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Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.

Menger KE, James AM, Cochemé HM, Harbour ME, Chouchani ET, Ding S, Fearnley IM, Partridge L, Murphy MP - Cell Rep (2015)

Bottom Line: Surprisingly, these cysteine residues did not become more oxidized with age.In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized.We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

View Article: PubMed Central - PubMed

Affiliation: MRC Mitochondrial Biology Unit, Cambridge CB2 0XY, UK; Institute of Ophthalmology, University College London, London EC1V 9EL, UK.

No MeSH data available.


Related in: MedlinePlus

Assessment of Protein Cysteine-Residue Redox State in Flies(A) Schematic showing how exposed cysteine residue can be reversibly oxidized and reduced by GSH/glutaredoxin (Grx) and Trx.(B) OxICAT methodology. Flies are rapidly frozen, and the heads and thoraces are homogenized in 100% TCA to separate solubilized protein from the exoskeleton and then diluted to 20% TCA to precipitate proteins. The protein homogenate is then reacted with the Light ICAT reagent (L-ICAT, red) to label reduced cysteine residues (Pr-SH). After reduction of reversibly oxidized cysteine residues (Pr-SX), these thiols are reacted with the heavy ICAT reagent (H-ICAT, blue). After tryptic digestion and enrichment of labeled peptides, the biotin tags are cleaved off before separation by liquid chromatography and analysis by mass spectrometry, enabling the peptide sequence and the ratio of heavy and light labeled cysteine-containing peptides to be determined simultaneously.(C) A typical chromatogram from control flies (UAS-cat/+). A cysteine peptide oxidized and reduced pair (retention time = 39 min) is highlighted.(D) Chromatograms for the heavy and light labeled peptide eluting at 39 min are shown. The percentage oxidation of that cysteine residue was determined (bar chart).(E) The peptide eluting at 39 min was identified by mass spectrometry as a component of thioredoxin reductase-1 (TrxR1). This gene encodes both a mitochondrial and a shorter cytoplasmic splice variant. The peptide could arise from either isoform but has been numbered as Cys142 from the mitochondrial isoform.See also Figure S1.
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fig1: Assessment of Protein Cysteine-Residue Redox State in Flies(A) Schematic showing how exposed cysteine residue can be reversibly oxidized and reduced by GSH/glutaredoxin (Grx) and Trx.(B) OxICAT methodology. Flies are rapidly frozen, and the heads and thoraces are homogenized in 100% TCA to separate solubilized protein from the exoskeleton and then diluted to 20% TCA to precipitate proteins. The protein homogenate is then reacted with the Light ICAT reagent (L-ICAT, red) to label reduced cysteine residues (Pr-SH). After reduction of reversibly oxidized cysteine residues (Pr-SX), these thiols are reacted with the heavy ICAT reagent (H-ICAT, blue). After tryptic digestion and enrichment of labeled peptides, the biotin tags are cleaved off before separation by liquid chromatography and analysis by mass spectrometry, enabling the peptide sequence and the ratio of heavy and light labeled cysteine-containing peptides to be determined simultaneously.(C) A typical chromatogram from control flies (UAS-cat/+). A cysteine peptide oxidized and reduced pair (retention time = 39 min) is highlighted.(D) Chromatograms for the heavy and light labeled peptide eluting at 39 min are shown. The percentage oxidation of that cysteine residue was determined (bar chart).(E) The peptide eluting at 39 min was identified by mass spectrometry as a component of thioredoxin reductase-1 (TrxR1). This gene encodes both a mitochondrial and a shorter cytoplasmic splice variant. The peptide could arise from either isoform but has been numbered as Cys142 from the mitochondrial isoform.See also Figure S1.

Mentions: To explore how aging and fasting affect redox state, we used the fruit fly Drosophila melanogaster and focused on reversible redox alterations to exposed cysteine residues. These often lack a clear structural or catalytic role and are a major, but underappreciated, component of the integrated response of the cell to redox alterations (Go and Jones, 2013; Murphy, 2012). Cysteine residues are the most abundant cellular thiol, and in the mitochondrial matrix, the concentration is ∼20- to 30-fold greater than glutathione (GSH) (Go and Jones, 2013; Requejo et al., 2010). A proportion of protein thiols are particularly reactive due to changes in pKa, accessibility and orientation wrought by the local environment (Go and Jones, 2013; Held and Gibson, 2012). Potential modifications to cysteine residues include disulfides, S-nitrosothiols, sulfenic acids, S-acylation, and S-thiolation, all of which can be reversed by the GSH/glutaredoxin and thioredoxin (Trx) systems (Murphy, 2012; Go and Jones, 2013; Held and Gibson, 2012). These changes are part of the bulk redox tone, and small changes to a large number of different cysteine residues are likely to buffer the cellular redox environment to cope with changes in redox couples and reactive species (Go and Jones, 2013). Protein cysteine residues can also prevent local damage by sequestering reactive species (Go and Jones, 2013). Finally, a proportion of protein cysteine residues will undergo reversible modifications that can alter protein activity, location, or function and thereby coordinate the transmission of redox signals (D’Autréaux and Toledano, 2007; Sobotta et al., 2015). Therefore, cysteine residues are central to the cellular response to environmental challenges through the bulk redox tone or by more specific contributions to antioxidant defenses and redox signaling (Go and Jones, 2013; Held and Gibson, 2012; Leichert et al., 2008; Murphy, 2012). Consequently, assessing shifts in redox state as well as the identities of individual cysteine residues that change will contribute to our understanding of how organisms respond to aging and fasting (Figure 1A).


Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.

