Ion channels are necessary for proper water and nutrient absorption in the intestine, thereby supporting cellular metabolism and organismal growth. While a role for Na+ co-transporters and pumps in intestinal nutrient absorption is well defined, how individual K+ uniporters function in this process is poorly understood. Mutations in the Caenorhabditis elegans genes drl-1 and flr-4, which encode two unique kinases that are components of a mitogen-activated protein kinase (MAPK) pathway, or the flr-1 Na+ ion channel, cause severe growth defects, reduced lipid storage, and a dramatic increase in autophagic lysosomes. Here, we show that a gain-of-function mutation in twk-26, which encodes a 2-pore domain K+ ion channel orthologous to human KCNK3, facilitates nutrient absorption and suppresses the metabolic and developmental defects caused by loss of DRL-1, FLR-4, or FLR-1 signaling. We reveal that these phenotypes likely arise from impaired intestinal amino acid absorption, which is restored upon activation of TWK-26. Furthermore, we show that loss of flr-4 disrupts intracellular and extracellular pH gradients, suggesting that the FLR-4 pathway may be necessary to maintain intestinal ion homeostasis and facilitate nutrient absorption. Importantly, the altered pH gradients in the flr-4 mutant are partially restored by the twk-26 gain-of-function mutation. Thus, this study uncovers a new role for the TWK-26 ion channel in governing intestinal physiology and metabolism.

Organisms allocate energetic resources between essential cellular processes to maintain homeostasis and, in turn, maximize fitness. The nutritional regulators of energy homeostasis have been studied in detail; however, how developmental signals might impinge on these pathways to govern metabolism is poorly understood. Here, we identify a non-canonical role for Hedgehog (Hh), a classic regulator of development, in maintaining intestinal lipid homeostasis in Caenorhabditis elegans. We demonstrate, using C. elegans and mouse hepatocytes, that Hh metabolic regulation does not occur through the canonical Hh transcription factor TRA-1/GLI, but rather via non-canonical signaling that engages mammalian target of rapamycin complex 2 (mTORC2). Hh mutants display impaired lipid homeostasis, decreased growth, and upregulation of autophagy factors, mimicking loss of mTORC2. Additionally, we find that Hh inhibits p38 MAPK signaling in parallel to mTORC2 activation to modulate lipid homeostasis. Our findings reveal a non-canonical role for Hh signaling in lipid metabolism via regulation of core homeostatic pathways.

Caenorhabditis elegans proliferates poorly in the presence of abundant Actinobacteria from its natural ecology, but it is unknown why. Here, we show how perturbed levels of hydrogen sulfide modulate the growth rate of both C. elegans and Actinobacteria. From a forward genetic selection, we find C. elegans mutants with faster growth on an Actinobacteria Microbacterium species and mutant alleles of conserved cystathionine gamma-lyase (cth-2/CTH) that improve growth rate. Conversely, null alleles of cth-2 cause developmental arrest of animals grown on Actinobacteria, but not on Proteobacteria, which can be rescued by exogenous H2S. We also find mutations in a leucine-rich-repeat gene that regulates cysteine and H2S production, lrr-2/LRRC58. We place lrr-2 in the animal sulfur metabolism pathway by demonstrating its role in post-translationally regulating levels of cysteine dioxygenase (cdo-1/CDO1). Exogenously supplied H2S inhibits the growth of Actinobacteria but not Proteobacteria. Thus, we conclude that the C. elegans-Actinobacteria interaction is mediated by H2S.

