Nagel Daniel, Krappmann Daniel
Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
Methods Mol Biol. 2015;1280:239-46. doi: 10.1007/978-1-4939-2422-6_13.
MALT1 (mucosa-associated lymphoid tissue protein 1) is a key regulator of antigen-induced NF-κB activation in the adaptive immune response. Activation of proteolytic activity of the MALT1 paracaspase was shown to boost the immune response. Additionally, MALT1 proteolytic activity is essential for the survival of MALT1-dependent lymphoma, such as the activated B-cell type (ABC) of diffuse large B-cell lymphoma (DLBCL) or MALT lymphoma. The functional impact of MALT1 paracaspase on T-cell activation and lymphomagenesis suggests that MALT1 is a promising therapeutic target for the treatment of autoimmune diseases and distinct lymphoma entities. To evaluate the requirement of MALT1 in further detail, direct measurement of its activity status is of great importance. We have established a fluorogenic cleavage assay which can be used to measure activity of recombinant and cellular MALT1. Here we describe the basis of the cleavage assay and include a detailed protocol for recombinant production of MALT1 and also the cellular immunoprecipitation of endogenous MALT1 to determine its proteolytic activity.
MALT1（黏膜相关淋巴组织蛋白1）是适应性免疫应答中抗原诱导的核因子κB（NF-κB）激活的关键调节因子。已证明MALT1副胱天蛋白酶的蛋白水解活性激活可增强免疫应答。此外，MALT1蛋白水解活性对于MALT1依赖性淋巴瘤的存活至关重要，例如弥漫性大B细胞淋巴瘤（DLBCL）的活化B细胞型（ABC）或MALT淋巴瘤。MALT1副胱天蛋白酶对T细胞活化和淋巴瘤发生的功能影响表明，MALT1是治疗自身免疫性疾病和不同淋巴瘤实体的有前景的治疗靶点。为了更详细地评估MALT1的需求，直接测量其活性状态非常重要。我们建立了一种荧光切割测定法，可用于测量重组和细胞MALT1的活性。在此，我们描述了切割测定法的基础，并包括重组生产MALT1的详细方案以及内源性MALT1的细胞免疫沉淀以确定其蛋白水解活性。
