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 (Fe²⁺). 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 Fe²⁺ ions and inhibits hydroxyl radical (HO·) generation via the Fenton reaction between Fe²⁺ and hydrogen peroxide (H₂O₂). Cytological analyses demonstrated that CASc* expression in both E. coli and cultured cells protected them from oxidative stress in the presence of Fe²⁺ and H₂O₂. 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.