Dual‐Proximity Labeling Strategy Identifies TrxR1 as a Cuproprotein that Regulates Apoptosis via Caspase‐3 S‐Nitrosylation

Abstract Copper (Cu) performs essential structural and functional roles in all aerobic organisms, but identifying natural cuproproteins through traditional affinity methods is hindered by the variety of copper ligand combinations and binding locations in proteins. Here, using ATOX1 as a bait, fused with the APEX2 or TurboID proximity labeling enzymes, with quantitative proteomics in cancer and normal cells (H1299 and HEK293T), we predicted 682 highly enriched candidate Cu‐binding proteins. Among them, we functionally validated the novel cuproprotein, thioredoxin reductase 1 (TrxR1). Through cryo‐electron microscopy (cryo‐EM), we resolved three copper‐binding sites across its N‐ and C‐terminal redox centers. Functional assays demonstrated that copper inhibits TrxR1 reductase activity and impairs the denitrosylation function of its substrate, thioredoxin 1 (Trx1), consequently inhibiting caspase‐3 cleavage and apoptosis. Beyond expanding the scope of annotated cuproproteins, this study provides a versatile strategy for cuproprotein discovery and defines the copper functions in modulating apoptosis through the TrxR1/Trx1 regulatory module.