API2-MALT1 protease activity controls posttranscriptional gene regulation. (A) Schematic depiction of API2-MALT1 protein and domains. The C689A mutation is the MALT1 PM and the E1031A is the TBM of MALT1. (B) Western blot analysis of BJAB cells overexpressing API2-MALT1B WT, PM, or TBM. Activation of the noncanonical NF-κB pathways was determined by p100 and p52 expression, and the activation of the MALT1 protease was shown by substrate cleavage and IκBNS expression. Nonspecific bands are marked with an asterisk. (C) Quantification of ΔMFI of the 6 × NF-κB–EGFP reporter in BJAB cells expressing API2-MALT1B WT, PM, or TBM. Data represent MFI of n = 3 replicates, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (D) GSVA quantifying the effects of API2-MALT1 WT and mutant expression on a published ABC DLBCL–derived NF-κB gene signature. (E) Heat map of differentially expressed genes in API2-MALT1 WT-, PM-, or TBM–expressing BJAB cells compared with parental cells. On the color scale, red denotes higher and blue denotes lower expression; n = 5 for each sample. Genes highlighted in green are MALT1-protease dependent and genes highlighted in blue are TRAF6-binding dependent. (F) Signed Padj plot comparing gene induction by API2-MALT1 PM– or TBM– vs API2-MALT1 WT–expressing BJAB cells. Genes in the top-left and bottom-right corner are strongly TRAF6-binding and MALT1-protease dependent, respectively. Annotated are the genes from the heatmap in panel E. (G) Transcriptional activation of NFKBIZ, NFKBID, and ZC3H12A was shown by qRT-PCR in BJAB cells expressing API2-MALT1B WT, PM, or TBM. Values were normalized to the control (mock) infected cells, n = 8, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ∗P < .05 and ∗∗P < .01. (H) Analysis of the inhibition effects of the MALT1 protease after MLT-748 (2 μM, 18 hours) on NFKBIZ, ZC3H12A, and NFKBID in BJAB cells expressing API2-MALT1B WT by qRT-PCR. n = 4, all error bars depict the mean ± SD, unpaired Student t test; ∗∗P < .01. (I-J) Luciferase reporter assay of the 3′ UTR of (I) NFKBIZ and (J) NFKBID in BJAB cells overexpressing API2-MALT1B WT, PM, and TBM (left). Stability of the reporter was determined after inhibition of the MALT1 protease with MLT-748 (right) in BJAB cells expressing API2-MALT1B WT. n = 4, all error bars depict the mean ± SD; ∗P < .05; ∗∗P < .01; and ∗∗∗∗P < .0001. (K) Scheme of posttranscriptional gene regulation by the MALT1 protease in BCR-addicted ABC DLBCL (upper panel) and API2-MALT1-driven MALT lymphomas (lower panel).

API2-MALT1 protease activity controls posttranscriptional gene regulation. (A) Schematic depiction of API2-MALT1 protein and domains. The C689A mutation is the MALT1 PM and the E1031A is the TBM of MALT1. (B) Western blot analysis of BJAB cells overexpressing API2-MALT1B WT, PM, or TBM. Activation of the noncanonical NF-κB pathways was determined by p100 and p52 expression, and the activation of the MALT1 protease was shown by substrate cleavage and IκBNS expression. Nonspecific bands are marked with an asterisk. (C) Quantification of ΔMFI of the 6 × NF-κB–EGFP reporter in BJAB cells expressing API2-MALT1B WT, PM, or TBM. Data represent MFI of n = 3 replicates, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (D) GSVA quantifying the effects of API2-MALT1 WT and mutant expression on a published ABC DLBCL–derived NF-κB gene signature. (E) Heat map of differentially expressed genes in API2-MALT1 WT-, PM-, or TBM–expressing BJAB cells compared with parental cells. On the color scale, red denotes higher and blue denotes lower expression; n = 5 for each sample. Genes highlighted in green are MALT1-protease dependent and genes highlighted in blue are TRAF6-binding dependent. (F) Signed Padj plot comparing gene induction by API2-MALT1 PM– or TBM– vs API2-MALT1 WT–expressing BJAB cells. Genes in the top-left and bottom-right corner are strongly TRAF6-binding and MALT1-protease dependent, respectively. Annotated are the genes from the heatmap in panel E. (G) Transcriptional activation of NFKBIZ, NFKBID, and ZC3H12A was shown by qRT-PCR in BJAB cells expressing API2-MALT1B WT, PM, or TBM. Values were normalized to the control (mock) infected cells, n = 8, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ∗P < .05 and ∗∗P < .01. (H) Analysis of the inhibition effects of the MALT1 protease after MLT-748 (2 μM, 18 hours) on NFKBIZ, ZC3H12A, and NFKBID in BJAB cells expressing API2-MALT1B WT by qRT-PCR. n = 4, all error bars depict the mean ± SD, unpaired Student t test; ∗∗P < .01. (I-J) Luciferase reporter assay of the 3′ UTR of (I) NFKBIZ and (J) NFKBID in BJAB cells overexpressing API2-MALT1B WT, PM, and TBM (left). Stability of the reporter was determined after inhibition of the MALT1 protease with MLT-748 (right) in BJAB cells expressing API2-MALT1B WT. n = 4, all error bars depict the mean ± SD; ∗P < .05; ∗∗P < .01; and ∗∗∗∗P < .0001. (K) Scheme of posttranscriptional gene regulation by the MALT1 protease in BCR-addicted ABC DLBCL (upper panel) and API2-MALT1-driven MALT lymphomas (lower panel).

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