Overexpression of an engineered artificial nuclease L29E myoglobin in breast cancer cells and its functional consequences.

Triple-negative breast cancer (TNBC) has a poor prognosis due to the lack of effective therapeutic targets, highlighting the urgent need for new intervention strategies. This study focuses on an engineered artificial nuclease, L29E myoglobin, which forms a heterodinuclear Mg2+-H2O-heme center upon Mg2+ binding and exhibits DNA cleavage activity. We overexpressed L29E Mb in the TNBC cell line MDAMB231 and systematically evaluated its effects on cellular physiology. The results showed that, in the presence of Mg2+, the overexpression of L29E Mb significantly induced DNA damage and loss of mitochondrial membrane potential, leading to G1-phase cell cycle arrest and activation of the caspase-9/caspase-3 cascade, and ultimately promoting apoptosis. These findings establish a proof-of-concept that the complex of Mg2+-L29E Mb may function as an artificial nuclease and trigger the mitochondrial apoptotic pathway, which provides a foundation for protein-based anticancer strategies.