Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Effects of redox state on the efficient uptake of cell permeable Peptide in Mammalian cells. Open Biochem J 2013;7:54-65

Date

08/07/2013

Pubmed ID

23919090

Pubmed Central ID

PMC3731798

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.

Author List

Squires S, Christians E, Riedel M, Timothy D, Rodesch CK, Marvin J, Benjamin I

Author

Ivor J. Benjamin MD Center Director, Professor in the Medicine department at Medical College of Wisconsin




jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a