請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10674
標題: | MALT1對BCL10切割的切點位置探討 Investigation on the cleavage site on BCL10 by MALT1 |
作者: | Wen-Hui Chuang 莊文蕙 |
指導教授: | 董馨蓮 |
關鍵字: | BCL10,MALT1,切割,白胺酸,精胺酸,胜肽, BCL10,MALT1,cleavage,Leucine,Arginine,peptides, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | BCL10與MALT1可互相結合且在許多的細胞之中皆有表現。在細胞受到刺激後可與不同的CARD-containing蛋白質形成CBM複合體,對於NF-κB引起的訊息傳導路徑中扮演不可或缺的角色。在本實驗室與他人之前的研究得知,利用TPA (12-O-tetradecanoly-phorbol-13-acetate) 及Ionomycin刺激Jurkat T cell後,MALT1可切割在BCL10的C端。本實驗室針對可疑的切點在BCL10 GFP做一連串的刪除實驗,推論切點位於BCL10第225個胺基酸白胺酸(Leucine),不同於2008年被報導的切點第228個胺基酸精胺酸(Arginine)。因此,本篇論文將以數種方法釐清被切割的BCL10其確切的切割點,並研究BCL10被切割後C端的小片段胜肽在細胞內所在的位置,進一步去探討可能所扮演的角色。
首先,本篇論文以DSS cross-linker鍵結BCL10抗體與protein G,用以純化產物,蛋白質來源為以TPA及Ionomycin刺激Jurkat T細胞的全長或內生性被切割的BCL10,結果顯示每次以2×108 細胞數進行純化,80 μg的BCL10抗體僅能結合5-10 ng BCL10。第二,構築質體FlagBCL10 GFP並與MALT1在293T細胞共同表現,以FLAG親和凝膠純化被切割的產物,結果顯示每10 μl凝膠可以結合約50 ng的切割後產物。純化的蛋白以LC-MS/MS進行分析,並先利用Asp-N內切酶處理樣品。Asp-N可切在aspartate的N端,針對BCL10被MALT1切割後可能出現的蛋白質處理Asp-N,全長BCL10、BCL10 1-225、BCL10 1-228分別可得到3196.5 daltons、2225.9 daltons及2625.2 daltons,皆可以LC-MS/MS準確分析結果。不幸的是,由於Asp-N切點前具有proline,目前仍無法分析這些片段。毛細管電泳亦被應用於分析切割後的C端胜肽RSRTVSRQ與TVSRQ,結果顯示雖然可以有效區分此2段C端胜肽,但靈敏度僅有5 ppm。 針對可疑的切點,以degenerate引子對Leu225及Arg228進行單點突變得到以下融合蛋白BCL10 L225A、BCL10 L225G、BCL10 L225Q、BCL10 L225K、BCL10 L225R、BCL10 L225E及BCL10 R228G,並在293T細胞中分別與MALT1共同表現,以釐清BCL10 Leu225與Arg228對於切割能力有無所扮演的角色。結果顯示除了BCL10 L225R與BCL10 L225K以外,所有的mutants皆無被切割現象,指出BCL10 Leu225與Arg228對於切割是重要的。有趣的是,將融合蛋白BCL10 L225R,R228G與MALT1共同表現於細胞中,仍具有被切割的能力。另外在BCL10 L225R上針對Phe222及Leu223進行點突變以分析BCL10對MALT1切割能力的影響,結果顯示僅有BCL10 F222A,L223T,L225R (FL-4) 仍舊保有被MALT1切割的能力,BCL10 F222H,L223E,L225R (FL-1)、BCL10 F222S,L223E,L225R (FL-2)、BCL10 F222E,L223A,L225R (FL-3)則無法被切割,對於這些融合蛋白影響MALT1的切割能力有無的機制將在內文中討論。 為了研究BCL10被切割後C端胜肽在細胞內所在的位置,我們欲直接將胜肽送入細胞以便觀察,但無法成功。因此在GFP的C端接上胜肽RSRTVSRQ得到融合蛋白GFP-RSRTVSRQ,結果顯示GFP-RSRTVSRQ如同GFP的表現,均勻分布在細胞中。而共同表現BCL10與GFP-RSRTVSRQ並不影響由BCL10所引起的NF-κB的活性。 BCL10 and MALT1 interacted with each other and were present in many cells. Upon stimulation, they associated with various CARD-containing proteins to form CBM complexes which then mediated the activation of NF-κB signaling pathway. Previously, we and others demonstrated that BCL10 was cleaved in the C-terminus by MALT1 in Jurkat T cells treated with TPA and ionomycin. In 2008, amino acid residue Arg228 of BCL10 was reported to be the cleavage site. Different from what was reported, our serial deletion mutants of BCL10 suggested a probable cleavage site at Leu225. In the present study, several proteomic methods will be utilized to elucidate the discrepancy. The location and the function of the cleaved C-terminus peptides will also be investigated. Firstly, anti-BCL10 antibodies were cross-linked to protein G sepharose chemically to generate an affinity column to purify either full length BCL10 or cleaved BCL10 from Jurkat T cells treated with TPA/Ionomycin or not. An average