Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity

Abstract

Previously, we developed cell-penetrating penta-peptides (CPP5s). In the present study, VPTLK and KLPVM, two representative CPP5s, were used to characterize the cell-penetration and protein-transduction activities of these small molecules. Various inhibitors of endocytosis and pinocytosis (chlorpromazine, cytochalasin D, Filipin III, amiloride, methyl-β-cyclodextrin, and nocodazole) were tested. Only cytochalasin D showed suppression of CPP5 entry, though the effect was partial. In addition, CPP5s were able to enter a proteoglycan-deficient CHO cell line. These results suggest that pinocytosis and endocytosis may play only a minor role in the cell entry of CPP5s. By mass spectrometry, we determined that the intracellular concentration of VPTLK ranged from 20 nM to 6.0 μM when the cells were cultured in medium containing 1 μM – 1.6 mM VPTLK. To determine the protein-transduction activity of CPP5s, the Tex-LoxP EG cell line, which has a Cre-inducible green fluorescent protein (GFP) gene, was used. VPTLK and KLPVM were added to the N-terminus of Cre, and these fusion proteins were added to the culture medium of Tex-LoxP EG cells. Both VPTLK-Cre and KLPVM-Cre were able to turn on GFP expression in these cells, suggesting that CPP5s have protein-transduction activity. Since CPP5s have very low cytotoxic activity, even at a concentration of 1.6 mM in the medium, CPP5s could be utilized as a new tool for drug delivery into cells.

Chemical-Functional Diversity in Cell-Penetrating Peptides

“…We evaluated the peptides composing these lowest values and concluded that they can often be explained by an incorrect descriptive conclusion of the authors. Possible reasons are that the classification was based on experiments without trypsinization, while also experiments with trypsinization of the cells were performed, or that much higher incubation concentrations than normally applied are used in order to reach cell-penetration, leading to low CP-responses as they are concentration corrected [17], [51]. Nevertheless, this observed consistency strengthens the value of our CP-response.…”

Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities

“…To investigate the role of the last four amino acids in the Hsp20 (IAVK) and the Hsp27 (LTVK) peptides, which we reasoned are necessary for cellular entry based on other cell permeable peptides [35], we scrambled the amino acid sequence of the four amino acids and tested their anti-apoptotic activity. The end-scrambled peptides did not enter the cells (Figures 8A and 8B) and failed to show protective effects against STS-induced apoptosis (Figure 8C).…”

“…The sequences of the two peptides are similar to that of the other cell permeable peptides [35]. Whether the cellular entry of the Hsp20 peptide was mediated through a transporter is unknown; however, it is likely as a recent report [29] showed that the αB-crystallin peptide is transported across the plasma membrane through an amino-acid transporter.…”

Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria

“…Cells were treated with the following endocytic inhibitors: nocodazole (2 μ m ), chlorporomazine hydrochloride (28 μ m ), methyl‐β‐cyclodextrin (MBC, 20 μ m ), cytochalasin D (10 μ m ) and low temperature (4 °C). The concentrations of the inhibitors were decided on the basis of a previously conducted study . The cells were preincubated with inhibitors for 30 min.…”