Figure 4. MALT1 E549 is required for MALT1 monoubiquitination.

(A) Strep-tagged MALT1 wildtype or its E549A mutant were co-expressed with VSV-tagged BCL10 in HEK293T cells, and monoubiquitination of immunoprecipitated MALT1 was assessed by immunoblot. The asterisk (*) indicates monoubiquitinated MALT1. (B) Strep-tagged MALT1 wildtype or its E549A mutant were expressed in HEK293T cells, treated overnight with (+) or without (−) 100 µM of the MALT1 inhibitor z-VRPR-fmk, and MALT1 monoubiquitination was assessed by immunoblot. (C, D) The protease activity of the indicated recombinant purified proteins was determined using the fluorogenic MALT1 substrate LVSR-amc in vitro in PBS (C) or in the presence of 0.5 M ammonium citrate (D). Statistical significance is indicated by stars. *≤0.05, **≤0.01, ***≤0.005 (two-tailed Student’s t-test). (E) Indicated Strep-tagged MALT1 wildtype or E549A constructs were co-expressed with the reporter construct eYFP-LVSR-eCFP in HEK293T cells, and reporter cleavage was assessed by anti-GFP immunoblot (which detects both eCFP and eYFP). (F) Recombinant wildtype or E549A-mutant MALT1-Ub fusion proteins corresponding to the protease domain and C-terminal extension (aa 333–824, MALT1(ΔNT)-Ub) were analyzed by size exclusion chromatography in presence of PBS and 10% glycerol, and presented as the absorbance at 280 nm (A₂₈₀) in milliarbitrary units’ (mAU). Downward arrowheads indicate the positions of the molecular weight standards ovalbumin (43 kDa) and bovine serum albumin (67 kDa). Data are representative of two (A, C, D and F) or three (B and E) independent experiments.