We applied mass-spectrometry-based quantitative proteomics to completely NER-deficient xpc-1(tm3886);csb-1(ok2335) double-mutant C. elegans. We analyzed synchronized worms at the first larvae stage (L1), 6 hr after UVB or mock treatment. Proteins were digested in solution followed by peptide identification and quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS) ( Figure 1 A). In total, more than 7,500 proteins were quantified at a false discovery rate (FDR) of less than 1% at the protein and peptide spectrum match level, of which more than 5,000 proteins were quantified between UV and untreated conditions at least in two out of three biological replicates. Excellent reproducibility (r > 0.95 for biological replicates) was determined with the Pearson correlation coefficient (r). Hierarchical clustering revealed strong segregation of different conditions indicating distinct proteomic changes and high data quality that allows for systematic analysis (see also Figure S1 ). By using a two-sided t test and correcting for multiple testing by estimating the FDR to 5% with a permutation-based algorithm, we identified about 1,000 significantly differentially expressed proteins, of which more than 550 proteins were more than 2-fold altered between UV and untreated conditions ( Table S1 ).

(B) Significantly increased (red; >1.5-fold up) or decreased (blue; >1.5-fold down) proteins (FDR < 5%) in the different subcellular compartments (see Table 1 for details on clusters).

To systematically analyze protein changes upon persistent UV lesions, we used Gene Ontology (GO) classification as well as UniProt (January 2016 release) and the C. elegans portal WormBase (version WS246). Abundance changes of significantly regulated proteins were observed in most of the subcellular compartments ( Figure 1 B). In the volcano plot, the logratio of UV-treated worms to untreated worms for each protein group is plotted against the respective –logp value ( Figure 1 C). In order to improve the annotations of C. elegans proteins and obtain insights into potential functions, we used BLAST search results (e < 10) of well-annotated human and mouse proteins. We used GO, Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) annotations provided by the UniProt database for C. elegans protein entries and the corresponding human orthologs raising protein annotations from ∼35% to ∼62% ( Figure S2 ).

We used 1D enrichment to identify groups of proteins that are involved in identical pathways, carry similar PFAM domains, or localize in the same compartment (e.g., categorical annotations). We visualized significantly regulated groups (Benjamini-Hochberg FDR < 0.02) by plotting the mean logratio of UV-treated to mock-treated worms for all proteins with the particular categorical annotation against the enrichment score ( Figure 1 D). Categories grouping proteins related to nuclear mechanisms and synaptic machinery showed a positive enrichment score, whereas categories related to protein synthesis and cellular metabolic processes showed a significant negative score ( Table S2 ). Overall, the systematic analysis indicates widespread changes of protein levels upon UV-induced DNA damage in C. elegans.

The significantly enriched upregulated proteins belonging to the nuclear GO category ( Figure 1 B; Table 1 ) includes chromatin remodelers (CHD-7, BAF-1, SWSN-4, SNFC-5, and LMN-1), transcription regulators (HMG-1.2, RTFO-1, STA-1, NONO-1, EMB-5, SPT-4, HCF-1, and SMK-1), and histone post-translational modifiers (SPR-5, HIL-2, HTZ-1, and HDA-3) associated with the epigenetic control of gene expression. The chromatin-associated proteins BAF-1, SWSN-4, and HCF-1 were previously shown to interact with the IIS effector DAF-16 to remodel chromatin and activate transcription (). Other transcription factors mediate specifically the response to DNA damage and oxidative stress (SMK-1) () or play a role in the UV-induced DNA damage response in mammalian cells (NONO-1) (). The upregulated proteins include transcription elongation, pre-mRNA processing proteins, and ribonucleoprotein (RNP) ( Table 1 ), in line with changes in spliceosome organization and the post-translational modifications of splicing factors, recently implicated in the DNA-damage response ().

Proteins belonging to the categories related to nuclear mechanisms such as chromatin remodelers, regulator of transcription, protein-DNA complex, and structures of the nuclear pore showed clear upregulation, consistent with chromatin remodeling modulating replication and transcription in response to DNA damage. In addition, the increased expression of members of the synaptic machinery and G protein signaling partners, belonging to plasma membrane and extracellular space categories, suggests that signals are released from genotoxically compromised cells that mediate the adaptation to the damage.

