Mechanism and specificity of the human paracaspase MALT1

Abstract

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (10(3)-10(4) M(-1)·s(-1)) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

Figure 1. Domain structure and purification of MALT1

(A) Domain structure of MALT1. (B and C) His-tagged full-length MALT1 (B) and the catalytic domain (amino acids 329–566) (C) were expressed in E. coli and purified using a Ni-NTA column. Lane 1, molecular mass markers; lane 2, total cell lysate; lane 3, soluble material after sonication; lane 4, the eluted protein fraction with full-length (FL) MALT1 (B) and catalytic domain (C) respectively, depicted by arrows. The band marked with an asterisk (*) was N-terminally sequenced and found to be the E. coli heat-shock protein DnaK. (D) Full-length and catalytic domain MALT1 were titrated with the depicted concentration range of the irreversible inhibitor z-VRPR-FMK. The linear portion of the titration curve was extrapolated to its intercept of the x-axis to reveal the active concentration of the respective MALT1 preparations.

Figure 2. MALT1 activation

(A–C) Different salts and buffer additives were screened for MALT1-activating potential. Full-length (150 nM) and catalytic domain (4 μM) MALT1 were assayed in 50 mM Hepes (pH 7.5), 10 mM DTT and 1 mM EDTA, supplemented as depicted (A and B) or in 50 mM Hepes, 100 mM NaCl and 0.9 M sodium citrate (pH 7.5) supplemented as depicted (C). (D) Full-length MALT1-WT (500 nM) was assayed in buffers with various citrate concentrations and supplemented with full-length catalytic mutant as depicted. The experiments were repeated three (A–C) or two (D) times respectively. Mean values and S.E.M. are shown.

Figure 3. Sodium citrate does not activate MALT1 via cleavage

Full-length MALT1-WT and -C464A (C/A) were activated in assay buffer plus EDTA for the indicated times at 37°C. Reactions were stopped by TCA precipitation and samples were resolved by SDS/PAGE. The identity of all bands was determined via N-terminal sequencing. His-rich, FKBP-type peptidylprolyl cis–trans isomerase slyD (histidine-rich binding protein). The asterisk marks a protein that could not be clearly identified due to its low intensity, but partial sequencing showed that it did not match the sequence of MALT1. The molecular mass in kDa is indicated on the left-hand side.

Figure 4. MALT1 substrate specificity

(A) MALT1 specificity was determined using a tetrapeptide P1 library consisting of Ac-LRSX-ACC (P1) and a P1-Arg PS-SCL (P2, P3 and P4). Full-length (130 nM) and catalytic domain MALT1 (5 μM) were analysed in assay buffer. Hydrolysis rates were determined and presented as a fraction of the maximal rate in each subset library position. The x-axes represent the amino acids depicted using the one-letter code (O, norleucine). (B and C) Cleavage of fluorigenic peptides of various lengths by full-length (B, 80 nM) or catalytic domain (C, 4 μM) was determined in assay buffer plus EDTA. The highest activity in each assay was set as 100%; all other activities are shown relative to this. All assays were repeated at least three times. Mean values and S.E.M. are shown.

Figure 5. Cleavage of CYLD by MALT1 and caspase 8

(A and B) FLAG-tagged human CYLD was purified from HEK-293 cells and incubated with 2-fold increasing concentrations of full-length MALT1-WT (A) or caspase 8-WT (B) respectively. Samples were resolved by SDS/PAGE, followed by Western blotting and detection with an antibody against the N-terminal domain of CYLD (top panels) and anti-FLAG antibody (bottom panels) respectively. Data are representative of three independent experiments. (C) Linear regression used to determine the k_cat/K_m values. Mean values and S.D. are shown. (D and E) Different FLAG-tagged CYLD mutants were purified as in (A) and incubated with the WT or catalytic mutant versions of full-length MALT1 (D) or caspase 8 (E) respectively at 1 μM. (F) Overview of CYLD domains and cleavage sites. CAP-Gly, cytoskeleton-associated protein glycine-rich domain; DUB, deubiquitinating enzyme domain.