In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1,2,3,4,5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6,7,8,9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.
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All relevant data are available and/or included with the manuscript or its Supplementary Information. RNA-sequencing data have been uploaded to the European Nucleotide Archive under the study accession PRJEB36386. Source data are provided with this paper.
The source code for the bioinformatics analysis of homologues is available at https://github.com/Ashafix/C_Elegans_Homologs.
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We thank M. Schölling for help with statistics based on the ordinal logistic regression model. We thank C. Kenyon for providing some C. elegans strains and J. Fares for providing NP717. JM103 E. coli strains were provided by R. V. Aroian. Some C. elegans strains and Microbacterium nematophilum were provided by the C. elegans Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), the International C. elegans Gene Knockout Consortium and the National BioResource Project (NBRP). This work was supported by funding from the DZNE (D.C.D.), Max Planck Society (R.J.S.), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (SFB 1035 to M. H., DA 1906/4-1 to D.C.D.) and a Marie Curie International Reintegration Grant (322120 to D.C.D.).
The authors declare no competing interests.
Peer review information Nature thanks Carmen Nussbaum and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Absence of coelomocytes causes LBP-2 to accumulate in pseudocoelom together with secreted GFP.
a, LBP-2::tagRFP expression pattern in body-wall muscles (day 2, n = 14 worms). b, c, Secreted LBP-2::tagRFP and secreted GFP colocalize in day 2 animals with coelomocytes (n = 16 worms) (b) and accumulate in animals without coelomocytes (n = 6) (c). Asterisk indicates pharyngeal GFP reporter in animal without coelomocytes. b, c, Secreted GFP exposure, 5 times shorter in c versus b, secreted LBP-2::tagRFP, identical exposure. d, LBP-2::tagRFP puncta in tail region (day 2, n = 15 worms; day 8, n = 14). Maximum projection. Scale bar, 20 μm. e, Quantification of LBP-2::tagRFP aggregation with age in the tail (n = 2 independent experiments). P values determined by two-sided Fisher’s exact test (day 8) and chi-square test (day 12). f, LBP-2 aggregates are separate from neurons (n = 27 worms). Scale bar, 20 μm. Single plane. g, Total protein stain of blot in Fig. 1j (n = 2 independent experiments) with fold changes quantified per fraction relative to levels in day 2. For blot source image, see Supplementary Fig. 1. Source data
a, Pie charts depict results from RNAi screen targeting genes encoding secreted factors and their effect on LBP-2 aggregation. b, Quantification of LBP-2::tagRFP aggregation with RNAi targeting top 13 candidates from egg in non-sterile background at day 4 (n = 1 independent experiment). Ctrl (−) is empty vector; ctrl (+) is rme-1 RNAi. P values determined by ordinal logistic regression and for lys-3 and tag-196 RNAi treatment by two-sided Fisher’s exact test. c, Maximum projection of head region of day 4 transgenics overexpressing LBP-2::tagRFP subjected to RNAi targeting a subset of ECRs (empty vector n = 8 worms, clec-1 n = 2, F56B6.6 n = 5, lys-3 n = 7, C36C5.5 n = 5). Scale bar, 20 μm. Laser intensity 8%. d, e, Downregulation of ECRs by RNAi does not change total levels of LBP-2::tagRFP (n = 2 independent experiments). Western blot detection of LBP-2::tagRFP in total fraction at day 4, 25 °C. Control 1, 2 and 3 are empty vector. Fold changes (in d) are normalized to total protein levels quantified by protein staining (e). For blot source images, see Supplementary Fig. 1. Source data
a, Quantification of animals without GFP-labelled coelomocytes in transgenic animals expressing secreted GFP (GS1912) treated with RNAi targeting 13 top candidates (n = 2 independent experiments). Ctrl (−) denotes empty vector; ctrl (+) denotes dyn-1 RNAi. b, LYS-7::tagRFP in young whole animal. Arrowheads indicate localization in coelomocytes, and asterisk indicates localization in anterior intestinal cells (top panel, n = 10 worms). LYS-7::tagRFP diffuse localization in head region of young animal (bottom left panel, n = 19) and puncta localized in head region of aged animal (bottom right panel, n = 14). Laser intensity 15%, maximum projection. Scale bar, 20 μm. c, Quantification of LYS-7::tagRFP aggregation with age (n = 2 independent experiments). d, Effect on LYS-7::tagRFP aggregation at day 6, 25 °C, with RNAi targeting top 13 candidates (n = 4 independent experiments). e, LYS-7 aggregation is reduced by CLEC-1 overexpression quantified at day 6 (n = 2 independent experiments). Ctrl indicates CLEC-1 non-overexpressing animals. P values determined by two-sided Fisher’s exact test with Benjamini–Hochberg correction. Source data
a, Overexpression of ECRs reduces LBP-2 aggregation (ssGFP n = 13 worms, LYS-3 n = 27, F56B6.6 n = 21, CLEC-1 n = 19, C36C5.5 n = 24). Scale bar, 20 μm. Maximum projection, laser intensity 8%. b, Secreted GFP does not accumulate in LBP-2 aggregates (n = 21 worms). Scale bar, 20 μm. Single plane. c, Coomassie staining of co-purification of C36C5.5 with LBP-2 (n = 2 independent experiments). Open arrow indicates LBP-2::tagRFP; closed arrow denotes C36C5.5::mVenus::histag. Three independent co-purification experiments with the same starting material are shown (elution 1–3).
