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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 賴明宗(Ming-Zong Lai) | |
| dc.contributor.author | Tzu-Sheng Hsu | en |
| dc.contributor.author | 徐子勝 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:58:22Z | - |
| dc.date.available | 2018-03-12 | |
| dc.date.copyright | 2015-03-12 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-11-28 | |
| dc.identifier.citation | Anandasabapathy, N., Ford, G.S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., Skrenta, H., Hollenhorst, M., Fathman, C.G., and Soares, L. (2003). GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18, 535-547.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55359 | - |
| dc.description.abstract | E3泛素連接酶對於誘發及維持T細胞去活化 (T cell anergy) 扮演重要的角色。誘發T細胞去活化過程中，去活化相關的E3泛素連接酶如Cbl-b, Itch和GRAIL的蛋白質表達量會上升，並透過降解T細胞活化訊息傳遞分子PKCθ和PLCγ1而抑制T細胞再活化。然而這些去活化相關的E3泛素連接酶如何彼此協同，進而維持T細胞去活化卻未完全清楚。
本篇研究中，我們探討Notch相關E3泛素連接酶deltex1 (DTX1) 在維持T細胞去活化的扮演的角色。我們發現，DTX1會結合PKCθ和PLCγ1並抑制其蛋白質表現。進一步的研究顯示，相似Cbl-b和Itch的作用，DTX1催化PKCθ單體泛素化，使得單體泛素化的PKCθ經由核內體-溶小體 (endosome-lysosome) 途徑而分解，處理蛋白酶體抑制劑並不會降低DTX1所調控PKCθ降解。此外，我們也觀察到DTX1會與Cbl-b結合並促進Cbl-b的表現。進一步的實驗證實，DTX1抑制PKCθ主導的Cbl-b降解並增加Cbl-b的蛋白質穩定。本篇的研究闡述了去活化相關的E3泛素連接酶在T細胞去活化中的協同作用：DTX1泛素化PKCθ並經由核內體-溶小體 (endosome-lysosome) 途徑調控PKCθ降解；DTX1透過調控PKCθ降解促進Cbl-b蛋白質穩定；DTX1與Cbl-b共同作用確保PKCθ的活性完全抑制。 | zh_TW |
| dc.description.abstract | Ubiquitin E3 ligases are associated with induction and maintance of T cell anergy. These E3 ligases, including Cbl-b, Itch, and GRAIL, attenuate T cell activation by targeting to signaling molecules such as PKCθ and PLCγ1 for degradetion. How these anergy-associated E3 ligases coordinate during T cell anergy remains incompletely understood. In this study, we found that Notch-related E3 ligase deltex1 (DTX1) also regulated the expression of PKCθ and PLCγ1. DTX1 interacted with PKCθ and PLCγ1 and promoted the degradation of PKCθ and PLCγ1. T cell anergy-induced downregulation of PKCθ was prevented in Dtx1-/- T cells, supporting the essential role of DTX1 in PKCθ downregulation. DTX1 promoted monoubiquitination of PKCθ, similar to Cbl-b and Itch. DTX1-directed PKCθ degradation was not prevented by proteasome inhibitor, but instead DTX1 directed the re-localization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results illustrate the coordination between E3 ligases during T cell anergy; DTX1-mediated PKCθ degradation further stabilizes Cbl-b and DTX1 acts with Cbl-b to assure a more completely silencing of PKCθ. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:58:22Z (GMT). No. of bitstreams: 1 ntu-103-D97449003-1.pdf: 7353927 bytes, checksum: fa0f6540d2e8f288c40fc555c1ff6616 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 縮寫表…..……………………………………………………………………1
