CARD9或CARMA1所組成的CBM複合體中MALT1蛋白水解酶功能差異的探討

Abstract

目前已經發現許多鷹架蛋白在由不同接受器刺激誘發的Nuclear Factor-κB (NF-κB)活化路經中扮演重要角色。在這些鷹架蛋白中，一種具有CARD (CAspase Recruitment Domain) 的鷹架蛋白家族於NF-κB的活化扮演重要角色。CARMA (CARD- and Membrane Associated guanylate kinase-like domain-containing proteins)蛋白在氮端具有CARD區域，後面接著一個Coiled-coil (CC) 區域、一個PDZ區域、一個SH3區域，以及一個Guanylate Kinase-like (GUK) 區域於碳端。CARMA蛋白透過CARD區域與兩個下游訊號分子結合形成複合體，一個為同樣具有CARD區域的蛋白質，BCL10 (B-cell lymphoma 10)；另一個為旁凋亡蛋白酶MALT1 (Mucosa-Associated Lymphoid tissue lymphoma Translocation protein 1)。全長的MALT1是一個不活化、單體的形態。當CARMA–BCL10–MALT1複合體 (又稱為CBM 複合體) 形成時，誘發MALT1進行寡聚合作用，活化其蛋白水解酶活性，並招募下游IKK複合體導致NF-κB的活化。含有CARMA1的CBM複合體是一個雛形及最被了解的signalsome。CARD9為另一個CARD蛋白，其與CARMA家族成員的結構相似。其在氮端與CARMA蛋白一樣具有CARD區域及一個CC區域，但缺少碳端的PDZ-SH3-GUK區域。CARD9同樣會與BCL10及MALT1形成複合體，而使NF-κB活化。然而，在具有CARD9的CBM複合體中其MALT1的蛋白水解活性仍未被研究透徹。在本篇研究中，以CARMA1、CARD9及把CARMA1不同區域與其相似蛋白CARD9置換的嵌合體CARD9-CARMA1蛋白來比較其形成聚集、與BCL10交互作用和活化NF-κB的能力。由實驗結果發現，CARD9在細胞質中形成多個大的聚集，CARMA1則形成puncta-like結構於細胞質以及細胞膜。將CARMA1的CARD或CARD-CC區域置換成CARD9，分別構築出CARD9-CARMA1嵌合體1及CARD9-CARMA1嵌合體2，影響表現量及型態。與BCL10共同表現，CARMA1、CARD9以及兩個嵌合體蛋白皆會與BCL10絲狀構造co-localization。利用NF-κB reporter assay分析，CARMA1具有明顯活化NF-κB的能力。CARD9以及兩個嵌合體蛋白發現具有中等的NF-κB活化能力。已經建立CARMA1-knock-down Jurkat T細胞並確認受MALT1所調控的BCL10及RelB切割現象會消失。在不遠的將來，CARMA1、CARD9以及兩個嵌合體蛋白將會用來研究活化MALT1蛋白酶活性的能力。

Many scaffold proteins have been shown to play a crucial role in activation of the nuclear factor-κB (NF-κB) following stimulation of different receptors. Among these scaffold proteins, a family of CARD (CAspase Recruitment Domain)-containing scaffold proteins plays critical roles in the activation of NF-κB. CARMA proteins (CARD- and Membrane Associated guanylate kinase-like domain-containing proteins) contain an N-terminal CARD domain, followed with a coiled-coil domain (CC), a PDZ domain, an SH3 domain, and a Guanylate Kinase-like (GUK) domain in the C-terminus. CARMA proteins, through their CARD domain, form a complex with two downstream signaling molecules, BCL10 (B-cell lymphoma 10), another CARD-containing protein, and paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1). Full length MALT1 is in an inactive, monomeric form. The formation of CARMA–BCL10–MALT1 complex (known as CBM complex) triggers oligomerization of MALT1, activates its proteolytic activity, and recruits the downstream IKK complex leading to activation of NF-κB. CARMA1-containing CBM complex is the prototype and the best characterized signalsome. CARD9, another CARD protein, is structurally similar to CARMA family members. It has an N-terminal CARD and a CC domain like CARMA proteins but lacks the C-terminal PDZ-SH3-GUK domain. CARD9 is also known to form a complex with BCL10 and MALT1 leading to activation of NF-κB. However, the proteolytic activity of MALT1 in the CARD9-containing CBM complex has not been fully characterized. In this study, CARMA1, CARD9 and chimeric CARD9-CARMA1 proteins containing distinct domain of CARMA1 replaced with counterpart from CARD9 were compared for their abilities to form aggregates, interact with BCL10 and activate NF-κB. While CARD9 formed multiple large aggregates in the cytoplasm, CARMA1 formed puncta-like structure both in the cytoplasm and along the cytoplasmic membrane. Replacement of CARD or CARD-CC in the CARMA1 with counterpart from CARD9, generating CARD9-CARMA1 chimera 1 and CARD9-CARMA1 chimera 2 respectively, affected the level and pattern of expression. With BCL10 coexpression, CARMA1, CARD9 and both chimeric proteins colocalized with BCL10 filaments. Using NF-κB reporter assay, CARMA1 was shown to induce an apparent NF-κB activation ability. CARD9 and both chimeric proteins exhibited moderate NF-κB activation ability. CARMA1-knock-down Jurkat cells were established and confirmed to lose their MALT1-mediated cleavage of BCL10 and RelB. In the near future, CARMA1, CARD9 and both chimeric proteins will be introduced and examined for their abilities to activate the protease activity of MALT1.