Monitoring Newly Synthesized Proteins over the Adult Life Span of <i>Caenorhabditis elegans</i>

Little is known regarding how the synthesis and degradation of individual proteins change during the life of an organism. Such knowledge is vital to understanding the aging process. To fill this knowledge gap, we monitored newly synthesized proteins on a proteome scale in <i>Caenorhabditis elegans</i> over time during adulthood using a stable-isotope labeling by amino acids in cell culture (SILAC)-based label-chase approach. For most proteins, the rate of appearance of newly synthesized protein was high during the first 5 days of adulthood, slowed down between the fifth and the 11th days, and then increased again after the 11th day. However, the magnitude of appearance rate differed significantly from protein to protein. For example, the appearance of newly synthesized protein was fast for proteins involved in embryonic development, transcription regulation, and lipid binding/transport, with >70% of these proteins newly synthesized by day 5 of adulthood, whereas it was slow for proteins involved in cellular assembly and motility, such as actin and myosin, with <70% of these proteins newly synthesized even on day 16. The late-life increase of newly synthesized protein was especially high for ribosomal proteins and ATP synthases. We also investigated the effect of RNAi-mediated knockdown of the <i>rpl-9 (</i>ribosomal protein), <i>atp-3 (</i>ATP synthase), and <i>ril-1 (</i>RNAi-induced longevity-1) genes and found that inhibiting the expression of <i>atp-3</i> and <i>ril-1</i> beginning in late adulthood is still effective to extend the life span of <i>C. elegans.</i

SIR-2.4, but not SIR-2.1, is required for stress-induced DAF-16 nuclear localization.

<p>TJ356 animals carrying an integrated <i>daf-16::gfp</i> array were fed either vector control or <i>sir-2.4</i> RNAi bacteria for at least one generation before being subjected to heat-shock or oxidative stress. (A) Images of TJ356 animals grown on control or <i>sir-2.4</i> RNAi bacteria after 15 min heat-shock. (B) Quantification of DAF-16::GFP nuclear accumulation in response to heat-shock (35°C for 15 min.) or oxidative stress (1.5 mM H₂O₂ for 1 hr). Worms were scored for the presence or absence of GFP accumulation within the intestinal nuclei (n = 120 or greater for all treatments). An animal was scored as having nuclear GFP if one or more intestinal nuclei contained DAF-16-GFP. (C–D) Time course analysis of DAF-16::GFP nuclear accumulation in response to stress. TJ356 or EQ200 [<i>sir-2.4(n5137)</i>; <i>daf-16::gfp</i>] animals grown on either control or <i>sir-2.1</i> RNAi bacteria were subjected to (C) heat-shock (35°C) or (D) oxidative stress (1.5 mM H₂O₂). Worms were scored for GFP accumulation within the head hypodermic nuclei at day 1 of adulthood (n = 30∼50) every 5–30 min.</p

Minimal impact of SIR-2.4 on IIS-induced longevity, DAF-16 nuclear localization induced by reduced IIS, and dauer formation.

<p>(A) Lifespan analysis of wild-type (N2) animals or <i>daf-2(e1370)</i> mutants grown on vector control bacteria (black or red) or <i>sir-2.4</i> RNAi bacteria (green or blue) at 20°C. (B) DAF-16 nuclear localization was assessed in TJ356 (expressing <i>daf-16::gfp</i> in WT background) or EQ200 (expressing <i>daf-16::gfp</i> in <i>sir-2.4(n5137)</i> background) animals. Animals were fed with either vector control or <i>daf-2</i> RNAi from the time of hatching. Worms were scored for the presence or absence of GFP accumulation within the head hypodermic nuclei as day 1 adult (n = 116 or greater) under unstressed condition. <i>P</i>-values were calculated by Pearson's chi-square test. (C) <i>daf-2(e1370)</i> mutants (P₀) were fed with control or <i>sir-2.4</i> RNAi bacteria at 20°C. F₁ eggs were then moved to 22°C for 72 hours prior to being scored for dauer formation (n = 336 or greater). <i>P</i>-values were calculated by Pearson's chi-square test.</p

SIR-2.4 inhibits CBP1-mediated DAF-16 acetylation.

