Squires Shayne, Christians Elisabeth, Riedel Michael, Timothy David, Rodesch Christopher K, Marvin James, Benjamin Ivor
Division of Cardiology, University of Utah School of Medicine, Salt Lake City, Utah, USA.
Open Biochem J. 2013 Jul 12;7:54-65. doi: 10.2174/1874091X20130531001. Print 2013.
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.
我们研究了一种通过二硫键与荧光团标记的货物肽相连的细胞穿透肽是否可用于探究细胞氧化还原状态的变化。构建了一个荧光共振能量转移（FRET）对，使货物肽用荧光素亚磷酰胺（FAM）标记，细胞穿透肽连接到淬灭剂上。使用活细胞延时成像观察培养细胞与FRET构建体的孵育情况，结果表明，当用还原剂N-乙酰半胱氨酸（NAC）处理细胞时，构建体的细胞摄取更早。使用酶标仪很容易在96孔板中培养的细胞中检测到FRET肽构建体。用各类还原剂或氧化剂处理细胞分别导致FAM荧光增加或减少。在用过氧化氢而非NAC处理的细胞中，FAM荧光的变化与氧化还原敏感的绿色荧光蛋白比率显著相关。与氧化细胞相比，细胞穿透肽在相对还原的细胞中摄取更快，这一事实增强了对细胞氧化还原状态相对变化的检测。我们得出结论，通过二硫键与可检测货物偶联的细胞穿透肽是评估细胞硫醇氧化还原状态相对变化的一种新颖且特异的方法。
