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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

OxICAT Analysis of Control Young Female D. melanogaster(A) Ion count for peptides plotted against percentage oxidation of the cysteine residue. The ion count is the log10 intensity of the sum of the heavy and light peptides. Data are the averages over three to five biological replicates. Red cross is cysteine residue 142 from TrxR1 (Figure 1C).(B) Distribution of total cysteine residue oxidation levels. Plotted are the means of the proportion of the total number of peptides containing unique cysteine residues in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Total unique peptides = 491.(C) Plasma membrane Na+/K+ ATPase. D. melanogaster Na+/K+ ATPase contains an α subunit and a β subunit with multiple isoforms. The monomeric structure from S. acanthias containing subunit α (green) and subunit β1 (yellow) is 77% and 25% homologous to the α and β2 subunits of D. melanogaster, which were detected by OxICAT. Cysteine residues on the S. acanthias structure present in homologous positions in the D. melanogaster α and β2 subunits are numbered. Cysteines observed by OxICAT are shown in red, and those not detected are blue. Disulfide cysteine partners are also labeled. The table shows the oxidation state of each cysteine in young control untreated flies.(D) Oxidation state of protein cysteine residues in mitochondria. Peptides from Figure 2B that are mitochondrial are plotted as the mean of the proportion of the total number of peptides in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Of 214 proteins identified in Figure 2A, 87 are mitochondrial, corresponding to 214 unique cysteine residues.(E) Comparison of peptides detected by OxICAT with transcript abundance. Whole-fly transcript intensity data were annotated to the head and thorax OxICAT dataset to characterize fly cysteines observable by mass spectrometry. Transcripts are subdivided by abundance into blocks that are √2 of the upper and lower bounds of the block immediately to the left. The percentage of both the observed OxICAT cysteine population (mean transcript abundance = 1,119; n = 849) and the total cysteine population (mean transcript abundance = 134; n = ∼135,000) falling within each transcript abundance block are on the y axis.See also Figure S2.
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fig2: OxICAT Analysis of Control Young Female D. melanogaster(A) Ion count for peptides plotted against percentage oxidation of the cysteine residue. The ion count is the log10 intensity of the sum of the heavy and light peptides. Data are the averages over three to five biological replicates. Red cross is cysteine residue 142 from TrxR1 (Figure 1C).(B) Distribution of total cysteine residue oxidation levels. Plotted are the means of the proportion of the total number of peptides containing unique cysteine residues in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Total unique peptides = 491.(C) Plasma membrane Na+/K+ ATPase. D. melanogaster Na+/K+ ATPase contains an α subunit and a β subunit with multiple isoforms. The monomeric structure from S. acanthias containing subunit α (green) and subunit β1 (yellow) is 77% and 25% homologous to the α and β2 subunits of D. melanogaster, which were detected by OxICAT. Cysteine residues on the S. acanthias structure present in homologous positions in the D. melanogaster α and β2 subunits are numbered. Cysteines observed by OxICAT are shown in red, and those not detected are blue. Disulfide cysteine partners are also labeled. The table shows the oxidation state of each cysteine in young control untreated flies.(D) Oxidation state of protein cysteine residues in mitochondria. Peptides from Figure 2B that are mitochondrial are plotted as the mean of the proportion of the total number of peptides in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Of 214 proteins identified in Figure 2A, 87 are mitochondrial, corresponding to 214 unique cysteine residues.(E) Comparison of peptides detected by OxICAT with transcript abundance. Whole-fly transcript intensity data were annotated to the head and thorax OxICAT dataset to characterize fly cysteines observable by mass spectrometry. Transcripts are subdivided by abundance into blocks that are √2 of the upper and lower bounds of the block immediately to the left. The percentage of both the observed OxICAT cysteine population (mean transcript abundance = 1,119; n = 849) and the total cysteine population (mean transcript abundance = 134; n = ∼135,000) falling within each transcript abundance block are on the y axis.See also Figure S2.

Mentions: In Figure 2A, the log10 intensity of the ion count for the peptide is plotted against the percentage oxidation of that cysteine residue. This shows there is no correlation between abundance and oxidation state that could indicate systematic bias in the methodology. We only considered cysteine residues that were labeled with both light and heavy ICAT labels in at least three biological replicates out of five. In control flies, we quantified the percentage oxidation of ∼537 cysteine residues, on 491 peptides, corresponding to 214 proteins (Figure 2A; Table S1). This compares favorably with previous OxICAT analyses, which based conclusions on ∼400 peptides from 290 proteins in yeast (Brandes et al., 2011), 170 peptides from 137 proteins in C. elegans (Knoefler et al., 2012), and 641 peptides from 333 proteins in mammalian cells (Go et al., 2011).


