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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 嚴欣勇 | zh_TW |
| dc.contributor.advisor | Hsin-Yung Yen | en |
| dc.contributor.author | 陳彥綾 | zh_TW |
| dc.contributor.author | Yen-Ling Chen | en |
| dc.date.accessioned | 2025-09-10T16:09:00Z | - |
| dc.date.available | 2025-09-11 | - |
| dc.date.copyright | 2025-09-10 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-07-31 | - |
| dc.identifier.citation | 1 Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805-820 (2010). https://doi.org:10.1016/j.cell.2010.01.022
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99393 | - |
| dc.description.abstract | 含NOD結構域、亮氨酸重複序列（LRR）與pyrin結構域（PYD）的蛋白質3（NLRP3）發炎體是先天免疫系統中的關鍵成分，負責調控與細胞焦亡（pyroptosis）相關的發炎性細胞激素釋放。NLRP3可感受來自病原相關分子模式（PAMPs）與損傷相關分子模式（DAMPs）等多種刺激後，組裝形成具mega-Dalton量級的大型複合體，進而招募並活化前體pro-caspase-1，促進介白素的成熟與分泌。儘管目前已廣泛研究NLRP3活化所造成的生理與免疫反應，但其初始活化所需的分子機制與關鍵調控因子，仍未被充分釐清。
為鑑別驅動NLRP3活化的關鍵因子，我們應用了高解析度native質譜（native mass spectrometry, nMS）技術，此技術能在溶液中直接監測多重蛋白質間與蛋白-配體間的交互作用。藉由比對活化前後NLRP3交互作用體（interactome）之變化，我們進一步探索其活化的潛在調控機制。首先，我們建立螢光標記NLRP3細胞影像平台，並證實異源表現的NLRP3在受到多種刺激後，仍具備形成puncta的能力，為其活化的表徵之一。其次，我們成功取得重組NLRP3的native質譜圖譜，並觀察到其在靜止狀態下會形成高階寡聚結構。第三，當NLRP3蛋白來自於經預先刺激的細胞時，僅觀察到單體與二聚體形式，顯示NLRP3活化伴隨高階寡聚體的解聚。綜合以上結果，我們推論NLRP3可能透過高階寡聚體的形成抑制其自體活化，而複合體的解離則為其活化的關鍵步驟。此外，我們也觀察到NLRP3的磷酸化狀態可能調控其聚集與活化能力。未來研究將著重於探討NLRP3的磷酸化與其寡聚體組成變化之間的關係，期望進一步揭示其活化的分子機制。 | zh_TW |
| dc.description.abstract | The NOD-, leucine-rich repeat (LRR)-, and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome is a critical component of the innate immune system, mediating pyroptosis-associated cytokine release. In response to a diverse array of stimuli, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), NLRP3 assembles into a mega-Dalton complex that recruits and activates pro-caspase-1, subsequently driving the maturation and secretion of interleukins. While the biological and physiological outcomes of NLRP3 activation have been extensively studied, the primary molecular mechanisms or key modulators directly triggering its activation remain elusively unclear.
To identify the key factors initiating NLRP3 activation, we employed high-resolution native mass spectrometry (nMS), a technique capable of directly monitoring multiplex protein-protein and protein-ligand interactions in solution. The idea is to probe the changes in NLRP3 interactome associated with its activation for further investigation. In summary, we first demonstrated that exogenously expressed NLRP3 retains its ability to form puncta—a hallmark of NLRP3 activation—in response to various stimuli. Secondly, we successfully acquired the native mass spectrum of recombinant NLRP3 and revealed its high order oligomerization at the rest state. Thirdly, only monomeric and dimeric NLRP3 was observed when proteins were purified from the cells pre-treated with the activation stimuli. Collectively, our results speculate that NLRP3 suppresses its auto-activation via formation of oligomeric complexes and disassembly of these complexes is essential for activation. Additionally, we observed that NLRP3 phosphorylation may regulate its assembly and activation. My future studies will focus on elucidating the relation between the stoichiometric modulation of NLRP3 and its phosphorylation, with the ultimate goal of advancing our understanding of the molecular mechanisms governing NLRP3 activation. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-09-10T16:09:00Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-09-10T16:09:00Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | ACKNOWLEDGEMENTS i
中文摘要 ii ABSTRACT iii CONTENTS v FIGURES x TABLES xiv ABBREVIATIONS xv Chapter 1 INTRODUCTION 1 1.1 Biological Function of Inflammasome 1 1.1.1 The Role of Inflammasome in Innate Immune System 1 1.1.2 The Role of NLRP3 Inflammasome in Diseases 4 1.2 Activation Mechanism of NLRP3 Inflammasome 6 1.2.1 Stage 1 : Priming and Licensing 6 1.2.2 Stage 2 : NLRP3 Activation in Response to DAMPs and PAMPs 7 1.2.3 The Assembly of NLRP3 Inflammasome and Its Downstream Signals 12 1.2.4 The Function of ATPase Activity of NLRP3 16 1.3 Structure and Function Investigation of NLRP3 18 1.3.1 NLRP3 Monomer and NLRP3 Auto-inhibitory Cage 18 1.3.2 The Active Structure of NLRP3 Inflammasome Disc 24 1.3.3 Structure Basis of NLRP3 Induce ASC Filament Formation 28 1.4 Role of Phosphorylation