2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656892摘要：在淋巴細胞或非淋巴細胞， MALT1 (mucosa-associated- lymphoid-tissue lymphoma-translocation gene 1)已經被確認是數條引發 NFB活化訊息傳遞鏈中的重要調節因子。事實上，MALT1 會跟 BCL10 結合，作用在 BCL10 的下游引發 NFB活化。蛋白質構造分析顯示，在 MALT1的 C 端有兩個在各種已知 caspase 或類 caspase蛋白質 ”metacaspase” 中重要及負責摧化的胺基酸 (Cys464 and His415)。MALT1的蛋白切割酵素活性已被證實在由抗原接受器引發的淋巴球活化及 ABC-DLBCL淋巴瘤的形成扮演重要角色。在細胞內大量表現 BCL10 會引發 MALT1形成多聚體而活化其切割蛋白質的能力。在這個過程中，一個在 SDS-PAGE移動較快速的MALT1 裂解產物經常被偵測到。這次的計劃中，我們想要探討下列問題： I. MALT1是否切割自己? -從 E. coli 中純化 wild-type 以及 catalytically-inactive 的重組 MALT1 蛋白質，在試管中進行反應，分析其切割能力 -利用定點突變(site-directed mutagenesis)定位(map)切割點 II. MALT1 自我切割的生物功能為何 ? -探討MALT1 切割後產物蛋白質切割的能力 -探討MALT1 切割後產物在活化 NFB路徑扮演的角色 III. 在生理狀態下探測 MALT1 是否自我切割 ? -利用多種已知可以透過CARMA-BCL10-MALT1多聚合體活化NFB的刺激物(如PMA/ionomycin, LPS, Zymosan)處理細胞並檢測 MALT1是否自我切割 ? -被報導十分依賴 NFB活性的 ABC-DLBCL 細胞也要加入檢測行列 我們的研究將提供生理狀況下發生在 MALT1 分子的變化及其機制的基本了解；希望經由這些資料的建立，提供一個用來了解黏膜相關組織淋巴瘤分子致病機轉的基礎，開發其在診斷、預後以及治療黏膜相關組織淋巴瘤的價值。<br> Abstract: MALT1 (mucosa-associated- lymphoid-tissue lymphoma-translocation gene 1) has essential roles in the activation of nuclear factor-B (NFB) by antigen receptors, receptors with immuno receptor tyrosine-based activation motifs (ITAMs) and some G-protein coupled receptors (GPCRs) expressed by both immune and non-immune cells. Structural analysis showed that MALT1 carboxyl terminus has a putative caspase-like domain The protease activity of the paracaspase MALT1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Overexpression of BCL10 induced oligomerization of MALT1 and triggered the proteolytic activity of MALT1. In the process, the appearance of a faster-migrating MALT1 breakdown product from lysates of cells co-transfected with MALT1 and BCL10 gene was consistently observed. In the present study, we would like to investigate the following issues: I. Is MALT1 able to process itself ? - In vitro cleavage assay: recombinant wild-type or catalytically-inactive MALT1 protein will be purified from E. coli and tested in the tube for its ability to process itself. - To map the cleavage site by generating mutations on candidate sites - In vivo transfection system to validate the in vitro findings II. What is(are) the biological consequence(s) of cleavage of MALT1 ? -Does the autocleavage product of MALT1 retain its proteolytic ability ? -Does the autocleavage product of MALT1 retain its activity on NF-B activation ? III. Can the cleavage of MALT1 occur under physiological settings ? -Multiple stimulators (such as PMA/ionomycin, LPS, Zymosan) known to activate NF-B signaling through CARMA-BCL10-MALT1 complex will be tested for their effects to induce the cleavage of MALT1. -ABC-DLBCL cells, characterized by its reliance on the oncogenic activation of the NFB pathway, will also be tested. Our study will provide a fundamental understanding of molecular events occurring on MALT1 and provide a basis from which to unravel the molecular pathogenesis of MALT lymphoma or DLBCL. We hope to explore their values in disease diagnosis, prognosis and treatment of MALT lymphoma or DLBCL.