The molecular mechanisms that govern the metabolic commitment to reproduction, which often occurs at the expense of somatic reserves, remain poorly understood. We identified the Caenorhabditis elegans F-box protein FBXL-5 as a negative regulator of maternal provisioning of vitellogenin lipoproteins, which mediate the transfer of intestinal lipids to the germline. Mutations in fbxl-5 partially suppress the vitellogenesis defects observed in the heterochronic mutants lin-4 and lin-29, both of which ectopically express fbxl-5 at the adult developmental stage. FBXL-5 functions in the intestine to negatively regulate expression of the vitellogenin genes; and consistently, intestine-specific over-expression of FBXL-5 is sufficient to inhibit vitellogenesis, restrict lipid accumulation, and shorten lifespan. Our epistasis analyses suggest that fbxl-5 functions in concert with cul-6, a cullin gene, and the Skp1-related gene skr-3 to regulate vitellogenesis. Additionally, fbxl-5 acts genetically upstream of rict-1, which encodes the core mTORC2 protein Rictor, to govern vitellogenesis. Together, our results reveal an unexpected role for a SCF ubiquitin-ligase complex in controlling intestinal lipid homeostasis by engaging mTORC2 signaling.

RNA interference (RNAi) is an evolutionarily conserved pathway that defends against viral infections in diverse organisms. Caenorhabditis elegans mutations that enhance RNAi have revealed pathways that may regulate antiviral defense. A genetic screen for C. elegans mutations that fail to up-regulate a defense response reporter transgene detected mutations that enhance RNAi to silence this reporter gene in the inositol polyphosphate multikinase impk-1 , the synMuv B gene lin-15B, and the pathogen defense response gene pals-22 . Using other assays for enhanced RNAi, we found that the impk-1 alleles and an ippk-1 gene inactivation of a later step in inositol hexaphosphate (IP 6 ) synthesis, and the lin-15B and pals-22 alleles enhance RNAi. IP 6 has been known for decades to bind and stabilize human adenosine deaminase that acts on RNA (ADAR) as well as the paralog tRNA editing ADAT. We show that the C. elegans IP 6 pathway is also required for mRNA and tRNA editing. Thus, a deficiency in two axes of RNA editing enhances the already potent C. elegans RNAi antiviral defense, suggesting adenosine to inosine RNA editing may normally moderate this siRNA antiviral defense pathway. The C. elegans IP 6 -deficient mutants are synthetic lethal with a set of enhanced RNAi mutants that act in the polyploid hypodermis to regulate collagen secretion and signaling from that tissue, implicating IP 6 signaling especially in this tissue. This enhanced antiviral RNAi response uses the C. elegans RIG-I-like receptor DRH-1 to activate the unfolded protein response (UPR). The production of primary siRNAs, rather than secondary siRNAs, contributes to this activation of the UPR through XBP-1 signaling. The gon-14 and pal-17 mutants that also emerged from this screen act in the mitochondrial defense pathway rather than by enhancing RNAi.

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction.

The serine/threonine protein kinase SGK-1 is a downstream target of mTOR complex 2 (mTORC2) and is a conserved regulator of growth and metabolism. In C. elegans , mutations in rict-1 , which encodes an essential component of mTORC2, impairs lipid homeostasis and growth; however, these defects are partially suppressed by an activating mutation in SGK-1 , E116K. Here, we describe a stronger gain-of-function mutation in sgk-1 , L112F, that was identified in a forward genetic screen for rict-1 suppressor mutations . This allele will be useful in further dissecting the mTORC2 pathway and provides new insight into the role of this conserved residue in regulating SGK-1 kinase activity.

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase ( cdo-1 ) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor ( hif-1 ). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1- dependent activation of cdo-1 occurs downstream of an H 2 S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H 2 S signal. H 2 S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