中文摘要………………………………………………………………………5 英文摘要………………………………………………………………………6 第一章 緒論 .……………………………………………………….…….7 一、T細胞活化….…………………………………………………......7 1. T細胞活化的訊息傳遞路徑………………………………...……7 2. PKCθ (Protein kinase C theta).……………………………..….…...8 二、T細胞去活化 (T cell anergy)…………………….……...……….10 1. T細胞去活化的訊息傳遞路徑………………………………..…10 2. 免疫耐受性相關E3泛素酶………………………………….….11 三、DTX1與Notch…………………………………………………...13 1. Notch受體訊息傳遞路徑……………………………………..…13 2. DTX1之簡介……………………………………………………..14 3. DTX1之蛋白質結構…………………………………………….14 4. DTX與Notch受體訊息傳遞…………………………………….15 四、研究動機與方向……………………………………………………16 第二章 材料與方法……………………………………………….…..…17 一、細胞株與細胞培養………………..……………………….…..…17 1. 細胞株培養………….…………………………………….…..…17 2. 小鼠T細胞純化.……………………………………….……..…17 3. 體外Th1 細胞的分化………………………………….….….…18 二、藥品與試劑 ………………………………………….….……..…18 三、質體與構築…………………………………….………….…...…19 1. DTX1及不同截切DTX1突變體表現質體之構築……..………19 2. PKCθ及不同截切PKCθ突變體表現質體之構築……….…...…20 3. Cbl-b及不同截切Cbl-b突變體表現質體之構築…………..…..20 4. pLL3.7-shDTX1、pLL3.7-shCbl-b、pLL3.7-shItch和pLL3.7- shPKCθ之構築………………21 5. pTRIP-HA-ubiquitin表現質體之構築……………….………..…21 6. 螢光結合蛋白質 EGFP-DTX1、EGFP-DTX1∆PR、mOrange-PKCθ和LAMP1-mcherry表現質體之構築………22 四、質體DNA的轉染 (Transfection) …………………………...…22 1. Calcium phosphate 轉染法……………….…………….……..…22 2. 陽離子轉染試劑Maestrofectin 轉染法.………….……….....…23 3. 反轉錄病毒感染法 (Retroviral infection) .…………...……...…23 4. 電穿孔法 (Eletroporation) .………………………...…….…..…24 五、T細胞免疫耐受性誘導 (Induction of T cell anergy) 和增殖分析(Proliferation assay)………………24 六、流式細胞儀分析與篩選 (Flow cytometry analysis and sorting)..24 七、共軛交顯微鏡觀察 (Confocal microscopy) 與數據分析...….....25 八、全細胞萃取液的製備…………………...………….……...…..…26 九、免疫沉澱法 (Immunoprecipitation)…...………….……..…....…26 1. 蛋白質結合免疫沉澱法…...……………...……….……...…..…26 2. 泛素化蛋白質免疫沉澱法...……………...……….……..…...…27 十、西方墨點法 (Western blot) ..……………...……….……..…...…27 1. 蛋白質凝膠的製備與電泳分析…………...……….……........…27 2. 雜合反應…………………………………...….……...…..…...…28 十一、資計統計分析 (Statistical analysis ) ……...……….………..…28 第三章 結果………..………………………………….……...…..…...…29 一、缺乏DTX1的T細胞去活化時PKCθ不會降減……………..…29 二、DTX1與PKCθ結合並促進PKC | |
| dc.language.iso | zh-TW | |
| dc.subject | T細胞去活化 | zh_TW |
| dc.subject | E3泛素連接? | zh_TW |
| dc.subject | PKCθ | en |
| dc.subject | Anergy | en |
| dc.subject | E3 ubiquitin ligase | en |
| dc.subject | deltex1 | en |
| dc.title | Deltex1在T細胞去活化機制調控之探討 | zh_TW |
| dc.title | Study on the Molecular Mechanism of Deltex1 in T cell Anergy | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 許秉寧(Ping-Ning Hsu),繆希椿(Shi-Chuen Jesse Miaw),王萬波(Won-Bo Wang),謝世良(Shie-Liang Edmond Hsieh) | |
| dc.subject.keyword | T細胞去活化,E3泛素連接?, | zh_TW |
| dc.subject.keyword | Anergy,E3 ubiquitin ligase,deltex1,PKCθ, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-11-28 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 免疫學研究所 | zh_TW |
| 顯示於系所單位： | 免疫學研究所 | |
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