探討與MALT1 death domain產生交互作用之蛋白質

Investigation on proteins interacting with MALT1 death domain

王庭恩(Ting-En Wang)

指導教授 ： 董馨蓮

國立臺灣大學/醫學院/微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.00780

摘要

從t(11；18)(q21；q21)染色體轉位引發的低惡性度黏膜相關淋巴組織淋巴癌病例中發現的MALT1為人體中唯一的paracaspase，起初發現其功能為一鷹架蛋白，在各種細胞中和CARD-containing protein及BCL10形成CBM複合體，活化下游分子進而啟動NF-κB訊息傳遞路徑。此外，陸續也發現MALT1的caspase-like domain和caspase結構相似，皆具有Cys-His diad，因此如caspase具有蛋白酶的功能，但其受質與caspase不同，MALT1會切在精胺酸的C端，目前已知的受質有BCL10(B cell lymphoma 10)、A20、CYLD(cylindromatosis)、NIK (NF-κB Inducing Kinase)、RelB、Regnase-1。
而MALT1要執行上述功能，都必須先有聚合作用之後而活化，因其蛋白質結構與initiator caspase很像，且MALT1的prodomain－death domain目前的功能還未知，我們認為其可能參與在MALT1的活化過程中，因此藉由酵母菌雙雜合篩選法去尋找和MALT1 death domain有交互作用的蛋白質。在兩次的篩選中，總共檢視了〖10〗^5個菌落，經過再次酵母菌轉型作用確認，最後得到了93個陽性的結果。
由於酵母菌雙雜合篩選法的偽陽性較高，因此需用別的方法加以測試，我們嘗試用雙分子螢光互補法(BiFC)做確認，然而只要在細胞同時表現質體pVN173與pVC155，沒有和其他有交互作用的蛋白質融合，就可以在細胞中觀察到螢光，所以無法用此法去確認我們找到的蛋白質是否可以和MALT1有交互作用。
在免疫螢光法中，以DNAJB6作為主要的研究對象，DNAJB6有兩種isoform－DNAJB6a與DNAJB6b，DNAJB6a主要分布在細胞核中，而DNAJB6b則分布在細胞質內，MALT1單獨表現時也只表現在細胞質中。將MALT1與DNAJB6b共同表現時，仍停留在細胞質中，但與DNAJB6a一起表現時，則可以觀察到MALT1有轉移到細胞核的現象，可證實MALT1的分布會受到DNAJB6的影響。而免疫共沉澱法的測試也證實了DNAJB6a及DNAJB6b確實與MALT1有交互作用。因此確認了DNAJB6和MALT1之間確實有交互作用的產生。

關鍵字

MALT1 ； DNAJB6a ； DNAJB6b ； 交互作用 ； 酵母菌雙雜合篩選法

並列摘要

MALT1 was originally discovered in cases of low grade MALT (Mucosa-Associated-Lymphoid-Tissue) lymphoma with t(11;18)(q21;q21) chromosomal translocation. MALT1 was found to be a scaffold protein. It forms a CBM complex with CARD-containing protein (CARMA1, CARMA3, CARD9) and BCL10 in various cells. CBM complex activates downstream molecules and NF-κB signaling transduction pathway. Amino acid sequence analysis revealed that MALT1 possesses Cys-His diad and shares sequence homology with caspase. MALT1 is the only paracaspase in human. It was not until 2008 that MALT1 was demonstrated to be able to proteolytically process protein substrates. So far, BCL10(B cell lymphoma 10), A20, CLYD(cylindromatosis), NIK (NF-κB Inducing Kinase), RelB and Regnase-1 were reported to be substrates of MALT1. MALT1 needs to be activated into oligomers to perform the above-mentioned functions. MALT1 contains a death domain in the N-terminus. Like initiator caspases, the involvement of the death domain in the MALT1 activation pathway was highly suspected. In this study, yeast two hybrid system was performed to identify proteins interacting with MALT1 death domain. One hundred thousand colonies were screened in two separate experiment. Ninety three clones, including DNAJB6a, UBE2C etc., were identified as positives. BiFC (Bimolecular fluorescence complementation) was exploited to inspect the interaction of MALT1 and the positive candidates. Cells transfecting with expression vectors pVN173 (containing the N-terminal fragment of GFP) and pVC155 (containing the C-terminal fragment of GFP) showed considerable amount of fluorescence intensity without any fusion moiety. The high background interfered with the scoring of true positive interaction. BiFC can’t confirm the interaction study. BiFC technique needed to be modified to be a convenient tool for interaction study. DNAJB6 expressed in cells in two isoforms–DNAJB6a and DNAJB6b. Immunofluorescence staining revealed that DNAJB6a was located in the nucleus whereas DNAJB6b was distributed in the cytoplasm as reported. MALT1 was located in the cytoplasm. However, in the presence of DNAJB6a, MALT1 was found to be distributed in the nucleus. Co-immunoprecipitation analysis also demonstrated the interaction of MALT1 with DNAJB6a.

並列關鍵字

MALT1 ； DNAJB6a ； DNAJB6b ； interaction ； yeast two hybrid

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