Similar to aged IIS mutant worms () and cells responding to DNA damage (), proteins belonging to the nuclear category implicated in DNA replication and cell-cycle progression (CDK-1, MCM-2, MCM-7, and RFC-4) were decreased in abundance upon UV treatment ( Table S1 ).

Increased proteins regulating translation, spliceosome assembly, and nuclear-cytoplasmic transport suggest an involvement of RNA biogenesis and translocation in the DNA damage response ( Table 1 ). The nuclear pore complex proteins (nucleoporins [NPPs]), together with Ran-GTPases, play an important role not only in nuclear import and export and nuclear envelope (NE) assembly dynamics but also in the localization of MEL-28 (), a structural NE component that regulates the distribution of the integral nuclear-envelope proteins EMR-1, LMN-1, LEM-2, and BAF-1 ( Table 1 ) (). These nuclear proteins provide an anchor attaching chromosomes to the nuclear membrane, are required for proper chromosome segregation (), and promote the reorganization of damaged chromatin upon UVC and ionizing radiation (IR)-induced DNA damage (). When responding to stress, BAF-1 is immobilized at the nuclear lamina, stabilizes the chromatin structure, and influences the gene expression via histone post-translational modifications (). Exposure of human cells to UV-induced DNA damage causes BAF-1 to dynamically interact with the histone H3/H4 ubiquitin ligase complex (CUL4-DDB-ROC1), facilitating the recruitment of repair proteins to the damaged DNA (). BAF-1 expression is regulated by transcription factors that modulate lifespan, including SKN-1, PHA-4, DAF-16, and ELT-3 ().

MEL-28 is downstream of the Ran cycle and is required for nuclear-envelope function and chromatin maintenance.

Increased extracellular proteins were mainly hormone carrier transthyretin (TTR)-related factors, also reported as elevated in aged C. elegans () and associated with neuroprotection in a murine AD model (). The extracellular Cu/Znsuperoxide dismutase SOD-4 and some lipid binding proteins and transporters (NRF-5, LBP-1, and EGL-3), which sequester respectively potentially toxic peroxidation products and toxic fatty acids (FAs), were also upregulated ( Table 1 ).

Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome.

We also observed upregulation of proteins belonging to plasma membrane and extracellular space, suggesting possible intra- or extracellular trafficking of signals from genotoxically compromised cells ( Figures 1 B and 1D). The plasma membrane category contains transmembrane channel proteins, ATPases, amino acid, ion and ATP transporters, and heterotrimeric G proteins (key regulators of G protein-coupled receptor [GPCR] signaling) ( Table 1 ). GPCR signaling has been implicated in fundamental aspects of development and behavior, including the synaptic transmission in the ventral cord motor neurons (). Excitable cells display the highest expression of heterotrimeric G proteins together with components of the endocytic pathway involved in the initial vesicles assembly (ARF-6, ARL-8, and DYN-1), vesicle fusion (SNAP-29 and AEX-3), and vesicles recycling through the endo-lysosomal system (ITSN-1 and SQST-1). The upregulation of those proteins might indicate neuronal signals responding to DNA damage ( Table 1 ). DNA repair defects have been linked to the impaired neuronal development in various human congenital progeroid syndromes, including CS, and to age-related neurodegenerative disorders such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) (). Consistent with neuronal developmental processes being affected by unrepaired DNA damage, we found also elevated levels of proteins implicated in axonal outgrowth (EAT-6, CAM-1, UNC-44, and TBB-4) and neuronal positioning during development (SAX-7, WRK-1, UNC-33, and UNC-37) ( Table S1 ).

