MAP-mediated nuclear delivery of a cargo protein.
Radiolabeled cytochrome c (Cyt c), either as a free protein or as cell penetrating peptide (CPP)-conjugates, was tested for cellular uptake and nuclear transport in Human embryonic kidney 293 (HEK293) cells and HeLa cells. Conjugation of Cyt c with either the amphipathic peptide model amphipathic peptide (MAP) or the cationic peptide oligoarginine via a disulfide linkage significantly increased the total internalization and nuclear localization of Cyt c in both cell lines, though to a greater extent following conjugation with MAP. The nuclear localization was also evaluated qualitatively by confocal laser scanning microscopy (CLSM). CLSM images depicted high amounts of colocalization of fluorescently labeled Cyt c-MAP and the nucleus in HEK293 cells. In addition, prevention of disulfide reduction at the cell surface, or comparison of reducible disulfide or non-reducible thioether MAP-conjugates, showed that maintenance of an intact conjugate using a stable linkage enhanced MAP-mediated nuclear delivery. Furthermore, nuclear transport of the MAP-cargo conjugate appears to be dependent on vesicle fusion events following internalization via endocytosis. The findings presented in this report demonstrate the MAP-mediated transport of a small protein such as Cyt c into the nuclear compartment, which can be used to improve current CPP-cargo delivery of macromolecules with nuclear biological functions.
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.
Randall
R
Kenien
Jennica L
JL
Zaro
Wei-Chiang
WC
Shen
D001120:Arginine / Q000737:chemistry; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D004337:Drug Carriers / Q000737:chemistry*; D004705:Endocytosis; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000737:chemistry*
D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D009842:Oligopeptides; D045304:Cytochromes c; D001120:Arginine