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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 戴榮湘 | |
| dc.contributor.author | Chien-Hsun Chu | en |
| dc.contributor.author | 朱建勳 | zh_TW |
| dc.date.accessioned | 2021-06-16T16:08:25Z | - |
| dc.date.available | 2015-09-24 | |
| dc.date.copyright | 2013-09-24 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-05-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62720 | - |
| dc.description.abstract | 陰道滴蟲細胞中轉錄因子Myb1及Myb2皆可結合鐵誘導基因ap65-1之啟動子調控其表現。本研究發現Myb2之入核控制是透過其結構正確性，以及Myb1之入核受其交互作用酵素TvCyPA1的影響。於陰道滴蟲細胞中過表現HA-fused Myb2或Myb1，利用IFA及western blotting可發現Myb2或Myb1在細胞中皆分布於細胞核。而突變Myb2序列內類似cNLS的鹼性胺基酸無法完全破壞入核能力，顯示其入核機制可能不同一般真核細胞，Myb2中具入核能力之序列為R2R3內胺基酸48-143區域，且若刪除此區域內helix 2~5或RC3任一皆會使Myb2無法進核；若突變helix 2中Isoleucine 74為proline而破壞其二級結構亦無法進核，顯示Myb2結構之完整性可能為入核所需。與Myb2結構組成類似的Myb1則發現相互作用之TvCyPA1蛋白質，可能為脯氨酸異構酵素(peptidyl prolyl cis-trans isomerase)，於陰道滴蟲細胞中過表現HA-CyPA1會促進Myb1入核，過表現HA-CyPA1-R63A突變蛋白會抑制Myb1入核。若突變Myb1之106GP107 dipeptide會破壞與TvCyPA1的結合，其中G106A突變會破壞入核能力但P107A突變卻增加入核比例，可能G106A使TvCyPA1無法結合Myb1導致無法進核，P107A使Myb1留在細胞質的限制消失而增加進核，Myb1之入核可能透過TvCyPA1轉換構型以調控。 | zh_TW |
| dc.description.abstract | In Trichomonas vaginalis, Myb proteins were previously shown to regulate transcription of an iron-inducible ap65-1 gene. In this research, a structure-related nuclear import signal of Myb2 transcription factor was studied, and a cyclophilin A-like protein, referred to as TvCyPA1, was identified to be a binding partner and nuclear localization regulator of Myb1. The HA-tagged Myb2 or Myb1 in T. vaginalis was localized to the nucleus as punctate signals. Myb2 was still localized to the nucleus with mutations in cNLS-like polybasic sequences, suggesting that cNLS may not function in T. vaginali. In Myb2, the sequence spanning amino acid residues 48 to 143, which is embedded within the R2R3 domains was essential and sufficient for protein nuclear import. Myb2 nuclear import was perturbed with point mutation of a conserved isoleucine (I74) in helix 2 to proline that slightly altered secondary structure and ternary folding of the R2 domain, suggesting that nuclear translocation of Myb2 requires a highly ordered structure. Myb1, which has a structure similar to Myb2 was found to interact with TvCyPA1, a cyclophilin A-like peptidyl-prolyl isomerase. Nuclear translocation of Myb1 was respectively enhanced or repressed by overexpressed HA-TvCyPA1 or HA-TvCyPA1-R63A, a catalytic mutant. Point mutation of G106 or P107 in the TvCyPA1-binding motif of Myb1 was demonstrated to disrupt its binding to TvCyPA1. Mutation of G106 in HA-tagged Myb1 resulted in cytoplasmic retention of the protein, while mutation of P107 lead to elevated nuclear translocation, but without affecting its repressive activity. These results suggest that TvCyPA1 may facilitate inter-conversion of the peptidylprolyl bond in the 106GP107 dipeptide to regulate the nuclear importation. | en |
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| dc.description.tableofcontents | 目錄
口試委員會審定書.....................................................i 誌謝.................................................................ii 縮寫表...............................................................iii 中文摘要...........................................................iv 英文摘要.............................................................v 前言.................................................................1 一、陰道滴蟲簡介....................................................1 二、鐵離子與陰道滴蟲致病因子........................................2 三、鐵調控相關Myb轉錄因子.........................................3 四、轉錄因子入核運輸機制............................................4 五、Cyclophilin酵素..................................................5 材料與方法...........................................................7 一、陰道滴蟲細胞的培養..............................................7 二、細胞轉殖與穩定蟲株篩選..........................................7 三、蟲株之保存與活化................................................7 四、陰道滴蟲蛋白質樣本製備..........................................8 五、細胞質與細胞核蛋白質之萃取(Subcellular fractionation).................8 六、轉殖質體之建構..................................................9 七、重組蛋白質之表現與純化.........................................13 八、GST-pull down分析..............................................14 九、SDS-PAGE與西方轉漬(Western Blot)...............................14 十、免疫螢光染色(Immunofluorescence assay，IFA)......................15 十一、CyPA酵素活性分析............................................15 第一章、Myb2結構與入核控制..........................................17 結果................................................................17 Myb2蛋白質表現與分布.............................................17 尋找Myb2之NLS位置. .............................................17 測試Myb2 NLS入核能力............................................18 Myb2結構與入核控制區.............................................19 討論................................................................20 GFP在Myb2研究的嘗試.............................................20 Myb2的nuclear localization domain結構與入核..........................21 Myb2 NLD內I74對入核機制的影響...................................22 第二章、Myb1入核與TvCyPA1酵素結合..................................24 結果................................................................24 Myb1之結合蛋白質CyPA1...........................................24 TvCyPA1和Myb1之in vitro交互作用..................................25 TvCyPA1之酵素活性................................................25 Myb1上之TvCyPA1結合位..........................................26 TvCyPA1與Myb1 的進核............................................27 討論................................................................28 Myb1與TvCyPA1的結合............................................28 探討Myb1內G-P dipeptide參與TvCyPA1結合的機制....................29 Myb1之G106與P107對入核的影響...................................31 TvCyPA1對Myb1入核的影響........................................31 CyPA1對寄生蟲研究的展望..........................................32 Myb1與Myb2入核機制相關性探討...................................33 附圖................................................................34 附表................................................................64 參考文獻............................................................67 圖目錄 圖一、過表現4HA-Myb2於陰道滴蟲.....................................34 圖二、Myb2鹼性胺基酸序列對入核之影響................................36 圖三、定義Myb2 NLS位置.............................................38 圖四、以TetR融合蛋白質測試Myb2 NLS區域.............................41 圖五、以TetR-Luc融合蛋白質測試Myb2 NLS區域.........................43 圖六、Myb2內部序列刪除以定義入核必要區域............................46 圖七、Myb2之I74胺基酸對入核的影響...................................49 圖八、Myb1交互作用蛋白質TvCyPA1之序列比對.........................51 圖九、以GST pull-down實驗檢驗Myb1、Myb2、Myb3和TvCyPA1 之In vitro交互作用...................................................53 圖十、In vitro peptidyl prolyl isomerase酵素活性測試........................55 圖十一、Myb1之G106及P107胺基酸對入核與TvCyPA1結合的影響............60 圖十二、TvCyPA1之R63胺基酸對其細胞分布以及對Myb1入核的影響..........62 表目錄 表一、陰道滴蟲Genome中可能的Importin相關蛋白質.......................64 表二、質體建構所用oligonucleotides序列..................................65 | |
| dc.language.iso | zh-TW | |
| dc.subject | 陰道滴蟲 | zh_TW |
| dc.subject | 轉錄因子 | zh_TW |
| dc.subject | 脯氨酸異構酶 | zh_TW |
| dc.subject | 核運送 | zh_TW |
| dc.subject | 入核機制 | zh_TW |
| dc.subject | Trichomonas vaginalis | en |
| dc.subject | Myb | en |
| dc.subject | cyclophilin | en |
| dc.subject | nuclear localization | en |
| dc.subject | NLS | en |
| dc.title | 陰道滴蟲Myb轉錄因子的入核運輸機制探討 | zh_TW |
| dc.title | Nuclear localization of Myb transcription factors in parasitic protozoan Trichomonas vaginalis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 翁秀貞,蕭信宏,陳青周,林文昌 | |
| dc.subject.keyword | 陰道滴蟲,轉錄因子,脯氨酸異構酶,核運送,入核機制, | zh_TW |
| dc.subject.keyword | Trichomonas vaginalis,Myb,cyclophilin,nuclear localization,NLS, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-05-14 | |
| 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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