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Efficient Delivery of Cell Impermeable Phosphopeptides by a Cyclic Peptide Amphiphile Containing Tryptophan and Arginine

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journal contribution
posted on 06.05.2013, 00:00 authored by Amir Nasrolahi Shirazi, Rakesh Kumar Tiwari, Donghoon Oh, Antara Banerjee, Arpita Yadav, Keykavous Parang
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.

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