title: Vesicle-to-cytosol transport of disulfide-linked cargo mediated by an amphipathic cell-penetrating peptide.
abstract: The effect of linker stability on the intracellular localization and apoptotic activity of cytochrome c (Cyt c) conjugated to an amphipathic cell-penetrating peptide (CPP), model amphipathic peptide (MAP), was tested in HeLa cells. While conjugates linked with a stable thioether cross-linkage were only found in the vesicular compartment, a portion of the conjugate linked with a reducible disulfide bond was also detected in the cytosol. The apoptotic function of the reducible and non-reducible Cyt c-MAP conjugates was also evaluated quantitatively using the caspase 3 and annexin V/propidium iodide detection assays in the presence of a proteasome inhibitor used to inhibit cytosolic Cyt c degradation. Analysis for early phase apoptosis revealed that linker stability was important for biological activity. Only the reducible disulfide-linked Cyt c-MAP conjugate, and not free Cyt c or thioether-linked Cyt c-MAP, initiated apoptosis in proteasome-inhibited cells which correlated with the cytosolic localization profiles of the proteins. The co-treatment of disulfide-linked Cyt c-MAP with a disulfide reduction inhibitor decreased the amount of Cyt c delivered to the cytosol, which correlated with a lack of apoptotic activity. These findings indicated the presence of a vesicle-to-cytosolic delivery process for disulfide-linked MAP conjugates, which can be used to improve CPP-based drug delivery systems transporting cargo to cytosolic sites.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Randall
authors.initials: R
authors.lastname: Kenien
authors.forename: Wei-Chiang
authors.initials: WC
authors.lastname: Shen
authors.forename: Jennica L
authors.initials: JL
authors.lastname: Zaro
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001692:Biological Transport; D053148:Caspase 3 / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004337:Drug Carriers / Q000737:chemistry*; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D011419:Propidium / Q000378:metabolism
chemical_list: D017304:Annexin A5; D057846:Cell-Penetrating Peptides; D003432:Cross-Linking Reagents; D004220:Disulfides; D004337:Drug Carriers; D011419:Propidium; D045304:Cytochromes c; D053148:Caspase 3