Abstract The TAT protein transduction domain (TAT‐PTD) is an effective tool for delivering therapeutic proteins into cells, yet its efficiency is often constrained by an incompletely understood intracellular fate. In this study, we identify a previously unrecognized proteolytic cascade that restricts the nuclear accumulation of TAT‐fusion proteins. After cellular uptake, TAT‐EGFP undergoes N‐terminal cleavage by matrix metalloproteinase‐3 (MMP‐3), an event that depends on an upstream calpain–MMP‐3 activation axis. This cleavage removes the intrinsic nuclear localization signal of the TAT‐PTD, trapping the protein in the cytoplasm and thereby abolishing its nuclear function. Importantly, this entire process was blocked by specific inhibitors of calpain or MMP‐3, which restored nuclear accumulation of the intact protein. In addition, site‐directed mutagenesis conferring resistance to cleavage, as observed in the ARA and AAR mutants, demonstrated that the two C‐terminal arginines of the TAT‐PTD are essential for this susceptibility. These findings elucidate, for the first time, a molecular mechanism underlying a key pathway that limits the nuclear delivery of TAT‐based vectors, providing a rational foundation for the design of cleavage‐resistant delivery systems with improved therapeutic efficacy.
KIM et al. (2026) studied this question.