FIGURE 1.
Two residues, Asp-27 and Arg-45, embedded in opposing differently charged surface patches, are essential for procaspase-1-mediated NF-κB activation. A, illustration of the electrostatic surface of the procaspase-1 CARD. The residues targeted for mutation are highlighted: Asp-27 in the acidic patch and Arg-45 in the basic patch. B, sequence alignment of the procaspase-1 CARD with INCA and ICEBERG, two procaspase-1-related CARD-only proteins that do not activate NF-κB. The residues targeted for mutation (bold) were changed into the corresponding residues in INCA or ICEBERG (red). C, HEK293T cells transiently transfected, as indicated, with 50 ng of plasmids encoding CARD mutants of FLAG-procaspase-1 C285A (catalytically inactive mutant) or FLAG-caspase-1 CARD-only, and the NF-κB dependent luciferase reporter plasmid. Cells were lysed 24 h after transfection, and NF-κB activity was measured. Equal aliquots of the lysates were analyzed by Western blotting to confirm efficient protein expression from the transfected plasmids with α-FLAG and α-actin antibodies as indicated. Data are representative of at least four independent experiments.