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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張?仁(Ching-Jin Chang) | |
dc.contributor.author | Hsin-Hui Hsieh | en |
dc.contributor.author | 謝欣蕙 | zh_TW |
dc.date.accessioned | 2021-06-15T13:54:47Z | - |
dc.date.available | 2020-08-31 | |
dc.date.copyright | 2015-08-31 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-31 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51876 | - |
dc.description.abstract | Tristetraprolin(TTP)是一個訊息核糖核酸降解因子(mRNA-destabilizing protein),在我們先前的研究中,發現到TTP可以結合至MAPK去磷酸酶(MKP-1)訊息核糖核酸上3’端非轉譯區(3’-untranslated region)的多腺嘌呤-尿嘧啶序列 (AU-rich elements),進而促使MKP-1訊息核糖核酸的降解,而TTP亦可自我調節本身訊息核糖核酸的穩定性。已知TTP的功能是受到本身蛋白質磷酸化程度所調控的,我們發現在 3T3-L1脂肪前驅細胞分化過程的早期當中,ERK是MAPKs訊息傳遞路徑中主要被活化的磷酸激酶,ERK除了會刺激TTP和MKP-1的表現也會增加TTP磷酸化的程度,而使用ERK抑制劑U0126則可以降低TTP的表現量及磷酸化程度,顯示TTP調控訊息核糖核酸穩定性的能力是會受到ERK訊息傳遞路徑所調控。故本篇論文旨在研究3T3-L1脂肪前驅細胞分化過程中ERK磷酸化TTP並藉此調控TTP活性的詳細作用機轉。首先,利用螢光素酶報導基因分析結合小干擾RNA(small interfering RNA)實驗以及蛋白質交互作用分析,我們發現到Ccr4-Not腺苷化酶複合蛋白(Ccr4-Not deadenylase complex) 中的支架蛋白(scaffold protein)Cnot1對於TTP的功能是不可或缺的。TTP需先透過與Cnot1結合才能與Ccr4-Not腺苷化酶複合蛋白結合,並進而達到其降解訊息核糖核酸的功能。我們也發現到TTP第220號絲氨酸的位置可以被ERK磷酸化,而被磷酸化後的TTP與14-3-3蛋白的結合會增強,故而導致TTP降解MKP-1訊息核糖核酸的能力降低。另外,利用質譜分析、蛋白質變異配合螢光素酶報導基因分析以及免疫沉澱分析,也證明了TTP位於C端的第316號絲氨酸亦可被ERK所磷酸化,該磷酸化會破壞TTP與Ccr4-Not複合蛋白的交互作用,且亦導致TTP降解MKP-1訊息核糖核酸的能力降低。綜合以上研究結果,我們成功地證明TTP可以藉由ERK訊息傳遞路徑的磷酸化修飾而改變其蛋白質交互作用的特性,進而調控MKP-1訊息核糖核酸的穩定性。我們的發現對於ERK訊息傳遞路徑、TTP和MKP-1這三者在3T3-L1脂肪前驅細胞分化早期的聯絡網路有了更清楚的認識。 | zh_TW |
dc.description.abstract | Previously, our lab has demonstrated that both tristetraprolin (TTP) and MKP-1 are immediate early genes, whose expression were activated and quickly subsided in response to ERK signaling during 3T3-L1 differentiation. Notably, TTP is known as an mRNA-destabilizing protein, which can bind to AU-rich elements (AREs) in the 3’UTR of mRNA and trigger mRNA destabilization. We also found the mRNA target of TTP includes MKP-1 and TTP itself. The function of TTP is tightly regulated by its protein phosphorylation status. The ERK signaling inhibitors U0126 decreased the expression and phosphorylation levels of TTP, indicating that TTP phosphorylation and its function are regulated by ERK signaling. Therefore, this thesis aims to explore the detailed mechanism of how TTP function is regulated by ERK signaling during adipogenesis. Here, we demonstrates that Cnot1, a scaffold protein of Ccr4–Not deadenylase complex, is an essential partner in TTP-mediated mRNA decay by functional luciferase reporter analysis combining with siRNA knockdown and protein-protein interactions analysis. Also, our results reveal that ERK can phosphorylate TTP at serine 220 and increases interaction between TTP with 14-3-3, thus impairing TTP-mediated mRNA decay. Moreover, using mass spectrometry analysis, functional reporter assay and co-immunoprecipitation assay combined with mutants, we demonstrated that TTP was phosphorylated at serine 316 in C-terminal conserved region by ERK signaling, which significantly perturbs Ccr4-Not complex recruitment and TTP mRNA-destabilizing activity. These findings provide a better understanding of how TTP regulate MKP-1 mRNA stability under the control of ERK signaling pathway during 3T3-L1 differentiation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T13:54:47Z (GMT). No. of bitstreams: 1 ntu-104-R02b46018-1.pdf: 2359280 bytes, checksum: a4e12b9bf78c1299036506818bf91c5c (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員會審定書 i
