Fig. 2: In silico analysis predicted two putative arginine methylation sites on procaspase-8 that are highly conserved and often mutated in human cancers.

A Analysis of conservation of procaspase-8 R233 and R435 positions. The corresponding procaspase-8 protein regions for the representative higher vertebrate organisms are shown. The species tree and time points of the lineage diversification is presented on the left. The amino acid residues corresponding to human R233 and R435 residues are highlighted. B The prevalence of caspase-8 mutations in cancer was analysed using COSMIC database. All mutations mapped to their position in caspase-8a primary structure are shown in the upper panel. Three most prevalent mutations at R233 and R435 are shown in the various cancers in the lower panel: R233Q missense mutation is shown in black, R233W is shown in dark green and R435* nonsense mutation is shown in yellow. C The location of R233 and R435 in the three-dimensional structure of the C-terminal part of procaspase-8a is shown. The latter is computed using Alphafold [51]. The active site cysteine of caspase-8 is shown in yellow, while R233 and R435 are shown in blue colors. D Putative methylation residues are shown in procaspase-8a primary structure (upper panel). Procaspase-8a-R233H-pcDNA3 (R233H), procaspase-8a-R435Q-pcDNA3 (R435Q) as well as procaspase-8a-WT-pcDNA3 (WT) were transfected into HeLa-CD95-C8-KO cells (low panel). The transfection of pcDNA3 (vector) and non-transfected cells (KO) were used as negative controls. The protein expression was analysed by Western Blot. E HeLa-CD95-C8-KO cells were transfected as in (D). Cytosolic and nuclear fractions were prepared. The analysis of PARP1 and FADD was used as a fractionation control. The fractionation was analysed by Western Blot. One representative fractionation out of three independent experiments is shown.