Effect of the Electrostatic Potential On the Internalization Mechanism of Cell Penetrating Peptides

In order to develop future therapeutic applications for cell penetrating peptides (CPPs), it is essential to characterize their internalization mechanisms, as they might affect the stability and the accessibility of the carried drug. In this work we studied the internalization mechanism in HeLa cells of a peptide extracted from the TIRAP protein (pepTIRAP), and pepTIRAP^ALA peptide, where some of the cationic amino acids were mutated by alanine. We analyzed the internalization experimental results and the electrostatic potential obtained for these peptides, to shed light on the internalization mechanism involved. We developed a mathematical model to process the internalization experimental data, to ultimately determine the internalization of the peptide or its nonspecifically association to the plasma membrane. When studying the internalization of pepTIRAP and pepTIRAP^ALA in HeLa cells, the experimental results suggest a stronger association of pepTIRAP to the plasma membrane, which might be related to the first stage of the transduction internalization mechanism. In fact, cell survival decreased when cells are cultured with a pepTIRAP concentration higher than 40 µM, effect which is not observed in the case of pepTIRAP^ALA. The internalization characteristics displayed by these peptides were interpretated using electrostatic potentials calculated from structural models built by comparative modeling. Even when those share similar 3D structures, their electrostatic potentials show noticeable differences. In particular, pepTIRAP structure displays a predominant positive potential over the surface when compared to pepTIRAPALA, which has more evenly distributed potential. We propose that the positive potential of pepTIRAP could interact with molecules on cell membrane such as proteoglycans and phospholipids heads, facilitating the formation of nucleation zones which are key in the transduction internalization mechanism. Finally, viability and internalization mechanism results are to be considered for potential therapeutic applications of these peptides in treating chronic inflammatory diseases.

This work was partially supported by FONDECYT Research Initiation Grants 11080016 and 11090268.