A large number of ribosomal proteins, including components of the small (40S), large (60S), and mitochondrial ribosome subunits, together with components of the translation machinery, were downregulated upon UV treatment ( Table 1 ). A similar drop was observed also for factors involved in protein homeostasis, localized between cytoplasm, mitochondria, endoplasmic reticulum (ER), peroxisomes, and for regulators of FA metabolism ( Figures 1 B and 1D; Table 1 ). This general decline in protein synthesis and dampening of metabolic processes upon UV treatment is consistent with previous reports from proteomic studies of aged animals (), supporting parallels between the DNA damage response and aging.

To monitor autophagy, we used a GFP-fusion transgene of the ubiquitin-like, microtubule-associated Atg8/LC3 ortholog LGG-1 required for autophagic vesicle growth (). Within 4–10 hr after UV treatment, we observed significantly increase of the lipidated LGG-1(II) form indicative of autophagy ( Figures 2 B and S3 ). To assess whether autophagy was required for withstanding DNA damage, we tested the UV sensitivity of two autophagy mutants, atg-3(bp412) and atg-9(bp564). We observed a significantly higher sensitivity of the autophagy mutants than of wild-type (WT) worms ( Figures 2 C and S4 ), suggesting that proteins involved in the formation of autophagosomes are essential for enduring DNA damage. The impaired UPS machinery and the increased autophagy activity are discussed below in the network analysis ( Figure 5 ).

Impairment of chaperones and UPS machinery give rise to misfolded proteins that that are imported into lysosomes during chaperone-mediated autophagy, or sequestered in autophagosomes during macroautophagy (). Upon DNA damage, we found an upregulation of macroautophagy sub-pathway members: ATG-3, ATG-18, and SQST-1, the p62 homologous. Autophagy, via the elimination of SQST-1, was recently implicated in the regulation of the DNA damage response via chromatin ubiquitination (). The decrease in protein synthesis, together with the impairment of protein refolding and degradation mechanisms and the decreased mitochondrial homeostasis, suggests general organismal energy depletion upon UV-induced DNA damage. Given the role of autophagy in degrading aberrant proteins and in recycling nutrients and energy, we hypothesized a proteostatic shift toward autophagy to allow the organism tolerating the consequences of impaired protein homeostasis ( Figure 2 A).

(C) WT, atg-3(bp412), and atg-9(bp564) L1 larvae were irradiated or mock treated, and developmental stages were evaluated 48 hr later. An average of three independent experiments per strain and dose is shown; >15 individuals were analyzed per experiment. Error bars denote standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (two-tailed t test compared with WT).

(B) Immunoblotting of the autophagy marker LGG-1(I) and LGG-1(II)::GFP. LGG-1 becomes lipidated after UV-induced DNA damage and starvation (representative of three independent experiment shown).

Impaired protein homeostasis has been suggested as a hallmark of aging (). Protein-fold stabilization restores the structure of misfolded polypeptides, or remove and degrade, via the proteasome or the lysosome, aberrant proteins. Upon UV treatment, many components of the proteostasis network, as chaperones, ubiquitin ligases, and members of the ubiquitin-proteasome system (UPS) machinery, together with ER, peroxisomal and mitochondrial homeostasis-related proteins were downregulated ( Table 1 ). This included the E3 ubiquitin ligase Y54E10A.11 that contains a RING-type-domain homologous to human TRAIP, implicated in the cellular UV response. Missense mutations in the TRAIP RING-domain have been identified in patients with premature aging syndromes (). Y54E10A.11 is a component of the ribosome quality-control complex (RQC), which recognizes stalled ribosome and associates with the 60S subunit, allowing the ubiquitination and extraction of incompletely synthesized nascent polypeptides (). The translation stress specifically sensed by the RQC is communicated to the transcription factor HSF-1 (), which in turn promotes lifespan extension (), suggesting a combined strategy to play a role in both longevity and stress responses.

Taken together, these observations suggest that amid persistent DNA damage worms reduce DNA replication and translation, thus potentially avoiding the production of aberrant proteins. Moreover, protein-refolding mechanisms are reduced, whereas autophagy is elevated, suggesting a rerouting of protein recycling as part of metabolic shift in response to the DNA damage.

