Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb 18;13(1):967.
doi: 10.1038/s41467-022-28599-9.

ATF-4 and hydrogen sulfide signalling mediate longevity in response to inhibition of translation or mTORC1

Cyril Statzer #  1 , Jin Meng #  2   3   4 , Richard Venz  1 , Monet Bland  2   3   4 , Stacey Robida-Stubbs  2   3   4 , Krina Patel  2   3   4 , Dunja Petrovic  5 , Raffaella Emsley  6 , Pengpeng Liu  7 , Ianessa Morantte  8 , Cole Haynes  7 , William B Mair  8 , Alban Longchamp  6 , Milos R Filipovic  5 , T Keith Blackwell  9   10   11 , Collin Y Ewald  12
Affiliations

ATF-4 and hydrogen sulfide signalling mediate longevity in response to inhibition of translation or mTORC1

Cyril Statzer et al. Nat Commun. .

Abstract

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H2S) production. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H2S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H2S levels, or enhancing mechanisms that H2S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. ATF-4 is preferentially translated under conditions of reduced global protein synthesis.
a Schematic diagram of the atf-4 mRNA and the Patf-4(uORF)::GFP reporter. UTRs are represented as empty boxes, exons as filled boxes, and the basic leucine zipper domain (bZIP) in red. b Representative images showing that reducing translation by administering 7.2 mM cycloheximide for 1 hour or 35 μg/ml tunicamycin (TM) for 4 h increased expression of transgenic Patf-4(uORF)::GFP in L4 stage animals. Quantification of GFP fluorescence intensity is shown in Supplementary Fig. 1c. Scale bar = 100 μm. c A 1 h pre-treatment with 0.7 μg/mlα-amanitin (RNA Pol II inhibitor) prevented 4 h of 35 μg/ml TM treatment from increasing atf-4 mRNA levels in L4 stage animals. Mean ± SEM. Three independent trials, measured in duplicates. P values are relative to untreated wild type (N2) determined by one sample t-test, two-tailed, hypothetical mean of 1. d A 1 h pre-treatment with 0.7 μg/mlα-amanitin did not prevent TM treatment from increasing levels of transgenic Patf-4(uORF)::GFP expression in L4 stage animals. Mean ± SEM. n > 30 animals examined over 2 independent experiments, One-way ANOVA with post hoc Tukey. e Stage-specific ribosome occupancy profiles of the endogenous atf-4 mRNA, along with quantification of relative uORF occupancy (f). Analysis of ribosomal profiling data revealed a decrease in ribosome occupancy on the endogenous atf-4 uORFs under unstressed conditions during late larval development. Mean ± SD, n = 2 biological replicates. Occupancy profiles were generated by assigning counts to the atf-4 transcript based on the number of raw reads at each position. Blue boxes indicate the atf-4 exons. One-way ANOVA post hoc Dunnett’s test.
Fig. 2
Fig. 2. ATF-4 mediates lifespan extension from translation inhibition.
a Adult-specific knockdown of ifg-1 extended the lifespan of WT animals but not atf-4(tm4397) mutants. b Adult-specific treatment with 25 μM cycloheximide increased lifespan dependent upon atf-4. c Representative western blots and quantification (d) showing that treatment with 35 μg/ml tunicamycin for 4 h dramatically increased eIF2α phosphorylation levels in L4 stage animals, while treatment with 7.2 mM cycloheximide for 1 h did not. Mean ± SEM. n = 3 biological replicates. One-way ANOVA with post hoc Tukey. e Representative western blots and quantification (f) showing the effects of adult-specific knockdown of eif-1.A, ifg-1, ragc-1, or rict-1 on eIF2α phosphorylation levels. Mean ± SEM. n = 3 biological replicates. One-way ANOVA with Dunnett’s post-test compared to EV. g Representative western blots of puromycin incorporation assay and quantification (h) showing that adult-specific knockdown of ifg-1, ragc-1, or rict-1 decreased translation. Mean ± SEM. n = 3 biological replicates. One-way ANOVA with Bonferroni post-test. i Quantification of GFP fluorescence showing that adult-specific ifg-1 knockdown increases expression of Patf-4(uORF)::GFP. j Adult-specific knockdown of ifg-1 comparably extended the lifespan of WT animals and eif-2α/y37e3.10(qd338) phosphorylation-defective mutants. For statistics and additional trials in (a), (b), and (j), see Supplementary Data 1. For western blots, source data are provided in Source data File.
