A Red Fluorescent Protein-Based Probe for Detection of Intracellular Reactive Sulfane Sulfur.

Luying Xun,Huaiwei Liu,Qingda Wang,Zimai Li,Yongzhen Xia

doi:10.3390/antiox9100985

Luying Xun, Huaiwei Liu + Show 3 more

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https://doi.org/10.3390/antiox9100985

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Abstract

Reactive sulfane sulfur, including persulfide and polysulfide, is a type of regular cellular component, playing an antioxidant role. Its function may be organelle-dependent; however, the shortage of probes for detecting organellar reactive sulfane sulfur has hindered further investigation. Herein, we reported a red fluorescent protein (mCherry)-based probe for specifically detecting intracellular reactive sulfane sulfur. By mutating two amino acid residues of mCherry (A150 and S151) to cysteine residues, we constructed a mCherry mutant, which reacted with reactive sulfane sulfur to form an intramolecular –Sn– bond (n ≥ 3). The bond largely decreased the intensity of 610 nm emission (excitation at 587 nm) and slightly increased the intensity of 466 nm emission (excitation at 406 nm). The 466/610 nm emission ratio was used to indicate the relative abundance of reactive sulfane sulfur. We then expressed this mutant in the cytoplasm and mitochondria of Saccharomyces cerevisiae. The 466/610 nm emission ratio revealed that mitochondria had a higher level of reactive sulfane sulfur than cytoplasm. Thus, the mCherry mutant can be used as a specific probe for detecting reactive sulfane sulfur in vivo.

Highlights

Sulfane sulfur, including persulfide and polysulfide, is commonly present in both eukaryotic and prokaryotic cells [1]
We recently developed a green fluorescent protein (GFP)-based reactive sulfane sulfur detection probe, psGFP1.1, which reacts with hydrogen polysulfide and alters its fluorescence [13]
The designing principle is to mutate two amino acid residues, whose locations are near the chromophore, to cysteine residues

Summary

Introduction

Sulfane sulfur, including persulfide and polysulfide, is commonly present in both eukaryotic and prokaryotic cells [1]. Increasing studies have demonstrated that it has important physiological functions, including antioxidation, anti-inflammation, angiogenesis, and cell signaling [2,3,4,5,6]. Cellular sulfane sulfur exists as hydrogen polysulfide (HSn H, n ≥ 2), organopolysulfide (RSn H, n ≥ 2), and dialkyl polysulfide (RSn R, n ≥ 3). They come in different reactivities with a rough order of HSn H > RSn H > RSn R [7,8]. HSn H and RSn H have both electrophilic and nucleophilic properties, and vividly react with reactive oxygen species (ROS), reactive nitrogen species (RNS), and signaling molecules at physiological pH (7.0~7.6)

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