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A specific fluorescent probe reveals compromised activity of methionine sulfoxide reductases in Parkinson's disease † † Electronic supplementary information (ESI) available: The characterization of probes, experimental procedures, supporting data and original spectra ( 1 H NMR, 13 C NMR, and MS) of the final 23 compounds. See DOI: 10.1039/c6sc04708d Click here for additional data file.

View Article: PubMed Central - PubMed

ABSTRACT

Oxidation of methionine residues to methionine sulfoxide (MetSO) may cause changes in protein structure and function, and may eventually lead to cell damage. Methionine sulfoxide reductases (Msrs) are the only known enzymes that catalyze the reduction of MetSO back to methionine by taking reducing equivalents from the thioredoxin system, and thus protect cells from oxidative damage. Nonetheless, a lack of convenient assays for the enzymes hampers the exploration of their functions. We report the discovery of Msr-blue, the first turn-on fluorescent probe for Msr with a >100-fold fluorescence increment from screening a rationally-designed small library. Intensive studies demonstrated the specific reduction of Msr-blue by the enzymes. Msr-blue is ready to determine Msr activity in biological samples and live cells. Importantly, we disclosed a decline of Msr activity in a Parkinson's model, thus providing a mechanistic linkage between the loss of function of Msrs and the development of neurodegeneration. The strategy for the discovery of Msr-blue would also provide guidance for developing novel probes with longer excitation/emission wavelengths and specific probes for Msr isoforms.

No MeSH data available.


Reduction of Msr-blue by Msr. (A) Effects of DTT in the reduction of 16 and Msr-blue. The probes (10 μM) were incubated with DTT (20 mM) and Msr A (3 μg ml–1, 120 nM) or DTT only (0–20 mM) for 3 h, and the fold of the fluorescence increment (F/F0) was determined. The excitation/emission wavelengths for Msr-blue and 16 are 335/438 nm and 420/490 nm, respectively. (B) Time-dependent activation of Msr-blue by Msr A. Msr-blue (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM). The emission spectra (λex = 335 nm) were recorded every 30 minutes. (C) Time- and dose-dependent increase of the fluorescence of Msr-blue. The probe was incubated with DTT (5 mM) and Msr A (0–3 μg ml–1, 0–120 nM), and F/F0 was determined (λex = 335 nm, λem = 438 nm). (D) Exclusive conversion of Msr-blue to the sulfide 15′. The probe (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM) for 6 h, and the mixture was analyzed by HPLC. (E) Fluorescence spectra of the pure sulfide 15′, the reaction mixture from (D) and the reaction mixture of Msr-blue (10 μM) incubated with DTT (5 mM) and mouse kidney protein extract for 2 h (λex = 335 nm). (F) Reduction of Msr-blue by Msr A and mouse kidney lysate. The probe was incubated with DTT (5 mM) in the presence of Msr A (3 μg ml–1, 120 nM) or mouse kidney lysate (29.2 mg protein per ml), and the formation of the sulfide 15′ was determined by HPLC. (G) Reduction of Msr-blue by a combination of Msr A and mouse kidney lysate. Msr-blue was incubated with DTT (5 mM) and Msr A (6 μg ml–1, 240 nM) for 6 h. Then, the mouse kidney lysate (29 mg protein per ml) was added and the incubation continued. The formation of the sulfide 15′ was determined by HPLC. All reactions were performed at 37 °C in TE buffer.
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fig2: Reduction of Msr-blue by Msr. (A) Effects of DTT in the reduction of 16 and Msr-blue. The probes (10 μM) were incubated with DTT (20 mM) and Msr A (3 μg ml–1, 120 nM) or DTT only (0–20 mM) for 3 h, and the fold of the fluorescence increment (F/F0) was determined. The excitation/emission wavelengths for Msr-blue and 16 are 335/438 nm and 420/490 nm, respectively. (B) Time-dependent activation of Msr-blue by Msr A. Msr-blue (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM). The emission spectra (λex = 335 nm) were recorded every 30 minutes. (C) Time- and dose-dependent increase of the fluorescence of Msr-blue. The probe was incubated with DTT (5 mM) and Msr A (0–3 μg ml–1, 0–120 nM), and F/F0 was determined (λex = 335 nm, λem = 438 nm). (D) Exclusive conversion of Msr-blue to the sulfide 15′. The probe (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM) for 6 h, and the mixture was analyzed by HPLC. (E) Fluorescence spectra of the pure sulfide 15′, the reaction mixture from (D) and the reaction mixture of Msr-blue (10 μM) incubated with DTT (5 mM) and mouse kidney protein extract for 2 h (λex = 335 nm). (F) Reduction of Msr-blue by Msr A and mouse kidney lysate. The probe was incubated with DTT (5 mM) in the presence of Msr A (3 μg ml–1, 120 nM) or mouse kidney lysate (29.2 mg protein per ml), and the formation of the sulfide 15′ was determined by HPLC. (G) Reduction of Msr-blue by a combination of Msr A and mouse kidney lysate. Msr-blue was incubated with DTT (5 mM) and Msr A (6 μg ml–1, 240 nM) for 6 h. Then, the mouse kidney lysate (29 mg protein per ml) was added and the incubation continued. The formation of the sulfide 15′ was determined by HPLC. All reactions were performed at 37 °C in TE buffer.

