title: Efficient delivery of cell impermeable phosphopeptides by a cyclic peptide amphiphile containing tryptophan and arginine.
abstract: Phosphopeptides are valuable reagent probes for studying protein-protein and protein-ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F'-GpYLPQTV, F'-NEpYTARQ, F'-AEEEIYGEFEAKKKK, F'-PEpYLGLD, F'-pYVNVQN-NH2, and F'-GpYEEI (F' = fluorescein), was evaluated in the presence or absence of a [WR]4, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]4 improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F'-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]4 and was found to be time-dependent. Confocal microscopy of a mixture of F'-PEpYLGLD and [WR]4 in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F'-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEpYLGLD and [WR]4. TEM results showed that the mixture of PEpYLGLD and [WR]4 formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]4 and PEpYLGLD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]4 can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States.
authors.forename: Amir
authors.initials: A
authors.lastname: Nasrolahi Shirazi
authors.forename: Rakesh Kumar
authors.initials: RK
authors.lastname: Tiwari
authors.forename: Donghoon
authors.initials: D
authors.lastname: Oh
authors.forename: Antara
authors.initials: A
authors.lastname: Banerjee
authors.forename: Arpita
authors.initials: A
authors.lastname: Yadav
authors.forename: Keykavous
authors.initials: K
authors.lastname: Parang
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001693:Biological Transport, Active; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D004337:Drug Carriers / Q000737:chemistry; D016503:Drug Delivery Systems; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D010456:Peptides, Cyclic / Q000737:chemistry; D010748:Phosphopeptides / Q000008:administration & dosage* / Q000737:chemistry* / Q000493:pharmacokinetics; D054730:Protein Interaction Domains and Motifs; D013501:Surface-Active Agents / Q000737:chemistry; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
chemical_list: D004337:Drug Carriers; D005456:Fluorescent Dyes; D010456:Peptides, Cyclic; D010748:Phosphopeptides; D013501:Surface-Active Agents; D014364:Tryptophan; D001120:Arginine
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