Cell Penetrating Activity of Bax Inhibiting Peptides (BIPs) and Its Utilization for Protein Transduction.

The cellular membrane has a strict regulation for molecular trafficking, and there is a strong need for the development of new technology for drug delivery into cells with minimum toxicity. Previously, we developed the cell permeable penta-peptides designed from Bax binding domain of Ku70 that are called “Bax Inhibiting Peptides (BIPs)” (i.e. VPMLK, VPTLK, VPALR). Among BIPs, VPTLK showed the best cell penetrating activity. We also found that one of the scrambled control mutant peptides (i.e. KLPVM) shows a strong cell permeable activity without an activity to bind and inhibit Bax-mediated cell death. We named these cell permeable peptides: BIP-derived cell permeable penta-peptides (BCPs). In this study, we determined the mechanism of membrane penetration by these penta-peptides. Human umbilical endothelial cells (HUVEC), Dami cells (human megakaryocytic cell line), and HeLa cells were used for the examination of cell entry of BCPs. Fluorescent dye (Fluorescein) was conjugated to the N-terminus of BCPs, and the cell entry of BCPs was monitored by FACS and confocal micropscope. BCP entered the cells in a dose dependent manner (10 uM-1.6 mM range was tested), and it did not show any significant toxicity even at 1.6 mM. The entry of BCP into the cells was detected within 15 minutes at 37C, and it reaches maximum approximately at 6 hrs of incubation. We found that BCPs entered the cells even at 0 or 4 C, suggesting that energy-independent mechanism is involved in the cell penetration of BCPs. We also tested whether BCPs has an ability to deliver a cargo protein (protein transduction activity) such as 30 kDa Green Fluorescent Protein across the cellular membrane. We used VPTLK and KLPVM for the examination of GFP delivery from the medium into the cell. Recombinant GFP proteins with and without VPTLK or KLPVM on the C-teminus were generated. The delivery of GFP was detected at a concentration as low as 20 nM within 6 hours of incubation at 37C. GFP without BCP was not imported into the cell, indicating that BCP has a cargo delivery activity (protein transduction). There are several bioactive proteins that can become effective therapeutics if they can be delivered into the cell. To be noted, BCPs are less toxic than the previously examined CPPs that had protein transduction activity, such as TAT peptide designed from the human immunodeficiency virus (HIV). BCPs did not show significant toxicity to bone marrow stem cells and endothelial cells, indicating that BCPs are effective tools for drug delivery in stem cells, and vasculature cells. BCPs may become a useful tool to deliver bioactive molecules into the cells, to protect cells from damage, to regulate cellular activity, and to control differentiation.