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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳小梨(Show-Li Chen) | |
| dc.contributor.author | Chia-Yang Lin | en |
| dc.contributor.author | 林嘉揚 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:50:20Z | - |
| dc.date.copyright | 2022-10-03 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85208 | - |
| dc.description.abstract | 核受體相互作用蛋白 (NRIP) 是一種攜鈣素結合蛋白，包含了一個 IQ motif和七個WD40 domains。在先前研究中，我們發現 NRIP是乙醯膽鹼受體複合物的結構性蛋白，並且與乙醯膽鹼受體蛋白有交互作用。乙醯膽鹼受體複合物表現在神經肌肉接合處（NMJ）上，透過參與乙醯膽鹼受體的聚集影響神經肌肉接合處的形成。此外，我們也觀察到肌肉NRIP基因剔除小鼠身上存在NMJ面積減少的異常表現。鑑於上述發現，我們認為NRIP作為乙醯膽鹼受體複合物的結構性蛋白，可以促進乙醯膽鹼受體的聚集，進而維持神經肌肉接合處的完整性。本篇研究中，我們想要進一步了解NRIP如何影響乙醯膽鹼受體聚集的形成。 首先，我們藉由蛋白質體外結合實驗和免疫沈澱法，以研究 NRIP structural domains與AChR的交互作用。實驗結果顯示含有WD6/7 domain的NRIP片段可以與AChR-α結合，而去除掉WD6/7 domain後，NRIP失去了與AChR-α的結合能力。因此，我們認為WD6/7 domain負責了NRIP與AChR的交互作用。接下來為了研究NRIP和AChR的結合與AChR聚集的形成是否相關，我們共轉染了不同EGFP-NRIP片段和mCherry-AChR-α到HEK293T 細胞中，觀察細胞內產生AChR聚集的數量。同時我們也將不同EGFP-NRIP片段轉染到 KO19細胞（NRIP剔除的C2C12細胞)中分析內生性AChR形成聚集的能力。實驗結果顯示，具有與 AChR 結合能力的 NRIP片段可以促進細胞內AChR的聚集，而去除掉WD6/7 domain後，細胞內形成聚集的數量顯著下降。總的來說，我們認為NRIP可以透過WD6/7domain與AChR進行交互作用，進而促進AChR聚集的產生。NRIP除了作為乙醯膽鹼受體結合蛋白外，還是一種肌動蛋白結合蛋白。在先前研究中，我們發現 NRIP與肌細胞融合相關，可以透過和肌動蛋白的結合來調控肌細胞融合的過程。為了進一步證實我們的想法，我們對肌肉NRIP基因剔除小鼠進行不同的NRIP 基因治療，並評估治療後小鼠的肌纖維大小。我們發現 NRIP、NRIP-C和NRIP-WD6/7可以有效增加肌纖維大小；WD6/7 domain，尤其是WD7 domain，在其中扮演了重要角色。上述結果證實NRIP通過WD6/7 domain參與了肌肉再生的過程。此外，我們發現NRIP還會與肌動蛋白、Tks5共同表現在C2C12 細胞中。肌動蛋白和Tks5是突觸足體的組成部分，而突觸足體是一種富含肌動蛋白的結構，負責AChR聚集的型態成熟。基於NRIP與肌動蛋白結合的作用，我們認為NRIP 可以通過與足體的肌動蛋白絲結合來影響突觸足體的形成。本篇研究中，我們將不同EGFP-NRIP片段轉染到 KO19細胞中，發現具有與肌動蛋白結合能力的 NRIP片段可以促進細胞內突觸足體的產生，而去除掉WD6/7 domain和IQ motif後細胞形成突觸足體的能力下降，其下降程度又以WD6/7 domain的去除較為嚴重。最後，我們將EGFP-NRIP和 FLAG-NRIP 共轉染到293T細胞中，透過免疫沈澱法觀察到NRIP在細胞內會產生交互作用，表明NRIP可能會以二聚體的形式表現在細胞中。總而言之，通過上述實驗，我們證實NRIP 是一種多功能蛋白質，可能以二聚體的結構表現在細胞中。NRIP作為乙醯膽鹼受體複合物的結構性蛋白，透過WD6/7 domain與乙醯膽鹼受體結合，可以促進乙醯膽鹼受體聚集的產生，進一步穩定神經肌肉接合處的形成。此外，NRIP 還可以作為肌動蛋白結合蛋白參與突觸足體的形成，並調控肌細胞融合的過程，從而產生更大的肌纖維。 | zh_TW |
| dc.description.abstract | Nuclear receptor interaction protein (NRIP), a calmodulin-binding protein that includes one IQ motif and seven WD40 domains, is an acetylcholine receptor (AChR) binding protein and acts as a scaffold protein to stabilize AChR-rapsyn-α-actinin (ACTN2) complex, which is associated with AChR clustering in NMJ formation. Additionally, muscle-restricted NRIP knockout (cKO) mice present decreased NMJ area, indicating that NRIP may be essential in the maintenance of NMJ integrity. Thus, we hypothesize that NRIP is essential for AChR clustering to impact NMJ formation. In this study, we would like to figure out which domain of NRIP is responsible for AChR binding and to determine whether the AChR binding is correlated with AChR cluster formation. First, we performed in vitro pull-down assays and co-immunoprecipitation assays to investigate the AChR binding ability of NRIP structural domains. We observed that NRIP mutants containing the WD6/7 domain could interact with AChR, while the deletion of the WD6/7 domain caused AChR binding ability defects. Taken together, WD40 6/7 was responsible for AChR binding. Next, we co-transfected mCherry-AChR-α and EGFP-NRIP mutants into HEK293T cells and then transfected EGFP-NRIP mutants into KO19 (NRIP-null C2C12) cells to evaluate the AChR clustering ability. We observed that NRIP mutants with AChR binding ability can form AChR clusters, while the loss of the WD6/7 domain caused AChR clustering defects. Collectively, the WD6/7 domain may be responsible for AChR clustering through binding with AChR. In addition, NRIP is also an actin-binding protein. We found that the binding of NRIP to actin is associated with myoblast fusion. To further confirm that, we evaluated the rescue effect of intramuscular NRIP gene therapy on the myofiber size of NRIP cKO mice, and found that NRIP had a significantly rescued effect on myofiber CSA. The WD6/7 