Arginine-Rich Cell-Penetrating Peptides Require Nucleolin and Cholesterol-Poor Subdomains for Translocation across Membranes

Abstract

Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesicles (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy-dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nucleolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.

Arginine‐enveloped virus inactivation and potential mechanisms

“…Loosening of lipid packing increases peptide uptake, indicating that arginine deforms the lipid bilayer for translocation . High cholesterol content in lipids decreases membrane fluidity and translocation of arginine . In the presence of the hydrophobic cation pyrenebutyrate, which is suspected to loosen lipid packing and increase membrane fluidity, arginine uptake increased in the lipid bilayer .…”

“…Therefore, UCHA may retain its structure in response to membrane defects, while CHA is denatured. Protein structure can also alter packing density of lipids at the protein–lipid interface . The differences in structure between UCHA and CHA influence the surrounding lipid packing density to affect arginine inactivation.…”

“…134,[138][139][140][141] High cholesterol content in lipids decreases membrane fluidity and translocation of arginine. 105,108,138 In the presence of the hydrophobic cation pyrenebutyrate, which is suspected to loosen lipid packing and increase membrane fluidity, arginine uptake increased in the lipid bilayer. 134,[139][140][141] Virus size can also affect membrane curvature and lipid packing density.…”

“…Protein structure can also alter packing density of lipids at the protein-lipid interface. 108 The differences in structure between UCHA and CHA influence the surrounding lipid packing density to affect arginine inactivation. The fact that arginine peptides and synergistic factors are known to affect lipid bilayers, in combination with viral inactivation data, support Hypothesis 2.…”

“…Considering that the magnitude of viral inactivation is contingent on protein structure, the synergistic effects of arginine may interact with proteins.There are multiple cases where a difference in membrane proteins, such as with UCHA and CHA influenza virus, could influence arginine interactions with the lipid bilayer. For example, protein structure can modify packing density of the surrounding lipids 108. With a looser lipid packing, arginine-lipid interactions would be enhanced to increase inactivation 108.…”