of 5-10 ng BCL10 could be generated from 2×108 cells using an affinity column made from 80μg antibody. Secondly, fusion construct FlagBCL10 GFP was generated. Co-transfection of FlagBCL10 GFP and MALT1 in 293T cells would generate truncated FlagBCL10 which could then be isolated by commercially available FLAG affinity gel. An average of 50 ng purified products could be generated from 10 μl packed gel. LC-MS/MS was utilized to identify the cleavage site. Protease, Asp-N, cleaved at the N-terminus of the amino acid residue aspartate. Treatment of full length BCL10 or BCL10 cleaved at Leu225 or at Arg228 with Asp-N would generate polypeptides with M.W. of 3196.5 daltons, 2225.9 daltons and 2625.2 daltons, respectively. These fragments should be easily distinguished by LC-MS/MS. Unfortunately, LC-MS/MS failed to detect these fragments so far. It might be due to the proline residue right before the Asp-N cleavage site. Capillary Zone Electrophoresis (CZE) was utilized to analyze the C-terminus peptides RSRTVSRQ and TVSRQ. The two peptides could be efficiently separated by CZE. The detection sensitivity was estimated to be 5 ppm. Site directed mutagenesis was employed to investigate the role of amino acid residues Leu225 and Arg228 of BCL10 played as a substrate of MALT1. Degenerate primers were utilized to generate the following mutants:BCL10 L225A, BCL10 L225G, BCL10 L225Q, BCL10 L225K, BCL10 L225R, BCL10 L225E, and BCL10 R228G. All mutant constructs were transfected into 293T cells along with the MALT1 expression vector and tested for their ability of being a proteolytic substrate. BCL10 L225K and BCL10 L225R could be processed by MALT1 as wild type BCL10. MALT1 failed to cleave the other mutants (BCL10 L225A, BCL10 L225G, BCL10 L225Q, BCL10 L225E, BCL10 R228G). These data suggested that both Leu225 and Arg228 were critical for cleavage. Interestingly, double mutants BCL10 L225R,R228G retained its ability to be cleaved by MALT1. Mutation at the amino acid residues Phe222 and Leu223 of BCL10 L225R affected its ability of being a substrate of MALT1. While BCL10 F222A,L223T,L225R (FL4) retained its ability as wild type BCL10, mutants BCL10 F222H,L223E,L225R (FL-1), BCL10F222S,L223E,L225R (FL-2) and BCL10F222E,L223A,L225R (FL-3) were not able to be processed. The mechanisms by which these mutations affected MALT-1-mediated cleavage on BCL10 will be discussed in the text. We failed to introduce the cleaved C-terminus polypeptides RSRTVSRQ into 293T cells. Therefore, the eight amino acid residues RSRTVSRQ at the C-terminus of BCL10 were fused at the C-terminus of GFP to investigate its effects. GFP-RSRTVSRQ showed a diffuse-distribution pattern as GFP. Expression of GFP-RSRTVSRQ did not have effect on BCL10-induced NF-κB activation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10674 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-99-1.pdf | 3.25 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。