Fig 1: BCL10 mutations are less dependent on upstream CARD11. A, Viability of HBL1 lymphoma cell lines transduced to express shRNA targeting CARD11 with two independent hairpins or nontargeting control. The indicated lines stably expressing WT and mutant BCL10 were transduced with lentiviruses expressing CARD11 shRNA along with YFP. The relative number of YFP+ live cells was plotted by normalizing them to day 4 (the YFP+ peak). ***, P < 0.001; ****, P < 0.0001. B, MALT1 activity using the MALT1 GloSensor reporter cells with CARD11 knockdown. The indicated MALT1 GloSensor cell lines were stably expressing WT and mutant BCL10, and then transduced with lentiviruses expressing nontargeting or two independent CARD11 hairpins coexpressing a YFP reporter. At day 4, cells were harvested for a MALT1 activity assay. Error bars, SEM with four biological replicates. ***, P < 0.001; ****, P < 0.0001; ns, not significant. C, NF-?B activity in lymphoma reporter cells with shCARD11. The HBL1 NF-?B reporter cells were stably expressing WT and mutant BCL10, and then transduced with lentiviruses expressing nontargeting or two independent CARD11 hairpins coexpressing a YFP reporter. NF-?B activity was measured 72 hours posttransduction. Error bars, SEM with four biological replicates. ****, P < 0.0001.
Fig 2: Cryo-EM structure of the BCL10E140X filament. A, Immunoblot analysis of BCL10 interactors performed by coimmunoprecipitating with anti-FLAG antibody in Raji cells overexpressing either FLAG-BCL10WT or FLAG-BCL10mutant protein. Samples were blotted for anti-FLAG, anti-MALT1, and anti-CARD11. Input was loaded with 1% of total cell lysate used for immunoprecipitation (IP). Anti-IgG antibody was used as a negative control for coimmunoprecipitation. B, Domain organization of the MBP-human BCL10 construct mapped with MALT1 previously defined and new binding sites. C, Domain organization of the human MALT1 construct. D, SDS-PAGE of MALT1 (Ig1–Ig2) pulldown by His-tagged BCL10 (165–233; left) and His-tagged BCL10 (116–164; right). *, A contaminant. E, SDS-PAGE of MALT1 (Ig1–Ig2) pulldown by different truncations of His-tagged BCL10. F, Negative stained EM micrographs of purified BCL10 WT filaments alone and with MALT1 (left) in comparison with BCL10E140X filaments alone and with MALT1 filaments (right) resulted in similar filaments. G, Cryo-EM structure of BCL10E140X–MALT1 DD filament at 4.3 Å fitted into the cryo-EM density map (left). The 4.3 Å structure is similar to the previously published BCL10WT CARD-MALT1 DD structure at 4.9 Å (right). However, BCL10E140X–MALT1 DD shows improved density for the MALT1 DD domain. EMDB, Electron Microscopy Data Bank; PDB, Protein Data Bank. H, Cryo-EM structure of BCL10E140X CARD and MALT1 DD (cyan) filament, emphasizing EM density for MALT1 DD. I, Monomeric BCL10E140X CARD–MALT1 DD (cyan) align to published monomeric BCL10WT CARD–MALT1 DD. J, Western blot for gel filtration fractions from HBL1 cells stably expressing FLAG-tagged BCL10 WT, R58Q and E140X. Different fractions were blotted with anti-FLAG and anti-MALT1 antibodies. BCL10E140X formed highly ordered oligomers migrating together with MALT1. K, MALT1 inhibits BCL10 filament formation through the BCL10 C-terminal binding site. Quenching polymerization was measured for purified Alexa488-labeled BCL10 WT, E140X, and R58Q at 3 µmol/L in the presence of increasing amounts of MALT1 (0, 1.5, 3, 6, and 12 µmol/L). The assay was initiated upon the addition of the 3C protease in order to remove the MBP tag from BCL10 WT, E140X, and R58Q for allowing filament polymerization. Quenching was monitored for 2 hours with 30-second intervals using a Neo BioTek plate reader and performed with three biological replicates. Titration of increasing doses of MALT1 suppressed filament polymerization of fluorescently labeled BCL10 WT and R58Q. However, increasing doses of MALT1 had very little effect on E140X filament polymerization.
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