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MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

Baens M, Bonsignore L, Somers R, Vanderheydt C, Weeks SD, Gunnarsson J, Nilsson E, Roth RG, Thome M, Marynen P - PLoS ONE (2014)

Bottom Line: MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity.Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits.Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium; Human Genome Laboratory, Center for Human Genetics, KU Leuven, Leuven, Belgium.

ABSTRACT
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

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Related in: MedlinePlus

MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells.A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q<0,001) in the pre-ranked down-regulated genes from JΔM-CA, JΔM-RA and RACA cells stimulated for 18 hrs with P hrs with P/I. Gene list depicted at the right side are NF-κB target genes down-regulated in JΔM-CA, JΔM-RA and RACA cells after 3 and 18 hrs stimulation with P hrs stimulation with P/I.
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pone-0103774-g007: MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells.A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q<0,001) in the pre-ranked down-regulated genes from JΔM-CA, JΔM-RA and RACA cells stimulated for 18 hrs with P hrs with P/I. Gene list depicted at the right side are NF-κB target genes down-regulated in JΔM-CA, JΔM-RA and RACA cells after 3 and 18 hrs stimulation with P hrs stimulation with P/I.

Mentions: So far, our data suggested that MALT1 auto-proteolysis affects neither IKK activation nor the nuclear translocation of NF-κB, despite a profound defect on the expression of the NF-κB target gene IL-2. Bi-allelic inactivation of endogenous MALT1 in R149A cells (JΔM-RA) further reduced IL-2 as well as CSF2 secretion to the basal levels observed in Jurkat T cells with bi-allelic MALT1 inactivation (JΔM) (Figure 7A). To assess whether these defects in IL-2 and CSF2 secretion were due to defects in transcription, we performed qRT-PCR analysis. A stimulus-dependent up-regulation of IL-2 and CSF2 mRNA levels was observed in the MALT1 expressing Jurkat T cells, while all Jurkat mutants were seriously hampered in their mRNA up-regulation (Figure 7B). Next we performed luciferase reporter assays with the different Jurkat clones. Cells expressing wild-type MALT1 showed a stimulus-dependent increase of gene reporter constructs containing the IL-2 promotor (IL-2p-Luc) or three NF-κB sites from the promoter of the kappa light chain of immunoglobulin (Igκ3-ConALuc), while this response was strongly impaired in JΔM-CA or JΔM-RA cells (Figure 7C). Together, these findings clearly support a role for MALT1 auto-proteolysis in regulating NF-κB transcriptional activity. To explore this further, we performed RNA sequencing for stimulated Jurkat MALT1 cells and the mutant RACA, JΔM-CA and JΔM-RA cells. Compared to the Jurkat MALT1 cells, the three mutant cell lines showed between 79 and 278 differentially expressed genes (DEGs) with a greater than two-fold change and a false discovery rate (FDR) q<0.001 after 3 or 18 hrs of stimulation with P/I (Table S2). qRT-PCR analysis performed for a selection of top ranked genes confirmed the effects observed by RNA sequencing (Table S3). Gene Set Enrichment Analysis (GSEA) indicated a significant enrichment of NF-κB target genes (http://www.bu.edu/nf-kb/gene-resources/target-genes) in the down-regulated genes of the datasets for RACA, JΔM-CA and JΔM-RA cells at 3 and 18 hrs of stimulation (FDR<0.001) (Figure 7D and Table S4). Ingenuity Pathway Analysis (Ingenuity Systems) linked the signatures of the RACA, JΔM-CA and JΔM-RA cells with reduced activation/proliferation of lymphocytes and inhibition of NF-κB signalling (Table S5). Collectively, these data demonstrate that MALT1 auto-processing is required to induce optimal transcription of NF-κB target genes in activated Jurkat T cells.


MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

Baens M, Bonsignore L, Somers R, Vanderheydt C, Weeks SD, Gunnarsson J, Nilsson E, Roth RG, Thome M, Marynen P - PLoS ONE (2014)

MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells.A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q<0,001) in the pre-ranked down-regulated genes from JΔM-CA, JΔM-RA and RACA cells stimulated for 18 hrs with P hrs with P/I. Gene list depicted at the right side are NF-κB target genes down-regulated in JΔM-CA, JΔM-RA and RACA cells after 3 and 18 hrs stimulation with P hrs stimulation with P/I.
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Related In: Results  -  Collection

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Show All Figures
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pone-0103774-g007: MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells.A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q<0,001) in the pre-ranked down-regulated genes from JΔM-CA, JΔM-RA and RACA cells stimulated for 18 hrs with P hrs with P/I. Gene list depicted at the right side are NF-κB target genes down-regulated in JΔM-CA, JΔM-RA and RACA cells after 3 and 18 hrs stimulation with P hrs stimulation with P/I.
Mentions: So far, our data suggested that MALT1 auto-proteolysis affects neither IKK activation nor the nuclear translocation of NF-κB, despite a profound defect on the expression of the NF-κB target gene IL-2. Bi-allelic inactivation of endogenous MALT1 in R149A cells (JΔM-RA) further reduced IL-2 as well as CSF2 secretion to the basal levels observed in Jurkat T cells with bi-allelic MALT1 inactivation (JΔM) (Figure 7A). To assess whether these defects in IL-2 and CSF2 secretion were due to defects in transcription, we performed qRT-PCR analysis. A stimulus-dependent up-regulation of IL-2 and CSF2 mRNA levels was observed in the MALT1 expressing Jurkat T cells, while all Jurkat mutants were seriously hampered in their mRNA up-regulation (Figure 7B). Next we performed luciferase reporter assays with the different Jurkat clones. Cells expressing wild-type MALT1 showed a stimulus-dependent increase of gene reporter constructs containing the IL-2 promotor (IL-2p-Luc) or three NF-κB sites from the promoter of the kappa light chain of immunoglobulin (Igκ3-ConALuc), while this response was strongly impaired in JΔM-CA or JΔM-RA cells (Figure 7C). Together, these findings clearly support a role for MALT1 auto-proteolysis in regulating NF-κB transcriptional activity. To explore this further, we performed RNA sequencing for stimulated Jurkat MALT1 cells and the mutant RACA, JΔM-CA and JΔM-RA cells. Compared to the Jurkat MALT1 cells, the three mutant cell lines showed between 79 and 278 differentially expressed genes (DEGs) with a greater than two-fold change and a false discovery rate (FDR) q<0.001 after 3 or 18 hrs of stimulation with P/I (Table S2). qRT-PCR analysis performed for a selection of top ranked genes confirmed the effects observed by RNA sequencing (Table S3). Gene Set Enrichment Analysis (GSEA) indicated a significant enrichment of NF-κB target genes (http://www.bu.edu/nf-kb/gene-resources/target-genes) in the down-regulated genes of the datasets for RACA, JΔM-CA and JΔM-RA cells at 3 and 18 hrs of stimulation (FDR<0.001) (Figure 7D and Table S4). Ingenuity Pathway Analysis (Ingenuity Systems) linked the signatures of the RACA, JΔM-CA and JΔM-RA cells with reduced activation/proliferation of lymphocytes and inhibition of NF-κB signalling (Table S5). Collectively, these data demonstrate that MALT1 auto-processing is required to induce optimal transcription of NF-κB target genes in activated Jurkat T cells.

Bottom Line: MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity.Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits.Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium; Human Genome Laboratory, Center for Human Genetics, KU Leuven, Leuven, Belgium.

ABSTRACT
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

Show MeSH
Related in: MedlinePlus