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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 余明俊 | |
| dc.contributor.author | Kuan-Ying Lee | en |
| dc.contributor.author | 李冠潁 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:42:45Z | - |
| dc.date.available | 2026-07-27 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50485 | - |
| dc.description.abstract | C型肝炎病毒（Hepatitis C virus, HCV）非結構性蛋白質5A (Non-structural protein 5A, NS5A)第235號絲胺酸（serine 235, S235）的磷酸化對病毒的複製來說是一個非常重要的轉譯後修飾作用。基於病毒學與醫學上的研究興趣，了解哪些激酶負責NS5A S235磷酸化是有其必要的。使用HEK293T細胞作為篩選平臺，我們發現第二型鈣調蛋白依賴激酶（Calmodulin-dependent kinase II, CaMKII）抑制劑及第一型酪蛋白激酶（Casein kinase I α, CKIα）抑制劑兩者皆在無細胞毒性的前提下能降低NS5A S235的磷酸化。然而在感染HCV的Huh7.5.1細胞中只有CKIα的gene knockdown能有效抑制NS5A S235的磷酸化及HCV的核醣核酸的數量，這顯示由CKIα所促使的NS5A S235磷酸化在HCV的複製中扮演關鍵的角色。HCV的複製通常需要許多宿主因子的協助，在病毒感染的細胞中一個稱為hVAP-A（人類囊泡相關膜蛋白相關蛋白A的宿主因子）的宿主因子在細胞質有聚集成簇的現象，然而這樣的現象卻在進行CKIα的gene knockdown後就消失了。上述的結果顯示在感染細胞中CKIα所介導的NS5A S235磷酸化能協助HCV複製複合體形成並幫助病毒的複製。我們最近也正在使用液相層析質譜儀來分析235磷酸化與不磷酸化之間不同的交互作用蛋白質差異。我們的分析結果將可為究竟有哪些蛋白質會參與在235磷酸化的NS5A上提供完整的說明。 | zh_TW |
| dc.description.abstract | Phosphorylation at serine 235 of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a critical post-translational modification required for viral replication. Because of virological and medical interests, it is necessary to identify the kinases responsible for NS5A S235 phosphorylation. Using HEK293T cells as a platform, we found both calmodulin-dependent kinase II (CaMKII) and casein kinase I α (CKIα) inhibitor reduced NS5A S235 phosphorylation without cytotoxicity. However, only shRNA-mediated CKIα knockdown reduced S235 phosphorylation and HCV RNA levels in the HCV-infected Huh7.5.1 cells, indicating a critical role CKIα-mediated NS5A S235 phosphorylation in HCV replication. HCV replication often requires host factors such as the vesicle-associated membrane protein-associated protein A (hVAP-A) that forms clusters in the HCV-infected cells. Upon CKIα knockdown, the hVAP-A clusters dispersed throughout the cytosol. The above data suggest that CKIα-mediated NS5A S235 phosphorylation facilitates HCV replication complex formation in the infected cells. We are currently using LC-MS/MS based protein mass spectrometry to profile the host protein factors that interact with S235D vs. S235A NS5A. Our findings will potentially provide a complete protein repertoire that participates in NS5A S235 phosphorylation-mediated HCV replication. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:42:45Z (GMT). No. of bitstreams: 1 ntu-105-R03442018-1.pdf: 1287452 bytes, checksum: ea2f720174c8aeb879319a6fce97c199 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | Contents
摘要 I Abstract II Introduction 1 Materials and Methods 5 Results 14 HEK293T cells recapitulated NS5A S235 phosphorylation as in the HCV-infected Huh7.5.1 cells 14 CaMKII and CKI was responsible for S235 phosphorylation without cytotoxicity in HEK293T 14 CKIα knockdown reduced S235 phosphorylation and HCV replication in Huh7.5.1 cells whereas CaMKIIδ or γ knockdown did not 16 S235 phosphorylation recruited hVAP-A to intracellular sites of HCV replication 17 LC-MS/MS analysis of S235 phosphorylation interactome 18 S235 phosphorylation promoted cell migration ability 19 Discussion 21 Figure Legends 25 References 30 | |
| dc.language.iso | en | |
| dc.subject | C型肝炎病毒 | zh_TW |
| dc.subject | 非結構性蛋白質5A | zh_TW |
| dc.subject | 絲胺酸235 | zh_TW |
| dc.subject | 人類囊泡相關膜蛋白相關蛋白A的宿主因子 | zh_TW |
| dc.subject | hVAP-A | en |
| dc.subject | S235 | en |
| dc.subject | NS5A | en |
| dc.subject | HCV | en |
| dc.subject | NS5A | en |
| dc.subject | S235 | en |
| dc.subject | HCV | en |
| dc.subject | hVAP-A | en |
| dc.title | 非結構性蛋白質5A絲氨酸235磷酸化介導C型 肝炎病毒複製之分子機制探討 | zh_TW |
| dc.title | Molecular Mechanisms of NS5A S235 Phosphorylation-Mediated Hepatitis C Virus Replication | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張鑫,劉旻禕 | |
| dc.subject.keyword | C型肝炎病毒,非結構性蛋白質5A,絲胺酸235,人類囊泡相關膜蛋白相關蛋白A的宿主因子, | zh_TW |
| dc.subject.keyword | HCV,NS5A,S235,hVAP-A, | en |
| dc.relation.page | 32 | |
| dc.identifier.doi | 10.6342/NTU201601420 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-27 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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