A CARD10-dependent tonic signalosome activates MALT1 paracaspase and regulates IL-17/TNF-a driven keratinocyte inflammation

The paracaspase MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) controls signaling downstream of several cell surface receptors, such as C-type lectin receptors on myeloid cells and antigen receptors on lymphocytes. Upon receptor engagement, MALT1, BCL10 (B-cell lymphoma/leukemia 10) and a CARD (Caspase recruitment domain) family member assemble into a ‘CBM’ complex, which is required to trigger MALT1 paracaspase activity and downstream transcriptional activation mechanisms (Meininger and Krappmann 2016; Rosebeck et al. 2011). Here, we found that CARD10 is highly expressed in proliferating keratinocytes and is responsible for a tonic level of paracaspase activity, driven by MALT1 isoform A. Furthermore, using the potent and selective MALT1 inhibitor MLT-827 (Bardet et al. 2018; Unterreiner et al. 2017), we reveal that MALT1 activity regulates pro-inflammatory responses downstream of IL-17/TNF-α

CARD10 cleavage by MALT1 restricts lung carcinoma growth in vivo

CARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that protein-kinase-C-induced CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity

Functional impact of C-terminal cleavage on MALT1 isoforms A and B.

<p><b>(A)</b> NF-κB luciferase reporter gene assay in HEK293 cells transfected with either, MALT1A WT, MALT1A-781, MALT1B WT, or MALT1B-770 in the absence or presence of CARD11-L244P. Luciferase activity was recorded after 24h. Data show the mean of triplicate determinations from 7 independent experiments. Statistical significance was calculated using the Student T-test. Western blot analysis, performed in parallel to control for protein expression, is shown below with samples from a representative experiment. An anti-tubulin immunoblot is provided as loading control. <b>(B)</b> CBM reconstitution was performed using MALT1A, MALT1B as well as their constitutively cleaved variants, together with the MALT1 protease substrate A20. Immunoblot analyses for MALT1 show the respective C-terminal auto-cleavage bands (white arrow heads). For A20, they show cleaved fragments as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref009" target="_blank">9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref022" target="_blank">22</a>]. A20p37 is a proteasome sensitive degradation product of A20 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref009" target="_blank">9</a>]. Its weak detection when MALT1A WT is used is consistent with high proteolytic activity resulting in early processing of A20 and disappearance of A20p37 by the time of harvest. A non-specific (NS) band detected by the anti-A20 antibody is provided as loading control.</p

TRAF6 induces auto-proteolysis-dependent down-regulation of MALT1.

<p><b>(A)</b> The CBMT reconstitution assay in HEK293 was performed with either MALT1A-WT, or the TRAF6-binding deficient MALT1A-4E/A mutant construct, in the presence of TRAF6-WT, TRAF6-C70A or TRAF6 289–522. Immunoblotting with anti-FLAG antibody is shown. An anti-tubulin immunoblot is provided as loading control. <b>(B)</b> The TM reconstitution assay in HEK293 cells was performed using MALT1A, MALT1B as well as their C-terminal truncated variants. Anti-FLAG Western Blot analyses show the respective C-terminal auto-cleavage bands (white arrow head), the respective N-terminal auto-cleavage bands (grey arrow head, p16) as well as the respective mono-ubiquitinated species detected in the presence of z-VRPR-fmk (black arrow head). They also provide TRAF6 co-expression levels (bottom panels). <b>(C)</b> CBM and TM reconstitution assays in HEK293 cells were performed in the presence of co-expressed CYLD, using MALT1A, MALT1B, as well as their C-terminal truncated variants. Anti-FLAG Western Blot analyses show MALT1 C-terminal auto-cleavage bands (white arrow heads) as well as CYLD full length (<i>fl</i>) and cleaved fragment (<i>cl</i>) levels. Anti-CYLD immunoblots are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.s007" target="_blank">S7C Fig</a>. <b>(D)</b> CBM and TM reconstitution assays in HEK293 cells were performed using MALT1A-WT, several point mutant forms thereof: R149A, R781A, C464A, K644R, as well as the MALT1A-781 truncated form mimicking constitutive C-terminal auto-cleavage. Immunoblots with anti-FLAG antibody are shown. Densitometry analysis of MALT1 signals as percentage of the signal obtained with MALT1A 1–781 is provided underneath. MALT1 C-terminal auto-cleavage is denoted with a white arrow head and mono-ubiquitination with a black arrow head. An anti-tubulin immunoblot provides loading controls for the both assays.</p

MALT1 auto-proteolysis and mono-ubiquitination mechanisms.