Fig. 3
Fig. 3. ATF-4 overexpression is sufficient to increase lifespan.
a Transgenic animals (wbmEx26 [Patf-4::ATF-4(gDNA)::GFP]) that overexpress ATF-4 (ATF-4OE) live longer compared to their non-transgenic siblings. b Pharyngeal pumping rate is similar at day 2 of adulthood between ATF-4OE (ldIs119 [Patf-4::ATF-4(gDNA)::GFP]) and WT, but higher in ATF-4OE at day 10 of adulthood, suggesting an improved healthspan. For the complete time-course of pharyngeal pumping rate during ageing, see Supplementary Data 2. Mean ± SEM. At least 5 worms were examined for each genotype and time point in one experiment. Unpaired two-tailed t-test. c MA (log ratio and mean average)-plot of RNA sequencing analysis comparing ATF-4OE(ldIs119) to WT, showing absolute log fold change (FC). In red, highlighted genes with FDR < 0.1 and log FC > 1 compared to WT. In black, genes with FDR > 0.1. Details are in Supplementary Data 3. d Validation by qRT-PCR of genes differentially expressed in ATF-4OE(ldIs119), using two new independent biological samples of about 200-215 animals each measured in duplicates. Mean ± SEM. P values relative to WT determined by one sample t-test, two-tailed, hypothetical mean of 1. The numbers of ATF4 binding sequences (-TGATG-), are indicated in Supplementary Data 4. The DAF-16 and SKN-1 transcription factor binding sites are based on ChIP data from www.modencode.org (Supplementary Data 5). e Longevity conferred by ATF-4OE(ldIs119) is abolished by knockdown of hsf-1, skn-1, or daf-16. Mean ± SEM of Kaplan–Meier survival plot. P-value determined by log-rank. n = 1 biological replicate. f The mitochondrial ATP translocase ant-1.3 is required for ATF-4 overexpression induced longevity. For statistical details and additional lifespan trials in (a), (e), and (f), see Supplementary Data 1.
Fig. 4
Fig. 4. ATF-4 overexpression increases H2S levels via cystathionine gamma-lyase, which is required for longevity.
a Heatmap of gene expression in ATF-4OE (ldIs119), wild type (N2), and atf-4(tm4397) showing genes whose orthologs are directly regulated by mammalian ATF4 (Details are in ‘Methods’, Supplementary Data 4). Absolute levels of expression were compared. Genes in light blue are predicted to be involved in the transsulfuration pathway, which is shown in (b). c ATF-4OE(ldIs119) showed higher cth-2 mRNA levels compared to WT by qRT-PCR. n = 3 independent biological samples in duplicates (each over 200 L4 worms). Mean ± SEM. P values relative to WT determined by one-sample t-test, two-tailed, a hypothetical mean of 1. d Quantification of CTH protein levels in ATF-4OE(ldIs119) compared to WT. n = 6 independent biological trials probed in 3 western blots. One-tailed t-test. e Western blots showing an ATF-4-induced increase in CTH levels was abolished by atf-4 or cth-2 knockdown. NS = non-specific band. f ATF-4 overexpression increases H2S production capacity in a cth-2-dependent manner. Additional biological trials are shown in Supplementary Fig. 4d. For H2S quantification, see Supplementary Data 12. g Representative fluorescent microscopy images and quantification showing that H2S levels in vivo were decreased in either atf-4(tm4397) or cth-2(mg599) mutants compared to WT. Data are represented as Mean ± SEM. n = 3 biological replicates of a total of 15 worms per condition. P values to WT are unpaired t-test, two-tailed. Scale bar = 50 μm. h Lifespan extension induced by ATF-4 overexpression depends upon cth-2. i Lifespan extension induced by ifg-1 knockdown requires cth-2. j Model for how ATF-4 promotes stress resistance and longevity. For statistical details and additional trials in (h) and (i), see Supplementary Data 7. For western blots, source data are provided in Source data File.
Fig. 5
Fig. 5. ATF-4 and CTH-2 regulate protein persulfidation levels.
