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標題: | 構築有持續性活化能力的MALT1 Construction of a constitutively active MALT1 |
作者: | Yi-Hsiu Fang 方怡琇 |
指導教授: | 董馨蓮 |
關鍵字: | CBM複合,oligomerization,paracaspase,NF-κB, CBM complex,paracaspase,oligomerization,NF-κB, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | T-cell receptor(TCR)接收抗原刺激後,BCL10與CARMA的CARD-CARD進行結合,此時MALT1因與BCL10結合而能藉助CARMA調控的聚合作用(oligomerization) ,進而吸引TRAF6結合且活化NF-κB以及具有切割蛋白質的能力,顯示聚合作用對MALT1執行功能的重要性。因此我們欲利用人工強迫聚合作用的方法了解其對於MALT1執行功的影響。
在293T細胞中,BCL10可作為促使MALT1進行聚合作用的活化因子。所以我們利用BCL10 truncations定義出可能足以進行聚合作用的CARD moiety,構築出CARD(106)-2Ig-CLD-myc、CARD(101)-2Ig-CLD-myc以及分別在CARD與CLD上進行突變的negative controls:CARD(106)L41R-2Ig-CLD-myc、CARD(106)-2Ig-CLDC464A-myc、CARD(101)L41R-2Ig-CLD-myc以及CARD(101)-2Ig-CLDC464A-myc。 接著利用與已經失去聚合能力卻還保留和MALT1結合區域及被切割位點的BCL10L41R-GFP共同轉染的方法當受質來確認我們構築的重組蛋白是否具有protease活性。結果顯示CARD(106)-2Ig-CLD-myc及CARD(101)-2Ig-CLD-myc的確具有protease活性。而CARD(106)-2Ig-CLDC464A-myc及CARD(101)-2Ig-CLDC464A-myc則因破壞掉CLD所以如預期無法切割受質。利用點突變破壞CARD moiety的CARD(106)L41R-2Ig-CLD-myc及CARD(101)L41R-2Ig-CLD-myc並未完全失活,仍具有些許protease活性,。接著透過共同免疫沉澱以及免疫螢光染色的實驗結果顯示與受質的結合對於受質被paracaspase CLD活性所切割是必要的條件之一。 另外我們也連接能進行雙聚合作用(dimerization)的GyraseB moiety給2Ig-CLD及△2Ig-CLD,構築出GyraseB-2Ig-CLD-myc和GyraseB-△2Ig-CLD-myc。利用Luciferase assay測定出兩者皆有顯著的NF-κB活化,代表GyraseB的連接促使聚合作用的發生。然而分別將BCL10-GFP或BCL10L41R-GFP與GyraseB-2Ig-CLD-myc共同轉染時,卻無法觀察到切割現象。Gyrase B moiety即使能貢獻聚合作用,但可能造成構型不適當的改變而影響受質進入CLD active site,而無觀察到預期的受質切割現象。 根據以上實驗結果,不同的強迫聚合作用可能活化MAL1不同的功能,細節會在本文中進一步的討論。 CARMA1-BCL10-MALT1(CBM) complex is central to antigen receptor-triggering NF-κB signaling of lymphocyte activation and thereby controls the expression and secretion of cytokines that are essential for lymphocyte proliferation. Upon activation, BCL10 acting as an upstream adaptor to recruit MALT1 into the context of oligomerized CBM signalsome. Oligomerized MALT1s are able to recruit TRAF6 and subsequently activate NF-κB. Also, oligomerized MALT1 has proteolytic activity. We are interested in how oligomerization controls the function of MALT1. My aim is to construct a constitutively active paracaspase by enforcing self-oligomerizaiton. BCL10 is an oligomerization activator in 293T cells. I fused N-terminal CARD of BCL10 with CLD (Caspase Like Domain) of MALT1, generating CARD(106)-2Ig-CLD-myc and CARD(101)-2Ig-CLD-myc respectively. Fusion constructs with mutations to abolish the catalytic activity CARD(106)-2Ig-CLDC464A-myc and CARD(101)-2Ig-CLDC464A-myc and mutations to disrupt the CARD function CARD(106)L41R-2Ig-CLD-myc and CARD(101)L41R-2Ig-CLD-myc were also generated to serve as negative controls. BCL10L41R-GFP, losing its oligomerization ability but retaining its ability to interact with MALT1, was utilized as a substrate. Cotransfection of BCL10L41R-GFP with variable fusion constructs was performed to test the proteolytic activity of these recombinant proteases. Both CARD(106)-2Ig-CLD-myc and CARD(101)-2Ig-CLD-myc showed prominant proteolytic activity. Catalytic inactive mutants CARD(106)-2Ig-CLDC464A-myc and CARD(101)-2Ig-CLDC464A-myc lose their ability to cleave BCL10L41R-GFP as expected. CARD(106)L41R-2Ig-CLD-myc and CARD(101) L41R--2Ig-CLD-myc showed less proteolytic activity than their wild type counterpart. Co-immunoprecipitation analysis and immunefluorescence staining assay confirmed that the interaction between the recombinant protease and its substrate was required for CLD-dependent cleavage. ATP-dependent dimerization domain of GyraseB was utilized as an oligomerization moiety in this study. Both GyraseB-2Ig-CLD-myc and GyraseB-△2Ig-CLD-myc were generated and tested. They showed significantly strong NF-κB activation ability as compared to MALT1. However, GyraseB-2Ig-CLD-myc was not able to cleave BCL10L41R-GFP or BCL10-GFP. Fusion constructs with different oligomerization moiety showed apparently distinct properties. The implication will be discussed in the text. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28430 |
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顯示於系所單位: | 微生物學科所 |
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