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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 余明俊(Ming-Jiun Yu) | |
dc.contributor.author | Shih-Chin Hsu | en |
dc.contributor.author | 許世勤 | zh_TW |
dc.date.accessioned | 2021-06-17T02:42:29Z | - |
dc.date.available | 2022-09-08 | |
dc.date.copyright | 2017-09-08 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68926 | - |
dc.description.abstract | C型肝炎病毒非結構蛋白NS5A被認為會透過連續磷酸化調控位於第一低複雜度序列 (low complexity sequence I) 區域中的絲氨酸群,使其在兩種磷酸化狀態,低度磷酸化與高度磷酸化間進行轉換。然而,這個論述缺乏直接證據。我首先利用專一性抗體測量於先前的磷酸化蛋白質體研究中發現的磷酸化位點絲氨酸222、235,及238的磷酸化。在被第二型C型肝炎病毒J6/JFH1所感染的人類肝腫瘤細胞株中,絲氨酸222的磷酸化幾乎不能被偵測到,然而絲氨酸235及238的磷酸化總是被偵測到,並且隨著感染時間平行發展,於細胞內部的空間分布上也總是高度重疊。絲氨酸235的丙氨酸變異 (S235A) 排除了絲氨酸238的磷酸化,然而絲氨酸238的丙氨酸變異 (S238A) 並不影響絲氨酸235的磷酸化,表示絲氨酸235的磷酸化可以獨立於絲氨酸238的磷酸化而發生,但絲氨酸238的磷酸化則依附於絲氨酸235的磷酸化。與此相同的是,當以免疫沉澱法配合免疫轉漬進行解析,發現帶有絲氨酸235磷酸化的分子可以不需要絲氨酸238的磷酸化而存在,但絲氨酸238的磷酸化只存在於具有絲氨酸235鄰酸化的NS5A分子中。S235A此變異型會導致病毒的複製無法進行,但S238A此變異型並不會影響病毒的複製,表示絲氨酸235的磷酸化是主要的NS5A磷酸化,用以負責病毒的複製。更重要的是,在非結構蛋白NS3至NS5A聚合蛋白表現載體 (NS3-NS5A expression construct) 所轉染 (transfection) 的人類胚胎腎臟細胞株HEK293T中,我發現只有當絲氨酸232變異為丙氨酸時 (S232A) 可以完全排除NS5A的高度磷酸化,表示絲氨酸232被用來啟動NS5A的高度磷酸化。與此一致的是,S232A排除了包含絲氨酸235及238磷酸化在內的高度磷酸化。有趣的是,絲氨酸232、235,及238所座落的位置,其序列恰好是第一型酪蛋白激酶 (casein kinase I ɑ) 的磷酸化偏好序列 (pS/pTXXS)。降低第一型酪蛋白激酶的表現量會同時降低絲氨酸235及238的磷酸化程度,但不會影響絲氨酸232的磷酸化。我的結論是,絲氨酸232的磷酸化會透過第一型酪蛋白激酶進行連續磷酸化調控絲氨酸235及238的磷酸化,並因此而造成高度磷酸化的出現。 | zh_TW |
dc.description.abstract | Hepatitis C virus NS5A protein is thought to switch between hypo- and hyper-phosphorylation states via sequential phosphorylation among a series of serine residues in the low complexity sequence I region; however direct evidence is missing. I used specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected whereas S235 and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecules. S235A blunted viral replication whereas S238A did not affect replication. S235 phosphorylation is the primary NS5A phosphorylation required for replication. Furthermore, I found that only at S232A mutation eliminated NS5A hyper-phosphorylation in NS3-NS5A vector-transfected HEK293T cells, indicating that S232 initiates NS5A hyper-phosphorylation. Consistently, S232A eliminated NS5A hyper-phosphorylation at S235 and S238. Intriguingly, S232, S235, and S238 located on the sequence consistent with the phosphorylation preference of casein kinase I ɑ (pS/pTXXS). Casein kinase I α knockdown reduced NS5A phosphorylation at S235 and S238 without affecting S232 phosphorylation. I conclude that S232 phosphorylation primes sequential NS5A phosphorylation at S235 and S238 by casein kinase I α. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:42:29Z (GMT). No. of bitstreams: 1 ntu-106-D99442004-1.pdf: 9840973 bytes, checksum: a8f14efcca50089391241c32eb0a05e5 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 口試委員審定書...I
誌謝........II 摘要........III Abstract.......V Contents.......VII List of Table.....XI List of Figures....XII Chapter 1: Introduction..............1 1.1 General information of hepatitis c virus...1 1.2 Advancement of research tools promotes the HCV study ...........................3 1.3 NS5A phosphorylation..............5 1.4 Regulation of NS5A phosphorylation.......7 1.5 Sequential phosphorylation............9 1.6 Specific aims...................11 1.6.1 To investigate the interdependence among serine 222, 