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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionOrganisms are continually exposed to environmental challenges that dramatically alter redox processes, changing the reduction potential of redox couples as well as the production of evanescent reactive species (). These redox changes can disrupt the molecular machinery of the organism, and consequently, cells contain short-term adaptive mechanisms and a parallel capacity for activating gene expression to maintain resilience. Two important environmental challenges that involve redox changes are aging and fasting.Aging correlates with changes in redox couples, increases in reactive species, and oxidative damage (), although their relationship with the mechanisms underlying aging has proven elusive. Fasting for 12–48 hr dramatically alters metabolic processes and is protective against ischemia-reperfusion injury () and alters signaling pathways in flies (). Intermittent starvation can be particularly effective in improving health and extending lifespan, and it may mediate some of the effects of dietary restriction (DR) (). In addition, it is not clear if DR slows changes that occur during aging or instead protects against their consequences for health and mortality (). While the molecular mechanisms underlying the benefits of fasting are obscure (), redox alterations are likely to be central.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 (). Cysteine residues are the most abundant cellular thiol, and in the mitochondrial matrix, the concentration is ∼20- to 30-fold greater than glutathione (GSH) (). A proportion of protein thiols are particularly reactive due to changes in pKa, accessibility and orientation wrought by the local environment (). 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 (). 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 (). Protein cysteine residues can also prevent local damage by sequestering reactive species (). 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 (). 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 (). 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 (href="/pmc/articles/PMC4508341/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figure 1A).href="/pmc/articles/PMC4508341/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=4508341_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC4508341/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC4508341/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1Assessment 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 href="#mmc1" rid="mmc1" class=" supplementary-material">Figure S1.