Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability

Takechi-Haraya, Y.; Ohgita, T.; Usui, A.; Nishitsuji, K.; Uchimura, K.; Abe, Y.; Kawano, R.; Konaklieva, M. I.; Reimund, M.; Remaley, A. T.; Sato, Y.; Izutsu, K.-i.; Saito, H.

doi:10.1101/2023.08.16.553537

bioRxiv · 10.1101/2023.08.16.553537

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability

Takechi-Haraya, Y.·Ohgita, T.·Usui, A.·Nishitsuji, K.·Uchimura, K.·Abe, Y.·Kawano, R.·Konaklieva, M. I.·Reimund, M.·Remaley, A. T.·Sato, Y.·Izutsu, K.-i.·Saito, H.

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Abstract

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide. We designed structurally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose -helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher -helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17 in HeLa cells. Partitioning into lipid membranes was more prominent for StchA2-17 than for A2-17 or StpA2-17; StchA2-17 remained on the cell membrane without cell penetration. Tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

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Takechi-Haraya, Y., Ohgita, T., Usui, A., Nishitsuji, K., Uchimura, K., Abe, Y., Kawano, R., Konaklieva, M. I., Reimund, M., Remaley, A. T., Sato, Y., Izutsu, K.-i., Saito, H.. 2023-08-18. Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability. https://doi.org/10.1101/2023.08.16.553537

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