摘要 ii Abstract iii Table of contents iv Abbreviation vi Chapter 1 Introduction 1 1.1 ARE-mediated mRNA decay 1 1.2 TTP family 2 1.3 The molecular mechanism of TTP-mediated mRNA degradation 2 1.4 TTP phosphorylation regulates its mRNA-destabilizing ability 4 1.5 The mRNA targets of TTP and the role of TTP in diseases 5 1.6 MAPKs and MKP-1 in adipogenesis 6 1.7 Specific aims 8 Chapter 2 Material and method 9 2.1 Plasmid constructs 9 2.2 Cell culture 10 2.3 Immunoprecipitation assay 10 2.4 Western blot analysis 11 2.5 Antibodies and chemicals 12 2.6 Dual luciferase reporter assay 12 2.7 Gene knockdown by siRNA 13 2.8 Mass spectrometry-based analysis 14 2.9 In vitro kinase assay 14 2.10 GST-tagged recombinant protein purification and GST pull-down assay 15 2.11 RNA isolation and reverse transcription 15 2.12 Real-time PCR 16 2.13 RNA half-life 17 2.14 Statistical analysis 17 Chapter 3 Results 18 3.1 MAPKs, MKP-1, and TTP during 3T3-L1 differentiation 18 3.2 TTP family requires Cnot1 for its mRNA-destabilizing ability 19 3.3 Identification of ERK signaling-mediated TTP phosphorylation sites 21 3.4 TTP phosphorylation at serine 220 by ERK regulates its function 22 3.5 TTP C-terminal phosphorylation by ERK signaling regulates its activity 24 Chapter 4 Discussion 26 Chapter 5 Figure 31 Figure 1. MAPKs, MKP-1, and TTP during 3T3-L1 differentiation 31 Figure 2. TTP family requires Cnot1 for its mRNA-destabilizing ability 34 Figure 3. Identification of ERK signaling-mediated TTP phosphorylation sites 39 Figure 4. TTP phosphorylation at serine 220 by ERK regulates its function 45 Figure 5. TTP C-terminal phosphorylation by ERK signaling regulates its activity 49 Chapter 6 Table 56 Table 1. Primers for generating mouse Cnot1 deletion constructs 56 Table 2. Primers for generating mouse TTP mutants 57 Table 3. Primers for real-time PCR 59 Table 4. TTP-associated proteins identified by MS/MS analysis 60 Table 5. Gene ontology analysis of TTP-associated proteins 65 Chapter 7 Supplementary information 68 Reference 71 | |
dc.language.iso | en | |
dc.title | 3T3-L1脂肪前驅細胞分化前期中TTP受ERK磷酸化之調控 | zh_TW |
dc.title | Phosphorylation of Tristetraprolin (TTP) by the ERK Signaling Pathway in the Early Differentiation of 3T3-L1 Preadipocyte | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張震東,果伽蘭,林照雄 | |
dc.subject.keyword | mRNA穩定性,鋅指蛋白36 (TTP),ERK訊息傳遞路徑,磷酸化,MAPK去磷酸?(MKP-1), | zh_TW |
dc.subject.keyword | mRNA stability,tristetraprolin,ERK signaling,MKP-1,phosphorylation, | en |
dc.relation.page | 75 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-31 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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