Extended Data Fig. 5 Extracellular proteostasis influences ageing and is differentially regulated during ageing.
a, F56B6.6-overexpressing and LYS-3-overexpressing animals are long-lived compared with non-overexpressing siblings (n = 2 independent experiments). b, Animals lacking coelomocytes are short-lived compared with control animals (n = 3 independent experiments). c, Secreted GFP (n = 2 biologically independent samples) and LBP-2 overexpression (n = 1 independent experiment) do not influence lifespan. P values were determined by log-rank test (a–c). For detailed values, see Extended Data Table 1. d, Changes in expression levels of ECR candidates with age (day 8 versus day 2 at 25 °C). Left, expression level in LBP-2 overexpressing sterile animals. Right, expression level in wild-type sterile animals. Data are means ± s.e.m. of n = 4 biologically independent samples. P values were determined by two-sided unpaired t-test with Welch’s correction. Source data
Extended Data Fig. 6 Impairing extracellular proteostasis accelerates intoxication-related mortality.
a, Expression level of LBP-2::tagRFP is not reduced after 3 h exposure to 100% Cry5B. Data are mean ± s.e.m. of n = 4 biologically independent samples. b, Quantification of LBP-2::tagRFP aggregation upon exposure to Microbacterium nematophilum and Bacillus atrophaeus at day 6 (n = 2 independent experiments). c, Expression level of four selected ECR candidates in unchallenged conditions with control pQE9 empty vector in kgb-1(km21) mutant versus wild-type background. d, Expression levels of eight selected ECRs with vhp-1 versus control RNAi. Data are mean ± s.e.m. of n = 3 (c) and n = 4 (d) biologically independent samples. e, Survival analysis of LBP-2::tagRFP transgenics on jnk-1(gk7) versus wild-type background subjected to 50% Cry5B (n = 2 independent experiments). f, Survival analysis of LBP-2::tagRFP transgenics subjected to 25% Cry5B with RNAi targeting selected ECRs (n = 2 independent experiments). g, Survival analysis of secreted GFP transgenics with versus without coelomocytes, subjected to 50% Cry5B (n = 4 independent experiments). h, Survival analysis of LBP-2::tagRFP transgenics versus N2 wild-type subjected to 50% Cry5B (n = 1 independent experiment). i, Survival analysis of LBP-2::tagRFP transgenics with and without secreted GFP overexpression subjected to 50% Cry5B (n = 2 biologically independent samples). j, Increased LBP-2::tagRFP aggregation at day 4 of adulthood during exposure to 25% Cry5B and treatment with RNAi targeting selected ECRs compared to empty vector (n = 2 independent experiments). P values determined by two-sided unpaired t-test with Welch’s correction (a, c, d), chi-square test (b) and two-sided Fisher’s exact test with Benjamini–Hochberg correction (j), and log-rank test with Bonferroni correction (e–i). For detailed values see Extended Data Tables 2, 3. Source data
Heat map of z-score-normalized expression data (rows correspond to genes, columns indicate samples). The dendrogram on the top shows that RNA-seq samples cluster by treatment (Cry5B versus control).
This file contains Supplementary Table 4 (List of 57 extracellular regulators of protein aggregation and predicted human orthologs); Supplementary Table 5 (the transgenic strains and alleles used); Supplementary Table 7 (Primer sequences for qRT-PCR); and Supplementary Figure 1 (Uncropped scans with size marker indications).
| Bioinformatic functional analysis of ECR candidates and their aggregation score.
| Enrichment analysis of ECRs among genes regulated by microbes. Excel list of ECRs significantly enriched in genes differentially regulated in response to microbes. Analysis was performed with WormExp (one-sided Fisher’s exact test).
| RNA sequencing of ECR overexpressing transgenics. Excel file with genes differentially regulated in at least two ECR overexpressing transgenics compared to control in Cry5B challenged conditions (sheet 1) or un-challenged conditions (sheet 2) (see Methods for statistics), enrichment analysis of genes upregulated during Cry5B related to microbes (sheet 3, WormExp one-sided Fisher’s exact test).
| List of predicted secreted coding genes tested by RNAi. Excel file list of genes with predicted signal peptide and without transmembrane domains knocked-down by RNAi to investigate their effect on LBP-2 aggregation.
| LBP-2 aggregates localize outside body-wall muscles. Confocal z-stack of C. elegans head with LBP-2 aggregates (magenta) and F-actin stained with phalloidin (green) at day 6 of adulthood. Representative video of n = 16 worms.
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Gallotta, I., Sandhu, A., Peters, M. et al. Extracellular proteostasis prevents aggregation during pathogenic attack. Nature 584, 410–414 (2020). https://doi.org/10.1038/s41586-020-2461-z
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