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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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MetROx response in subcellular compartments and physiological stimuli in HEK293 cells(a) Representative images of MetROx fluorescence in different cellular compartments. Scale bars represent 20 μm. (b) MetROx fluorescence ratio in different regions of interest was corrected by the ratio of the C129S MetROx targeted to the same compartment. Raw data are shown in Supplementary Fig. 13 (n = 13–27, *p < 0.05, **p < 0.005). (c) Corrected fluorescence ratio of MetROx in control (RFP-expressing) and MICAL1-expressing cells. Raw data are shown in Supplementary Fig. 14 (n = 17–26, p < 0.05). (d) Kinetics of MetROx response to serum stimulation. Raw fluorescent (F) and pseudocolored ratio (R) images of MetROx (upper panel) and kinetics of fluorescence (lower panel) of MetROx- and C129S MetROx-expressing cells subjected to 6 h of serum starvation followed by 10% serum treatment (↑) (n = 3). Scale bars represent 20 μm. (e) Kinetics of MetROx response to insulin. Raw fluorescent (F) and pseudocolored (R) ratio image-series of MetROx (upper panel), and kinetics of fluorescence (lower panel) of MetROx and C129S MetROx expressing cells subjected to 6 h serum starvation followed by 1.4 μM insulin treatment (↑) (n = 3). Scale bars represent 20 μm. Data presented are the means ± SD and are representative of 3 replicates.
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Figure 6: MetROx response in subcellular compartments and physiological stimuli in HEK293 cells(a) Representative images of MetROx fluorescence in different cellular compartments. Scale bars represent 20 μm. (b) MetROx fluorescence ratio in different regions of interest was corrected by the ratio of the C129S MetROx targeted to the same compartment. Raw data are shown in Supplementary Fig. 13 (n = 13–27, *p < 0.05, **p < 0.005). (c) Corrected fluorescence ratio of MetROx in control (RFP-expressing) and MICAL1-expressing cells. Raw data are shown in Supplementary Fig. 14 (n = 17–26, p < 0.05). (d) Kinetics of MetROx response to serum stimulation. Raw fluorescent (F) and pseudocolored ratio (R) images of MetROx (upper panel) and kinetics of fluorescence (lower panel) of MetROx- and C129S MetROx-expressing cells subjected to 6 h of serum starvation followed by 10% serum treatment (↑) (n = 3). Scale bars represent 20 μm. (e) Kinetics of MetROx response to insulin. Raw fluorescent (F) and pseudocolored (R) ratio image-series of MetROx (upper panel), and kinetics of fluorescence (lower panel) of MetROx and C129S MetROx expressing cells subjected to 6 h serum starvation followed by 1.4 μM insulin treatment (↑) (n = 3). Scale bars represent 20 μm. Data presented are the means ± SD and are representative of 3 replicates.

Mentions: We further tested whether MetROx could detect protein-bound MetO in vivo and whether it was able to detect MetO under physiologically relevant conditions. In order to estimate Met-R-O variation detected by MetROx in HEK293 cells, we determined the value of the ratio for the fully reduced and oxidized sensor using saturating concentrations of DTT and MetO, and developed an empiric scale similarly to the E. coli assays (Supplementary Fig. 12). We further targeted MetROx and its inactive form to various cellular compartments (Fig. 6a). The corrected fluorescence ratios (Fig. 6b, Supplementary Fig. 13) allowed estimating MetROx oxidized fractions: ~ 0.3, ~0.1, ~0.7 and ~0.9 in the cytosol, nucleus, mitochondrial matrix and ER, respectively, suggesting that cytosol and nucleus had less than 100 μM Met-R-O whereas mitochondria and ER had 5–10 fold more Met-R-O. Next, to oxidize intracellular proteins, we overexpressed an RFP-fused constitutively active MICAL1 along with MetROx (Fig. 6c, Supplementary Fig. 14). A significant decrease in the fluorescence ratio was detected in MICAL1-overexpressing cells, but not in RFP-expressing control cells, indicating that MetROx detected Met-R-O generated by MICAL1 on actin and possibly other proteins. Since many growth factor and hormone signaling pathways and the regulation of the actin cytoskeleton dynamics involve redox mediators2, we tested whether serum stimulation of HEK293 cells could trigger Met oxidation. Following 6 h of serum starvation, 10% FBS induced a rapid and reversible oxidation of the sensor, showing that this treatment led to a transient increase in Met-R-O (Fig. 6d). We further tested whether a prototypical redox signaling process, the insulin pathway, could induce Met oxidation. Following serum starvation, 1.4 μM insulin induced a rapid oxidation of the sensor (Fig. 6e). Neither short-term (<12h) serum starvation nor cell confluency changed the MetROx ratio significantly (Supplementary Fig. 15).


Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors.

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

MetROx response in subcellular compartments and physiological stimuli in HEK293 cells(a) Representative images of MetROx fluorescence in different cellular compartments. Scale bars represent 20 μm. (b) MetROx fluorescence ratio in different regions of interest was corrected by the ratio of the C129S MetROx targeted to the same compartment. Raw data are shown in Supplementary Fig. 13 (n = 13–27, *p < 0.05, **p < 0.005). (c) Corrected fluorescence ratio of MetROx in control (RFP-expressing) and MICAL1-expressing cells. Raw data are shown in Supplementary Fig. 14 (n = 17–26, p < 0.05). (d) Kinetics of MetROx response to serum stimulation. Raw fluorescent (F) and pseudocolored ratio (R) images of MetROx (upper panel) and kinetics of fluorescence (lower panel) of MetROx- and C129S MetROx-expressing cells subjected to 6 h of serum starvation followed by 10% serum treatment (↑) (n = 3). Scale bars represent 20 μm. (e) Kinetics of MetROx response to insulin. Raw fluorescent (F) and pseudocolored (R) ratio image-series of MetROx (upper panel), and kinetics of fluorescence (lower panel) of MetROx and C129S MetROx expressing cells subjected to 6 h serum starvation followed by 1.4 μM insulin treatment (↑) (n = 3). Scale bars represent 20 μm. Data presented are the means ± SD and are representative of 3 replicates.
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Figure 6: MetROx response in subcellular compartments and physiological stimuli in HEK293 cells(a) Representative images of MetROx fluorescence in different cellular compartments. Scale bars represent 20 μm. (b) MetROx fluorescence ratio in different regions of interest was corrected by the ratio of the C129S MetROx targeted to the same compartment. Raw data are shown in Supplementary Fig. 13 (n = 13–27, *p < 0.05, **p < 0.005). (c) Corrected fluorescence ratio of MetROx in control (RFP-expressing) and MICAL1-expressing cells. Raw data are shown in Supplementary Fig. 14 (n = 17–26, p < 0.05). (d) Kinetics of MetROx response to serum stimulation. Raw fluorescent (F) and pseudocolored ratio (R) images of MetROx (upper panel) and kinetics of fluorescence (lower panel) of MetROx- and C129S MetROx-expressing cells subjected to 6 h of serum starvation followed by 10% serum treatment (↑) (n = 3). Scale bars represent 20 μm. (e) Kinetics of MetROx response to insulin. Raw fluorescent (F) and pseudocolored (R) ratio image-series of MetROx (upper panel), and kinetics of fluorescence (lower panel) of MetROx and C129S MetROx expressing cells subjected to 6 h serum starvation followed by 1.4 μM insulin treatment (↑) (n = 3). Scale bars represent 20 μm. Data presented are the means ± SD and are representative of 3 replicates.
Mentions: We further tested whether MetROx could detect protein-bound MetO in vivo and whether it was able to detect MetO under physiologically relevant conditions. In order to estimate Met-R-O variation detected by MetROx in HEK293 cells, we determined the value of the ratio for the fully reduced and oxidized sensor using saturating concentrations of DTT and MetO, and developed an empiric scale similarly to the E. coli assays (Supplementary Fig. 12). We further targeted MetROx and its inactive form to various cellular compartments (Fig. 6a). The corrected fluorescence ratios (Fig. 6b, Supplementary Fig. 13) allowed estimating MetROx oxidized fractions: ~ 0.3, ~0.1, ~0.7 and ~0.9 in the cytosol, nucleus, mitochondrial matrix and ER, respectively, suggesting that cytosol and nucleus had less than 100 μM Met-R-O whereas mitochondria and ER had 5–10 fold more Met-R-O. Next, to oxidize intracellular proteins, we overexpressed an RFP-fused constitutively active MICAL1 along with MetROx (Fig. 6c, Supplementary Fig. 14). A significant decrease in the fluorescence ratio was detected in MICAL1-overexpressing cells, but not in RFP-expressing control cells, indicating that MetROx detected Met-R-O generated by MICAL1 on actin and possibly other proteins. Since many growth factor and hormone signaling pathways and the regulation of the actin cytoskeleton dynamics involve redox mediators2, we tested whether serum stimulation of HEK293 cells could trigger Met oxidation. Following 6 h of serum starvation, 10% FBS induced a rapid and reversible oxidation of the sensor, showing that this treatment led to a transient increase in Met-R-O (Fig. 6d). We further tested whether a prototypical redox signaling process, the insulin pathway, could induce Met oxidation. Following serum starvation, 1.4 μM insulin induced a rapid oxidation of the sensor (Fig. 6e). Neither short-term (<12h) serum starvation nor cell confluency changed the MetROx ratio significantly (Supplementary Fig. 15).

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