Translation and autophagy are regulated, in parallel to IIS signaling, by the target of rapamycin (CeTOR) LET-363 in complex with the raptor protein DAF-15 to influence cell growth and longevity (). Upon UV treatment, autophagy-related proteins as well as CeTOR and IIS pathway members were elevated ( Table S1 ), reminiscent of the increase of the same members of those pathways during aging (). Autophagy has been reported to mobilize lipids via the breakdown of lipid droplets (lipophagy) (). Upon UV treatment, we observed a decrease of proteins involved in lipid metabolism and localized between the cytoplasm, ER, peroxisomes and mitochondria ( Table 1 ), similar to their decrease during C. elegans aging ().

To address the role of transcriptional responses to UV-induced DNA damage, we compared the proteomes with previously published transcriptome data of xpa-1 mutants as NER deficient as the xpc-1;csb-1 mutants (and phenotypically identical in response to UV irradiation) (). We found a significant moderate positive correlation (r = 0.347) between the significantly changed transcripts and proteins upon UV treatment ( Figure 3 A; Table S3 ), suggesting that the expression of only a part of proteins can be explained by transcription, while a large fraction is subject to post-transcriptional regulation (see later discussion).

(A–C) Correlation analyses (A) between proteome of xpc-1;csb-1 double mutants (FDR < 5%) and transcriptome of xpa-1 mutants after UV treatment (similarly regulated proteins and genes in red and green; specific protein clusters are detailed in Table S3 ), (B) between proteins detected in xpc-1;csb-1 double mutants upon UV treatment versus aging in WT worms (p < 2.2 × 10for the three Pearson correlation coefficients, r), and (C) between proteins changed in abundance of at least 2-fold (FDR < 5%) in xpc-1;csb-1 double mutants upon UV treatment versus starvation.

To address whether proteome changes in response to DNA damage might bear similarities to those occurring during aging, we conducted a correlation analysis between proteomes of UV-treated xpc-1;csb-1 double mutants, unable to repair the UV-induced DNA damage, and WT worms during aging (). Indeed, the proteomes of UV-treated NER deficient animals and WT worms during aging were positively correlated (day 12, r = 0.26; day 27, r = 0.34; Figure 3 B), suggesting that the regulation at the protein level upon persistent DNA damage bears similarities to proteome alterations during aging. The significant positive correlations were more striking when we compared the DNA damage responses of L1 larvae with those of aging adult animals. The similarly regulated processes revealed a general enrichment of factors involved in FA metabolism, oxidative stress response, UPR, and IIS.

L1 larvae arrest their growth not only upon genotoxic treatment but also for extended periods of time in the absence of food and resume developmental growth only when food becomes available. We have previously found similar and contrasting transcription responses between starvation conditions and UV-induced DNA damage (). In parallel to UV treatment, we also performed starvation experiment in xpc-1;csb-1 double mutants: three independent biological replicates were analyzed, with excellent reproducibility (r > 0.95 for biological replicates) (see also Figure S1 ). We obtained a positive Pearson correlation between the proteomes of UV-irradiated and starved animals (r = 0.77) ( Figure 3 C). The similarities were composed of proteins associated with chromatin, vesicle/neurotransmitter trafficking including heterotrimeric G proteins implicated in the starvation-induced activation of the Ras-MAP kinase pathway (), and metabolic pathways involved in the synthesis and use of carbohydrate, amino acid, and lipids ( Tables 1 and S1 ). Key enzymes involved in FA biosynthesis ( Figure 4 A) and playing important roles in FA accumulation and consumption during lifespan () were downregulated ( Tables 1 and S1 ). The expression of the same class of genes related to lipid metabolism has been found significantly decreased in the UV-irradiated and photoaged human skin, suggesting that inhibition of de novo lipid synthesis could have a detrimental effect, leading also to collagen destruction () ( Table S1 ).

(C and D) Changes in the amount of three SLs subclasses (ceramides, sphingomyelins, and glucosylceramides) (C) and of the five glycerophospholipids subclasses (phosphatidylcholine [PC], phosphatidylethanolamine [PE], PI, phosphatidylserine [PS], and phosphatidylglycerol [PG]) (D) assessed by MS analysis.

