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Quantitative Mass Spectrometry Identifies Insulin Signaling Targets in C. elegans

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Science  03 Aug 2007:
Vol. 317, Issue 5838, pp. 660-663
DOI: 10.1126/science.1139952

Abstract

DAF-2, an insulin receptor–like protein, regulates metabolism, development, and aging in Caenorhabditis elegans. In a quantitative proteomic study, we identified 86 proteins that were more or less abundant in long-lived daf-2 mutant worms than in wild-type worms. Genetic studies on a subset of these proteins indicated that they act in one or more processes regulated by DAF-2, including entry into the dauer developmental stage and aging. In particular, we discovered a compensatory mechanism activated in response to reduced DAF-2 signaling, which involves the protein phosphatase calcineurin.

The insulin signaling pathway and insulinlike growth factor–1 (IGF-1) signaling pathway are conserved from invertebrates to mammals (1, 2). DAF-2, the sole homolog of the insulin receptor or IGF-1 receptor in C. elegans, controls the expression of presumably a large number of downstream targets by negatively regulating DAF-16, a FoxO transcription factor (38). In this study, we integrated technological advancements in quantitative mass spectrometry (MS) (911), including labeling multicellular organisms with the 15N stable isotope, to identify DAF-2 signaling targets. We made direct measurements of proteins as opposed to mRNAs, which are not final gene products. Our method allows for the identification of targets not regulated at the transcription level. The MS techniques demonstrated in this study can be readily used to determine protein abundance changes as a result of genetic or pharmacological perturbations.

We prepared lysates from wild-type (WT), daf-16(mu86) null, or daf-2(e1370ts) worms in their first day of adulthood, a period important for regulation of longevity by DAF-2 signaling (8). These lysate samples were mixed at a 1:1 ratio with a reference sample in which all proteins were labeled with 15N (atomic enrichment of 15N ≥ 96%) [(12) and fig. S1]. This reference sample was a lysate of WT worms fed with bacteria that were grown in a medium enriched in 15N. Soluble proteins (S100 fraction) from the lysate mixtures were then digested and analyzed by MS. A total of 1685 proteins were identified. Using its 15N-labeled counterpart as a reference, we determined the relative abundance of each unlabeled (i.e., 14N-labeled) protein with a modified version of the RelEx software (10). We also assessed the relative abundance of individual proteins by spectral counting (SC), in which we counted how many times the unlabeled version of a protein was identified by the fragmentation spectra of its peptides. Spectral counts correlate with protein abundance (11). We detected little difference between the daf-16 and WT samples (fig. S2A), reflecting the fact that daf-16 and WT adult worms were phenotypically similar under our experimental conditions (3, 4, 68). In contrast, we observed many protein abundance changes in the daf-2 mutants as compared with those in WT worms (fig. S2B), with an obvious correlation (i) between the RelEx and SC measurements (r = 0.59) and (ii) between the two daf-2 samples (r = 0.83, fig. S3). Using stringent criteria that emphasize high-quality measurements and consistent changes (table S1), we identified 86 proteins that were differentially expressed in the S100 fraction of the daf-2 sample (tables S2 and S3). Of these proteins, 47 were more abundant and 39 were less abundant than those in the WT sample. A subset of these proteins corresponds to known DAF-16 target genes, including superoxide dismutase sod-3 (7, 13).

To assess the quality of the quantitative MS results, we used selected reaction monitoring (SRM) to validate a subset of these results. For SRM, we measured the intensity of a specific fragment ion from a specific precursor ion (12). SRM is suitable for monitoring selected peptides at multiple time points. We reasoned that if a protein is truly regulated in daf-2 mutants, then the difference in its abundance between the WT and the daf-2 mutant animals should increase gradually during the temperature shift from 15°C (permissive for e1370) to 20°C (semi-permissive) and 25°C (nonpermissive). This was indeed the case with the three proteins that we monitored (Fig. 1, A to C). Western blotting results also correlated directly with MS results for nine proteins tested (Fig. 1, D to L, and fig. S4). Thus, the SRM and Western blotting results confirmed the accuracy of the quantitative proteomic results.

Fig. 1.