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)

OxICAT Analysis of Control Young Female D. melanogaster(A) Ion count for peptides plotted against percentage oxidation of the cysteine residue. The ion count is the log10 intensity of the sum of the heavy and light peptides. Data are the averages over three to five biological replicates. Red cross is cysteine residue 142 from TrxR1 (Figure 1C).(B) Distribution of total cysteine residue oxidation levels. Plotted are the means of the proportion of the total number of peptides containing unique cysteine residues in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Total unique peptides = 491.(C) Plasma membrane Na+/K+ ATPase. D. melanogaster Na+/K+ ATPase contains an α subunit and a β subunit with multiple isoforms. The monomeric structure from S. acanthias containing subunit α (green) and subunit β1 (yellow) is 77% and 25% homologous to the α and β2 subunits of D. melanogaster, which were detected by OxICAT. Cysteine residues on the S. acanthias structure present in homologous positions in the D. melanogaster α and β2 subunits are numbered. Cysteines observed by OxICAT are shown in red, and those not detected are blue. Disulfide cysteine partners are also labeled. The table shows the oxidation state of each cysteine in young control untreated flies.(D) Oxidation state of protein cysteine residues in mitochondria. Peptides from Figure 2B that are mitochondrial are plotted as the mean of the proportion of the total number of peptides in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Of 214 proteins identified in Figure 2A, 87 are mitochondrial, corresponding to 214 unique cysteine residues.(E) Comparison of peptides detected by OxICAT with transcript abundance. Whole-fly transcript intensity data were annotated to the head and thorax OxICAT dataset to characterize fly cysteines observable by mass spectrometry. Transcripts are subdivided by abundance into blocks that are √2 of the upper and lower bounds of the block immediately to the left. The percentage of both the observed OxICAT cysteine population (mean transcript abundance = 1,119; n = 849) and the total cysteine population (mean transcript abundance = 134; n = ∼135,000) falling within each transcript abundance block are on the y axis.See also Figure S2.
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fig2: OxICAT Analysis of Control Young Female D. melanogaster(A) Ion count for peptides plotted against percentage oxidation of the cysteine residue. The ion count is the log10 intensity of the sum of the heavy and light peptides. Data are the averages over three to five biological replicates. Red cross is cysteine residue 142 from TrxR1 (Figure 1C).(B) Distribution of total cysteine residue oxidation levels. Plotted are the means of the proportion of the total number of peptides containing unique cysteine residues in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Total unique peptides = 491.(C) Plasma membrane Na+/K+ ATPase. D. melanogaster Na+/K+ ATPase contains an α subunit and a β subunit with multiple isoforms. The monomeric structure from S. acanthias containing subunit α (green) and subunit β1 (yellow) is 77% and 25% homologous to the α and β2 subunits of D. melanogaster, which were detected by OxICAT. Cysteine residues on the S. acanthias structure present in homologous positions in the D. melanogaster α and β2 subunits are numbered. Cysteines observed by OxICAT are shown in red, and those not detected are blue. Disulfide cysteine partners are also labeled. The table shows the oxidation state of each cysteine in young control untreated flies.(D) Oxidation state of protein cysteine residues in mitochondria. Peptides from Figure 2B that are mitochondrial are plotted as the mean of the proportion of the total number of peptides in each 5% quantile of percentage oxidation across five biological replicates (mean ± SEM). Of 214 proteins identified in Figure 2A, 87 are mitochondrial, corresponding to 214 unique cysteine residues.(E) Comparison of peptides detected by OxICAT with transcript abundance. Whole-fly transcript intensity data were annotated to the head and thorax OxICAT dataset to characterize fly cysteines observable by mass spectrometry. Transcripts are subdivided by abundance into blocks that are √2 of the upper and lower bounds of the block immediately to the left. The percentage of both the observed OxICAT cysteine population (mean transcript abundance = 1,119; n = 849) and the total cysteine population (mean transcript abundance = 134; n = ∼135,000) falling within each transcript abundance block are on the y axis.See also Figure S2.
Mentions: In Figure 2A, the log10 intensity of the ion count for the peptide is plotted against the percentage oxidation of that cysteine residue. This shows there is no correlation between abundance and oxidation state that could indicate systematic bias in the methodology. We only considered cysteine residues that were labeled with both light and heavy ICAT labels in at least three biological replicates out of five. In control flies, we quantified the percentage oxidation of ∼537 cysteine residues, on 491 peptides, corresponding to 214 proteins (Figure 2A; Table S1). This compares favorably with previous OxICAT analyses, which based conclusions on ∼400 peptides from 290 proteins in yeast (Brandes et al., 2011), 170 peptides from 137 proteins in C. elegans (Knoefler et al., 2012), and 641 peptides from 333 proteins in mammalian cells (Go et al., 2011).

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