的位置，以進一步和下游訊息傳遞蛋白質MAVS作用。RIG-I及MDA5會和下游蛋白質MAVS作用進而活化下游第一型干擾素訊息傳遞路徑。目前對於RIG-I活化路徑的研究較為完整及深入，但是對於MDA5活化路徑的探討卻是相對較少。在此研究當中，我們發現14-3-3η對於MDA5活化下游第一型干擾素傳遞路徑具有非常重要的功能。藉由免疫共同沉澱法發現有不同的14-3-3蛋白質亞型可以與MDA5的N端結構域CARDs結合，然而對於MDA5之C端結構域(C-terminal domain)卻沒有結合能力。此外，於細胞中異位表現(ectopic expression)14-3-3η可增強MDA5傳遞路徑所誘發的第一型干擾素啟動子活性(promoter activity)，且此一增強作用是與異位表現的14-3-3η蛋白質表現量呈正相關。而在Huh7肝癌細胞中抑制(knock-down)內源性的14-3-3η蛋白質表現量，對於異位表現全長MDA5或MDA5 N端所引發的第一型干擾素啟動子活性上升的現象會產生抑制作用。為了近一步探討14-3-3η蛋白質對於MDA5傳遞路徑的影響，我們將MDA5 N端序列中預測的14-3-3蛋白質結合基序(binding motif)進行突變，並且發現如果將MDA5第88個胺基酸由絲胺酸(serine)改為天門冬胺酸(aspartic acid)，除了會使MDA5無法正常活化外，也會使14-3-3蛋白質無法和MDA5結合。最後藉由細胞離析方法(cell fractionation)得知內源性MDA5會因為異位表現的N-MDA5而活化細胞第一型干擾素訊息傳遞路徑，並從細胞質移動到粒線體相關區域，而之前發現可以與MDA5結合的14-3-3 蛋白質也同樣可以分布於粒線體相關區域。總結來說，在此研究當中我們發現了14-3-3η蛋白質在MDA5傳遞路徑當中扮演重要的角色。 | zh_TW |
| dc.description.abstract | During RNA virus infection, RIG-I like receptors (RLRs), such as Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated protein 5 (MDA5), recognize cytoplasmic viral RNA and activate downstream adaptor proteins to produce type I interferon (IFN). It is known that RIG-I preferentially binds to short dsRNA (<200 bp) whereas MDA5 binds to dsRNA longer than 2000 base pairs. It has been reported that upon viral infection, RIG-I interacts with the chaperon protein 14-3-3ε, which facilitates translocation of RIG-I to mitochondrial associated membrane (MAM) for interaction with mitochondrial antiviral-signaling protein (MAVS), which is the common adaptor protein for both RIG-I-dependent and MDA5-dependent signaling to induce type I IFN. However, the molecular mechanisms of MDA5 activation remain less understood. Here we show that 14-3-3η is crucial for MDA5-dependent type I IFN induction. We found that several 14-3-3 isoforms may be co-immunoprecipitated with MDA5 through the CARDs (N-MDA5) but not the C-terminus of MDA5 (C-MDA5). Furthermore, ectopic expression of 14-3-3η, but not other isoforms, could enhance MDA5-dependent activation of IFNβ promoter in a dose-dependent manner. Knock-down of 14-3-3η in Huh7 cells also led to the attenuation of the IFNβ promoter activities which were facilitated by ectopic expression of MDA5 or N-MDA5.
