c‐ FLIP : A pseudoprotease with emerging metal‐binding activity

The transition from catalytically active enzymes to inactive pseudoenzymes has been recognized as an evolutionary strategy through which certain proteins acquire novel cellular functions. Cellular FLICE‐like inhibitory protein (c‐FLIP; gene symbol CFLAR ) is one such pseudoenzyme, which exemplifies how evolutionary divergence yields unexpected functional outcomes. During evolution, c‐FLIP diverged from its ancestral caspase‐8 (Casp8) via amino acid substitutions at the active site of its protease domain, resulting in enzymatic inactivity and anti‐apoptotic function. Intriguingly, our structural and phylogenetic analyses revealed an evolutionarily conserved LYR triad within the pseudoprotease domain (CASc*) of c‐FLIP, suggesting a potential interaction with ferrous ions (Fe 2+ ). We investigated whether c‐FLIP could have evolved beyond its canonical apoptotic role to acquire a novel iron‐binding function. Biochemical analyses confirmed that CASc* binds to Fe 2+ ions and inhibits hydroxyl radical (HO·) generation via the Fenton reaction between Fe 2+ and hydrogen peroxide (H 2 O 2 ). Cytological analyses demonstrated that CASc* expression in both E. coli and cultured cells protected them from oxidative stress in the presence of Fe 2+ and H 2 O 2 . Furthermore, we identified a secondary promoter within intron 5 of the CFLAR gene that selectively drives expression of the CASc‐only isoform in certain fetal‐derived cell lines (e.g., HUVECs). These results reveal a unique adaptation among caspase‐family proteins, highlighting c‐FLIP's dual role in iron‐dependent redox regulation and apoptosis signaling. This study provides new insights into how the pseudoenzyme acquires a new function during evolution and into the defensive role of the c‐FLIP isoform in iron‐dependent oxidative stress.