Effects of Redox State on the Efficient Uptake of Cell Permeable Peptide in Mammalian Cells

David Timothy,Elisabeth Christians,Shayne Squires,Ivor Benjamin,Michael Riedel,Christopher K Rodesch,James Marvin

doi:10.2174/1874091x20130531001

Abstract

We investigated whether a cell-penetrating peptide linked via a disulfide bond to a fluorophore-labeled cargo peptide can be used to interrogate changes in cellular redox state. A fluorescence resonance energy transfer (FRET) pair was constructed so that the cargo peptide was labeled with fluorescein amidite (FAM) and the cell-penetrating peptide was attached to a quencher. Incubation of cells in culture with the FRET construct was visualized using live-cell, time-lapse imaging, which demonstrated earlier cellular uptake of the construct when cells were treated with the reducing agent n-acetylcysteine (NAC). The FRET peptide construct was easily detected in cells cultured in 96-well plates using a plate-reader. Treatment of cells with various classes of reducing or oxidizing agents resulted in an increase or decrease in FAM fluorescence, respectively. Changes in FAM fluorescence correlated significantly with redox-sensitive green fluorescent protein ratios in cells treated with hydrogen peroxide but not NAC. Detection of relative changes in cellular redox state was enhanced by the fact that uptake of the cell-penetrating peptide occurred more quickly in relatively reduced compared with oxidized cells. We conclude that cell-penetrating peptides coupled via disulfide bonds to detectable cargo is a novel and specific approach for assessment of relative changes in cellular thiol redox state.

Highlights

Cell penetrating peptides (CPP) exhibit unique properties for translocation across cellular membranes and nonendocytic uptake into mammalian cells, boosting the popularity of their possible applications in diagnostics and therapeutics [1]
Because the emission signal of fluorescein amidite (FAM) is quenched by nearby to 5(6) carboxytetramethylrhodamine (TAMRA), reduction of the disulfide bond joining the two moieties of reductide triggers separation and achieves readable FAM fluorescence
The presence of added GSSG in the glutathione pool resulted in slower development of reductide fluorescence and a decrease in maximum fluorescence achieved by 20 minutes (Fig. 1B), suggesting that the rate of peptide reduction depends on the GSSG reduction potential as calculated using the Nernst equation

Summary

Introduction

Cell penetrating peptides (CPP) exhibit unique properties for translocation across cellular membranes and nonendocytic uptake into mammalian cells, boosting the popularity of their possible applications in diagnostics and therapeutics [1]. In the course of studying its interaction with the plasma membrane, investigators unexpectedly discovered the cell-penetrating property of MAP and paved the way for research into the mechanism(s) that govern peptide translocation into mammalian cells [4]. The mechanisms of cell peptide internalization and localization remain under active investigation. A cellular penetration mechanism was originally inferred to be nonendocytic based upon observed uptake at 0°C and following energy depletion [4]. Peptide uptake was decreased but not abolished after treatment of the cells with 2-deoxyglucose, motivating the inference that uptake is mediated by both energy-dependent and -independent mechanisms. The subcellular distribution of MAP has been reported to include both cytosolic and nuclear compartments [7]

Methods

Results

Conclusion