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A GFP-based ratiometric sensor for cellular methionine oxidation.

Nikita Kuldyushev1, Roland Schönherr1, Ina Coburger1

  • 1Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, 07745, Jena, Germany.

Talanta
|March 11, 2022
PubMed
Summary
This summary is machine-generated.

Researchers developed a genetically encoded probe for methionine oxidation (GEPMO) to detect this modification in living cells. This breakthrough allows real-time monitoring of methionine oxidation, crucial for understanding aging and disease.

Keywords:
Fluorescence sensorGEPMOGFPMethionine oxidationMethionine sulfoxideOxidative stress

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Methionine oxidation is a reversible post-translational modification impacting protein function.
  • This modification is linked to aging and various degenerative diseases.
  • Detecting methionine oxidation in living systems has been a significant challenge.

Purpose of the Study:

  • To develop a novel, genetically encoded probe for detecting methionine oxidation in living cells.
  • To create a versatile and integrating sensor for real-time monitoring of methionine oxidation.
  • To enable subcellular resolution of methionine oxidation.

Main Methods:

  • Development of a genetically encoded probe for methionine oxidation (GEPMO) using super-folder green fluorescent protein (sfGFP).
  • sfGFP variant engineered with methionine at position 147, exhibiting ratiometric fluorescence change upon oxidation.
  • Expression of GEPMO in mammalian cells and S. cerevisiae for live-cell imaging and FACS analysis.
  • Creation of subcellularly targeted variants (membrane, mitochondria).

Main Results:

  • GEPMO successfully detects and reports methionine oxidation in living mammalian cells and S. cerevisiae.
  • The probe exhibits ratiometric fluorescence changes upon methionine oxidation, detectable with standard laser wavelengths.
  • Subcellular variants provide localized detection of methionine oxidation, including in mitochondria.
  • Sensor expression is homogeneous and suitable for live-cell imaging and FACS.

Conclusions:

  • GEPMO is a specific, versatile, and integrating sensor for methionine oxidation in living cells.
  • This tool overcomes previous limitations in detecting methionine oxidation in vivo.
  • GEPMO enables new research avenues into the roles of methionine oxidation in biological processes and diseases.
  • Subcellular targeting expands the utility of GEPMO for localized oxidation studies.