Membrane Repair Proteins as Negative Regulators of Cytosolic Delivery Using Attenuated Cationic Lytic Peptide L17E and Cell-Penetrating Peptides: Differences and Similarities.

Cytosolic delivery of functional macromolecules is beneficial for intracellular targeting. Although numerous intracellular delivery methods have been developed, the biological factors that govern their efficacy remain poorly understood, thereby limiting further advancement of existing approaches. L17E is an attenuated cationic amphiphilic lytic (ACAL) peptide developed by our research group that facilitates the cytosolic delivery of macromolecules, including antibodies and functional proteins. The L17E peptide exhibits varying levels of cytosolic delivery even within a single cell line, suggesting heterogeneity in the cellular sensitivity to the delivery process. Based on the hypothesis that the specific proteins contribute to this variability, HeLa cells were sorted into L17E-sensitive and L17E-insensitive populations. Comparative proteome analysis of the membrane fractions of these two groups revealed that annexin A2, a membrane repair-related protein, was more abundant in L17E-insensitive cells. Time-lapse imaging and knockdown experiments indicated that annexin A2 negatively regulates L17E-mediated cytosolic delivery by sealing the plasma membrane regions permeabilized by the L17E peptide. To assess whether membrane repair factors also influence cytosolic delivery via conventional cell-penetrating peptides (CPPs), we examined the roles of repair-related proteins. We found that the endosomal sorting complex required for transport (ESCRT) suppressed the cytosolic translocation of the CPPs, whereas annexin A2 did not. These findings suggest that the L17E peptide and the CPPs utilize distinct membrane repair-associated pathways during delivery and that the expression levels of these repair factors affect delivery efficiency. Modulating such factors may therefore provide a strategy for enhancing peptide-based cytosolic delivery systems.