Abstract
Cell-penetrating peptides (CPPs) are able to be taken up by cells and can deliver macromolecular cargos. However, the mechanism of this internalization is not yet fully understood. Recent theories suggest that the binding of cationic CPPs to negatively charged extracellular glycosaminoglycans, such as heparan sulfate (HS), is a possible mechanism of cellular uptake (CU). Our group has screened the CU activities of 54 systematically designed amphiphilic α-helical peptides in HeLa cells. Notably, a mutation in even a single residue significantly alters the CU ability of a peptide. To determine the structure-CU activity relationship of CPPs, four peptides, which contain a difference in one or two amino acids (i.e., Arg/Glu and Ala/Phe), were chosen from our CPP library to examine their interactions with HS. Fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering analysis indicated that the HS-binding affinities and HS-clustering abilities of the four CPPs correlated well with their CU activities in HeLa and A549 cells. The heat capacities of the CPPs, determined using ITC and binding free energy decomposition analyses in molecular dynamics simulations, revealed that electrostatic interactions were more dominant in the HS-binding processes of Arg-containing peptides in comparison to Glu-containing peptides, whereas hydrophobic contributions were the primary mode of interaction of Phe-containing peptides in comparison to Ala-containing peptides. Furthermore, it was implied that hydrophobic interactions may be more favourable than electrostatic interactions during the CU process.
Highlights
Cell-penetrating peptides (CPPs) are generally short basic peptides (8–30 residues) that have demonstrated effective internalization into diverse cell lines
Because it is expected that the α-helical content of a CPP will be enhanced when the amphiphilic CPP binds to heparan sulfate (HS), the circular dichroism (CD) analysis yielded an identical order of secondary structure variation of the four designed CPPs in the presence of HS (Fig. 2b)
The experimentally determined HS-binding affinities of the CPPs are closely correlated to their cellular uptake (CU) activities in HeLa and A549 cell lines, which are in the order RF ≈ EF > RA > EA
Summary
Cell-penetrating peptides (CPPs) are generally short basic peptides (8–30 residues) that have demonstrated effective internalization into diverse cell lines These peptides are of great interest due to their potential as cell-delivery vectors for macromolecular drugs (e.g. proteins, genes, etc.) that would otherwise not cross the cell membrane. The mechanism of this internalization is not yet fully understood Both an endocytic process[1,2,3,4,5,6,7,8,9,10,11,12] and direct translocation across the plasma membrane[13,14,15,16,17,18,19,20] have been proposed as CPP uptake mechanisms. A combined mechanism of both endocytic process and direct translocation has been recently proposed.[21,22,23,24,25] Recent theories suggest that the
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
