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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳美如(Mei-Ru Chen) | |
dc.contributor.author | Guan-Ting Liu | en |
dc.contributor.author | 劉冠婷 | zh_TW |
dc.date.accessioned | 2021-06-08T01:22:17Z | - |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-06 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18730 | - |
dc.description.abstract | ESCRT (Endosomal Sorting Complexes Required for Transport) 機制已知會參與在細胞中膜剪切的過程,以及許多病毒在細胞質膜出芽離開細胞的過程。在先前的研究中,我們發現EB病毒會藉由內核膜蛋白BFRF1與ESCRT的橋接分子Alix作用,幫助核膜衍生液泡結構的形成,可助於已包裹完成的病毒衣殼 (nucleocapsid) 離開細胞核。在本篇研究中,進一步探討BFRF1協同ESCRT蛋白調控核膜結構產生液泡的分子機制。透過時程分析蛋白分佈,我們發現BFRF1會先後吸引不同的ESCRT分子到細胞核膜,其中 Alix 與 TSG101會先後靠近核,進而幫助液泡結構的形成。更進一步利用結構域突變株研究BFRF1與Alix結合區,透過共同免疫沉澱法、麩胺基硫轉移酶沉降及螢光染色訊號,發現在BFRF1胺基酸61-69之中,存在著一段非典型的Alix結合區。BFRF1上面的這一段Alix結合區主要會與Alix的Bro domain有交互作用,且處理核酸酶之後,不會影響BFRF1與Alix的結合。進一步,我們也探討BFRF1造成的液泡結構幫助細胞核巨分子運送的可能性。當細胞表現BFRF1時可將細胞核中不正常累積的錯誤摺疊蛋白運送到細胞質中,進而藉由自噬作用將這些錯誤摺疊蛋白進行降解。暗示著細胞中原本可能存在一條路徑,可以調控細胞核與細胞質之間巨分子的運輸及不正常堆積蛋白的降解。這篇研究提供了EB病毒BFRF1引發核膜衍生液泡的機制及其對物質由細胞核到質運送之調控。 | zh_TW |
dc.description.abstract | The cellular endosomal sorting complex required for transport (ESCRT) machinery mediates membrane scission and cytoplasmic budding of various viruses. Previously, we showed that Epstein-Bar virus (EBV) BFRF1 recruits ESCRT component Alix to the nuclear envelope-associated membrane for virus maturation. In this study, the mechanism used by BFRF1 for modulating nuclear envelope was further characterized. ESCRT components were recruited sequentially to the nuclear envelope along with BFRF1 expression by time-course analysis. Alix was recruited at the beginning of nuclear envelope modulation, whereas the other ESCRT component TSG101 was recruited to nuclear rim at late stages for cytoplasmic vesicle release. By serial deletion and site-directed mutagenesis, a nonconventional late domain on BFRF1 was identified to mediate the Bro and PRR domains of Alix recruitment. Remarkably, depletion of nuclear acid did not abolish the associations between BFRF1 and Bro domain, suggesting BFRF1 is directly interact with Aix Bro domains. Investigating the possible capability of BFRF1-mediated vesicles for delivering nuclear component, we found that expression of BFRF1 reduced the nuclear accumulation of protein aggregates. The reduced nuclear aggregates was recovered or enhanced by autophagy inhibitor or inducer, suggesting cytoplasmic autophagy system is involved in the nuclear envelope-directed molecule transport. Overall, this study provides understandings not only for virus mediated nuclear envelope modulation, but also the mechanism of nucleocytoplasmic transport. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:22:17Z (GMT). No. of bitstreams: 1 ntu-103-R01445105-1.pdf: 7349714 bytes, checksum: edafecdea6e942be11e0c39a72d79317 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 致謝 I
中文摘要 II Abstract III Contents IV 1. Introduction 1 1.1. Epstein-Barr Virus 1 1.1.1. Classification, structure and associated diseases 1 1.1.2. EBV life cycle 1 1.2. Nuclear egress of Herpesviruses 2 1.2.1. Nuclear membrane of eukaryotic cells 2 1.2.2. Escape of herpesvirus form the nucleus 3 1.2.3. EBV nuclear membrane associated protein BFRF1 4 1.3. The ESCRT machinery 5 1.3.1. ESCRTs in cellular processes 5 1.3.2. The compositions of ESCRTs 5 1.3.3. ESCRTs in viral budding 6 1.3.4. The role of ESCRTs in herpesvirus maturation 7 1.4. Cellular protein quality control and aggresome-related diseases 8 1.4.1. Aggresome formation and clearance 9 1.4.2. Autophagy 10 1.5. Specific aims of this study 11 2. Materials and methods 12 3. Results 18 3.1. The late domain mapping of EBV BFRF1 18 3.1.1. Different ESCRT components involved in BFRF1-induced vesicle formation 18 3.1.2. Mapping of Alix interacting domain on EBV BFRF1 19 3.1.2.1. BFRFl and serial deletion mutants exhibits different expressing pattern 19 3.1.2.2. BFRF1 WT and point mutants exhibit different expressing pattern 21 3.1.2.3. Virus release was affected by BFRF1 LD1_2LA 24 3.1.3. BFRF1-interacting regions on Alix 25 3.1.3.1. The interaction between EBV BFRF1 and the Bro domain of