Menger KE, James AM, Cochemé HM, Harbour ME, Chouchani ET, Ding S, Fearnley IM, Partridge L, Murphy MP - Cell Rep (2015)

Assessment of Protein Cysteine-Residue Redox State in Flies(A) Schematic showing how exposed cysteine residue can be reversibly oxidized and reduced by GSH/glutaredoxin (Grx) and Trx.(B) OxICAT methodology. Flies are rapidly frozen, and the heads and thoraces are homogenized in 100% TCA to separate solubilized protein from the exoskeleton and then diluted to 20% TCA to precipitate proteins. The protein homogenate is then reacted with the Light ICAT reagent (L-ICAT, red) to label reduced cysteine residues (Pr-SH). After reduction of reversibly oxidized cysteine residues (Pr-SX), these thiols are reacted with the heavy ICAT reagent (H-ICAT, blue). After tryptic digestion and enrichment of labeled peptides, the biotin tags are cleaved off before separation by liquid chromatography and analysis by mass spectrometry, enabling the peptide sequence and the ratio of heavy and light labeled cysteine-containing peptides to be determined simultaneously.(C) A typical chromatogram from control flies (UAS-cat/+). A cysteine peptide oxidized and reduced pair (retention time = 39 min) is highlighted.(D) Chromatograms for the heavy and light labeled peptide eluting at 39 min are shown. The percentage oxidation of that cysteine residue was determined (bar chart).(E) The peptide eluting at 39 min was identified by mass spectrometry as a component of thioredoxin reductase-1 (TrxR1). This gene encodes both a mitochondrial and a shorter cytoplasmic splice variant. The peptide could arise from either isoform but has been numbered as Cys142 from the mitochondrial isoform.See also Figure S1.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508341&req=5

fig1: Assessment of Protein Cysteine-Residue Redox State in Flies(A) Schematic showing how exposed cysteine residue can be reversibly oxidized and reduced by GSH/glutaredoxin (Grx) and Trx.(B) OxICAT methodology. Flies are rapidly frozen, and the heads and thoraces are homogenized in 100% TCA to separate solubilized protein from the exoskeleton and then diluted to 20% TCA to precipitate proteins. The protein homogenate is then reacted with the Light ICAT reagent (L-ICAT, red) to label reduced cysteine residues (Pr-SH). After reduction of reversibly oxidized cysteine residues (Pr-SX), these thiols are reacted with the heavy ICAT reagent (H-ICAT, blue). After tryptic digestion and enrichment of labeled peptides, the biotin tags are cleaved off before separation by liquid chromatography and analysis by mass spectrometry, enabling the peptide sequence and the ratio of heavy and light labeled cysteine-containing peptides to be determined simultaneously.(C) A typical chromatogram from control flies (UAS-cat/+). A cysteine peptide oxidized and reduced pair (retention time = 39 min) is highlighted.(D) Chromatograms for the heavy and light labeled peptide eluting at 39 min are shown. The percentage oxidation of that cysteine residue was determined (bar chart).(E) The peptide eluting at 39 min was identified by mass spectrometry as a component of thioredoxin reductase-1 (TrxR1). This gene encodes both a mitochondrial and a shorter cytoplasmic splice variant. The peptide could arise from either isoform but has been numbered as Cys142 from the mitochondrial isoform.See also Figure S1.
Mentions: To explore how aging and fasting affect redox state, we used the fruit fly Drosophila melanogaster and focused on reversible redox alterations to exposed cysteine residues. These often lack a clear structural or catalytic role and are a major, but underappreciated, component of the integrated response of the cell to redox alterations (Go and Jones, 2013; Murphy, 2012). Cysteine residues are the most abundant cellular thiol, and in the mitochondrial matrix, the concentration is ∼20- to 30-fold greater than glutathione (GSH) (Go and Jones, 2013; Requejo et al., 2010). A proportion of protein thiols are particularly reactive due to changes in pKa, accessibility and orientation wrought by the local environment (Go and Jones, 2013; Held and Gibson, 2012). Potential modifications to cysteine residues include disulfides, S-nitrosothiols, sulfenic acids, S-acylation, and S-thiolation, all of which can be reversed by the GSH/glutaredoxin and thioredoxin (Trx) systems (Murphy, 2012; Go and Jones, 2013; Held and Gibson, 2012). These changes are part of the bulk redox tone, and small changes to a large number of different cysteine residues are likely to buffer the cellular redox environment to cope with changes in redox couples and reactive species (Go and Jones, 2013). Protein cysteine residues can also prevent local damage by sequestering reactive species (Go and Jones, 2013). Finally, a proportion of protein cysteine residues will undergo reversible modifications that can alter protein activity, location, or function and thereby coordinate the transmission of redox signals (D’Autréaux and Toledano, 2007; Sobotta et al., 2015). Therefore, cysteine residues are central to the cellular response to environmental challenges through the bulk redox tone or by more specific contributions to antioxidant defenses and redox signaling (Go and Jones, 2013; Held and Gibson, 2012; Leichert et al., 2008; Murphy, 2012). Consequently, assessing shifts in redox state as well as the identities of individual cysteine residues that change will contribute to our understanding of how organisms respond to aging and fasting (Figure 1A).

Bottom Line: Surprisingly, these cysteine residues did not become more oxidized with age.In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized.We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

View Article: PubMed Central - PubMed

Affiliation: MRC Mitochondrial Biology Unit, Cambridge CB2 0XY, UK; Institute of Ophthalmology, University College London, London EC1V 9EL, UK.

No MeSH data available.


Related in: MedlinePlus