<p><b>(A)</b> The TM reconstitution assay performed with MALT1A-C464A, MALT1A-R149A, MALT1A-R781A and combinations thereof. Immunoblotting with anti-FLAG antibody is shown. MALT1 C-terminal auto-cleavage is denoted with a white arrow head, N-terminal auto-cleavage with a grey arrow head (p16) and mono-ubiquitination of MALT1A-C464A with a black arrow head. * denotes a faint band that migrates above p16, which we observed in lanes loaded with MALT1A-C464A samples. <b>(B)</b> TM and CBM reconstitution assays were performed with MALT1A-C464A, MALT1A-K644R and the combination of the two. Immunoblots with anti-FLAG antibody are shown. MALT1 C-terminal auto-cleavage is denoted with white arrow heads, N-terminal auto-cleavage with a grey arrow head (p16) and mono-ubiquitination of MALT1A C464A with black arrow heads. The immunoblot with anti-BCL10 antibody (ep605y) shows reduced phosphorylated BCL10 when MALT1A-C464A and MALT1A-K644R are co-expressed, indicative of MALT1 protease activity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.g002" target="_blank">Fig 2A</a>).</p

Post-translational modifications of MALT1 in human lymphocytes.

<p><b>(A)</b> OCI-Ly3 cells were grown for 4 days in the presence or absence of 50 μM z-VRPR-fmk. Cells were harvested for MALT1 immunoprecipitation and lysate analysis using an anti-MALT1 antibody (H-300), as described in the Methods section. The MALT1 faster and slower migrating species are indicated with white and black arrow heads, respectively. The BCL10 antibody recognizing more specifically the uncleaved form of BCL10 (ep606y) was reported already [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref038" target="_blank">38</a>]. Densitometry of MALT1and BCL10 signal intensities was measured in 3 independent experiments and is shown as Mean ± SD. Cells were counted at the end of each experiment and counts are represented as Mean ± SD (N = 3). Statistical significance was calculated using the Student T-test. <b>(B)</b> Jurkat cells were stimulated with PMA (10 ng/ml) anti-CD28 (1 μM) and anti-CD3 (1 μM) in the presence of 5 μM MG-132, for various times before full cell extraction and analysis of MALT1 (MT1/410 antibody) and BCL10 (antibodies specified on the figure) by immunoblotting. The MALT1 faster and slower migrating species are indicated with a white and a black arrow head, respectively. <b>(C)</b> Primary human CD3 T cells were stimulated with PMA (10 ng/ml) and Ionomycin (1 μM) in the presence of 5 μM MG-132, for various times before full cell extraction and analysis of MALT1 (MT1/410 antibody). The MALT1 faster and slower migrating species are indicated with a white and a black arrow head, respectively. Anti-tubulin immunoblots are provided as loading controls.</p

Schematic representation of MALT1 isoforms and variants thereof.

<p>The two reported human MALT1 isoforms A (NP_006776.1) and B (NP_776216.1) are depicted. The drawings show the different domains, the auto-cleavage sites (red arrows), the catalytic cysteine residue (black), the reported ubiquitination site (green) and shows the regions required for binding BCL10 (grey) and TRAF6 (blue). The truncated variants of the two isoforms, used or discussed in the present work, are represented in the shaded boxes at the bottom, with predicted molecular weights. The 11-amino acid deletion of isoform B is depicted by a red triangle. Ig, Immunoglobulin-like domain.</p

MALT1 auto-cleavage at R149 is induced by TRAF6.

