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Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors.

Tarrago L, Péterfi Z, Lee BC, Michel T, Gladyshev VN - Nat. Chem. Biol. (2015)

Bottom Line: Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells.In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins.The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.

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Characterization of MetROx in HEK293 cells(a) Time-series of raw fluorescent (F) and pseudocolored (R) ratio images of MetROx- and C129S MetROx-expressing cells subjected to 5 mM MetO. Scale bars represent 20 μm. (b) Kinetics of MetROx fluorescence in cells subjected to MetO (0.1 – 10 mM) detected by single cell live-microscopy. Background was subtracted and fluorescence ratios were normalized by the value at t = 0 s (n = 6–28). (c) Kinetics of fluorescence changes in MetROx-expressing cells treated with 250 μM MetO (↓), followed by washing or 1 mM DTT treatment (↑). (d) MetROx response to H2O2 in HEK293 cells. Time course analysis of HEK293 cells expressing MetROx and subjected to the indicated concentrations of H2O2. The fluorescence ratios were normalized by the value at t = 0 s. Data presented are the means (n = 6–8) ± SD and are representative of 3 replicates.
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Figure 5: Characterization of MetROx in HEK293 cells(a) Time-series of raw fluorescent (F) and pseudocolored (R) ratio images of MetROx- and C129S MetROx-expressing cells subjected to 5 mM MetO. Scale bars represent 20 μm. (b) Kinetics of MetROx fluorescence in cells subjected to MetO (0.1 – 10 mM) detected by single cell live-microscopy. Background was subtracted and fluorescence ratios were normalized by the value at t = 0 s (n = 6–28). (c) Kinetics of fluorescence changes in MetROx-expressing cells treated with 250 μM MetO (↓), followed by washing or 1 mM DTT treatment (↑). (d) MetROx response to H2O2 in HEK293 cells. Time course analysis of HEK293 cells expressing MetROx and subjected to the indicated concentrations of H2O2. The fluorescence ratios were normalized by the value at t = 0 s. Data presented are the means (n = 6–8) ± SD and are representative of 3 replicates.

Mentions: To characterize the use of the sensors in mammalian cells, we transfected HEK293 cells with MetSOx and MetROx expression vectors and followed the fluorescence changes by single cell fluorescence microscopy. Both sensors were expressed and detected by fluorescence. However, MetSOx showed very low fluorescence intensity and was not further characterized. As in bacteria, the addition of MetO induced a dose-dependent decrease in the fluorescence ratio of MetROx, but not of its inactive form (Fig. 5a, b). Subsequent treatment of cells with DTT or removal of MetO led to the reduction of the sensor showing that MetROx was fully functional in HEK293 cells (Fig. 5c). When MetROx expressing cells were treated with exogenous H2O2, changes in the fluorescence ratio were observed only at very high concentrations of the oxidant (Fig. 5d), arguing against the direct oxidation of the sensor by H2O2.


Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors.

Tarrago L, Péterfi Z, Lee BC, Michel T, Gladyshev VN - Nat. Chem. Biol. (2015)

Characterization of MetROx in HEK293 cells(a) Time-series of raw fluorescent (F) and pseudocolored (R) ratio images of MetROx- and C129S MetROx-expressing cells subjected to 5 mM MetO. Scale bars represent 20 μm. (b) Kinetics of MetROx fluorescence in cells subjected to MetO (0.1 – 10 mM) detected by single cell live-microscopy. Background was subtracted and fluorescence ratios were normalized by the value at t = 0 s (n = 6–28). (c) Kinetics of fluorescence changes in MetROx-expressing cells treated with 250 μM MetO (↓), followed by washing or 1 mM DTT treatment (↑). (d) MetROx response to H2O2 in HEK293 cells. Time course analysis of HEK293 cells expressing MetROx and subjected to the indicated concentrations of H2O2. The fluorescence ratios were normalized by the value at t = 0 s. Data presented are the means (n = 6–8) ± SD and are representative of 3 replicates.
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Figure 5: Characterization of MetROx in HEK293 cells(a) Time-series of raw fluorescent (F) and pseudocolored (R) ratio images of MetROx- and C129S MetROx-expressing cells subjected to 5 mM MetO. Scale bars represent 20 μm. (b) Kinetics of MetROx fluorescence in cells subjected to MetO (0.1 – 10 mM) detected by single cell live-microscopy. Background was subtracted and fluorescence ratios were normalized by the value at t = 0 s (n = 6–28). (c) Kinetics of fluorescence changes in MetROx-expressing cells treated with 250 μM MetO (↓), followed by washing or 1 mM DTT treatment (↑). (d) MetROx response to H2O2 in HEK293 cells. Time course analysis of HEK293 cells expressing MetROx and subjected to the indicated concentrations of H2O2. The fluorescence ratios were normalized by the value at t = 0 s. Data presented are the means (n = 6–8) ± SD and are representative of 3 replicates.
Mentions: To characterize the use of the sensors in mammalian cells, we transfected HEK293 cells with MetSOx and MetROx expression vectors and followed the fluorescence changes by single cell fluorescence microscopy. Both sensors were expressed and detected by fluorescence. However, MetSOx showed very low fluorescence intensity and was not further characterized. As in bacteria, the addition of MetO induced a dose-dependent decrease in the fluorescence ratio of MetROx, but not of its inactive form (Fig. 5a, b). Subsequent treatment of cells with DTT or removal of MetO led to the reduction of the sensor showing that MetROx was fully functional in HEK293 cells (Fig. 5c). When MetROx expressing cells were treated with exogenous H2O2, changes in the fluorescence ratio were observed only at very high concentrations of the oxidant (Fig. 5d), arguing against the direct oxidation of the sensor by H2O2.

Bottom Line: Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells.In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins.The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.

Show MeSH
Related in: MedlinePlus