Mentions: Firstly, we performed experiments to confirm the response of both probes to DTT by using varying concentrations of the reductant. As shown in Fig. 2A, high concentrations of DTT cause a significant elevation in the fluorescence signal of 16, while DTT appears to affect 15 much less. In addition, compound 16 shows only about a maximum 10-fold increase in emission under Msr A catalysis (Fig. S1†). Considering the potential interference of DTT with 16 and the lower fluorescence increase of 16 under enzyme catalysis, we thus decided to focus on the probe 15 in the following experiments. The DTT concentration was fixed at 5 mM in the following assays, as DTT at this concentration causes negligible interference in the fluorescence signal. Since the probe 15 emits blue fluorescence after activation by Msr A, we termed it as Msr-blue. Msr-blue responded to Msr A in both a time- and dose-dependent manner (Fig. 2B and C), and more than a 100-fold increase in the emission was observed. HPLC analysis of the reaction demonstrated that Msr-blue was converted to its corresponding sulfide (15′) under catalysis by either the purified Msr A or a cell lysate (Fig. 2D and S2†). When incubation of Msr-blue (10 μM) with DTT and Msr A in TE buffer (50 mM Tris–HCl, 1 mM EDTA, pH 7.4) took place for 6 h at 37 °C, the desired sulfide 15′ was found in the reaction mixture with ∼40% conversion. At the time point of analysis, the remaining amount of Msr-blue (6.2 μM) plus the product 15′ (3.8 μM) was equal to the amount of the starting Msr-blue (10 μM), indicating that the probe was exclusively converted to the expected sulfide 15′. It should be noted that Msr-blue is a racemic mixture containing both S-epimers and R-epimers, and Msr A should only convert the S-epimer to the sulfide 15′. Incubation of Msr-blue with mouse kidney lysate gave similar results as those for the purified enzyme, yet with a lower conversion rate of the probe (∼14% conversion, Fig. S2†). The sulfide 15′ and the reaction mixtures which were catalyzed by purified Msr A and by the cell lysate gave similar fluorescence profiles (Fig. 2E), supporting the conversion of Msr-blue to the sulfide 15′ under Msr catalysis. Taken together, we firmly concluded herein that the probe Msr-blue could be reduced by Msr to the corresponding sulfide, leading to a more than 100-fold increase in the fluorescence signal.