domain, especially domain7, was important for the rescued effect. Moreover, NRIP co-localized with F-actin and Tks5, which are structural components of synaptic podosomes. The synaptic podosome is an F-actin-enriched structure, which is responsible for AChR cluster morphological maturation. Hence, we hypothesized that NRIP could affect the synaptic podosome formation by binding to actin filaments of podosomes. Here, we evaluated the percentages of clusters containing synaptic podosomes in KO19 (NRIP-null C2C12) cells transfected with NRIP mutants and found that the loss of actin-binding domains, WD6/7 and IQ motif, resulted in a significant loss of synaptic podosome formation. Last, we performed an immunoprecipitation assay by co-transfecting EGFP-NRIP and FLAG-NRIP to HEK293T cells and verified an association between NRIP molecules, indicating that NRIP would form an oligomer, such as a dimer in cells. In summary, we demonstrate that NRIP is a multi-functional protein. It may form dimers and acts as a scaffold protein of the AChR complex, participates in AChR clustering through its binding with AChR by WD6/7 domains, and further stabilizes the NMJ formation. Besides, NRIP also acts as an actin-binding protein to form synaptic podosomes and facilitate myoblast fusion which results in larger myofibers. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:50:20Z (GMT). No. of bitstreams: 1 U0001-0308202214034900.pdf: 11429742 bytes, checksum: feaa797bea6d7c3fa9d81d3541f4d367 (MD5) Previous issue date: 2022 | 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 The characteristics of Nuclear receptor interaction protein (NRIP) 1 1.2 The characteristics of the neuromuscular junction (NMJ) 3 1.3 The characteristics of acetylcholine receptor (AChR) and cluster formation 4 1.4 The role of NRIP at neuromuscular junction 6 1.5 The role of NRIP for skeletal muscle function 7 1.6 The mechanism of myoblast fusion during myogenesis 10 1.7 The characteristics and functions of the WD40 domain 12 1.8 The characteristics and functions of synaptic podosomes 13 1.9 Aims of study 15 Chapter 2 METHODS AND MATERIALS 18 2.1 Plasmid construction 18 2.2 Protein production 18 2.3 Cell culture 19 2.4 Cell transfection 20 2.5 In vitro pull-down assay 20 2.6 Protein extraction and western blot analysis 21 2.7 Immunoprecipitation assay 22 2.8 Immunofluorescence staining of cells 22 2.9 Immunofluorescence staining of tissues for CSA analysis 23 2.10 Statistical analysis 24 Chapter 3 RESULTS 25 3.1 Mapping NRIP domains for interaction with AChR-α in vitro 25 3.2 Mapping NRIP domains with interaction with AChR-α in cultured cells 27 3.3 Mapping of NRIP structural domains for AChR cluster formation in 293T cells 28 3.4 Mapping of NRIP structural domains for endogenous AChR cluster formation in KO19 cells 30 3.5 The rescue effect of intramuscular NRIP gene therapy on the myofiber size (cross-sectional area) of NRIP cKO mice 33 3.6 NRIP affected the formation of synaptic podosomes 36 3.7 NRIP can form dimers as a multi-functional protein 38 Chapter 4 DISCUSSION 40 Chapter 5 FIGURES 50 Chapter 6 SUPPLEMENTARY 68 Chapter 7 APPENDIX 70 Chapter 8 REFERENCES 72 | |
| dc.language.iso | en | |
| dc.title | 探討NRIP 如何影響乙醯膽鹼受體聚集形成與肌纖維大小 | zh_TW |
| dc.title | To study how NRIP affects acetylcholine receptor (AChR) cluster formation and myofiber size | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃祥博(Hsiang-Po Huang),曾秀如(Shiou-Ru Tzeng) | |
| dc.subject.keyword | 核受體交互作用蛋白,乙醯膽鹼受體,肌動蛋白,肌管形成,足體,WD40 domain, | zh_TW |
| dc.subject.keyword | NRIP,AChR,actin,myotube formation,podosome,WD40 domain, | en |
| dc.relation.page | 79 | |
| dc.identifier.doi | 10.6342/NTU202202007 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-08-03 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2027-08-03 | - |
| 顯示於系所單位： | 微生物學科所 | |
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