Disruption of SETD3-mediated histidine-73 methylation by the BWCFF-associated β-actin G74S mutation.

Histidine-73 methylation of β-actin by SETD3 modulates ATPase activity, filament assembly, and protein interactions. The pathogenic G74S mutation in cytoskeletal β-actin, associated with Baraitser-Winter cerebrofrontofacial syndrome (BWCFF), alters the adjacent phosphate sensor loop, disrupting SETD3-mediated methylation. Molecular docking indicates that SETD3 undergoes structural rearrangements to accommodate the mutant β-actin, leading to reduced catalytic efficiency. Enzymatic assays confirm slower turnover of mutant actin peptides, while mass spectrometry reveals decreased histidine-73 methylation in both recombinant mutant β-actin and patient-derived fibroblasts. This perturbance of SETD3-mediated methylation likely generates β-actin pools with distinct methylation states, varying across cell types and developmental stages, thereby impairing cytoskeletal dynamics and contributing to BWCFF pathology. Impact statement This study reveals that the BWCFF-linked G74S mutation in β-actin disrupts SETD3-mediated histidine-73 methylation, impairing a critical post-translational modification. It provides the first direct mechanistic link between a cytoskeletal actinopathy and altered methylation, highlighting potential targets for therapeutic intervention in β-actin-related developmental disorders.