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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.


Decline of Msr activity in a PD model. (A) Imaging Msr activity in live PC 12 cells treated with or without 6-OHDA. (B) Determination of Msr activity in PC 12 cell lysates. PC 12 cells were treated as described in (A), and the time-dependent reduction of Msr-blue by the lysates was determined (λex = 335 nm). (C) Determination of Msr A protein level in the lysates prepared in (B) by Western blots.
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fig5: Decline of Msr activity in a PD model. (A) Imaging Msr activity in live PC 12 cells treated with or without 6-OHDA. (B) Determination of Msr activity in PC 12 cell lysates. PC 12 cells were treated as described in (A), and the time-dependent reduction of Msr-blue by the lysates was determined (λex = 335 nm). (C) Determination of Msr A protein level in the lysates prepared in (B) by Western blots.

Mentions: Due to the critical roles of Msrs in defending against oxidative stress and preventing accumulation of misfolded proteins, a compromised function of Msr has been linked to the development of aging-associated disorders like neurodegenerative diseases.4,40–42 In line with this observation, an overexpression of Msr A confers benefits against the development of PD,40,41,43 while knockout of the enzyme enhances the progression of neurodegeneration.42 Markesbery et al. reported the decrease of Msr A activity in brains that have been affected by Alzheimer's disease.44 However the activity of Msrs in PD tissues or models is unknown. 6-Hydroxydopamine (6-OHDA) is a well-established neurotoxin used to generate PD models in animals or cells.45,46 We employed the 6-OHDA-treated PC 12 cells as a cellular model of PD47–49 and applied Msr-blue to probe the function of Msrs in the cells. As illustrated in Fig. 5A, 6-OHDA treatment causes a remarkable decline in the enzyme activity, revealed by the decrease in the fluorescence intensity of the cells. As the activity of Msrs is supported by the thioredoxin system in cells, we thus further determined their activity in the lysate by supplying DTT as a reductant to exclude the possible alteration of function by the thioredoxin system. Consistent with the Msr-blue imaging results, the activity of Msr decreased by ∼30% in the drug-treated cells compared to the control groups (Fig. 5B). Analysis of the protein levels of Msr A indicated no significant alteration of the protein expression after 50 μM of 6-OHDA treatment, yet a slight decrease after 100 μM of 6-OHDA treatment (Fig. 5C). Taken together, with the aid of Msr-blue, a decline of the Msr activity in a PD model was disclosed for the first time.


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.
Decline of Msr activity in a PD model. (A) Imaging Msr activity in live PC 12 cells treated with or without 6-OHDA. (B) Determination of Msr activity in PC 12 cell lysates. PC 12 cells were treated as described in (A), and the time-dependent reduction of Msr-blue by the lysates was determined (λex = 335 nm). (C) Determination of Msr A protein level in the lysates prepared in (B) by Western blots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382841&req=5

fig5: Decline of Msr activity in a PD model. (A) Imaging Msr activity in live PC 12 cells treated with or without 6-OHDA. (B) Determination of Msr activity in PC 12 cell lysates. PC 12 cells were treated as described in (A), and the time-dependent reduction of Msr-blue by the lysates was determined (λex = 335 nm). (C) Determination of Msr A protein level in the lysates prepared in (B) by Western blots.
Mentions: Due to the critical roles of Msrs in defending against oxidative stress and preventing accumulation of misfolded proteins, a compromised function of Msr has been linked to the development of aging-associated disorders like neurodegenerative diseases.4,40–42 In line with this observation, an overexpression of Msr A confers benefits against the development of PD,40,41,43 while knockout of the enzyme enhances the progression of neurodegeneration.42 Markesbery et al. reported the decrease of Msr A activity in brains that have been affected by Alzheimer's disease.44 However the activity of Msrs in PD tissues or models is unknown. 6-Hydroxydopamine (6-OHDA) is a well-established neurotoxin used to generate PD models in animals or cells.45,46 We employed the 6-OHDA-treated PC 12 cells as a cellular model of PD47–49 and applied Msr-blue to probe the function of Msrs in the cells. As illustrated in Fig. 5A, 6-OHDA treatment causes a remarkable decline in the enzyme activity, revealed by the decrease in the fluorescence intensity of the cells. As the activity of Msrs is supported by the thioredoxin system in cells, we thus further determined their activity in the lysate by supplying DTT as a reductant to exclude the possible alteration of function by the thioredoxin system. Consistent with the Msr-blue imaging results, the activity of Msr decreased by ∼30% in the drug-treated cells compared to the control groups (Fig. 5B). Analysis of the protein levels of Msr A indicated no significant alteration of the protein expression after 50 μM of 6-OHDA treatment, yet a slight decrease after 100 μM of 6-OHDA treatment (Fig. 5C). Taken together, with the aid of Msr-blue, a decline of the Msr activity in a PD model was disclosed for the first time.

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.