Alix is independent of Nucleic acid 26 3.1.3.2. BFRF1 directly interacts with Alix through Alix Bro, V and PRR domains in vitro 27 3.1.3.3 The interactions between BFRF1-Alix Bro domain is nucleic acid independent in vitro 28 3.1.4. The minimal domain of BFRF1 that can interact with Alix in vitro 28 3.1.5. The TSG101 binding site on BFRF1 28 3.2. Export of the nuclear proteins by BFRF1-mediated vesicles 30 3.2.1. EBV BFRF1 is capable of redistributing the nuclear protein GFP-GCP170* into cytoplasm 30 3.2.2. EBV BFRF1 also affects the cytoplasmic distribution of Htt99Q protein 31 3.2.3. The BFRF1 mutants are defective for the vesicle formation and GFP-GCP170* transport into cytoplasm 31 3.2.4. BFRF1 enhances the delivery of nuclear aggregates into cytoplasm in a dose-dependent manner 32 3.2.5. BFRF1 promotes the delivery of preformed nuclear aggragates 32 3.2.6. The inhibition of autophagosome-lysosome fusion induces GFP-GCP170* accumulation in the cytoplasm of BFRF1 expressing cells 33 3.2.7. The clearance of GFP-GCP170* is enhanced by autophagy inducer rapamycin 34 3.2.8. MG132 treatment affect the GFP-GCP170* aggregation and BFRF1-induced vesicle formation 34 3.2.9. The aggresome-related protein are involved in the BFRF1-mediated clearance of nuclear aggregates 35 3.2.10. Other pathogenic polyQ proteins are also affected by BFRF1 36 3.2.11. The clearance of nuclear aggregates in neuron cells 36 3.2.12. Alix is involved in BFRF1-induced vesicle formation and GFP-GCP170*transport into cytoplasm 37 3.2.13. The autophagy component Atg5 is not involved in the BFRF1-induce vesicle formation 37 4. Discussion 39 4.1 The late domain mapping of EBV BFRF1 39 4.2 Export of the nuclear proteins by BFRF1-mediated vesicles 42 5. Figures 46 Fig. A. Composition and molecular interactions of the ESCRT machinery 46 Fig. B. Schematic model for HDAC6-dependent clearance of ubiquitinated protein aggregates by QC autophagy 47 Fig. 1. Time lapse images of BFRF1-induced vesicle formation. 48 Fig. 2. The expression patterns of WT BFRF1 at different time points were colocalized with emerin under confocal microscopy analysis 49 Fig. 3. The expression patterns of WT BFRF1 at different time points were colocalized with Alix under confocal microscopy 50 Fig. 4. The expression patterns of WT BFRF1 at different time points were colocalized with Alix under confocal microscopy 51 Fig. 5. The hypothetical model of BFRF1-mediated vesicle formation 52 Fig. 6. Functional domains of EBV BFRF1 protein and the summary of mutants used in this study 53 Fig. 7. Subcellular distributions of emerin in cells expressing various deletion mutants of BFRF1 54 Fig. 8. Subcellular distributions of Alix in cells expressing various deletion mutants of BFRF1 55 Fig. 9. Subcellular fractionation of Alix in cells expressing putative late domain (pLD) mutants of BFRF1 56 Fig. 10. Enlarged putative late domains (pLD) of BFRF1 57 Fig. 11. Subcellular distributions of emerin in cells expressing point mutation mutants of BFRF1 58 Fig. 12. Subcellular distributions of Alix in cells expressing point mutation mutants of BFRF1 59 Fig. 13. Quantitation of the colocalization signal of Alix and BFRF1 mutants 60 Fig. 14. Subcellular fractionations of Alix and TSG101 in cells expressing different point mutation mutants of BFRF1 61 Fig. 15. The interaction between Alix and point mutation mutants of BFRF1 62 Fig. 16. Mutation of the putative L-domain disrupted the BFRF1-Alix interaction. 63 Fig. 17. Mutation of the putative late domain disrupted the interaction between BFRF1 and Alix. 64 Fig. 18. The expression pattern of WT BFRF1 or various mutants were colocalized with calnexin under confocal microscopy 65 Fig. 19. BFRF1 LD1_2LA was defective for virus release 66 Fig. 20. The BFRF1 LD1_2LA mutant expression level decreased at 48 h post transfection 67 Fig. 21. Treatment of chloroquine and rapamycin treatment did not affect BFRF1 LD1_2LA expression level 68 Fig. 22. The interaction of HA-BFRF1 WT or various deletion mutants with three functional domain of Alix 69 Fig. 23. The interaction between EBV BFRF1 and the Bro domain of Alix is independent of nucleic acid 70 Fig. 24. The interaction of LD1_2LA mutant with three functional domain of Alix 71 Fig. 25. Amino acids 270-290 of BFRF1 are important for BFRF1-Alix_PRR interaction. 