A specific fluorescent probe reveals compromised activity of methionine sulfoxide reductases in Parkinson's disease † † Electronic supplementary information (ESI) available: The characterization of probes, experimental procedures, supporting data and original spectra ( 1 H NMR, 13 C NMR, and MS) of the final 23 compounds. See DOI: 10.1039/c6sc04708d Click here for additional data file.
Reduction of Msr-blue by Msr. (A) Effects of DTT in the reduction of 16 and Msr-blue. The probes (10 μM) were incubated with DTT (20 mM) and Msr A (3 μg ml–1, 120 nM) or DTT only (0–20 mM) for 3 h, and the fold of the fluorescence increment (F/F0) was determined. The excitation/emission wavelengths for Msr-blue and 16 are 335/438 nm and 420/490 nm, respectively. (B) Time-dependent activation of Msr-blue by Msr A. Msr-blue (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM). The emission spectra (λex = 335 nm) were recorded every 30 minutes. (C) Time- and dose-dependent increase of the fluorescence of Msr-blue. The probe was incubated with DTT (5 mM) and Msr A (0–3 μg ml–1, 0–120 nM), and F/F0 was determined (λex = 335 nm, λem = 438 nm). (D) Exclusive conversion of Msr-blue to the sulfide 15′. The probe (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM) for 6 h, and the mixture was analyzed by HPLC. (E) Fluorescence spectra of the pure sulfide 15′, the reaction mixture from (D) and the reaction mixture of Msr-blue (10 μM) incubated with DTT (5 mM) and mouse kidney protein extract for 2 h (λex = 335 nm). (F) Reduction of Msr-blue by Msr A and mouse kidney lysate. The probe was incubated with DTT (5 mM) in the presence of Msr A (3 μg ml–1, 120 nM) or mouse kidney lysate (29.2 mg protein per ml), and the formation of the sulfide 15′ was determined by HPLC. (G) Reduction of Msr-blue by a combination of Msr A and mouse kidney lysate. Msr-blue was incubated with DTT (5 mM) and Msr A (6 μg ml–1, 240 nM) for 6 h. Then, the mouse kidney lysate (29 mg protein per ml) was added and the incubation continued. The formation of the sulfide 15′ was determined by HPLC. All reactions were performed at 37 °C in TE buffer.
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fig2: Reduction of Msr-blue by Msr. (A) Effects of DTT in the reduction of 16 and Msr-blue. The probes (10 μM) were incubated with DTT (20 mM) and Msr A (3 μg ml–1, 120 nM) or DTT only (0–20 mM) for 3 h, and the fold of the fluorescence increment (F/F0) was determined. The excitation/emission wavelengths for Msr-blue and 16 are 335/438 nm and 420/490 nm, respectively. (B) Time-dependent activation of Msr-blue by Msr A. Msr-blue (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM). The emission spectra (λex = 335 nm) were recorded every 30 minutes. (C) Time- and dose-dependent increase of the fluorescence of Msr-blue. The probe was incubated with DTT (5 mM) and Msr A (0–3 μg ml–1, 0–120 nM), and F/F0 was determined (λex = 335 nm, λem = 438 nm). (D) Exclusive conversion of Msr-blue to the sulfide 15′. The probe (10 μM) was incubated with DTT (5 mM) and Msr A (3 μg ml–1, 120 nM) for 6 h, and the mixture was analyzed by HPLC. (E) Fluorescence spectra of the pure sulfide 15′, the reaction mixture from (D) and the reaction mixture of Msr-blue (10 μM) incubated with DTT (5 mM) and mouse kidney protein extract for 2 h (λex = 335 nm). (F) Reduction of Msr-blue by Msr A and mouse kidney lysate. The probe was incubated with DTT (5 mM) in the presence of Msr A (3 μg ml–1, 120 nM) or mouse kidney lysate (29.2 mg protein per ml), and the formation of the sulfide 15′ was determined by HPLC. (G) Reduction of Msr-blue by a combination of Msr A and mouse kidney lysate. Msr-blue was incubated with DTT (5 mM) and Msr A (6 μg ml–1, 240 nM) for 6 h. Then, the mouse kidney lysate (29 mg protein per ml) was added and the incubation continued. The formation of the sulfide 15′ was determined by HPLC. All reactions were performed at 37 °C in TE buffer.
Mentions: Firstly, we performed experiments to confirm the response of both probes to DTT by using varying concentrations of the reductant. As shown in Fig. 2A, high concentrations of DTT cause a significant elevation in the fluorescence signal of 16, while DTT appears to affect 15 much less. In addition, compound 16 shows only about a maximum 10-fold increase in emission under Msr A catalysis (Fig. S1†). Considering the potential interference of DTT with 16 and the lower fluorescence increase of 16 under enzyme catalysis, we thus decided to focus on the probe 15 in the following experiments. The DTT concentration was fixed at 5 mM in the following assays, as DTT at this concentration causes negligible interference in the fluorescence signal. Since the probe 15 emits blue fluorescence after activation by Msr A, we termed it as Msr-blue. Msr-blue responded to Msr A in both a time- and dose-dependent manner (Fig. 2B and C), and more than a 100-fold increase in the emission was observed. HPLC analysis of the reaction demonstrated that Msr-blue was converted to its corresponding sulfide (15′) under catalysis by either the purified Msr A or a cell lysate (Fig. 2D and S2†). When incubation of Msr-blue (10 μM) with DTT and Msr A in TE buffer (50 mM Tris–HCl, 1 mM EDTA, pH 7.4) took place for 6 h at 37 °C, the desired sulfide 15′ was found in the reaction mixture with ∼40% conversion. At the time point of analysis, the remaining amount of Msr-blue (6.2 μM) plus the product 15′ (3.8 μM) was equal to the amount of the starting Msr-blue (10 μM), indicating that the probe was exclusively converted to the expected sulfide 15′. It should be noted that Msr-blue is a racemic mixture containing both S-epimers and R-epimers, and Msr A should only convert the S-epimer to the sulfide 15′. Incubation of Msr-blue with mouse kidney lysate gave similar results as those for the purified enzyme, yet with a lower conversion rate of the probe (∼14% conversion, Fig. S2†). The sulfide 15′ and the reaction mixtures which were catalyzed by purified Msr A and by the cell lysate gave similar fluorescence profiles (Fig. 2E), supporting the conversion of Msr-blue to the sulfide 15′ under Msr catalysis. Taken together, we firmly concluded herein that the probe Msr-blue could be reduced by Msr to the corresponding sulfide, leading to a more than 100-fold increase in the fluorescence signal.

View Article: PubMed Central - PubMed

ABSTRACT

Oxidation of methionine residues to methionine sulfoxide (MetSO) may cause changes in protein structure and function, and may eventually lead to cell damage. Methionine sulfoxide reductases (Msrs) are the only known enzymes that catalyze the reduction of MetSO back to methionine by taking reducing equivalents from the thioredoxin system, and thus protect cells from oxidative damage. Nonetheless, a lack of convenient assays for the enzymes hampers the exploration of their functions. We report the discovery of Msr-blue, the first turn-on fluorescent probe for Msr with a >100-fold fluorescence increment from screening a rationally-designed small library. Intensive studies demonstrated the specific reduction of Msr-blue by the enzymes. Msr-blue is ready to determine Msr activity in biological samples and live cells. Importantly, we disclosed a decline of Msr activity in a Parkinson's model, thus providing a mechanistic linkage between the loss of function of Msrs and the development of neurodegeneration. The strategy for the discovery of Msr-blue would also provide guidance for developing novel probes with longer excitation/emission wavelengths and specific probes for Msr isoforms.

No MeSH data available.