Abstract
BackgroundMALT1 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.MethodsVarious 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.ResultsBCL10GFP 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.ConclusionsWe 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
Highlights
MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity
BCL10 was fused at the C-terminus with green fluorescence protein, generating BCL10GFP fusion protein, to study the determinants required for MALT1 cleavage in HEK293T cells
BCL10GFP was proteolytically processed by MALT1 as BCL10 BCL10 was phosphorylated intracellularly and presented as multiple slowly-migrating forms in the SDS/polyacrylamide gel (Figure 1A, lane 1; Figure 1B, lane 1)
Summary
MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity. MALT1, together with BCL10, has been shown to couple with different CARMA (CARD containing MAGUK protein) family protein to assemble into the CBM complex to that a mechanistic connection between MALT1 protease activity and NF-κB activation was demonstrated by Coornaert et al [12]. They provided evidences that NFκB signaling was enhanced from A20 cleavage by MALT1 [12]. The structure of human MALT1 paracaspase bonded with a peptide inhibitor was published [16,17] Both studies suggested for Arg invariant at the P1 position and preference for a hydrophobic residue at P4 position. BCL10 was fused at the C-terminus with green fluorescence protein, generating BCL10GFP fusion protein, to study the determinants required for MALT1 cleavage in HEK293T cells
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