235, and 238 phosphorylation..........11 1.6.2 To investigate the initiation of NS5A sequential phosphorylation....................11 Chapter 2: Materials and Methods...........13 2.1 Cell culture....................13 2.2 Antibodies and reagents..............13 2.3 HCV infection...................14 2.4 Plasmids and transfection............14 2.5 Immunoblotting..................15 2.6 Immunoprecipitation...............16 2.7 Replicon transfection..............16 2.8 Protein kinase CKIɑ inhibitor treatment....16 2.9 Gene specific knockdown.............17 Chapter 3: Results..................18 3.1 Serine 235 is the primary NS5A hyper-phosphorylation site responsible for HCV replication........18 3.1.1 NS5A S235 phosphorylation and S238 phosphorylation occurred in parallel.................18 3.1.2 S235 phosphorylation was a prerequisite to S238 phosphorylation....................19 3.1.3 S235 phosphorylation could exist alone whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule...............21 3.1.4 S235 and S238 phosphorylated NS5A co-localized with double stranded RNA..................23 3.1.5 Casein kinase I α (CKIα) was responsible for S235 and S238 phosphorylation...............25 3.2 Phosphorylation at serine 232 initiates sequential phosphorylation....................25 3.2.1 Phosphorylation-ablated alanine mutation at serine 232 ablated NS5A hyper-phosphorylation........25 3.2.2 NS5A phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells .............................26 3.2.3 Alanine mutations revealed sequential NS5A phosphorylation cascade from S232 through S35 to S238 .............................27 3.2.4 S232 phosphorylated NS5A did not rescue phosphorylation at S235 or S238 in trans.......28 3.2.5 CKIɑ mediated NS5A phosphorylation at S235 and S238 but not S232......................29 Chapter 4: Discussion.................30 4.1 S235 phosphorylated NS5A is the primary NS5A species required for HCV replication.............30 4.2 CKIɑ mediates NS5A hyper-phosphorylation through sequential phosphorylation cascade..........31 4.3 S232 phosphorylation initiates sequential phosphorylation at LCS I region............33 4.4 S232 phosphorylated NS5A is less in NS5A species .............................35 4.5 Modulation of S232 phosphorylation regulates hyper-phosphorylation level.................36 4.6 Further investigation of S232 phosphorylation initiation is promise to unveil the regulatory circuit of sequential phosphorylation...............37 Table..........................39 Figures.........................40 Appendix.........................56 References........................58 Publications.......................74 | |
dc.language.iso | en | |
dc.title | C型肝炎病毒非結構蛋白質NS5A連續磷酸化的機制 | zh_TW |
dc.title | Mechanisms of Sequential Phosphorylation of the Hepatitis C Virus Non-Structural Protein NS5A | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),楊宏志(Hung-Chih Yang),施嘉和(Chia-Ho Shih),張典顯(Tien-Hsien Chang) | |
dc.subject.keyword | C型肝炎病毒,非結構蛋白質5A,連續磷酸化, | zh_TW |
dc.subject.keyword | Hepatitis C Virus,Non-Structural Protein 5A,Sequential Phosphorylation, | en |
dc.relation.page | 74 | |
dc.identifier.doi | 10.6342/NTU201703533 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-16 | |
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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