- 作者: Shin-Yi Jou, Chien-Chih Chang, Chun-Hsien Wu, Mei-Ru Chen, Ching-Hwa Tsai, Wen-Hui Chuang, Yun-Hui Chen, Ann-Lii Cheng and Shin-Lian Doong
- 作者服務機構: Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taiwan, R.O.C.
- 中文摘要: --
- 英文摘要:
Background: MALT1 belongs to a family of paracaspase and modulates NF-kappaB 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.
Methods: 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.
Results: 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.
Conclusions: 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 - 中文關鍵字: --
- 英文關鍵字: Paracaspase, MALT1, BCL10GFP, in vivo, processing