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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳小梨 | zh_TW |
| dc.contributor.advisor | Show-Li Chen | en |
| dc.contributor.author | 黃慧儀 | zh_TW |
| dc.contributor.author | Hui Yee Ng | en |
| dc.date.accessioned | 2024-08-27T16:11:33Z | - |
| dc.date.available | 2024-08-28 | - |
| dc.date.copyright | 2024-08-27 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-03 | - |
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Deficiency of nuclear receptor interaction protein leads to cardiomyopathy by disrupting sarcomere structure and mitochondrial respiration. Journal of Molecular and Cellular Cardiology, 137, 9-24. Zhang, Y., Ye, J., Chen, D., Zhao, X., Xiao, X., Tai, S., et al. (2006). Differential expression profiling between the relative normal and dystrophic muscle tissues from the same LGMD patient. Journal of Translational Medicine, 4(1), 53. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95064 | - |
| dc.description.abstract | 核受體相互作用蛋白 (NRIP)，它也可以稱為 DCAF6 (DDB1 及 CUL4 相關因子 6) 或 IQWD1。NRIP由 860 個氨基酸組成，目前已知NRIP有七個 WD40 domain再加上一個IQ結構模體 (motif)。我們之前的研究發現MyoG是NRIP調控的目標，因為在全身性NRIP基因剔除 (NRIP-gKO) 模式小鼠中，受傷後第6天的MyoG mRNA和蛋白質, 其表達量皆低於野生型小鼠。另外，肌肉特異性NRIP條件性基因敲除 (NRIP-cKO) 小鼠顯示出肌肉異常和運動功能受損。在NRIP-cKO小鼠中，MyoG在神經肌肉接合處 (NMJ) 的突觸核中的表達量下降。通過MyoG過表達可以改善NRIP-cKO小鼠的異常表型，表示NRIP可以通過MyoG表達逆行支持脊髓運動神經元的NMJ功能。鑑於NRIP在肌肉分化中的多重角色及其與MyoG調控的關聯性，我們旨在確定NRIP是否作為轉錄共激活因子來增強MyoG基因表達。
我們首先構建了一個驅動熒光素酶報告基因的MyoG啟動子質粒(MyoG_P-luc2) 並進行了熒光素酶測定，以檢查NRIP對MyoG啟動子活性的影響。為了研究NRIP是否作為MyoG的轉錄共激活因子，我們探討了NRIP是否與MyoG已知的轉錄因子MyoD和MEF2D協同作用來調控MyoG轉錄。結果表明，NRIP和MyoD在增強MyoG_P-luc2活性中具有協同效應。 接著，我們想要了解NRIP和MyoD的協同效應是否通過蛋白質-蛋白質相互作用介導。因此，我們進行了蛋白質拉下 (pull-down) 實驗以探討NRIP對MyoD的結合能力。我們也使用免疫沉澱實驗確認C2C12肌母細胞中內源性NRIP和MyoD的這兩個蛋白是否有交互作用。兩項實驗結果皆顯示NRIP能與MyoD相互作用，這表明MyoG的轉錄激活可以通過NRIP和MyoD的結合來介導。 此外，我們檢查了NRIP、MyoD和MyoG的蛋白質表達，以深入了解在肌肉分化過程中關鍵轉錄因子和共調控因子的時間調控。研究結果顯示，MyoD的表達在分化的第3天增加，表明MyoD在此階段作為轉錄因子的功能。NRIP在分化初期增加並保持在高水平，使其能夠作為MyoD的轉錄共激活因子，從而激活MyoG的轉錄，並在整個分化過程中穩定增加。 為了研究NRIP的哪個特定區域或基序對其轉錄共激活效應負責，我們測試不同的NRIP截短構建體與MyoD對MyoG_P-luc2的轉錄共激活效應。我們發現全長NRIP (NRIP-FL)和IQ基序缺失的NRIP突變體 (NRIP-ΔIQ)，兩者都包含所有七個WD40區域，對於MyoG啟動子的有效轉錄共激活是必需的。這一結果進一步得到了AlphaFold 3結構預測的支持，該預測顯示MyoD與NRIP的WD40區域頂表面相互作用。 總而言之，我們發現NRIP在調控MyoG表達中作為MyoD的轉錄共激活因子並證明了NRIP是一種新型的MyoD結合蛋白。我們的發現提供了一個新的視角，展示了NRIP通過與MyoD相互作用調控MyoG的機制，說明了NRIP在肌肉分化中的作用。 | zh_TW |
| dc.description.abstract | Nuclear receptor interaction protein (NRIP) can be referred to as IQWD1 or DDB1 and CUL4 associated factor 6 and DDB1 (DCAF6). NRIP consists of 860 amino acids; the known functional domain in NRIP includes seven WD40 domains and an IQ motif. Our previous research found that MyoG was identified as a target for NRIP regulation, with significantly lower mRNA and protein levels of myogenin (MyoG) in global NRIP-knockout (NRIP-gKO) mice compared to wild-type mice at six-day post-muscle injury.
