在試管中MALT1蛋白質切割反應系統的建立

Abstract

T-cell receptor(TCR)或B-cell receptor(BCR)在接受抗原刺激後，CARMA1和BCL10以及MALT1三種蛋白質形成的CBM複合體，會參與活化NF-κB的訊息傳導，缺一不可。其中MALT1是一種paracaspase，其結構和序列和caspase有極高同源性及相似性，但是在生理功能上卻有著極大差異。在2000年時，Uren et al. 第一個報導MALT1的論文，指出MALT1無法切割caspase的已知受質，曾分析其3D立體結構，指出MALT1可能對於非帶價胺基酸有較好的專一性，而非caspase在P1位置上的帶負價胺基酸----天冬門胺酸(Asp)。長久以來，大家都不清楚MALT1是否真正具有蛋白酶切割能力，更不清楚其受質為何。 我們先前實驗室的研究發現，共同轉染BCL10和MALT1時，發現BCL10有被誘導切割的現象；利用一系列刪除構築(deletion constructs)來研究切割位置，發現BCL10一旦失去Leucine225這個胺基酸後就無法被切割，之後將Leucine225突變成天冬門胺酸(Aspartate)，同樣看不到被切割的現象，這樣的結果和Uren在2000年發表的論點吻合。但2008年有兩篇論文報導MALT1具切割BCL10和A20的能力，利用蛋白質序列比對(alignment)並說明MALT1能夠切割在P1位置帶正電胺基酸----精胺酸，雖然切割兩者的有無對於NF-κB不會造成嚴重影響。 本篇論文主要目的是建立MALT1的in vitro cleavage assay，以BCL10當作切割模型，探討MALT1的生化切割特性。首先在in vivo下分別共同轉染MALT1、MALT1-H415、MALT1-C464、或MALT-C539與BCL10GFP入293T，確定MALT1及MALT-C539可以進行切割，一旦MALT1的catalytic diad (H415-C464)突變即失去蛋白切割活性。之後將系統移到細菌內，大量表現MALT1及BCL10，利用His-tag純化將MALT1及BCL10純化出來。為了assay MALT1的活性及其專一性，除了使用BCL10蛋白質，還有廣泛被拿來assay caspase活性的發射螢光胜肽，模擬BCL10L225或R228切割位置。我們發現，in vitro下MALT1必需要在1.0M ammonium citrate才能切割BCL10。 針對BCL10可能切割位置進行一系列點突變，構築並表現BCL10L225A、BCL10L225E、BCL10L225G、BCL10L225Q、BCL10L225R、BCL10L225T、BCL10R228G及BCL10R228I蛋白質。結果發現MALT1可以對BCL10L225R及BCL10L225T進行切割，但其他的突變蛋白質皆無法被切割。另一方面使用模擬BCL10切位L225的發射螢光胜肽 Ac-FLPL-AMC及R228 Ac-LRSR-AMC與MALT1作用進行實驗。實驗結果暗示著，MALT1似乎對於P1 site為正電胺基酸有較好的親和性，但是不論L225或R228對於切割與否都相當重要。 除了切點位置的突變構築外，也試圖在in vitro下探討，如果BCL10的CARD發生構形改變--BCL10L41R，或是BCL10失去和MALT1結合作用—BCL10Δ107~119對於MALT1切割的影響。在in vivo中，BCL10L41R及BCL10Δ107~119皆無法觀察到被MALT1切割的現象。實驗結果發現BCL10L41R及BCL10Δ107~119皆能夠被切割，暗示著BCL10的C端序列保持不變，在高鹽(1.0 M ammonium citrate)情況下，雖然切割能力下降許多，但MALT1都能夠進行切割。

MALT1 (mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1) acts as an adaptor protein with proteolytic activity that controls T/B-cell activation by regulating key molecules in T/B-cell-receptor-induced signaling pathways. It plays a central role in MALT lymphoma by its interaction with BCL10 and enforced activation of NF-κB signaling. Computer assisted amino acid sequence analysis indicated that MALT1 shared sequence similarity with caspases. However, MALT1 was not able to cleave substrates of caspases. It was, therefore, defined as a “paracaspase”. A 3D model of the “caspase-like domain” of MALT1 predicted its specificity toward uncharged residues in the P1 position. Previous study in our laboratory demonstrated the proteolytic cleavage of BCL10 by MALT1 in 293T cells. Serial deletion constructs were constructed to map the probable cleavage site. Since deletion of single amino acid residue--Leu225 of BCL10 abolished its ability to be cleaved, suggesting a probable cleavage site at Leu225. The result was consistent with the hypothesis that MALT1 might show specificity toward uncharged resides in the P1 site. In 2008, MALT1 was reported to cleave A20 and BCL10 with specificity toward basic amino acid residue--Arginine. In the present study, we would like to set up an in vitro proteolytic cleavage assay of MALT1 to characterize its substrate specificity. His-tag fusion constructs of full length MALT1 and catalytic inactive mutants (MALT1-H415A；MALT1-C464A) were generated. Expression of His-tag MALT1 and mutants were performed in Arctic expression competent E. coli cell. Though major proportion of MALT1 were in insoluble inclusion bodies, we were able to purify sufficient soluble form of MALT1 with Ni-Column. Purified His-tag BCL10 proteins from BL21 E. coli expression system were utilized as substrates in the in vitro cleavage assay. Two reported assay buffers were tested (Buffer1：50mM MES pH 6.8，150mM NaCl，10%(wt/vol) sucrose，0.1%(wt/vol) CHAPS，10mM dithiothreitol，1 M ammonium citrate；Buffer2：50 mM Tris-HCl，pH 7.4， 60 mM NaCl，10 mM KCl，20 mM MgCl2，100 mM CaCl2 and 10 mM dithiothreitol). The reaction was performed at 30℃ for 16 hours. BCL10 was found to be processed by wild-type MALT1 but not catalytic inactive mutants in buffer1. The removal of ammonium citrate completely abolish the reaction. Site-directed mutagenesis was performed 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：BCL10L225A、BCL10L225E、BCL10L225G、BCL10L225Q、BCL10L225R、BCL10L225T、BCL10R228G and BCL10R228I. MALT1 was found to be able to cleave BCL10L225R and BCL10L225T, albeit at a less efficient level as compared to wild-type BCL10. All the other mutants (BCL10L225A、BCL10L225E、BCL10L225G、BCL10L225Q、BCL10R228G and BCL10R228I) lost their abilities of being processed by MALT1. BCL10-derived fluorogenic peptides--Ac-FLPL-AMC and Ac-LRSR-AMC were also tested in the in vitro assay for being substrates. A time-dependent in the fluoroscence intensity was observed in the reaction using Ac-LRSR-AMC but not using Ac-FLPL-AMC, suggesting its specificity toward basic amino acid residue in P1 position. Caspase recruitment domain (CARD) mutant (BCL10L41R) of BCL10 failed to interact with other CARD-containing proteins. Deletion of amino acid residues 107~119 of BCL10 (BCL10Δ107~119) resulted in the poor interaction with MALT1. In vivo, MALT1 failed to cleave both mutants. However, in the presence of ammonium citrate, incubation of purified MALT1 with BCL10L41R or BCL10Δ107~119 in vitro resulted in cleavage albeit at a less efficient level.