Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors.

Yuta Hatori,Toshio Seyama,Sachiye Inouye,Yuichiro Sato,Takanori Kubo,Reiko Akagi

doi:10.3390/antiox9020129

Yuta Hatori, Toshio Seyama + Show 4 more

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https://doi.org/10.3390/antiox9020129

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Abstract

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

Highlights

Glutathione plays a major role in protecting cells against oxidative insults, such as free radicals, reactive oxygen species (ROS), and hydrogen peroxide [1]
Oxidatively modified protein thiols are reduced by glutaredoxins, which depend upon glutathione [4]
To apply the established sensor for assessing intracellular patterns of glutathione redox status, it was critical that the free cytosolic form of Grx1-roGFP2 was diffused within the cytosol, potentially resulting in the mixing of overall cytosolic information

Summary

Introduction

Glutathione plays a major role in protecting cells against oxidative insults, such as free radicals, reactive oxygen species (ROS), and hydrogen peroxide [1]. Oxidatively modified protein thiols are reduced by glutaredoxins, which depend upon glutathione [4]. The ratio of oxidized and reduced glutathione (GSSG/GSH) serves as a quantitative measure of oxidative insult. Recent evidence suggests that oxidized glutathione reacts with protein thiols to induce oxidative protein modification (i.e., disulfide formation and/or glutathionylation) [5,6,7,8]. Maintaining the redox balance of cellular glutathione is essential for normal homeostasis. Multiple techniques have been exploited to estimate the redox balance of cellular glutathione [9,10,11,12,13,14]

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