Additionally, muscle-specific NRIP conditional knockout (NRIP-cKO) mice exhibited muscular abnormalities and impaired motor function. MyoG expression in NRIP-cKO mice was downregulated at the synaptic nuclei in the neuromuscular junction (NMJ). The abnormal phenotype in NRIP-cKO mice could be rescued through MyoG overexpression, indicating that NRIP can act as a trophic factor that retrogradely supports motor neuron survival via MyoG expression. Given the multifaceted role of NRIP in the muscle differentiation process and its association with MyoG expression, we aim to determine if NRIP acts as a transcription coactivator to enhance MyoG gene expression. First, we constructed a myogenin promoter-driven luciferase reporter plasmid (MyoG_P-luc2) and performed a luciferase assay to examine the effect of NRIP on MyoG promoter activity. To study if NRIP functions as a transcription coactivator of MyoG, we investigated whether NRIP acts synergistically with MyoD and MEF2D, known transcription factors of MyoG, in regulating MyoG transcription. The results indicated that NRIP and MyoD exhibit a synergistic effect in enhancing MyoG_P-luc2 activity. Next, we sought to determine if the synergistic effect of NRIP and MyoD is mediated through protein-protein interaction. Therefore, we performed in vitro pull-down assays to investigate whether NRIP can interact with MyoD. The interaction of endogenous NRIP and MyoD in C2C12 myoblast cells was also examined using immunoprecipitation assays. NRIP was discovered to have protein interaction with MyoD both in vitro and in C2C12 myoblasts, indicating that the transcriptional activation of MyoG may be facilitated by the binding of NRIP to MyoD. Additionally, we investigated the protein expression of NRIP, MyoD, and MyoG to better understand the temporal regulation of key transcription factors and coregulators during muscle differentiation. Our results showed that MyoD levels increase on day 3 of differentiation, indicating that MyoD can function as a transcription factor at this stage. NRIP increases early in differentiation and remains elevated, allowing it to act as a transcription coactivator for MyoD, thereby activating MyoG transcription, which consistently increases throughout differentiation. To investigate which specific domain or motif is responsible for the transcription coactivator effect of NRIP, we tested the trans-cofactor effect of different NRIP truncation constructs with MyoD on MyoG_P-luc2. We found that full-length NRIP (NRIP-FL) and IQ motif-deletion NRIP mutant (NRIP-ΔIQ), both containing all seven WD40 domains, are required for effective transcriptional coactivation of the MyoG_P-luc2. AlphaFold 3 structural predictions further supported this result, revealing that MyoD interacts with the amino acid residues on the top surface of the WD40 domain of NRIP. In conclusion, we found that NRIP acts as a transcription coactivator of MyoD in regulating MyoG expression. NRIP was also identified as a novel MyoD-binding protein. Our findings provide a new perspective on NRIP-regulated MyoG through MyoD interaction, illustrating the role of NRIP in muscle differentiation. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-08-27T16:11:33Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-08-27T16:11:33Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 III Abstract V Chapter 1 INTRODUCTION 1 1.1 Attributes of the Nuclear Receptor Interaction Protein (NRIP) 1 1.2 The contribution of NRIP in the functioning of skeletal muscles 3 1.3 The involvement of NRIP at the neuromuscular junction system 4 1.4 Molecular control of muscle formation 5 1.5 Myogenin's involvement in skeletal muscle functionality and the development of neuromuscular junctions 7 1.6 NRIP's influence on myogenin regulation 9 1.7 Aim of the study 10 Chapter 2 METHODS AND MATERIALS 11 2.1 Plasmid construction 11 2.2 Cell culture 12 2.3 Cell transfection 12 2.4 Luciferase assay 12 2.5 Recombinant protein production 13 2.6 In vitro pull-down assay 14 2.7 Protein extraction and western blot analysis 15 2.8 Nuclear protein extraction 16 2.9 Immunoprecipitation 17 2.10 AlphaFold 3 protein structure prediction 18 2.11 Statistical analysis 18 Chapter 3 RESULTS 19 3.1 Construction of myogenin promoter-driving luciferase reporter plasmid (MyoG_P-luc2) 19 3.2 NRIP and MyoD act synergistically to enhance MyoG_P activity 21 3.3 NRIP directly interacts with MyoD in vitro 23 3.4 Endogenous NRIP reciprocally interacts with MyoD in C2C12 cells 24 3.5 NRIP, MyoD, and myogenin expression profile during the differentiation of C2C12 26 3.6 The mutant analysis of NRIP for interacting with MyoD enhances MyoG promoter activity 26 3.7 AlphaFold 3 predicted structure of MyoD 28 3.8 AlphaFold 3 predicted structure of NRIP 30 3.9 AlphaFold 3 predicted structure of NRIP-MyoD complex 32 Chapter 4 DISCUSSION 36 Chapter 5 FIGURES 45 Chapter 6 SUPPLEMENTARY 64 Chapter 7 APPENDIX 71 Chapter 8 REFERENCES 73 | - |
| dc.language.iso | en | - |
| dc.subject | WD40 | zh_TW |
| dc.subject | 轉錄共激活因子 | zh_TW |
| dc.subject | 核受體結合蛋白 | zh_TW |
| dc.subject | MyoD | zh_TW |
| dc.subject | 肌细胞生成蛋白 | zh_TW |
| dc.subject | transcription coactivator | en |
| dc.subject | WD40 | en |
| dc.subject | myogenin | en |
| dc.subject | MyoD | en |
| dc.subject | NRIP | en |
| dc.title | 探討NRIP在調控肌細胞生成蛋白的基因表現中所扮演的角色 | zh_TW |
| dc.title | Role of NRIP in the regulation of myogenin gene expression | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 李明學;黃祥博;陳伯翰 | zh_TW |
| dc.contributor.oralexamcommittee | Ming-Shyue Lee;Hsiang-Po Huang;Po-Han Chen | en |
| dc.subject.keyword | 核受體結合蛋白,MyoD,肌细胞生成蛋白,WD40,轉錄共激活因子, | zh_TW |
| dc.subject.keyword | NRIP,MyoD,myogenin,WD40,transcription coactivator, | en |
| dc.relation.page | 77 | - |
| dc.identifier.doi | 10.6342/NTU202402820 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-08-05 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| dc.date.embargo-lift | 2026-08-10 | - |
| 顯示於系所單位： | 微生物學科所 | |
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