Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

Tarrago, Lionel; Péterfi, Zalán; Lee, Byeong Cheol; Michel, Thomas; Gladyshev, Vadim N.

doi:10.1038/nchembio.1787

Cited by 51 publications

(72 citation statements)

References 40 publications

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“…In the case of MetSOx, the spectrum presents two peaks of excitation with maxima at 410 nm and 500 nm and a single peak of emission at 510–516 nm, and the reaction with MetO lead to an increase in fluorescence intensity at 410 nm and a decrease at 500 nm with an isosbestic point at 447 nm. Thus, the 500/410 nm fluorescence intensity ratio decreases with the oxidation of MetROx [48]. To efficiently monitor MetO oxidation using MetSOx and/or MetROx, the experimental setting should allow to directly measure the fluorescence intensity for the two excitation wavelengths upon emission at ~510 nm, with a fluorimeter, or alternatively, to record fluorescence emission intensity for the two excitation wavelengths (~410 nm and ~500 nm) with a microscope.…”

Section: Theoreticalmentioning

confidence: 99%

“…Fluorescence intensity increases or decreases with the increase in pH when excited at ~500 nm, or at ~400–425 nm, respectively [48]. Reduced and oxidized forms of MetSOx have pKa of 8.5 and 9.5, respectively, and reduced and oxidized forms of MetROx exhibit pKa of 7.7 and 8.6, respectively.…”

Section: Theoreticalmentioning

confidence: 99%

“…The importance of MetO as a redox biomarker in numerous physiological and pathological conditions, the limitations of existing methods and the fact that no known chemicals or antibodies react specifically with MetO, prompted us to develop genetically encoded fluorescent sensors to dynamically monitor MetO formation and reduction in living cells [48]. The two created sensors, MetSOx and MetROx, are derived from yeast MSRA and MSRB, ensuring specificity for the S and R diastereomers of MetO, respectively.…”

Section: Introductionmentioning

confidence: 99%

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Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide

Péterfi

Tarrago

Gladyshev

2016

Methods

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In cells, physiological and pathophysiological conditions may lead to the formation of methionine sulfoxide (MetO). This oxidative modification of methionine exists in the form of two diastereomers, R and S, and may occur in both free amino acid and proteins. MetO is reduced back to methionine by methionine sulfoxide reductases (MSRs). Methionine oxidation was thought to be a nonspecific modification affecting protein functions and methionine availability. However, recent findings suggest that cyclic methionine oxidation and reduction is a posttranslational modification that actively regulates protein function akin to redox regulation by cysteine oxidation and phosphorylation. Methionine oxidation is thus an important mechanism that could play out in various physiological contexts. However, detecting MetO generation and MSR functions remains challenging because of the lack of tools and reagents to detect and quantify this protein modification. We recently developed two genetically encoded diasterospecific fluorescent sensors, MetSOx and MetROx, to dynamically monitor MetO in living cells. Here, we provide a detailed procedure for their use in bacterial and mammalian cells using fluorimetric and fluorescent imaging approaches. This method can be adapted to dynamically monitor methionine oxidation in various cell types and under various conditions.

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Section: Theoreticalmentioning

confidence: 99%

Section: Theoreticalmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide

Péterfi

Tarrago

Gladyshev

2016

Methods

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“…As MSRB1 is intricately linked with Se metabolism, studies on Se and selenoproteins may require quantification of Met- R -O levels and MSR activity in cells. In this chapter, we describe the procedures that allow determining Met- R -O levels and its changes in living cells using the genetically-encoded ratiometric fluorescent biosensor MetROx [14, 15]. We also provide protocols for quantification of MSRA, MSRB and total MSR activities in protein extracts using specific substrates and an HPLC-based procedure.…”

Section: Introductionmentioning

confidence: 99%

Monitoring of Methionine Sulfoxide Content and Methionine Sulfoxide Reductase Activity

Tarrago¹,

Oheix²,

Péterfi³

et al. 2017

Methods in Molecular Biology

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The sulfur-containing amino acid methionine (Met) plays critical roles in protein synthesis, methylation, and sulfur metabolism. Both in its free form and in the form of an amino acid residue, it can be oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Organisms evolved methionine sulfoxide reductases (MSRs) to reduce MetO to Met, with the MSRs type A (MSRA) and type B (MSRB) being specific for the S and R forms of MetO, respectively. In mammals, the selenoprotein MSRB1 plays an important protein repair function, and its expression is tightly regulated by dietary selenium. In this chapter, we describe a protocol for determining the concentration of protein-based Met-R-O and its analysis in HEK293 cells using a genetically-encoded ratiometric fluorescent biosensor MetROx. We also describe the procedure for quantifying MSR activities in cell extracts using specific substrates and a reverse phase HPLC-based method.

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“…Other studies from the Gladyshev lab used methionine sulfoxide reduction to reveal reactive oxygen species (ROS)-dependent and -independent components of aging (36), to identify methionine restriction as a general strategy for lifespan control (41), and to develop fluorescent ratiometric sensors for in vivo monitoring of methionine sulfoxide (61). They also recently discovered a new exciting mechanism of regulation of protein function: reversible, sitespecific methionine-R-sulfoxidation (40).…”

Section: Selenoprotein Redox Functionsmentioning

confidence: 99%

Redox Pioneer: Professor Vadim N. Gladyshev

Hatfield

2016

Antioxidants & Redox Signaling

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Professor Vadim N. Gladyshev is recognized here as a Redox Pioneer, because he has published an article on antioxidant/redox biology that has been cited more than 1000 times and 29 articles that have been cited more than 100 times. Gladyshev is world renowned for his characterization of the human selenoproteome encoded by 25 genes, identification of the majority of known selenoprotein genes in the three domains of life, and discoveries related to thiol oxidoreductases and mechanisms of redox control. Gladyshev's first faculty position was in the Department of Biochemistry, the University of Nebraska. There, he was a Charles Bessey Professor and Director of the Redox Biology Center. He then moved to the Department of Medicine at Brigham and Women's Hospital, Harvard Medical School, where he is Professor of Medicine and Director of the Center for Redox Medicine. His discoveries in redox biology relate to selenoenzymes, such as methionine sulfoxide reductases and thioredoxin reductases, and various thiol oxidoreductases. He is responsible for the genome-wide identification of catalytic redox-active cysteines and for advancing our understanding of the general use of cysteines by proteins. In addition, Gladyshev has characterized hydrogen peroxide metabolism and signaling and regulation of protein function by methionine-R-sulfoxidation. He has also made important contributions in the areas of aging and lifespan control and pioneered applications of comparative genomics in redox biology, selenium biology, and aging. Gladyshev's discoveries have had a profound impact on redox biology and the role of redox control in health and disease. He is a true Redox Pioneer. Antioxid. Redox Signal. 25, 1-9.

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Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

Cited by 51 publications

References 40 publications

Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide

Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide

Monitoring of Methionine Sulfoxide Content and Methionine Sulfoxide Reductase Activity

Redox Pioneer: Professor Vadim N. Gladyshev

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