Calpain– <scp>MMP</scp> 3 activation restricts nuclear uptake during protein transduction domain <scp>TAT</scp> ‐mediated protein delivery

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.