(A) Key members of the FA biosynthetic coupled to SL and phospholipid metabolic pathways were significantly decreased in abundance in xpc-1;csb-1 double mutants upon starvation and UV treatment.

UV decreases the synthesis of free fatty acids and triglycerides in the epidermis of human skin in vivo, contributing to development of skin photoaging.

Another major component of cellular membranes is the lipid class of glycerophospholipids, synthesized from the intermediate phosphatidic acid, through a series of reduction and acylation reactions ( Figure 4 A). Phosphatidic acid is dephosphorylated to yield DAG, which is converted into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which can be both intermediates for the formation of phosphatidylserine (PS). PS and phosphatidylinositol (PI) are generally synthesized from cytidine diphosphatediacylglycerol (CDP-DAG), substrate for the synthesis of phosphatidylglycerol (PG) and cardiolipin (). Quantitative glycerophospholipids MS profiling, upon starvation and UV treatment, showed a change of the DAG downstream products, indicating a preferential direction in the phospholipid synthesis ( Figures 4 D and S4 ). Upon UV, the PC and the PC-derived PS were increased, whereas PE was reduced. In contrast, upon starvation, the PE and the PE-derived PS were elevated, whereas PC was decreased. Other CDP-DAG-derived phospholipids (PI and PG) were not changed, except for a significant reduction of PG in response to starvation ( Figures 4 D and S4 ). Taken together, these observations suggest that the worms respond to persistent DNA damage by a metabolic shift reminiscent of adaptations during starvation () and aging ().

Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome.

The downstream products of these FA biosynthetic pathways are normally used to synthesize more complex lipids: saturated FAs (SFAs) serve as building blocks for the sphingolipids (SLs), whereas both SFAs and unsaturated FAs (UFAs) are incorporated into glycerophospholipids ( Figure 4 A). SLs are highly conserved components of cell membranes having regulatory roles in growth control and aging in a wide range of organisms (). SL works as an intermediate for the production of ceramide (Cer), a key product for the synthesis of glucosylceramide and sphingomyelin (SM) () ( Figure 4 A). Cer is produced from SM in UV- and IR-treated mammalian cells (), whereas an increased synthesis of SM from Cer is associated to accelerated development and aging (). Similarly to aging studies, MS-based quantitative SL profiling showed a general increase in SM and decrease in Cer upon both treatments ( Figures 4 C and S4 ), potentially as a consequence of the impaired SFAs biosynthesis. elo-5 mutants, deficient for monomethyl branched chain FA (mmBCFA) synthesis, arrest development similar to starved L1 larvae () and could be rescued by SFA-derived SLs, d17iso-glucosylceramides (d17iso-GlcCer), together with downstream factors of the CeTOR pathway (). Intriguingly, upon UV treatment, we observed an increased abundance of members of the CeTOR pathway ( Table S1 ) and of d17iso-GlcCer ( Figures 4 C and S4 ). In line with a previous study () reporting stable mmBCFA levels in starved L1 larvae, we also observed stable levels of d17iso-GlcCer species upon starvation ( Figures 4 C and S4 ). The role of the GlcCer/TOR pathway in promoting development independently from the IIS and DAF-7/TGFβ-signaling () suggests that it regulates the developmental response to UV-induced DNA damage.

A branched-chain fatty acid is involved in post-embryonic growth control in parallel to the insulin receptor pathway and its biosynthesis is feedback-regulated in C. elegans.

A branched-chain fatty acid is involved in post-embryonic growth control in parallel to the insulin receptor pathway and its biosynthesis is feedback-regulated in C. elegans.

Prompted by the alterations in FA biosynthesis enzymes, we next traced lipid profiles of xpc-1;csb-1 double mutants upon UV treatment and starvation by using thin-layer chromatography (TLC) and MS. We observed a decrease in triacylglycerols, the storage form of FAs ( Figures 4 B and S4 ) that is consistent with the worms’ deriving energy from degradation of fat stored to survive stress such as food deprivation ().

Proteome and Phosphoproteome-Coupled Analysis to Build a Regulatory Network