a Schematic diagram showing that the thiol group (–SH) of reactive cysteine residues in proteins can undertake various redox states. Sulfenylation (–SOH) can be reversed, particularly efficiently through the intermediate of persulfidation (–SSH), but sulfinylation (–SO2H) is reversible only within peroxiredoxins and sulfonylation (–SO3H) is irreversible,. Arrows in orange indicate oxidation processes while those in blue indicate reduction processes. b Representative fluorescent images and quantification (c) showing that ATF-4OE exhibited higher persulfidation levels, while cth-2(mg599) animals exhibited lower global persulfidation levels, compared to WT. Data are represented as Mean ± SEM. n = 3 biological replicates of at least 10 worms per condition. Scale bar = 50 μm. F.I. = fluorescent intensity. P values to WT are unpaired t-test, two-tailed.
Fig. 6
Fig. 6. ATF-4 is essential for longevity from reduced mTORC1 activity.
a Inhibition of mTORC1 by either raga-1 or ragc-1 knockdown led to preferential translation of ATF-4. RNAi treatments were initiated at the L4 stage, with GFP intensity scored at day 3 of adulthood. b Inhibition of mTORC1 or mTORC2 by knockdown of ragc-1 or rict-1, respectively, leads to preferential translation of ATF-4. Similar effects were observed in WT and eif-2α(qd338) mutants. c Post-development knockdown of raga-1 or rict-1 extends lifespan in both WT and eif-2α(qd338) mutants. d Mutation in raga-1 increases lifespan in an atf-4-dependent manner. e Reducing mTORC1 signalling by adulthood specific raga-1 knockdown improves healthspan dependent upon atf-4, as assessed by pharyngeal pumping rate. Mean ± S.E.M. P values relative to WT of the corresponding day with One-way ANOVA with post hoc Dunnett’s multiple comparisons test. f Adult-specific knockdown raga-1 increases oxidative stress resistance (2 mM tert-butyl hydrogen peroxide (tBOOH)) in an atf-4-dependent manner. RNAi was started at the L4 stage, and stress resistance was measured at day 3 of adulthood with the lifespan machine (See Supplementary Data 10 for details). g Rapamycin treatment during adulthood extends lifespan independently of atf-4. h Adult-specific knockdown of the mTORC2 subunit rict-1 extends lifespan in an atf-4-dependent manner. i Adult-specific inactivation of both mTORC1 and mTORC2 increases lifespan independently of atf-4. For statistical details and additional trials in (a), (b), see Supplementary Data 9. For statistical details and additional lifespan trials in (c), (d), (g)-(i), see Supplementary Data 1.
Fig. 7
Fig. 7. Longevity from mTOR inhibition upregulates H2S and requires cth-2.
a Assay of C. elegans lysates showing that raga-1 mutation increased H2S production capacity in an atf-4-dependent manner. Two additional independent biological trials are shown in Supplementary Fig. 6d. b Assay of C. elegans lysates showing that rict-1 mutation increased H2S production capacity in an atf-4-dependent manner. An additional independent biological trial is in Supplementary Fig. 6e. c Longevity of raga-1(ok386) mutants is ablated by cth-2 knockdown. This particular experiment was performed at 25 °C. d Longevity induced by adult-specific knockdown of either raga-1 or rict-1 depends upon cth-2. e, f Representative images showing persulfidation levels in WT (N2), cth-2 (mg599), atf-4 (tm4397), raga-1 (ok386), and raga-1;atf-4 mutants. Data are represented as Mean ± SEM. n = 3 biological replicates of at least 10 worms per condition. Scale bar = 50 μm. P values to WT are unpaired t-test, two-tailed. g Inhibition of mTORC1 promotes longevity by increasing ATF-4 expression and stimulating H2S production. For H2S quantification in (a, b), see Supplementary Data 12. For statistical details and additional lifespan trials in (c, d), see Supplementary Data 1.
See this image and copyright information in PMC

References

    1. Kenyon CJ. The genetics of ageing. Nature. 2010;464:504–512. - PubMed
    1. Blackwell TK, Sewell AK, Wu Z, Han M. TOR Signaling in Caenorhabditis elegans development, metabolism, and aging. Genetics. 2019;213:329–360. - PMC - PubMed
    1. Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 2020;21:183–203. - PMC - PubMed
    1. Ben-Sahra I, Manning BD. mTORC1 signaling and the metabolic control of cell growth. Curr. Opin. Cell Biol. 2017;45:72–82. - PMC - PubMed
    1. Bjedov I, et al. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab. 2010;11:35–46. - PMC - PubMed

Publication types

MeSH terms

Substances