To further investigate the essential role of 14-3-3 proteins in MDA5-dependent signaling pathway, we co-immunoprecipitated 14-3-3 proteins with different N-MDA5 mutants and found that a defective mutant form of N-MDA5, N-MDA5-S88D could not interact with 14-3-3 proteins. Besides, in our study we also found that endogenous MDA5 could be translocated from cytosol to mitochondrial membrane fraction in response to the activation of type I IFN signaling pathway due to ectopically expressing N-MDA5. In conclusion, we define 14-3-3η as a novel molecule which is involved in the MDA5-dependent IFNβ signaling pathway. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:33:21Z (GMT). No. of bitstreams: 1 ntu-105-R03424002-1.pdf: 2724499 bytes, checksum: 3d8fe7287ace90ec2a1871d1bbd12b74 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 I
Abstract III Table of Contents V List of Figures VIII List of Table X Chapter 1: Introduction 1 1.1 Discovery of Melanoma Differentiation-Associated Protein 5 1 1.2 Structure of RIG-I Like Receptors 2 1.3 Innate Immune System: Interferon Induction and Signaling Pathways 4 1.4 The Mechanisms for RIG-I and MDA5 Activation 7 1.5 The 14-3-3 Chaperon Proteins Control Intracellular Protein Localizations 9 1.6 Specific Aims 12 Chapter 2: Materials and Methods 13 2.1 Materials 13 2.1.1 Cell Lines 13 2.1.2 Competent Cells 13 2.1.3 Viruses 13 2.1.4 Plasmids 13 2.1.5 Reagents 15 2.1.6 Enzymes 16 2.1.7 Antibodies 17 2.1.8 Commercial Kits 18 2.2 Methods 18 2.2.1 Cell Culture 18 2.2.2 Making Transformation Competent Cells 19 2.2.3 Construction of Expression Plasmids 20 2.2.4 Bacterial Transformation 21 2.2.5 DNA Plasmids Transfection 22 2.2.6 RNA Ligands Transfection 23 2.2.7 Western Blot 24 2.2.8 Protein-Protein Co Immunoprecipitation 26 2.2.9 Sendai Virus Cultivation 28 2.2.10 Sendai Virus Infection 29 2.2.11 Luciferase Assay 30 2.2.12 In vitro RNA Ligands Preparation 30 2.2.13 Cell Fractionation Assay 31 2.2.14 Selection of 14-3-3η Knock-Down Huh7 Cells Stable Clones 32 2.2.15 Phosphatase Assay 33 Chapter 3: Results 34 3.1 Differential Activation of MDA5 by Long Double Stranded RNA Stimulations 34 3.2 The Intra-Molecular Characteristics of MDA5 and RIG-I in IFNβ Signaling Pathway 35 3.3 Identification of MDA5-interacting 14-3-3 Isoforms 37 3.4 The Distribution of MDA5 and 14-3-3 Proteins in Cells 40 3.5 The Role of 14-3-3 Isoforms in MDA5-Dependent IFNβ Induction Pathway 41 3.6 The Properties of Predicted 14-3-3 Proteins Binding Motif with MDA5 CARD Region 44 Chapter 4: Discussion 46 Chapter 5: References 53 | |
| dc.language.iso | en | |
| dc.subject | 黑色素瘤分化相關蛋白質-5 | zh_TW |
| dc.subject | 第一型干擾素傳遞路徑 | zh_TW |
| dc.subject | 14-3-3蛋白質家族 | zh_TW |
| dc.subject | 第一型干擾素傳遞路徑 | zh_TW |
| dc.subject | 14-3-3蛋白質家族 | zh_TW |
| dc.subject | 黑色素瘤分化相關蛋白質-5 | zh_TW |
| dc.subject | MDA5 | en |
| dc.subject | MDA5 | en |
| dc.subject | Type I IFN signaling | en |
| dc.subject | 14-3-3 proteins | en |
| dc.subject | Type I IFN signaling | en |
| dc.subject | 14-3-3 proteins | en |
| dc.title | 探討14-3-3蛋白質家族對黑色素瘤分化相關蛋白質-5 (MDA5)活化之調控 | zh_TW |
| dc.title | Controls of MDA5 Activation by The 14-3-3 Protein Family | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 高全良(Chuan-Liang Kao),張淑媛(Sui-Yuan Chang),郭瑞琳(Rei-Lin Kuo) | |
| dc.subject.keyword | 黑色素瘤分化相關蛋白質-5,第一型干擾素傳遞路徑,14-3-3蛋白質家族, | zh_TW |
| dc.subject.keyword | MDA5,Type I IFN signaling,14-3-3 proteins, | en |
| dc.relation.page | 80 | |
| dc.identifier.doi | 10.6342/NTU201601258 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-02 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf 未授權公開取用 | 2.66 MB | Adobe PDF |
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