title: BCL10GFP fusion protein as a substrate for analysis of determinants required for mucosa-associated lymphoid tissue 1 (MALT1)-mediated cleavage.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No.1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan.
authors.forename: Shin-Yi
authors.initials: SY
authors.lastname: Jou
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Chang
authors.forename: Chun-Hsien
authors.initials: CH
authors.lastname: Wu
authors.forename: Mei-Ru
authors.initials: MR
authors.lastname: Chen
authors.forename: Ching-Hwa
authors.initials: CH
authors.lastname: Tsai
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Chuang
authors.forename: Yun-Hui
authors.initials: YH
authors.lastname: Chen
authors.forename: Ann-Lii
authors.initials: AL
authors.lastname: Cheng
authors.forename: Shin-Lian
authors.initials: SL
authors.lastname: Doong
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D000074018:B-Cell CLL-Lymphoma 10 Protein; D020169:Caspases / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D017384:Sequence Deletion; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000074018:B-Cell CLL-Lymphoma 10 Protein; C118591:BCL10 protein, human; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D049452:Green Fluorescent Proteins; D020169:Caspases; C400109:MALT1 protein, human; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein