Mechanism of uptake of C105Y, a novel cell-penetrating peptide.

C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359-374 of alpha1-antitrypsin, enhances gene expression from DNA nanoparticles. To investigate how this enhancement occurs, C105Y was fluorescently labeled to study its uptake and intracellular trafficking. When human hepatoma cells (HuH7) were incubated with fluorescently labeled C105Y for as little as 3 min, C105Y displayed nuclear and cytoplasmic staining with enrichment of fluorescent signal in the nucleus and nucleolus. Uptake and nucleolar localization were observed with the short sequence PFVYLI, but not with SIPPEVKFNK, and the D-isomer was readily taken up into cells but not into the nucleus. We found that the C105Y peptide is routed to the nucleolus very rapidly in an energy-dependent fashion, whereas membrane translocation and nuclear localization are energy-independent. When we tested the involvement of known endocytosis pathways in uptake and trafficking of this peptide, we demonstrated that C105Y peptide is internalized by a clathrin- and caveolin-independent pathway, although lipid raft-mediated endocytosis may play a role in peptide intracellular trafficking. Efficient energy-independent cell entry with rapid nuclear localization probably accounts for enhancement of gene expression from inclusion of C105Y into DNA nanoparticles.