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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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MALT1 undergoes auto-proteolysis in vitro.A) Features of F-STII-MALT1 and LZ-MALT1. F: Flag epitope, STII: StrepII-tag, 6H: 6-Histidine tag, LZ: Leuzine zipper. B) Top: In vitro cleavage of the fluorogenic tetratpeptide substrate Ac-LVSR-AMC (50 µM) by F-STII-MALT1 in increasing concentrations of the cosmotropic salt NH4-citrate (0.2, 0.4, 0.6, and 0.8 M M), by F-STII-MALT1 in 0.8M NH4-citrate buffer in the presence of the MALT1 protease inhibitors z-VRPR-fmk (10 µM) and z-LVSR-fmk (10 µM) and by LZ-MALT1 or LZ-MALT1-C464A in 0.8 M NH M NH4-citrate buffer. The barchart shows cleavage activity as Fluorescence Units (FU) increase/min. Results are expressed as means ± SD (n = 3). Bottom: enzymatic reactions were analysed by immunoblotting with a-MALT1-C, a-p76 neo-epitope and a-MALT1-N.
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pone-0103774-g003: MALT1 undergoes auto-proteolysis in vitro.A) Features of F-STII-MALT1 and LZ-MALT1. F: Flag epitope, STII: StrepII-tag, 6H: 6-Histidine tag, LZ: Leuzine zipper. B) Top: In vitro cleavage of the fluorogenic tetratpeptide substrate Ac-LVSR-AMC (50 µM) by F-STII-MALT1 in increasing concentrations of the cosmotropic salt NH4-citrate (0.2, 0.4, 0.6, and 0.8 M M), by F-STII-MALT1 in 0.8M NH4-citrate buffer in the presence of the MALT1 protease inhibitors z-VRPR-fmk (10 µM) and z-LVSR-fmk (10 µM) and by LZ-MALT1 or LZ-MALT1-C464A in 0.8 M NH M NH4-citrate buffer. The barchart shows cleavage activity as Fluorescence Units (FU) increase/min. Results are expressed as means ± SD (n = 3). Bottom: enzymatic reactions were analysed by immunoblotting with a-MALT1-C, a-p76 neo-epitope and a-MALT1-N.

Mentions: Thus far MALT1 cleavage was observed in cellular assays, which do not discriminate between a direct, auto-proteolytic event and an indirect cleavage event, which could be mediated by a protease that is activated by MALT1-mediated processing. To test the possibility of MALT1 auto-proteolysis we therefore performed in vitro cleavage assays. The recombinant MALT1 we used represents the full length MALT1 with the Flag-epitope and the StrepII-tag at its N terminus (F-STII-MALT1, Figure 3A). This construct was purified from stably transduced HKB11 cells via Strep-Tactin affinity chromatography. The in vitro proteolytic activity of F-STII–MALT1 was assessed by incubation with the fluorogenic tetrapeptide substrate Ac-LVSR-AMC in paracaspase assay buffer [33]. Increasing concentrations of the cosmotropic salt NH4-citrate gradually increased MALT1 cleavage activity and release of free AMC, which could be completely blocked by the MALT1 tetrapeptide inhibitors z-VRPR-fmk and z-LVSR-fmk (Figure 3B, top). Immunoblots of the in vitro reactions further demonstrated generation of the p76 and p19 cleavage fragments of MALT1 with an efficiency that mimicked the pattern of MALT1 protease activity observed in the LVSR-AMC protease assay (Figure 3B and S3, bottom).


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 undergoes auto-proteolysis in vitro.A) Features of F-STII-MALT1 and LZ-MALT1. F: Flag epitope, STII: StrepII-tag, 6H: 6-Histidine tag, LZ: Leuzine zipper. B) Top: In vitro cleavage of the fluorogenic tetratpeptide substrate Ac-LVSR-AMC (50 µM) by F-STII-MALT1 in increasing concentrations of the cosmotropic salt NH4-citrate (0.2, 0.4, 0.6, and 0.8 M M), by F-STII-MALT1 in 0.8M NH4-citrate buffer in the presence of the MALT1 protease inhibitors z-VRPR-fmk (10 µM) and z-LVSR-fmk (10 µM) and by LZ-MALT1 or LZ-MALT1-C464A in 0.8 M NH M NH4-citrate buffer. The barchart shows cleavage activity as Fluorescence Units (FU) increase/min. Results are expressed as means ± SD (n = 3). Bottom: enzymatic reactions were analysed by immunoblotting with a-MALT1-C, a-p76 neo-epitope and a-MALT1-N.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126661&req=5

pone-0103774-g003: MALT1 undergoes auto-proteolysis in vitro.A) Features of F-STII-MALT1 and LZ-MALT1. F: Flag epitope, STII: StrepII-tag, 6H: 6-Histidine tag, LZ: Leuzine zipper. B) Top: In vitro cleavage of the fluorogenic tetratpeptide substrate Ac-LVSR-AMC (50 µM) by F-STII-MALT1 in increasing concentrations of the cosmotropic salt NH4-citrate (0.2, 0.4, 0.6, and 0.8 M M), by F-STII-MALT1 in 0.8M NH4-citrate buffer in the presence of the MALT1 protease inhibitors z-VRPR-fmk (10 µM) and z-LVSR-fmk (10 µM) and by LZ-MALT1 or LZ-MALT1-C464A in 0.8 M NH M NH4-citrate buffer. The barchart shows cleavage activity as Fluorescence Units (FU) increase/min. Results are expressed as means ± SD (n = 3). Bottom: enzymatic reactions were analysed by immunoblotting with a-MALT1-C, a-p76 neo-epitope and a-MALT1-N.
Mentions: Thus far MALT1 cleavage was observed in cellular assays, which do not discriminate between a direct, auto-proteolytic event and an indirect cleavage event, which could be mediated by a protease that is activated by MALT1-mediated processing. To test the possibility of MALT1 auto-proteolysis we therefore performed in vitro cleavage assays. The recombinant MALT1 we used represents the full length MALT1 with the Flag-epitope and the StrepII-tag at its N terminus (F-STII-MALT1, Figure 3A). This construct was purified from stably transduced HKB11 cells via Strep-Tactin affinity chromatography. The in vitro proteolytic activity of F-STII–MALT1 was assessed by incubation with the fluorogenic tetrapeptide substrate Ac-LVSR-AMC in paracaspase assay buffer [33]. Increasing concentrations of the cosmotropic salt NH4-citrate gradually increased MALT1 cleavage activity and release of free AMC, which could be completely blocked by the MALT1 tetrapeptide inhibitors z-VRPR-fmk and z-LVSR-fmk (Figure 3B, top). Immunoblots of the in vitro reactions further demonstrated generation of the p76 and p19 cleavage fragments of MALT1 with an efficiency that mimicked the pattern of MALT1 protease activity observed in the LVSR-AMC protease assay (Figure 3B and S3, bottom).

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