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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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Response of recombinant MetSOx and MetROx to DTT and various oxidantsKinetics of reactivity of MetSOx (a) and MetROx (b) with MetO-containing substrates and DTT. Reduced sensors (1 μM) were incubated with free L-Met-R,S-O (100 μM, or 1,000 μM for MetSOx and MetROx, respectively), oxidized β-casein (10 μM), N-acetyl-L-Met-R,S-O (10 μM), PBS or DTT (1 mM), and the reduced inactive mutants (C25S MetSOx or C129S MetROx) were incubated with N-acetyl-L-Met-R,S-O (10 μM). F505 nm/F425 nm and F500 nm/F410 nm ratios (R) were normalized to the value at t = 0 (R0). Response of recombinant MetSOx (c) and MetROx (d) to DTT and various oxidants. Sensors (1 μM), partially reduced, were incubated with DTT (1 mM), oxidized β-casein (10 μM), NaOCl (10 μM), oxidized glutathione (GSSG, 100 μM) and H2O2 (100 μM). Variations in the ratio F505 nm/F425 nm or F500 nm/F410 nm (R) were normalized to the value at t = 0 (R0). In panels a, b, c and d, arrows indicate the addition of reagent. The data presented are representative of 3 replicates. (e) Comparison of MetSOx and MetROx reactivity towards MICAL1-oxidized actin. Sensors (0.12 μM) were incubated without actin (“No actin”), with 1.4 μM non-treated actin (“+ actin”) or with 1.4 μM MICAL1-oxidized actin (“+ oxidized actin”) for 15 min. Fluorescence excitation ratios (F505 nm/F425 nm for MetSOx and F500 nm/F410 nm for MetROx) were determined from spectra recorded at 530 nm emission wavelength. The data presented are the means (n = 3) ± SD. The data presented are representative of 3 replicates.
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Figure 2: Response of recombinant MetSOx and MetROx to DTT and various oxidantsKinetics of reactivity of MetSOx (a) and MetROx (b) with MetO-containing substrates and DTT. Reduced sensors (1 μM) were incubated with free L-Met-R,S-O (100 μM, or 1,000 μM for MetSOx and MetROx, respectively), oxidized β-casein (10 μM), N-acetyl-L-Met-R,S-O (10 μM), PBS or DTT (1 mM), and the reduced inactive mutants (C25S MetSOx or C129S MetROx) were incubated with N-acetyl-L-Met-R,S-O (10 μM). F505 nm/F425 nm and F500 nm/F410 nm ratios (R) were normalized to the value at t = 0 (R0). Response of recombinant MetSOx (c) and MetROx (d) to DTT and various oxidants. Sensors (1 μM), partially reduced, were incubated with DTT (1 mM), oxidized β-casein (10 μM), NaOCl (10 μM), oxidized glutathione (GSSG, 100 μM) and H2O2 (100 μM). Variations in the ratio F505 nm/F425 nm or F500 nm/F410 nm (R) were normalized to the value at t = 0 (R0). In panels a, b, c and d, arrows indicate the addition of reagent. The data presented are representative of 3 replicates. (e) Comparison of MetSOx and MetROx reactivity towards MICAL1-oxidized actin. Sensors (0.12 μM) were incubated without actin (“No actin”), with 1.4 μM non-treated actin (“+ actin”) or with 1.4 μM MICAL1-oxidized actin (“+ oxidized actin”) for 15 min. Fluorescence excitation ratios (F505 nm/F425 nm for MetSOx and F500 nm/F410 nm for MetROx) were determined from spectra recorded at 530 nm emission wavelength. The data presented are the means (n = 3) ± SD. The data presented are representative of 3 replicates.

Mentions: When sensors were incubated with free Met, no change in the fluorescence ratio was observed, whereas incubation with free MetO, N-acetyl-MetO (a substrate mimicking MetO in proteins) and oxidized β-casein induced significant changes in both sensors (Fig. 2a, b, Supplementary Fig. 4). Kinetic measurements of sensor oxidation upon reaction with MetO-containing substrates showed fast reactivity, whereas no changes were observed for the inactive Cys-to-Ser forms. DTT treatment induced a decrease and increase in the fluorescence ratios of MetSOx and MetROx, respectively, indicating that this reducing agent could reduce both sensors (Fig. 2a, b).


Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors.

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

Response of recombinant MetSOx and MetROx to DTT and various oxidantsKinetics of reactivity of MetSOx (a) and MetROx (b) with MetO-containing substrates and DTT. Reduced sensors (1 μM) were incubated with free L-Met-R,S-O (100 μM, or 1,000 μM for MetSOx and MetROx, respectively), oxidized β-casein (10 μM), N-acetyl-L-Met-R,S-O (10 μM), PBS or DTT (1 mM), and the reduced inactive mutants (C25S MetSOx or C129S MetROx) were incubated with N-acetyl-L-Met-R,S-O (10 μM). F505 nm/F425 nm and F500 nm/F410 nm ratios (R) were normalized to the value at t = 0 (R0). Response of recombinant MetSOx (c) and MetROx (d) to DTT and various oxidants. Sensors (1 μM), partially reduced, were incubated with DTT (1 mM), oxidized β-casein (10 μM), NaOCl (10 μM), oxidized glutathione (GSSG, 100 μM) and H2O2 (100 μM). Variations in the ratio F505 nm/F425 nm or F500 nm/F410 nm (R) were normalized to the value at t = 0 (R0). In panels a, b, c and d, arrows indicate the addition of reagent. The data presented are representative of 3 replicates. (e) Comparison of MetSOx and MetROx reactivity towards MICAL1-oxidized actin. Sensors (0.12 μM) were incubated without actin (“No actin”), with 1.4 μM non-treated actin (“+ actin”) or with 1.4 μM MICAL1-oxidized actin (“+ oxidized actin”) for 15 min. Fluorescence excitation ratios (F505 nm/F425 nm for MetSOx and F500 nm/F410 nm for MetROx) were determined from spectra recorded at 530 nm emission wavelength. The data presented are the means (n = 3) ± SD. The data presented are representative of 3 replicates.
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Figure 2: Response of recombinant MetSOx and MetROx to DTT and various oxidantsKinetics of reactivity of MetSOx (a) and MetROx (b) with MetO-containing substrates and DTT. Reduced sensors (1 μM) were incubated with free L-Met-R,S-O (100 μM, or 1,000 μM for MetSOx and MetROx, respectively), oxidized β-casein (10 μM), N-acetyl-L-Met-R,S-O (10 μM), PBS or DTT (1 mM), and the reduced inactive mutants (C25S MetSOx or C129S MetROx) were incubated with N-acetyl-L-Met-R,S-O (10 μM). F505 nm/F425 nm and F500 nm/F410 nm ratios (R) were normalized to the value at t = 0 (R0). Response of recombinant MetSOx (c) and MetROx (d) to DTT and various oxidants. Sensors (1 μM), partially reduced, were incubated with DTT (1 mM), oxidized β-casein (10 μM), NaOCl (10 μM), oxidized glutathione (GSSG, 100 μM) and H2O2 (100 μM). Variations in the ratio F505 nm/F425 nm or F500 nm/F410 nm (R) were normalized to the value at t = 0 (R0). In panels a, b, c and d, arrows indicate the addition of reagent. The data presented are representative of 3 replicates. (e) Comparison of MetSOx and MetROx reactivity towards MICAL1-oxidized actin. Sensors (0.12 μM) were incubated without actin (“No actin”), with 1.4 μM non-treated actin (“+ actin”) or with 1.4 μM MICAL1-oxidized actin (“+ oxidized actin”) for 15 min. Fluorescence excitation ratios (F505 nm/F425 nm for MetSOx and F500 nm/F410 nm for MetROx) were determined from spectra recorded at 530 nm emission wavelength. The data presented are the means (n = 3) ± SD. The data presented are representative of 3 replicates.
Mentions: When sensors were incubated with free Met, no change in the fluorescence ratio was observed, whereas incubation with free MetO, N-acetyl-MetO (a substrate mimicking MetO in proteins) and oxidized β-casein induced significant changes in both sensors (Fig. 2a, b, Supplementary Fig. 4). Kinetic measurements of sensor oxidation upon reaction with MetO-containing substrates showed fast reactivity, whereas no changes were observed for the inactive Cys-to-Ser forms. DTT treatment induced a decrease and increase in the fluorescence ratios of MetSOx and MetROx, respectively, indicating that this reducing agent could reduce both sensors (Fig. 2a, b).

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