Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No.1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan.
J Biomed Sci. 2012 Oct 5;19(1):85. doi: 10.1186/1423-0127-19-85.
MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity. MALT1 cleaves protein substrates after a positively charged Arginine residue. BCL10, a 233 amino acids polypeptide, is identified as one of the MALT1 proteolytic substrates. MALT1 cleaves BCL10 at the C-terminal end of Arg228. A mere 5 amino acids difference between the substrate and the proteolytic product made it difficult to tell whether the cleavage event took place by using a simple western blot analysis. Here, BCL10GFP was constructed and utilized to examine the specificity and domain determinants for MALT1 cleavage in cells.
Various BCL10GFP constructs were transfected into HEK293T cell with MALT1 construct by using calcium phosphate-DNA precipitation method. Lysates of transfectants were resolved by SDS/PAGE and analyzed by western blot analysis.
BCL10GFP was proteolytically processed by MALT1 as BCL10. The integrity of caspase recruitment domain (CARD) and MALT1-interacting domain on BCL10 were required for MALT1 proteolytic activity. Besides the invariant P1 cleavage site Arg228, P4 Leu225 played a role in defining BCL10 as a good substrate for MALT1.
We offered a way of monitoring the catalytic activity of MALT1 in HEK293T cells using BCL10GFP as a substrate. BCL10GFP can be utilized as a convenient tool for studying the determinants for efficient MALT1 cleavage in HEK293T cells.
MALT1 属于衔接酶家族，通过其支架功能和蛋白水解活性调节 NF-κB 信号通路。MALT1 在正电荷精氨酸残基之后切割蛋白底物。BCL10 是一种 233 个氨基酸的多肽，被鉴定为 MALT1 蛋白水解底物之一。MALT1 在 Arg228 的 C 末端切割 BCL10。由于底物和蛋白水解产物之间仅相差 5 个氨基酸，因此很难通过简单的 Western blot 分析来判断切割事件是否发生。在这里，构建了 BCL10GFP 并用于在细胞中检查 MALT1 切割的特异性和结构域决定因素。
通过磷酸钙-DNA 沉淀法将各种 BCL10GFP 构建体与 MALT1 构建体共转染到 HEK293T 细胞中。转染子的裂解物通过 SDS/PAGE 分离，并通过 Western blot 分析进行分析。
BCL10GFP 被 MALT1 蛋白水解加工为 BCL10。BCL10 的半胱天冬酶募集结构域（CARD）和 MALT1 相互作用结构域的完整性对于 MALT1 蛋白水解活性是必需的。除了不变的 P1 切割位点 Arg228 外，P4 Leu225 在确定 BCL10 作为 MALT1 的良好底物方面起作用。
我们提供了一种使用 BCL10GFP 作为底物在 HEK293T 细胞中监测 MALT1 催化活性的方法。BCL10GFP 可作为研究在 HEK293T 细胞中有效切割 MALT1 的决定因素的便捷工具。