in NLRP3 Inflammasome Activation 30 1.4.1 NLRP3 Inflammasome Regulation by Phosphorylation 30 1.5 Mass Spectrometry Analysis for Studying NLRP3 32 1.5.1 Analytical Principles of Native Mass Spectrometry 32 1.5.2 Sample Preparation for Native Mass Spectrometry 35 1.5.3 Q Exactive UHMR Hybrid Quadrupole-Orbitrap Mass Spectrometer 36 1.5.4 Analytical Principles of Phosphoproteomics 39 Chapter 2 Research Aim 42 Chapter 3 MATERIAL AND METHOD 43 3.1 Protein Expression 47 3.1.1 Construct Design 47 3.1.2 Bacmid Preparation and Baculovirus generation 48 3.1.3 Transient Expression by Baculovirus Infection 51 3.1.4 Transient Expression by PEI transfection 52 3.1.5 Transient Expression by Expi293TM Expression System Kit 53 3.1.6 Immunoblotting 55 3.2 Protein Production 57 3.2.1 Purification of MBP-NLRP3 Without Stimulation 57 3.2.2 Purification of MBP-NLRP3 with Gramicidin Treatment 58 3.2.3 Purification of hNLRP3-mVenus by FLAG and Strep Tags 59 3.2.4 Caspae-1 p35 C285A Purification 60 3.3 Confocal Images of NLRP3 Puncta Formation 62 3.3.1 Protein Expression and Inflammasome Stimulation 62 3.3.2 Immunostaining and Fluorescence Microscopy 63 3.3.3 Quantification of Cells with NLRP3 Puncta 63 3.3.4 Fluorescence Recovery After Photobleaching (FRAP) Assay 64 3.4 Native Mass Spectrometry (nMS) Analysis 65 3.5 Phosphorylation profiling of NLRP3 66 3.5.1 Phosphoproteomics Sample Preparation 66 3.5.2 TiO2 Enrichment 68 3.5.3 LC-MS/MS and Data Analysis 69 3.5.4 Label-Free Quantification of Phosphopeptide Abundance 70 3.5.5 Point Mutation of Phosphorylation Sites 71 Chapter 4 RESULT 73 4.1 Optimization of NLRP3 Expression and Purification 73 4.1.1 Strategy of Protein Engineering of NLRP3 73 4.1.2 Optimization of Engineered NLRP3 Expression 76 4.2 Investigation of NLRP3 Puncta Formation and the Impact of Protein Engineering 89 4.3 Live-cell Imaging Reveals Distinct Morphologies of NLRP3 Puncta and Phase Separation Behavior 96 4.4 Subcellular Fractionation and Soluble NLRP3 Purification for Structural Characterization 102 4.4.1 Separate Cellular Components by Differential Centrifugation 102 4.4.2 Purification of Engineered NLRP3 106 4.5 Investigation of NLRP3 Stoichiometry by nMS 112 4.5.1 Native Mass Spectrometry Analysis of Engineered NLRP3 Constructs 112 4.5.2 Changes of Stoichiometry Relate to NLRP3 Activation 116 4.5.3 Discovery of NLRP3 Disassembly Mediated by ATP 119 4.6 Phosphorylation Landscape Analysis of NLRP3 Inflammasome 123 4.6.1 Identification of New Phosphorylation Sites of NLRP3 123 4.6.2 Studying the Changes of Phosphorylation after NLRP3 Activation 126 4.6.3 Effects of Phosphorylation Site Mutations on NLRP3 Activity 130 4.7 Establishment of NLRP3 In-vitro Activity Assay 133 4.7.1 PYD-Caspase-1 Expression Condition Optimization 136 4.7.2 In-vitro Assay for NLRP3 Activation 140 4.7.3 Signal Amplification by Caspase-1 p35 C285A 144 Chapter 5 DISCUSSION 153 5.1 Summary of Major Findings 153 5.2 NLRP3 Puncta Formation and Evidence for LLPS-like Behavior 156 5.3 Native Mass Spectrometry Reveals a Phosphorylation-Linked Mechanism of ATP-Induced NLRP3 Complex Dissociation 159 REFERENCE 161 SUPPLEMENTORY TABLES 171 | - |
| dc.language.iso | en | - |
| dc.subject | native質譜 | zh_TW |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | 寡聚化 | zh_TW |
| dc.subject | 發炎體 | zh_TW |
| dc.subject | NLRP3 | zh_TW |
| dc.subject | NLRP3 | en |
| dc.subject | inflammasome | en |
| dc.subject | oligomerization | en |
| dc.subject | phosphorylation | en |
| dc.subject | native mass spectrometry | en |
| dc.title | 質譜分析揭示NLRP3發炎體早期活化過程中的化學計量調控與磷酸化的關聯性 | zh_TW |
| dc.title | Mass spectrometry reveals the stoichiometric regulation and phosphorylation associated with early stage activation of NLRP3 inflammasome | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳瑞華;邱繼輝;曾文逸 | zh_TW |
| dc.contributor.oralexamcommittee | Ruey-Hwa Chen;Kay-Hooi Khoo;Wen-Yi Tseng | en |
| dc.subject.keyword | NLRP3,發炎體,寡聚化,磷酸化,native質譜, | zh_TW |
| dc.subject.keyword | NLRP3,inflammasome,oligomerization,phosphorylation,native mass spectrometry, | en |
| dc.relation.page | 178 | - |
| dc.identifier.doi | 10.6342/NTU202502903 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-08-02 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2030-07-29 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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