Protein Transduction in Human Cells Is Enhanced by Cell-Penetrating Peptides Fused with an Endosomolytic HA2 Sequence


Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs.


Biological Sciences


National Institutes of Health (U.S.)


This work was supported by Award Number R15EB009530 from the National Institutes of Health (Y.-W.H.).

Keywords and Phrases

Hemagglutinin; Hemagglutinin 2; Hybrid Protein; Red Fluorescent Protein; Unclassified Drug; Amino Acid Sequence; Cytoplasm; Flow Cytometry; Human Cell; Imaging; Live Cell Imaging; Protein Analysis; Protein Transduction; Protein Transport; Cell Line, Tumor; Cell-Penetrating Peptides; Endocytosis; Endosomes; Hemagglutinins, Viral; Humans; Luminescent Proteins; Plasmids; Recombinant Fusion Proteins; Escherichia coli; Orthomyxoviridae; Cytotoxicity; Endosomal Escape; Membrane Fusion

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Article - Journal

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© 2012 Elsevier, All rights reserved.

Publication Date

01 Oct 2012

PubMed ID