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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐立中(Li-Chung Hsu) | |
| dc.contributor.author | Ting-Yu Liu | en |
| dc.contributor.author | 劉亭妤 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:40:16Z | - |
| dc.date.available | 2021-07-10T21:40:16Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76912 | - |
| dc.description.abstract | 在先天免疫系統中，NLRP3發炎小體(Inflammasome)是一群蛋白質複合體，在宿主抵抗病原體感染時扮演重要的角色。活化的發炎小體會誘發第一型半胱天冬酶(caspase-1)活化，並促使第一型介白素β (IL-1β)以及第十八型介白素(IL-18)等細胞激素成為具有生物活性的型態，同時引發一種發炎性程序性的細胞凋亡－稱為細胞焦亡(pyroptosis)。然而，當NLRP3發炎小體失調時，則會導致許多疾病，包括自體發炎疾病、糖尿病、痛風以及癌症。在我們實驗室先前的研究發現一個核糖核酸解旋酶27 (DDX27)，在類鐸受體2(TLR2)以及類鐸受體4(TLR4)的刺激下，能夠負調控NLRP3發炎小體活化。在我們以及其他研究中發現，NLRP3發炎小體能夠與DDX27和另一個核糖核酸解旋酶3X (DDX3X)結合，而DDX3X是組成壓力顆粒 (stress granules)的其中一個分子。在本篇研究，我們將探討DDX27是否會影響NLRP3與DDX3X結合，導致影響到NLRP3發炎小體活化以及壓力顆粒的組成。我們研究發現，藉由人類胚腎細胞株HEK293T外源性表達或是小鼠骨髓細胞分化而成的巨噬細胞(BMDMs)內源性表達的系統中，DDX27會抑制NLRP3與DDX3X結合。此外，DDX27會增加藉由偏亞砷酸鈉(Sodium (meta)arsenite)所引發的壓力顆粒形成，證明DDX27會與DDX3X競爭NLRP3，導致負向調控NLRP3發炎小體活化以及正向調控壓力顆粒的組成。 透過BLAST生物資訊學分析，我們發現結核分歧桿菌(Mtb)中的核糖核酸解旋酶DeaD的DEAD-box區域與DDX3X和DDX27之DEAD-box區域的胺基酸有超過30%的相似性。由於NLRP3發炎小體對於宿主防禦結核分歧桿菌感染非常重要，我們推測，結核分歧桿菌是否能透過DeaD來調控NLRP3發炎小體使其能夠在宿主細胞中存活。我們建構了海洋分歧桿菌(M. marinum)表達DeaD以及恥垢分歧桿菌(M. smegmatis) dead缺失突變，用於感染人類巨噬細胞THP-1。我們發現，在感染人類巨噬細胞THP-1後，表達DeaD的海洋分歧桿菌可以促進NLRP3發炎小體的活化以及細胞死亡，但在腫瘤壞死因子(TNF-α)則沒有改變。相反的，恥垢分歧桿菌dead缺失突變則會抑制NLRP3發炎小體的活化以及細胞死亡。我們也證實，在人類巨噬細胞THP-1中感染帶有表達DeaD的海洋分歧桿菌會增加NLRP3與DDX3X的結合，然而，感染恥垢分歧桿菌dead缺失突變則會降低NLRP3與DDX3X的結合。這些研究結果顯示，分歧桿菌中的DEAD蛋白質在調節NLRP3發炎小體的活化以及宿主細胞死亡中扮演重要的角色。 | zh_TW |
| dc.description.abstract | The NLRP3 inflammasome, a multi-protein heteromeric complex, plays a vital role in host defenses against pathogenic infection. Activation of the NLRP3 inflammasome induces caspase-1 activation, which then facilitates pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 processing and initiates a form of inflammatory programmed cell death called -pyroptosis. However, dysregulation of NLRP3 inflammasome activation has been associated with a variety of human diseases, including autoinflammatory diseases, diabetes, gout, and cancers. We previous identified a DEAD-box RNA helicase, DDX27, which negatively regulates NLRP3 inflammasome activation upon TLR2 or TLR4 engagements. Our and other studies demonstrate that NLRP3 can bind to DDX27 and another DEAD-box RNA helicase, DDX3X, a component of stress granules. In this study, we aim to investigate whether DDX27 influences NLRP3 binding to DDX3X, leading to regulation of NLRP3 inflammasome activation and the assembly of stress granules. We found that the interaction of NLRP3 and DDX3X was suppressed by DDX27 when exogenously expressed in HEK293T cells or endogenously expressed in bone marrow derived macrophages (BMDMs). In addition, DDX27 increased the formation of stress granules by arsenite, suggesting that DDX27 competes for NLRP3 with DDX3X, resulting in negatively regulating NLRP3 inflammasome activation and positively regulating the assembly of stress granules. Through the BLAST (Basic Local Alignment Search Tool) analysis, we found that the DEAD-box of ATP-dependent RNA helicase DeaD in Mycobacterium tuberculosis (Mtb) shares more than 30% amino acid identity with that of DDX3X and DDX27. Since the NLRP3 inflammasome was critical for the host to defend against Mtb infection, we speculated whether Mtb modulates NLRP3 inflammasome activation by DeaD for its survival in the host. We constructed M. marinum (over)expressing DeaD box and M. smegmatis mc2 155 dead deletion mutant, and used to infect THP-1 macrophages. We found that M. marinum expressing DeaD promoted NLRP3 inflammasome activation and cell death, but there was no change in TNF-α production compared with control. In contrast, deletion of dead in M. smegmatis mc2 155 suppressed NLRP3 activation and cell death in THP-1 macrophages. We also revealed that M. marinum (over)expressing DeaD increased the interaction of NLRP3 and DDX3X, whereas dead-deleted M. smegmatis decreased NLRP3 binding to DDX3X in THP-1 macrophages. These data together indicate that mycobacterium DEAD-box containing proteins play a role in regulation of NLRP3 inflammasome activation and host cell death. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:40:16Z (GMT). No. of bitstreams: 1 U0001-1008202012332300.pdf: 3044750 bytes, checksum: 48e9bfc1c2e5d0de9ea8f19a08ac08ca (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 II 中文摘要 III Abstract V Contents VII Introduction 1 1. Pattern recognition receptors 1 2. Inflammasomes 2 3. NLR family pyrin domain containing 3 (NLRP3) inflammasome 3 4. The mechanisms of the NLRP3 inflammasome activation 5 5. DEAD-box protein family of RNA helicase 6 6. DEAD-Box Helicase 27 (DDX27) 7 7. DEAD-Box Helicase 3 (DDX3) 8 8. Mycobacterium tuberculosis 8 9. Crosstalk between Mycobacterium tuberculosis and the NLRP3 inflammasome 9 Specific Aims 11 Materials and methods 12 1. Reagents and Antibodies 12 2. Plasmids 13 3. Mice 15 4. Cell culture 16 5. Bacterial strains and growth conditions 17 6. Construction of M.marinum NTUH-M6094 (over)expressing DeaD strain 18 7. Construction of M. smegmatis mc2 155 with dead deletion mutant 19 8. Infection of THP-1 cells by M. marinum and M. smegmatis 20 9. Transfection 20 10. Preparation of mouse bone marrow derived macrophages (BMDMs) 21 11. Lentivirus production 21 12. Immunoblotting 23 13. Detection of secreted cytokines and caspase-1 24 14. Immunofluorescence 25 15. Immunoprecipitation 26 16. Enzyme-Linked Immunosorbent Assays (ELISA) 26 17. Detection of Lactate Dehydrogenase (LDH) Release 29 18. Statistical Analysis 30 Results 31 1. DDX27 blocked the interaction of NLRP3 and DDX3X. 31 2. The interaction of NLRP3 and DDX3X was increased in Ddx27+/- BMDMs after NLRP3 inflammasome activation. 32 3. DDX27 affected arsenite-induced stress granules formation upon NLRP3 inflammasome activation. 33 4. M. marinum expressing DeaD induced more IL-1β and secretion and increased cell death in THP-1 macrophages. 35 5. Deletion of dead in M. smegmatis mc2 155 triggered less IL-1β release and cell death in THP-1 macrophages. 36 6. Mycobactium DeaD influenced the interaction of NLRP3 and DDX3X in THP-1 macrophages 37 Discussion 39 References 43 Figures 50 Figure 1. DDX27 diminished the interaction of NLRP3 and DDX3X in HEK293T cells. 50 Figure 2. Knockdown of DDX27 affected the protein level of DDX3X in THP-1 cells. 51 Figure 3. The interaction of NLRP3 and DDX3X was increased in Ddx27+/- macrophages after LPS+ATP stimulation. 52 Figure 5. THP-1 macrophages infected with M. marinum expressing DeaD secreted increased IL-1β and lactate dehydrogenase (LDH). 55 Figure 6. THP-1 macrophages infected dead-deleted in M. smegmatis mc2 155 secreted decreased IL-1β and lactate dehydrogenase (LDH). 57 Figure 7. The interaction of NLRP3 and DDX3X in THP-1 macrophages was affected after infection with DeaD (over)expressing M. marinum or dead-deleted M. smegmatis. 59 Figure S1. Depletion of DDX27 enhances NLRP3 inflammasome activation and IL-1 secretion in macrophages. 60 Figure S2. DDX27 specifically associated with NLRP3 but not other NLRs or AIM2 in HEK293T cells. 62 Figure S3. DDX27 Associated with NLRP3, but not ASC nor Caspase-1. 63 Figure S4. The DEAD domain of DDX27 interacts with the LRR domain of NLRP3. 64 Figure S5. Generation of M. marinum expressing DeaD and dead-deleted M. smegmatis strains. 66 | |
| dc.language.iso | en | |
| dc.subject | 結核分歧桿菌 | zh_TW |
| dc.subject | NLRP3發炎小體 | zh_TW |
| dc.subject | 核糖核酸解旋酶27 | zh_TW |
| dc.subject | 核糖核酸解旋酶3X | zh_TW |
| dc.subject | NLRP3 inflammasome | en |
| dc.subject | Mycobacterium tuberculosis | en |
| dc.subject | DEAD-box RNA helicase 3 | en |
| dc.subject | DEAD-box RNA helicase 27 | en |
| dc.title | 具有DEAD-box的蛋白於NLRP3發炎小體活化與分歧桿菌感染中所扮演的角色 | zh_TW |
| dc.title | The roles of DEAD-box containing proteins in regulation of NLRP3 inflammasome activation and mycobacterial infections | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林琬琬(Wan-Wan Lin),楊性芳(Hsin-Fang Yang-Yen),嚴仲陽(Jong-Young Yen) | |
| dc.subject.keyword | NLRP3發炎小體,核糖核酸解旋酶27,核糖核酸解旋酶3X,結核分歧桿菌, | zh_TW |
| dc.subject.keyword | NLRP3 inflammasome,DEAD-box RNA helicase 27,DEAD-box RNA helicase 3,Mycobacterium tuberculosis, | en |
| dc.relation.page | 66 | |
| dc.identifier.doi | 10.6342/NTU202002783 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-17 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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