Carbonylated proteins are eliminated during reproduction in C. elegans
Jérôme Goudeau
Ecole Normale Supérieure de Lyon – CNRS – Université de Lyon Claude Bernard, Molecular Biology of the Cell Laboratory/UMR5239, 46, Allée d’Italie, 69364, Lyon Cedex 07, France
Search for more papers by this authorHugo Aguilaniu
Ecole Normale Supérieure de Lyon – CNRS – Université de Lyon Claude Bernard, Molecular Biology of the Cell Laboratory/UMR5239, 46, Allée d’Italie, 69364, Lyon Cedex 07, France
Search for more papers by this authorJérôme Goudeau
Ecole Normale Supérieure de Lyon – CNRS – Université de Lyon Claude Bernard, Molecular Biology of the Cell Laboratory/UMR5239, 46, Allée d’Italie, 69364, Lyon Cedex 07, France
Search for more papers by this authorHugo Aguilaniu
Ecole Normale Supérieure de Lyon – CNRS – Université de Lyon Claude Bernard, Molecular Biology of the Cell Laboratory/UMR5239, 46, Allée d’Italie, 69364, Lyon Cedex 07, France
Search for more papers by this authorSummary
Oxidatively damaged proteins accumulate with age in many species (Stadtman (1992) Science257, 1220–1224). This means that damage must be reset at the time of reproduction. To visualize this resetting in the roundworm Caenorhabditis elegans, a novel immunofluorescence technique that allows the detection of carbonylated proteins in situ was developed. The application of this technique revealed that carbonylated proteins are eliminated during C. elegans reproduction. This purging occurs abruptly within the germline at the time of oocyte maturation. Surprisingly, the germline was markedly more oxidized than the surrounding somatic tissues. Because distinct mechanisms have been proposed to explain damage elimination in yeast and mice (Aguilaniu et al. (2003) Science299, 1751–1753; Hernebring et al. (2006) Proc Natl Acad Sci USA103, 7700–7705), possible common mechanisms between worms and one of these systems were tested. The results show that, unlike in yeast (Aguilaniu et al. (2003) Science299, 1751–1753; Erjavec et al. (2008) Proc Natl Acad Sci USA105, 18764–18769), the elimination of carbonylated proteins in worms does not require the presence of the longevity-ensuring gene, SIR-2.1. However, similar to findings in mice (Hernebring et al. (2006) Proc Natl Acad Sci USA103, 7700–7705), proteasome activity in the germline is required for the resetting of carbonylated proteins during reproduction in C. elegans. Thus, oxidatively damaged proteins are eliminated during reproduction in worms through the proteasome. This finding suggests that the resetting of damaged proteins during reproduction is conserved, therefore validating the use of C. elegans as a model to study the molecular basis of damage elimination.
Supporting Information
Fig. S1 Example animals where protein carbonyls distribution can be assessed in somatic and germline tissues simultaneously. Left panels represent the tail of day-2 adult worms.
Fig. S2 Staining of oxidized proteins performed on animals at day-2 and day-5 of adulthood. Immuno-staining protocols are not strictly quantitative when samples are prepared on different slides. This type of comparison is therefore prone to error and cannot be rigorously quantified. Rather, we present trends.
Fig. S3 More example of the resetting of oxidized proteins within the gonad of four independent experiments.
Fig. S4 (A) Detection on the proteasome activity on extract from wild type animals treated with either the empty vector or pbs-1 RNAi for 48 h. Activities were measured over 30 min at 20 degrees in the absence or the presence of MG132, a specific inhibitor of the proteasome. To calculate the activity of the proteasome of each sample, signal obtained in the presence of MG132 (unspecific) was subtracted from the signal obtained with untreated animals. Fluorescence was measured with a temperature-controlled microplate fluorimetric reader (see experimental procedures). (B) Graphic representation of the proteasome activity obtained after 30 min of incubation of protein extracts obtained from wild type worms treated with either the empty vector (EV), pbs-1 or pas-6 RNAi with a fluorescent proteasome substrate (on average the activity is lowered by 40–55%. errors bars are sd. Unpaired two-tailed t-test; P-value<0.0001 for both treatment when compared to wild type). Our data shows that pbs-1 and pas-6 RNAi lowers the activity of the proteasome. All experiments were repeated at least two times on independent extracts. Error bars depict SD.
Fig. S5 Panel A shows the lifespan of wild type animals fed with either the empty vector or pbs-1 RNAi (mean lifespan of 16 and 10.5 days respectively, P < 0.0001). Panel B shows that the lifespan of rrf-1(pk1417) P0 animals treated with either pbs-1 RNAi or empty vector (Mean lifespan of 10.5 and 18 days, respectively, P < 0.0001). Panel C shows that the lifespan of rrf-1(pk1417) F1 animals treated either pbs-1 RNAi or empty vector (Mean lifespan of 9.5 and 17.5 days, respectively, P < 0.0001)) issued from P0 animals treated with the empty vector. Panel D shows that the lifespan of rrf-1(pk1417) F1 animals treated either pbs-1 RNAi or empty vector (Mean lifespan of 9.5 and 16.5 days, respectively, P < 0.0001) issued from P0 animals treated with pbs-1 RNAi. Lifespan analyses were performed at least twice independently. P values were calculated using the log rank (Mantel-Cox) analyses.
Table S1 Statistical analysis of the resetting of carbonylated proteins during reproduction in C.elegans.
Data S1 Supplementary material for detailed protocol.
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