Mechanism of Uptake of C105Y, a Novel Cell-penetrating Peptide

Abstract

C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359 -374 of ␣1-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 SIPPEVK-FNK, 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.

Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

“…This mutation severely reduces EC tropism of this strain, indicating an important sequence motif within the C-terminal 11 amino acids. One distinctive feature is the C-terminal pentapeptide PNLIV, which is conserved between HCMV and CCMV and resembles a previously published cell-penetrating peptide (17). Actually, loss of this pentapeptide severely reduced EC tropism, supporting the idea that this motif is essential.…”

“…The C-terminal amino acids PNLIV of pUL130 bear a striking similarity to cell-penetrating pentapeptides (CPPs) described in the literature (17,32). CPPs are thought to mediate endocytic uptake, which is also the proposed entry mechanism of HCMV in ECs (3,20).…”

“…A frameshift mutation that changes the last 11 amino acids (aa) of pUL130 is the most prominent difference between the poorly endotheliotropic HCMV strain Towne and the highly endotheliotropic strain HCMV-TB40-BAC4 in this region (8,11,27). Rhee and Davis have described a cell-penetrating pentapeptide (CPP) motif (PFVYLI) mediating internaliza-tion by endocytosis, which is clathrin and caveolin independent but may involve lipid rafts (17). Not only do the last five amino acids of pUL130 (PNLIV) bear a striking similarity to this motif, but also the entry of HCMV into ECs has been reported to occur by an endocytic pathway (20,23).…”

“…We thus hypothesized for UL130 that a scrambled mutant (PNLIV changed to PINVL) should still be able to mediate endocytosis of HCMV in ECs. To test these assumptions we generated a series of mutant viruses where the PNLIV motif was either deleted, scrambled (PNLIV changed to PINVL), or exchanged against a known CPP (PFVYLI [17]) and characterized them with regard to EC infectivity.…”

Imaging Presynaptic Exocytosis in Corticostriatal Slices

“…These specialized qualities are bestowed by a positively charged head, which prevents membrane permeability, and a lipophilic tail of varying length that defines the dye's spectral properties and washout kinetics (11). The compounds can also, like horseradish peroxidase, be used to form an electron dense reaction product (12) and a fixable derivative is available (13).…”