A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans

Abstract

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

Keywords: C. elegans; cullin-RING ubiquitin ligase complex; heat shock; intracellular pathogen response; proteostasis.

Fig. 1.

CUL-6 expression in the intestine or in the pharynx promotes thermotolerance. (A) Confocal fluorescence images of L4 animals with cul-6::GFP transgenes driven either from the endogenous promoter and expressed from a multicopy in the case of cul-6p::cul-6::GFP (29), or by the vha-6 or myo-2 promoter and integrated with MosSCI. (B) Survival of animals after 2 h of 37.5 °C heat shock, followed by 24 h at 20 °C (hereafter referred to as “heat shock treatment”). Strains were tested in triplicate experiments, with three plates per experiment, 30 animals per plate. The genotypes myo-2p::APX::GFP and jyIs8[pals-5p::GFP; myo-2p::mCherry] were tested as controls for myo-2p-driven expression. Each dot represents a plate and different shapes represent the experimental replicates done on different days. Mean fraction alive of the nine replicates is indicated by the black bar with errors bars as SD. ***P < 0.001, one-way ANOVA with Tukey’s post hoc multiple comparisons test. DIC, differential interference contrast.

Fig. 2.

Coimmunoprecipitation mass spectrometry analysis identifies binding partners for CUL-6. Volcano plot of proteins significantly enriched in CUL-6 IP compared to F42A10.5 IP (A) or to GFP IP (B). Proteins significantly more abundant compared to either of the control IPs (GFP alone control or F42A10.5 control, at adjusted P < 0.05 and log2 fold change >1) were considered interacting proteins (Dataset S1). Gray dots indicate nonsignificant proteins, red dots indicate significant proteins, green dots indicate significant SCF proteins, and blue dots indicate significant proteasome subunits.

Fig. 3.

RING domain protein RCS-1 (C28G1.5) promotes thermotolerance in pals-22 mutants. (A) Phylogenetic relationships of RCS-1 protein with TRIM23 homolog proteins (in red), canonical RBX proteins (in black), and known TRIM proteins (in blue; all are C. elegans except noted human gene). The tree was built from a protein alignment using the Bayesian Markov chain Monte Carlo (MCMC) method. Posterior probabilities are indicated on the branches. (B) Confocal fluorescence images of L4 animals with rcs-1::GFP driven by the endogenous promoter and expressed from a multicopy array (29). (C) rcs-1 isoforms and exon/intron structures. Protein domains are colored in green and yellow. jy84 and jy105 are deletion alleles. (D and E) Survival of animals after heat shock treatment. Strains were tested in triplicate experiments, with three plates per experiment, 30 animals per plate. Each dot represents a plate, and different shapes represent the experimental replicates done on different days. Mean fraction alive of the nine replicates is indicated by the black bar with errors bars as SD. ***P < 0.001, n.s., not significant, P > 0.05, one-way ANOVA with Tukey’s post hoc multiple comparisons test. DIC, differential interference contrast.

Fig. 4.

SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance in pals-22 mutants. (A) skr-3, skr-4, and skr-5 gene exon/intron structures. ok365 and ok3068 are deletion alleles, gk759439 is a premature stop mutation. (B) Survival of animals after heat shock treatment. ***P < 0.001, n.s., not significant, P > 0.05, one-way ANOVA with Tukey’s post hoc multiple comparisons test. n = 9 replicates per strain. (C) Confocal fluorescence images of L4 animals with skr-5::GFP driven by the endogenous promoter and expressed from a multicopy array (29). (D) Survival of animals after heat shock treatment. ***P < 0.001, **P < 0.01, one-way ANOVA with Tukey’s post hoc multiple comparisons test. n = 7 replicates per strain. (E) Survival of animals after heat shock treatment. skr-5 and pals-22; skr-5 mutants were fed on (R)OP50 expressing either L4440 (control vector) or skr-3 RNAi. ***P < 0.001, two-way ANOVA with Sidak’s multiple comparisons test. n = 9 replicates per condition. For B, D, and E strains were tested in triplicate experiments, 30 animals per plate. Mean fraction alive of the replicates is indicated by the black bar with errors bars as SD. Each dot represents a plate, and different shapes represent the experimental replicates done on different days. DIC, differential interference contrast.

Fig. 5.

FBXA-158 and FBXA-75 promote thermotolerance in pals-22 mutants. (A) Survival of animals after heat shock treatment. pals-22 mutants were fed on (R)OP50 expressing either L4440 (control vector) or RNAi for the indicated genes. pals-22 mutants were tested in duplicate experiments, with two plates per experiment, 30 animals per plate. ***P < 0.001, **P < 0.01, n.s., not significant, P > 0.05, one-way ANOVA with Tukey’s post hoc multiple comparisons test. (B) Survival of animals after heat shock treatment. Wild-type and vha-6p::cul-6 animals were fed on (R)OP50 expressing either L4440 (control vector), cul-6, or fbxa-158 RNAi. Strains were tested in quadruplicate experiments, with three plates per experiment, 30 animals per plate. ***P < 0.001, *P < 0.05, two-way ANOVA with Sidak’s multiple comparisons test. (C) fbxa-158 isoforms and exon/intron structures. jy145 and jy146 are deletion alleles. (D) Survival of animals after heat shock treatment. ***P < 0.001, n.s. P > 0.05, one-way ANOVA with Tukey’s post hoc multiple comparisons test. (E) Survival of animals after heat shock treatment. Wild-type and vha-6p::cul-6 animals were fed on (R)OP50 expressing either L4440 (control vector), cul-6, or fbxa-75 RNAi. Strains were tested in triplicate experiments, with three plates per experiment, 30 animals per plate. **P < 0.01, *P < 0.05, two-way ANOVA with Sidak’s multiple comparisons test. (F) fbxa-75 exon/intron structure. jy143 and jy144 are deletion alleles. (G) Survival of animals after heat shock treatment. ***P < 0.001, **P < 0.01, n.s. P > 0.05, one-way ANOVA with Tukey’s post hoc multiple comparisons test. For A, B, D, E, and G each dot represents a plate, and different shapes represent the experimental replicates done on different days. Mean fraction alive of the replicates is indicated by the black bar with errors bars as SD.

Fig. 6.

Model for a RCS-1/CUL-6/SKR/FBXA-158 or FBXA-75 ubiquitin ligase that promotes proteostasis. Cullin-RING ligase components are transcriptionally up-regulated as part of the intracellular pathogen response. CUL-6 acts together with the RING domain protein RCS-1, functionally redundant Skp-related proteins SKR-3, SKR-4, and SKR-5, and both FBXA-75 and FBXA-158 to promote thermotolerance and improved proteostasis. CUL-6 also requires neddylation by NED-8. FBXA-158 and FBXA-75 may act sequentially to ubiquitinate the same target, or alternatively may have distinct targets.