Establishment of a MALT1-Mediated Proteolytic Cleavage Assay in vitro
| Autor: | Chien-Chih Chang, 張健治 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 MALT1 (mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1) acts as an adaptor protein with proteolytic activity that controls T/B-cell activation by regulating key molecules in T/B-cell-receptor-induced signaling pathways. It plays a central role in MALT lymphoma by its interaction with BCL10 and enforced activation of NF-κB signaling. Computer assisted amino acid sequence analysis indicated that MALT1 shared sequence similarity with caspases. However, MALT1 was not able to cleave substrates of caspases. It was, therefore, defined as a “paracaspase”. A 3D model of the “caspase-like domain” of MALT1 predicted its specificity toward uncharged residues in the P1 position. Previous study in our laboratory demonstrated the proteolytic cleavage of BCL10 by MALT1 in 293T cells. Serial deletion constructs were constructed to map the probable cleavage site. Since deletion of single amino acid residue--Leu225 of BCL10 abolished its ability to be cleaved, suggesting a probable cleavage site at Leu225. The result was consistent with the hypothesis that MALT1 might show specificity toward uncharged resides in the P1 site. In 2008, MALT1 was reported to cleave A20 and BCL10 with specificity toward basic amino acid residue--Arginine. In the present study, we would like to set up an in vitro proteolytic cleavage assay of MALT1 to characterize its substrate specificity. His-tag fusion constructs of full length MALT1 and catalytic inactive mutants (MALT1-H415A；MALT1-C464A) were generated. Expression of His-tag MALT1 and mutants were performed in Arctic expression competent E. coli cell. Though major proportion of MALT1 were in insoluble inclusion bodies, we were able to purify sufficient soluble form of MALT1 with Ni-Column. Purified His-tag BCL10 proteins from BL21 E. coli expression system were utilized as substrates in the in vitro cleavage assay. Two reported assay buffers were tested (Buffer1：50mM MES pH 6.8，150mM NaCl，10%(wt/vol) sucrose，0.1%(wt/vol) CHAPS，10mM dithiothreitol，1 M ammonium citrate；Buffer2：50 mM Tris-HCl，pH 7.4， 60 mM NaCl，10 mM KCl，20 mM MgCl2，100 mM CaCl2 and 10 mM dithiothreitol). The reaction was performed at 30℃ for 16 hours. BCL10 was found to be processed by wild-type MALT1 but not catalytic inactive mutants in buffer1. The removal of ammonium citrate completely abolish the reaction. Site-directed mutagenesis was performed to investigate the role of amino acid residues Leu225 and Arg228 of BCL10 played as a substrate of MALT1. Degenerate primers were utilized to generate the following mutants：BCL10L225A、BCL10L225E、BCL10L225G、BCL10L225Q、BCL10L225R、BCL10L225T、BCL10R228G and BCL10R228I. MALT1 was found to be able to cleave BCL10L225R and BCL10L225T, albeit at a less efficient level as compared to wild-type BCL10. All the other mutants (BCL10L225A、BCL10L225E、BCL10L225G、BCL10L225Q、BCL10R228G and BCL10R228I) lost their abilities of being processed by MALT1. BCL10-derived fluorogenic peptides--Ac-FLPL-AMC and Ac-LRSR-AMC were also tested in the in vitro assay for being substrates. A time-dependent in the fluoroscence intensity was observed in the reaction using Ac-LRSR-AMC but not using Ac-FLPL-AMC, suggesting its specificity toward basic amino acid residue in P1 position. Caspase recruitment domain (CARD) mutant (BCL10L41R) of BCL10 failed to interact with other CARD-containing proteins. Deletion of amino acid residues 107~119 of BCL10 (BCL10Δ107~119) resulted in the poor interaction with MALT1. In vivo, MALT1 failed to cleave both mutants. However, in the presence of ammonium citrate, incubation of purified MALT1 with BCL10L41R or BCL10Δ107~119 in vitro resulted in cleavage albeit at a less efficient level. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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