Confirmation of quantitative proteomic results by SRM and Western blotting. (A to C) SRM quantitation of the abundance of F15E11.1/14, D2096.8, and Y19D10B.7/F15E11.13 in WT (N2) and daf-2 mutant animals after a temperature shift from 15° to 20°C for 3 hours and then to 25°C for 20 hours. Samples were collected (at times indicated), lysed, and mixed at a 1:1 ratio with a 15N-labeled standard derived from WT worms. The S100 fractions were analyzed. (D to L) The amounts of nine proteins in the S100 fraction of WT, daf-16(mu86), or daf-2(e1370ts) worms were visualized by Western blotting. Shown below the blot of each protein is the relative abundance of that protein normalized to the WT amount as determined by RelEx with the use of a 15N-labeled standard [(D) to (K)] or by normalized SC (L).

Other researchers have previously identified DAF-16 target genes that barely overlapped between studies (1316). To help clarify this issue, we compared results (tables S2 and S3) and found that ours matched best with a microarray analysis by Murphy et al. (13). In total, only 35 proteins identified in this study were identified in previous studies (tables S2 and S3). The remaining 51 proteins may be previously unrecognized targets of the DAF-2 signaling pathway.

To understand the protein abundance changes in the context of protein function, we adopted GoMiner software (17) to classify the differentially expressed proteins on the basis of their annotation in the gene ontology database (table S2). Proteins whose abundance decreased in daf-2 mutant animals were enriched for functions in translation elongation and lipid transport. In contrast, proteins that became more abundant in daf-2 mutant animals were enriched for functions in amino acid biosynthesis, oxygen and reactive oxygen species metabolism, and carbohydrate metabolism [including gluconeogenesis and the glyoxylate cycle (GC), a modified version of the tricarboxylic acid cycle].

To determine whether the signaling targets identified in this study function in DAF-2–dependent processes such as dauer formation, we used RNA interference (RNAi) to analyze a subset of these targets (Fig. 2A). We found that npa-1 RNAi suppressed dauer formation in daf-2 mutants as strongly as did daf-16 RNAi. npa-1 encodes a polyprotein precursor for multiple fatty acid– binding and retinol-binding proteins (www.wormbase.org). This result suggests that NPA-1, which has increased abundance in the daf-2 mutant (tables S2 and S3), plays a critical role in dauer formation. Several genes functioning in carbohydrate metabolism also seem to be important for daf-2 dauer formation (Fig. 2A).

Fig. 2.

Function of a subset of the identified DAF-2 signaling targets in life-span regulation, dauer formation, or both. (A) Suppression of dauer formation of daf-2(e1370ts) animals by RNAi of npa-1 and several genes involved in carbohydrate metabolism. Percentages of dauers were averaged from four (for Ctrl, daf-16, R11A5.4, and gpd-2 RNAi) or two (for RNAi of the remaining genes) independent experiments (n > 110 animals in each experiment). Error bars indicate SE. Ctrl, control. (B and C) Effects of fbp-1, gpd-2, R11A5.4, and aco-2 RNAi on the life span of daf-2(e1370) mutant animals (one experiment, n ≥ 39 animals). (D) Effects of RNAi of inf-1, a translation initiation factor, and eft-2, an elongation factor, on the life span of WT worms (one experiment, n ≥ 80 animals). All P values are less than 0.05 (log-rank tests) between sample and control.

Given the importance of insulin signaling and IGF-1 signaling in aging, we asked whether these targets play a role in life-span regulation, and we focused on a subset of proteins involved in gluconeogenesis, GC, or translation. RNAi of some of the genes that we tested showed no significant effect on life span (table S4). However, RNAi of R11A5.4 (phosphoenolpyruvate carboxykinase), fbp-1 (fructose bisphosphatase), gpd-2 (glyceraldehyde-3-phosphate dehydrogenase), and aco-2 (aconitase) further extended the life span of daf-2 mutants (P < 0.05, log-rank test) (Fig. 2, B and C) and so did RNAi of gpi-1 (glucose phosphate isomerase) (18). RNAi of eft-2 (an elongation factor) or inf-1 (an initiation factor) shortened the life span of WT worms (P < 0.05, log-rank test) (Fig. 2D). Because R11A5.4, FBP-1, GPD-2, ACO-2, and GPI-1 showed increased abundance in the daf-2 mutant, whereas EFT-2 and INF-1 showed decreased abundance (table S2), these results suggest that abundance changes of proteins involved in carbohydrate metabolism or translation do not necessarily contribute to the longevity of daf-2 mutants. Instead, such changes may reflect a compensatory mechanism or mechanisms activated by a reduction in DAF-2 signaling.