<p><b>(A)</b> MALT1A was expressed in HEK293 cells, either alone, or together with CARD11-L244P, or as part of a CBM reconstitution assay (transfection ratio 1:1:1:1 using a control plasmid), or a TM reconstitution assay (TRAF6 + MALT1 (transfection ratio 1:1:2 with “2” referring to a control plasmid), or a CBMT reconstitution assay (transfection ratio 1:1:1:1). Western Blot analyses with anti-FLAG and anti-MALT1 (MT1/410) antibodies are shown, displaying MALT1 auto-cleaved fragments (C-terminal, white arrow head; N-terminal depicted as p16/p76, grey arrow heads). Loading controls with an anti-tubulin antibody are also provided. <b>(B)</b> The CBMT reconstitution assay was performed as in (A) using either TRAF6-WT, or TRAF6-C70A, or TRAF6 289–522. Anti-FLAG Western Blot analyses are shown, displaying MALT1 auto-cleaved fragments (C-terminal, white arrow heads; N-terminal p16, grey arrow heads) as well as MALT1 mono-ubiquitinated species (black arrow heads). An anti-cleaved BCL10 immunoblot providing evidence for proteolytic activity of MALT1 is also shown together with a non-specific (NS) band detected by the BCL10 antibody, as loading control.</p

Influence of TRAF6 on MALT1 function, scaffolding, self-cleavage reactions and protein turnover.

<p>The model represents the capacity of MALT1 isoforms (M) to interact with their scaffolding partners CARD11 (C), BCL10 (B) and TRAF6 (T) and how self-cleavage reactions impact this capacity. Upon CARD11 activation resulting in CBM assembly, proteolytic competence in MALT1 is turned on and persists throughout the entire MALT1 activation process (indicated with scissors). MALT1 substrates identified to date can all be cleaved as part of CBM or TM complexes (data not shown). Only for MALT1B is proteolytic function severely impaired after self-cleavage at R781. Self-cleavage of MALT1A at R149 is favored by strong interaction with TRAF6 and represents a feed-forward mechanism that releases a highly functional MT complex from a transient CBMT complex and actively contributes to MALT1 turnover. This mechanism is less favored in MALT1B which cannot interact strongly with TRAF6. Self-cleavage of MALT1A at R781 weakens the interaction with TRAF6. In MALT1B, cleavage at the equivalent site (R770) abrogates interaction with TRAF6, “locking” the truncated MALT1 in the CBM complex.</p

Ectopic CBM reconstitution triggers MALT1 auto-cleavage and ubiquitination.

<p><b>(A)</b> Constituents of the CBM complex ─ CARD11-L244P, BCL10 and MALT1 (N-terminal FLAG) ─ were ectopically expressed in HEK293 cells individually or in combination (CBM reconstitution assay). 24h after transfection, lysates were analyzed with anti-CARD11, anti-BCL10 (ep605y), anti-MALT1 (ep603y) and anti-FLAG antibodies. BCL10 cleaved by MALT1 is indicated with Δ5. Phosphorylated BCL10 species are indicated with (P). The white arrow head points to a faster migrating species of MALT1. <b>(B)</b> CBM reconstitution was performed in the presence of z-VRPR-fmk, a covalent peptidic inhibitor of MALT1 protease (10) or by using a catalytically deficient MALT1-C464A mutant form. The white arrow head points to a faster migrating species of MALT1, the black one to a slower running species. <b>(C)</b> MALT1A-824 (WT), R781A, 1–781 and R800A expression constructs were submitted to a CBM reconstitution assay and analyzed by Western Blot with an anti-FLAG antibody to detect MALT1. This identified the main self-cleavage product (white arrow) to result from cleavage at R781. <b>(D)</b> MALT1A-824 WT, C464A and C464A/K644R expression constructs were submitted to a CBM reconstitution assay and analyzed by Western Blot with an anti-FLAG antibody to detect MALT1. This showed that the slower migrating species of MALT1 in our experiments (black arrow) corresponds to the K644-monoubiquitinated MALT1 species identified by Thome and coll. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref019" target="_blank">19</a>]. <b>(E)</b> MALT1A and MALT1B both undergo C-terminal cleavage and ubiquitination. CBM reconstitution was performed with MALT1A-824, R781-cleaved MALT1A (MALT1A-781), MALT1B-813 and R770-cleaved MALT1B (MALT1B-770), in the absence or presence of z-VRPR-fmk. The white arrow head points to faster migrating species of MALT1A and B, the black one to a slower running species of these two isoforms.</p