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BCL10GFP fusion protein as a substrate for analysis of determinants required for Mucosa-Associated Lymphoid Tissue 1 (MALT1)-mediated cleavage
1 Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No.1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan
2 Department of Oncology and Internal Medicine, National Taiwan University Hospital, No.7, Chung San South Road, Taipei, Taiwan
3 Cancer Research Center, College of Medicine, National Taiwan University, Taipei, Taiwan
Journal of Biomedical Science 2012, 19:85 doi:10.1186/1423-0127-19-85Published: 5 October 2012
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