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. 2016 Sep 13;16(11):3028-3040.
doi: 10.1016/j.celrep.2016.07.088.

FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

Ineke Dhondt  1 , Vladislav A Petyuk  2 , Huaihan Cai  1 , Lieselot Vandemeulebroucke  1 , Andy Vierstraete  1 , Richard D Smith  2 , Geert Depuydt  3 , Bart P Braeckman  4
Affiliations

FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

Ineke Dhondt et al. Cell Rep. .

Abstract

Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditiselegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

Keywords: (15)N-metabolic labeling; Caenorhabditis elegans; FOXO/DAF-16; aging; daf-2 mutant; protein turnover.

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Figures

Figure 1
Figure 1. Study Design
A15N metabolic labeling approach was performed to study individual protein turnover rates in long-lived daf-2 C. elegans. Adult worms were sampled at regular time points after pulsing with 15N-labeled E. coli. Samples were blocked based on the time of the pulse, randomized, and blindly analyzed using LC-MS/MS. The resulting datasets were processed using a custom R package. Protein half-lives of 245 overlapping proteins between reference and daf-2 worms were estimated for five and four biological replicates, respectively.
Figure 2
Figure 2. Slowdown of Protein Turnover in daf-2
(A) Volcano plot representing peptides with upregulated (red dots), downregulated (blue dots), and unchanged (black dots) turnover and their corresponding change in peptide half-life in daf-2. p Values were obtained from the moderated t test implemented in the limma R package and adjusted for multiple testing according to Benjamini and Hochberg's method. (B) Histogram showing the distribution of protein half-lives for reference and daf-2 worms. The median protein half-life is 103 hr for the reference strain and 173 hr for daf-2. Source data are available in Table S1.
Figure 3
Figure 3. Changes in Protein Turnover and Abundance Do Not Correlate in daf-2
Scatterplot illustrating the fold change (log2) of protein abundance and protein turnover in daf-2 relative to the reference strain (origin). Significant changes in protein turnover (p < 0.05) are signified with closed circles. p values were obtained from the moderated t test implemented in the limma R package and adjusted for multiple testing according to Benjamini and Hochberg's method. Ribo, ribosomal; Mito, mitochondrial; Cytosk, cytoskeletal; ER; Lyso, lysosomal; Nucl, nuclear; Cytopl, cytoplasmic; Extracell, extracellular; Chap, chaperones; Unclass, unclassified proteins. Inset: case study of SODH-1 and ALH-1 demonstrating discordance between transcript (McElwee et al., 2007) and protein (Depuydt et al., 2013) abundance versus protein half-life (fold change [log2]) in daf-2. Source data are available in Table S2.
Figure 4
Figure 4. Protein Dynamics in Subcellular Compartments
(A and B) Scatterplot comparing significant (A) and non-significant (B) changes of protein half-lives in daf-2 versus the reference strain. Functional groups are color-coded and summarized in pie chart insets. p Values were obtained from the moderated t test implemented in the limma R package and adjusted for multiple testing according to Benjamini and Hochberg's method. Error bars are SEM. (C and D) Protein half-lives in different cellular compartments with differentiation of membrane-bound proteins (open symbols) and free proteins (filled symbols). Significantly changed half-lives are represented in (C) (two-sample Student's t test, p < 0.05), and non-significant changes are depicted in (D). Source data are available in Table S4.
Figure 5
Figure 5. Individual Protein Half-Lives of daf-2 and Reference Replicates and Estimation of Mitochondrial Turnover Rates with the Photoswitchable Reporter Dendra2
(A–C, E–H, and J–M) Heatmap representation of individual protein half-lives of daf-2 and reference replicates. The colors indicate a relative decrease (red) or increase (blue) of protein half-life compared with the median of the reference strain (white, 103 hr). Color limits signify the 5th (41 hr) and the 95th (291 hr) percentile. Each tile represents a biological replicate. p Values were obtained from the moderated t test implemented in the limma R package and adjusted for multiple testing according to Benjamini and Hochberg's method. Asterisks indicate significant differences between both strains. *p < 0.05, **p < 0.01, ***p < 0.001, respectively. (D) Confocal images of jrIs5 transgenic worms expressing Dendra2 under the constitutive rps-0 promotor and targeted to the mitochondria with the mitochondrial localization signal of gas-1. Left: overview of Dendra2-expressing worms. Scale bar, 100 μm. Right: detail of the midsection of the body. Scale bar, 10 μm. (I) Linear regression of the decline in red Dendra2 fluorescence after photoconversion in reference versus daf-2 strains (F-test, p < 0.0001). Source data are available in Table S4.
Figure 6
Figure 6. Organization of the Rough Endoplasmic Reticulum
(A and B) Transmission electron micrographs of intestinal cells of reference (A) and daf-2 worms (B). Arrows indicate the RER. int, intestine; lys, lysosome; m, mitochondrion; g, glycogen; lip, lipid. Scale bars, 1 μm.
Figure 7
Figure 7. Validation of the SILeNCe Method with the Dendra2 Approach
(A and B) Protein half-lives of RLA-1 and ASP-4, estimated via SILeNCe pulse labeling (A) and the Dendra2 pulse-chase method (B). Red fluorescence decline was determined in glp-4 daf-2 worms expressing RLA-1∷Dendra2 and ASP-4∷Dendra2 translation reporters and treated with daf-16 (reference) and empty vector (L4440) RNAi (daf-2) (minimum six individually tracked worms per condition; two-sample Student's t test; p = 0.0019 and p = 0.6278 for RLA-1 and ASP-4, respectively). Error bars are SEM. (C and D) Representative confocal images of glp-4 daf-2;rla-1p∷rla-1∷Dendra2 (C) and glp-4 daf-2; asp-4p∷asp-4∷Dendra2 (D) transgenic worms grown on daf-16 RNAi (reference) and empty vector L4440 (daf-2). Images were taken 96 hr (RLA-1) and 48 hr (ASP-4) after photoconversion. Scale bars, 20 μm.
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