Another protein exemplifying a compensatory mechanism is TAX-6 or CNA-1, the C. elegans calcineurin A protein that is the catalytic subunit of a serine or threonine protein phosphatase. The activity of calcineurin A requires the regulatory subunit calcineurin B (CNB-1 in C. elegans) (19). TAX-6 and CNB-1 are expressed in multiple cell types (20, 21) including intestinal epithelial cells (Fig. 3, A to F), a site of action for DAF-16 (22). Neither tax-6 nor cnb-1 has been implicated in the DAF-2 signaling pathway. Both genes lack the DAF-16 binding element (13) in a 1.5-kb region upstream of the start codon.

Fig. 3.

Identification of TAX-6 as a target and a positive regulator of DAF-2 signaling. (A to F) TAX-6::GFP was expressed in neurons (open arrows) and in the cytoplasm (solid arrows) and nucleus (arrowheads) of intestinal cells. tax-6(p675); Ex[pAK13] animals were imaged at magnifications 100× [(A) to (C)] and 400× [(D) to (F)]. DIC, differential interference contrast. (G) Expression of TAX-6::GFP in the intestine of animals treated with control, daf-2, or tax-6 RNAi. The intensity of cytoplasmic and nuclear TAX-6::GFP in the intestine was scored separately. Percentages of worms expressing strong (+++), intermediate (++), weak (+), very weak (+/–), or background (–) TAX-6::GFP in the intestine were averaged from three experiments (n ≥ 39 animals in each experiment). (H) Effects of the tax-6(p675) mutation on dauer formation and a synthetic effect with daf-2 RNAi. WT (“1” and “2”), daf-16 (mu86) (“3” and “4”), and tax-6(p675) (“5” and “6”) animals were treated with daf-2 (“2,”“4,” and “6”) or control RNAi (“1,”“3,” and “5”) at 27°C (black, one experiment) or 25°C (gray, average of two experiments) (n > 120 animals in each experiment). (I) daf-16(mu86) suppressed tax-6(p675) dauer formation (average of two experiments, n > 300 animals per experiment). (J) daf-16(mu86) suppressed the long life span of tax-6(p675) mutant animals (one experiment, n ≥ 80 animals, P < 0.01, log-rank test). (K) cnb-1 mutants displayed an extended life span (one experiment, n ≥ 80 animals, P < 0.01, log-rank test).

Our MS result indicated a higher TAX-6 abundance in daf-2 mutant worms as compared to WT worms (table S2). This was verified by Western blotting (Fig. 1L) and by comparing the green fluorescent protein (GFP) signal in animals expressing a TAX-6::GFP fusion protein (Fig. 3G) (20). When these worms were treated with daf-2 RNAi, cytoplasmic as well as nuclear TAX-6::GFP increased in intestinal cells.

Both tax-6 and cnb-1 loss-of-function mutants displayed phenotypes that were similar to but weaker than those of daf-2 mutants, such as an extended life span and increased propensity to entering the dauer phase (Fig. 3, H to K, fig. S5, and table S4). tax-6(p675) acted synergistically with daf-2(RNAi) (Fig. 3H), and daf-16(mu86) partially suppressed tax-6(p675) (Fig. 3, I and J). Thus, the TAX-6 and CNB-1 complex (i) facilitates DAF-2 signaling in life-span regulation and dauer formation and (ii) acts in parallel to DAF-16 or acts both upstream of and in parallel to DAF-16. Further epistasis analysis suggested that tax-6 acts upstream of or in parallel to age-1, which encodes a phosphatidylinositol 3-kinase downstream of daf-2 (see supporting online material text).

Although TAX-6 facilitates DAF-2 signaling, TAX-6 itself is regulated by DAF-2 because reduced DAF-2 signaling results in increased abundance of TAX-6. Thus, TAX-6 is part of a feedback loop that acts to maintain DAF-2 signaling at normal levels. This again suggests a compensatory mechanism. We propose that the life span of daf-2 mutants results from two types of changes: One change extends life span (e.g., SOD-3) and the other, represented by the compensatory mechanism(s) involving TAX-6 and several proteins functioning in carbohydrate metabolism or translation, shortens life span. Thus, inhibition of the compensatory mechanism can further extend the life span of daf-2 mutants (e. g., by aco-2 RNAi). Further investigation of the compensatory mechanism and DAF-2 signaling targets identified here is likely to aid the research of diabetes and aging in mammals.

Supporting Online Material

www.sciencemag.org/cgi/content/full/317/5838/660/DC1

Materials and Methods

SOM Text

Figs. S1 to S5

Tables S1 to S4

References

References and Notes

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