<p>(A) DAF-16 nuclear localization was assessed in TJ356 (expressing <i>daf-16::gfp</i> in WT background) or EQ200 (expressing <i>daf-16::gfp</i> in <i>sir-2.4(n5137)</i> background) animals. Animals (n = 90 or greater) were scored for DAF-16::GFP nuclear translocation as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002948#pgen-1002948-g004" target="_blank">Figure 4B</a>. (B) <i>cbp-1</i> KD decreases DAF-16 acetylation. DAF-16 acetylation was assessed in TJ356 worms grown on either control or <i>cbp-1</i> RNAi by acetyl-lysine immunoprecipitation followed by GFP immunoblot. (C) SIR-2.4 blocks CBP1-dependent DAF-16 acetylation <i>in vitro</i>. Purified DAF-16 was incubated with CBP in the presence of WT SIR-2.4 or the SIR-2.4 NA mutant at 37°C. DAF-16 acetylation levels were assessed as described in (B).</p

SIR-2.4 interacts with DAF-16 and promotes DAF-16 deacetylation and function independently of catalytic activity.

<p>(A) <i>sir-2.4</i> deletion promotes DAF-16 hyperacetylation. DAF-16 acetylation was assessed in control or <i>sir-2.4</i> KO worms by acetyl-lysine immunoprecipitation followed by GFP immunoblot. (B) SIR-2.4 and DAF-16 interact. Plasmids encoding FLAG-tagged SIR-2.4 and HA-tagged DAF-16 were transfected into 293T cells as indicated (GFP, negative control). Immunoprecipitation and immunoblotting were performed as shown. (C) Rescue of DAF-16 nuclear localization with a catalysis-defective <i>sir-2.4</i> mutant. Stable transgenic strains of <i>sir-2.4(n5137)</i> were generated expressing either wild-type SIR-2.4 or the <i>sir-2.4</i> N124A mutant. Worms were scored for GFP accumulation within the head hypodermic nuclei as day 1 adult (n = 50 or greater) after 20 min of heat-shock at 35°C. <i>P</i>-values were calculated by Pearson's chi-square test.</p

SIR-2.4 is required for optimal DAF-16–dependent gene expression.

<p>Wild-type N2 animals fed on either vector control or <i>sir-2.4</i> RNAi bacteria from the time of hatching were exposed to 10 mM H₂O₂ for 80 min. Relative mRNA levels of SOD-3, HSP-16.1, DOD-3, DOD-24, C32H11.4, and INS-7 were measured by quantitative RT-PCR and the means of three different sample sets are shown. Relative mRNA levels were normalized against ACT-1 (beta-actin). Error bars: ± STD. Statistical significance as determined by two-tailed t-test is shown in the table below; significant differences are represented in black font.</p

Model: SIR-2.4 promotes DAF-16 deacetylation and function during stress.

<p>See text for details.</p

SIR-2.4 promotes stress resistance.

<p>(A) Wild-type N2 or <i>sir-2.4(n5137)</i> worms grown on vector control or <i>daf-16</i> RNAi bacteria were subjected to heat-shock at 35°C and scored for viability every 1-2 hours. (B) Wild-type N2 or <i>daf-16(mu86)</i> worms grown on vector control or <i>sir-2.4</i> RNAi bacteria were treated with 1.5 mM H₂O₂ and scored for viability every 1–2 hours. Data are mean survival±SEM, in hours for (A–B). ***, <i>p</i><0.001; ns, <i>p</i>>0.05. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002948#pgen.1002948.s006" target="_blank">Table S1</a> for statistical analysis. (C) AM140 worms expressing a poly-Q tract (Q35::YFP) were seeded on the RNAi bacteria indicated and scored for poly-Q induced paralysis every other day. Data are mean time to paralysis in days±SEM. ***, <i>p</i><0.001; **, <i>p</i><0.01; ns, <i>p</i>>0.05.</p