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.
Department of Natural Resources and Environmental Studies, National Dong Hwa University, No. 1, Sec. 2, Da-Hsueh Road, Shoufeng, Hualien 97401, Taiwan.
Ji-Sing
JS
Liou
Betty Revon
BR
Liu
Adam L
AL
Martin
Yue-Wern
YW
Huang
Huey-Jenn
HJ
Chiang
Han-Jung
HJ
Lee
D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism* / Q000473:pathology*; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D057846:Cell-Penetrating Peptides; D006389:Hemagglutinins, Viral; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus