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Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor

Nature Chemical Biology volume 13, pages 10451052 (2017) | Download Citation

Abstract

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor—TrxRFP1—to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

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Acknowledgements

We acknowledge funding from the National Institutes of Health (R01GM118675 and R21EB021651) and the National Science Foundation (CHE-1351933).

Author information

Affiliations

  1. The Environmental Toxicology Graduate Program, University of California Riverside, Riverside, California, USA.

    • Yichong Fan
    •  & Hui-wang Ai
  2. Department of Chemistry, University of California Riverside, Riverside, California, USA.

    • Merna Makar
    • , Michael X Wang
    •  & Hui-wang Ai

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Contributions

H.A. conceived and supervised the project. Y.F. performed all experiments, except for that M.M. assisted Y.F. with cell culture and transfection and M.X.W. assisted Y.F. with plasmid preparation and SDS–PAGE. H.A. and Y.F. analyzed the data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hui-wang Ai.

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DOI

https://doi.org/10.1038/nchembio.2417

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