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  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 微生物學科所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52301
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor董馨蓮
dc.contributor.authorZih-Hsuan Liuen
dc.contributor.author劉子瑄zh_TW
dc.date.accessioned2021-06-15T16:11:23Z-
dc.date.available2020-09-25
dc.date.copyright2015-09-25
dc.date.issued2015
dc.date.submitted2015-08-18
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29. Baens, M., L. Bonsignore, R. Somers, C. Vanderheydt, S.D. Weeks, J. Gunnarsson, E. Nilsson, R.G. Roth, M. Thome, and P. Marynen, MALT1 auto-proteolysis is essential for NF-kappaB-dependent gene transcription in activated lymphocytes. PLoS One, 2014. 9(8): p. e103774.
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55. 蕭雅今,API2-MALT1 融合蛋白質對BCL10 蛋白質之影響,國立台灣大學,2006 年
56. 陳雯華,BCL10 蛋白質在細胞核質位移之研究,國立台灣大學,2004 年
57. 周欣儀,BCL10 磷酸化位置與MALT1 誘導切割位確認的研究,國立台灣大學,2005 年
58. 方怡琇,構築有持續性活化能力的MALT1,國立台灣大學,2011 年
dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52301-
dc.description.abstract當 T 細胞受體接受到抗原刺激時,會活化CBM complex (CARMA1-BCL10-MALT1) 並且將訊號往下傳來活化NF-κB。其中MALT1 具有兩種功能,其中一個功能是當作鷹架蛋白可以幫助訊息傳遞,另一個是功能蛋白酶水解的功能可以切割受質,其中一個受質為BCL10。在觀察MALT1 蛋白酶水解時發現MALT1 不只會切割BCL10 也會造成BCL10 表現增加,本篇論文主要探討MALT1 在蛋白質恆定的調控中扮演的角色為何。在293T 細胞中大量表現MALT1 的情況下會增加BCL10-GFP 蛋白質量,MALT1 catalytic site mutants (MALT1 C464A) 依然可以增加BCL10-GFP,顯示MALT1 不需要經由蛋白酶水解功能來調控BCL10-GFP。MALT1 主要是透過前面兩個Ig-like domain 與BCL10 結合,我們也發現到MALT1只要有前面兩個Ig-like domain 就足以有使BCL10-GFP 蛋白質增加的能力。而BCL10-GFP 也必須保有與MALT1 結合的位置(107-119 胺基酸),MALT1 才會有增進BCL10-GFP 的現象。在U2932 細胞將MALT1 knockdown 發現BCL10 減少與磷酸化p62 S403 增加的情形。從RNA 層級去檢查,MALT1 knockdown 的U2932細胞BCL10 RNA 不會受影響,表示MALT1 主要是調控在蛋白質層級。另外,在MALT1 knockdown 的U2932 細胞也發現BCL10 降解的速度增加,顯示MALT1 對於BCL10 的穩定度是很重要的。在U2932 細胞內以TPA/Ionomycin 刺激後藉由BCL10 切割與磷酸化p-IKBα的情況來觀察NFκB 訊號傳遞。以TPA/Ionomycin刺激MALT1 knockdown 的U2932 細胞會造成BCL10 切割與磷酸化p-IKBα較不明顯,顯示NFκB 的訊號受到影響。在293T 細胞內大量表現MALT1 也會促使其他形成聚集蛋白質表現,例如:GFP 250、Htt-exon1-99Q-GFP、GFP-GCP170*。在293T 細胞加入蛋白質體酶抑制劑(protease inhibitor) MG132 後發現並對於聚集型蛋白質影響不明顯,但是加入自噬作用抑制劑(autophagy inhibitor) 3MA 後會使聚集型蛋白質表現增加,因此暗示MALT1 對於這些聚集型的蛋白質中扮演調控角色。zh_TW
dc.description.abstractWhen T cell receptor is stimulated with antigen, the CARMA1 recruite MALT1 and BCL10 to form CBM complex which then mediate the activation of NFκB signaling pathway.There are two functions of MALT1, one is scaffold protein, and the other is protease activity. One of substrates of MALT1 is BCL10. Previously in the lab, MALT1 not only cleaved BCL10 but also enhanced its protein expression.In this study, the role of MALT1 in protein homeostasis was investigated.Overexpression of MALT1 in 293T cell enhanced the level of BCL10-GFP expression.MALT1 catalytic site mutants (MALT1 C464A) also enhanced the expression of BCL10 GFP. Catalytic activity of MALT1 was not essential for the enhancement effect.MALT1 was reported to interacte with BCL10 through the first and second Ig-like domain.The first and second Ig-like domains of MALT1 were sufficient for the enhancement effect.MALT1 enhanced the level of BCL10-GFP expression when BCL10 conserved the interacting domain (107-119 amino acid) with MALT1.In U2932 cell lines, knockdown of MALT1 decreased the endogenous BCL10 expression and increased serine 403 phosphorylation of p62.The RNA levels of BCL10 were not different among U2932 wild type cells and U2932 MALT1 knockdown cells.Knockdown of MALT1 accelerated the degradation of BCL10 in U2932 cells,implying that MALT1 was important for the stability of BCL10.NFκB signaling pathway was activated in U2932 cells treated with TPA/Ionomycin.BCL10 cleavage and the amounts of phosphorylated IKBα,indicators of NFκB activation,were reduced in MALT1-knockdown U2932 cells treated with TPA/Ionomycin.These results confirmed the role of MALT1 in NFκB signaling pathway.In 293T cell, overexpression of MALT1enhance the level of aggregated protein accumulation such as GFP 250、Htt-exon1-99QGFP、GFP-GCP170*.Autophagy inhibitor such as 3MA enhanced the expression of these aggregated proteins, implying that autophagy might play roles in regulating expression of these aggregated proteins.en
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dc.description.tableofcontents口試委員會審定書........................................................................................................... i
致謝 .................................................................................................................................. ii
中文摘要 ......................................................................................................................... iii
目錄 .................................................................................................................................. v
圖表目錄 ........................................................................................................................ vii