72 Fig. 26. Amino acids 270-290 of BFRF1 are critical for BFRF1 to induce vesicle formation 73 Fig. 27. Subcellular distributions of Alix domains in cells expressing BFRF1 LD1_2LA or d(270-290) mutant 74 Fig. 28. EBV BFRF1 interacts with the Bro, V and PRR domains of cellular Alix in vitro. 75 Fig. 29. The presence of nucleic acid is crucial for EBV BFRF1 to interact with Alix V or PRR domain in vitro 76 Fig. 30. EBV BFRF1directlt interacted witj Bro domain of Alix 77 Fig. 31. Subcellular distributions of cellular TSG101 in cells expressing different EBV BFRF1 deletion mutants 78 Fig. 32. Protein-protein interaction among Alix, TSG101 and EBV BFRF1 mutants 79 Fig. 33. K63P and LD1_2LA mutant of EBV BFRF1 are colocalized with TSG101 at the nuclear rim and perinuclear region of transfected cells 80 Fig. 34. The hypothetical model of EBV BFRF1 functional domain for the ESCRT protein interaction and vesicle formation 81 Fig. 35. Expression of EBV BFRF1 reduces the nuclear aggregation of GFP-GCP170* but not cytoplasmic Htt99Q aggregates 82 Fig. 36. BFRF1 mutants interfere the vesicle formation and GFP-GCP170* transport into cytoplasm 83 Fig. 37. Expression of BFRF1 reduces the protein amount of GFP-GCP170* in a dose-dependent manner 84 Fig. 38. BFRF1 promotes the clearance of pre-formed nuclear aggregates 85 Fig. 39. Inhibition of autophagy by CQ treatment attenuates the ability of BFRF1 to clear nuclear GFP-GCP170* 86 Fig. 40. GFP-GCP170* was accumulated in the cytoplasm of CQ-treated BFRF1 expressing cells 87 Fig. 41. CQ treatment enhances the colocalization of GFP-GCP170* and LC3A in BFRF1 expressing cells 88 Fig. 42. Rapamycin treatment enhanced GFP-GCP170* clearance in BFRF1 coexpressing cells 89 Fig. 43. MG132 treatment affects the GFP-GCP170* aggregation and BFRF1-induced vesicle formation 90 Fig. 44. Quantification of GFP-GCP170*-induced aggregates in cells with BFRF1 expression 91 Fig. 45. The autophagy selective substrate p62 colocalized with GFP-GCP170* in BFRF1-induced vesicles 92 Fig. 46. The ubiquitin-selective autophagy marker HDAC6 partially colocalized with GFP-GCP-170* in BFRF1 expressing cells 93 Fig. 47. The autophagy-related molecule HSP70 highly colocalized with BFRF1-induced vesicles 94 Fig. 48. Amountsof polyQ proteins was decreased in BFRF1 expressing cells. 95 Fig. 49. BFRF1-inducd vesicles enhance the clearance of Htt81Q-NLS-GFP aggregates 96 Fig. 50. The aggregation of GFP-GCP170* and Htt-axon1-99Q-GFP in N2A cells 97 Fig. 51. Autophagy contributes to the BFRF1-meidated clearance of GFP-GCP170* in N2A cells 98 Fig. 52. Cellular Alix associates weakly with EBV BFRF1 and GFP-GCP170* 99 Fig. 53. Overexpression of Alix did not affect the aggregate formation of GFP-GCP170* in the cytoplasm of N2A cells 100 Fig. 54. Subcellular distribution of GFP-GCP170 is independent of Alix in N2A cells 101 Fig. 55. Knockdown of autophagy essential component, Atg5, showed minor effect on the vesicles formation of BFRF1 102 Fig. 56. The BFRF1-induced vesicle formation and nucleocytoplasmic transport of nuclear aggregates is independent of cellular Atg5 103 Fig. 57. The hypothetical model of BFRF1-mediated nuclear aggregates transport and clearance 104 6. References 105 | |
dc.language.iso | en | |
dc.title | EB 病毒 BFRF1 引發核膜衍生液泡的機制及其對物質由細胞核到質運送之調控 | zh_TW |
dc.title | The mechanism of EBV BFRF1 induced nuclear envelope-associated vesicle formation and its regulatory role on nucleocytoplasmic transport | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳儀莊(Yi-Juang Chern),陳士隆(Steve S-L Chen),史有伶(Yu-Ling Shih),李重霈(Chung-Pei Lee) | |
dc.subject.keyword | EB病毒,BFRF1,Alix,TSG101,液泡結構形成,自噬,細胞核與細胞質間的運輸, | zh_TW |
dc.subject.keyword | EBV,BFRF1,Alix,TSG101,vesicle formation,autophagy,nulear aggregates,nucleocytoplasmic transport, | en |
dc.relation.page | 110 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-08-06 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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