The cellular entry mechanism and potential target of the novel bioactive peptide AFYRW derived from Fagopyrum tataricum

The novel bioactive peptide AFYRW derived from Tartary buckwheat (Fagopyrum tataricum Gaertn.) with strong antioxidant capacity was identified in our previous research, but its cellular entry and targeting mechanism remain unknown. Herein, we employed endocytosis pharmacological inhibition methodologies and determined that AFYRW localized primarily in the nucleus, and NH₄Cl, chloroquine (CQ), and nystatin exposure resulted in a significantly reduced uptake by HepG2 cells. Our results indicate that the cellular uptake of AFYRW depends on caveolae-mediated endocytosis that requires a low pH, but does not involve macropinocytosis or clathrin-mediated endocytosis. In addition, biotin-streptavidin affinity purification was used to screen for interacting proteins, and protein spectrum analysis in vitro identified poly (ADP-ribose) polymerase-1 (PARP-1) as a potential target. Subsequent in silico molecular docking supports the presence of a peptide–protein interaction between AFYRW and PARP-1. Our findings suggest that the novel bioactive peptide AFYRW could be a potential inhibitor of PARP-1.