Chapter 1 序論 .......................................................................................................... 1
1.1 MALT1 (Moucosa-Associated-Lymphoid-TissueLymphoma-
Translocation Gene1)................................................................................................ 1
1.2 MALT1 當作鷹架蛋白幫助NF-κB 訊號傳遞....................................... 1
1.3 MALT1 具有蛋白酶活性的功能............................................................ 2
1.3.1 BCL10 .............................................................................................. 3
1.3.2 A20 ................................................................................................... 3
1.3.3 CYLD ............................................................................................... 3
1.3.4 NIK................................................................................................... 4
1.3.5 RelB.................................................................................................. 4
1.3.6 Regnase-1 ......................................................................................... 4
1.3.7 MALT1 ............................................................................................. 5
1.3.8 Roquin-1 ........................................................................................... 5
1.3.9 LIMA-1α........................................................................................... 5
1.4 MALT1 活化mTOR 訊號路徑............................................................... 6
Chapter 2 材料與方法 .............................................................................................. 7
2.1 製備勝任細胞 (Preparation of competent cells)..................................... 7
2.2 細菌轉型 (Bacteria transformation)........................................................ 7
2.3 小量質體製備 (Mini plasmid preparation)............................................. 7
2.4 大量質體製備 (Maxi Plasmid Preparation)............................................ 8
2.5 細胞蛋白質萃取 (Protein Lysate Extraction)......................................... 9
2.6 蛋白質定量 (Protein quantification analysis) ......................................... 9
2.7 蛋白質電泳 (SDS-polyacrylamide gel electrophoresis) ......................... 9
2.8 西方墨點法 (Western blotting analysis) ............................................... 10
2.9 細胞培養 (cell culture).......................................................................... 10
2.9.1 培養方法 ........................................................................................ 10
2.9.2 HEK (Human Embryonic Kidney) 293T 細胞.............................. 10
2.9.3 U2932 細胞.....................................................................................11
2.10 暫時性基因轉染-磷酸鈣轉染(Transient Transfection-Calcium
Phosphate) ...............................................................................................................11
2.11 製備慢病毒載體顆粒 (Lentiviral Vector Particles Packaging) .............11
vi
2.12 建立以慢病毒載體系統主導之核醣核酸干擾作用-慢病毒感染
(Establishment of Lentiviral Vector System-Mediated RNA interference- Lentiviral
infection) .................................................................................................................12
2.13 免疫螢光試驗 (immunofluorescence assay) ........................................ 12
2.14 MTT 細胞增殖試驗(MTT cell proliferation assay) ........................... 12
2.15 細胞 RNA 萃取(RNA extraction)........................................................ 13
2.16 反轉錄反應 (Reverse Transcription ) ................................................... 13
2.17 聚合酶鏈鎖反應 (polymerase chain reaction)...................................... 14
2.18 質體 ........................................................................................................ 15
Chapter 3 實驗結果 ................................................................................................ 16
Chapter 4 討論 ........................................................................................................ 20
Chapter 5 結果圖表 ................................................................................................ 25
Chapter 6 附錄圖表 ................................................................................................ 41
參考文獻 ........................................................................................................................ 45
dc.language.isozh-TW
dc.titleMALT1在蛋白質恆定中扮演的角色zh_TW
dc.titleThe role of MALT1 in protein homeostasisen
dc.typeThesis
dc.date.schoolyear103-2
dc.description.degree碩士
dc.contributor.oralexamcommittee陳美如,張正琪,李明學
dc.subject.keywordMALT1,BCL10,蛋白質恆定,zh_TW
dc.subject.keywordMALT1,BCL10,proten homeostasis,en
dc.relation.page50
dc.rights.note有償授權
dc.date.accepted2015-08-18
dc.contributor.author-college醫學院zh_TW
dc.contributor.author-dept微生物學研究所zh_TW
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