ABSTRACTIon channels are necessary for proper water and nutrient absorption in the intestine, which supports cellular metabolism and organismal growth. While a role for Na+co-transporters and pumps in intestinal nutrient absorption is well defined, how individual K+uniporters function to maintain ion homeostasis is poorly understood. UsingCaenorhabditis elegans, we show that a gain-of-function mutation intwk-26, which encodes a two-pore domain K+ion channel orthologous to human KCNK3, facilitates nutrient absorption and suppresses the metabolic and developmental defects displayed by impaired intestinal MAP Kinase (MAPK) signaling. Mutations indrl-1andflr-4,which encode two components of this MAPK pathway, cause severe growth defects, reduced lipid storage, and a dramatic increase in autophagic lysosomes, which mirror dietary restriction phenotypes. Additionally, these MAPK mutants display structural defects of the intestine and an impaired defecation motor program. We find that activation of TWK-26 reverses the dietary restriction-like state of the MAPK mutants by restoring intestinal nutrient absorption without correcting the intestinal bloating or defecation defects. This study provides unique insight into the mechanisms by which intestinal K+ion channels support intestinal metabolic homeostasis.

Animals integrate developmental and nutritional signals before committing crucial resources to growth and reproduction; however, the pathways that perceive and respond to these inputs remain poorly understood. Here, we demonstrate that DRL-1 and FLR-4, which share similarity with mammalian mitogen-activated protein kinases, maintain lipid homeostasis in the C. elegans intestine. DRL-1 and FLR-4 function in a protein complex at the plasma membrane to promote development, as mutations in drl-1 or flr-4 confer slow growth, small body size, and impaired lipid homeostasis. To identify factors that oppose DRL-1/FLR-4, we performed a forward genetic screen for suppressors of the drl-1 mutant phenotypes and identified mutations in flr-2 and fshr-1, which encode the orthologues of follicle stimulating hormone and its putative G protein–coupled receptor, respectively. In the absence of DRL-1/FLR-4, neuronal FLR-2 acts through intestinal FSHR-1 and protein kinase A signaling to restrict growth. Furthermore, we show that opposing signaling through DRL-1 and FLR-2 coordinates TIR-1 oligomerization, which modulates downstream p38/PMK-1 activity, lipid homeostasis, and development. Finally, we identify a surprising noncanonical role for the developmental transcription factor PHA-4/FOXA in the intestine where it restricts growth in response to impaired DRL-1 signaling. Our work uncovers a complex multi-tissue signaling network that converges on p38 signaling to maintain homeostasis during development.

Summary The rate at which organisms grow is influenced by their biotic environment. The nematode Caenorhabditis elegans grows slower in the presence of Actinobacteria , but it is unknown why. Here, we show how perturbed levels of hydrogen sulfide and cysteine modulate the growth rate of C. elegans on Actinobacteria . Using an unbiased forward genetic screen of C. elegans we discovered alleles of the conserved cystathionine gamma-lyase ( cth-2/CTH) that improved animal growth rate on Actinobacteria . Conversely, null alleles of cth-2 cause developmental arrest of animals grown on Actinobacteria , which can be rescued by exogenous H 2 S. We also discovered a leucine rich repeat gene that regulates cysteine and H 2 S production, lrr-2/LRRC58. A wild isolate of C. elegans that naturally grows well on Actinobacteria has a mutant allele of lrr-2 , suggesting this sulfur metabolism pathway is important for the regulation of animal growth rate in its natural ecological context. We propose a model in which wild-type worms use sulfurous compounds to promote growth of their favored bacterial food sources by inhibiting Actinobacteria growth. This strategy becomes a liability when Actinobacteria are the sole food source but can be bypassed by mutations in sulfur metabolism. This study reveals how the homeostasis of sulfurous compounds controls the growth rate of animals in an ecological context.

ABSTRACT Animals integrate developmental and nutritional signals before committing crucial resources to growth and reproduction; however, the pathways that perceive and respond to these inputs remain poorly understood. Here, we demonstrate that DRL-1 and FLR-4, which share similarity with mammalian mitogen-activated protein kinases, maintain lipid homeostasis in the C. elegans intestine. DRL-1 and FLR-4 function in a protein complex at the plasma membrane to promote development, as mutations in drl-1 or flr-4 confer slow growth, small body size, and impaired lipid homeostasis. To identify factors that oppose DRL-1/FLR-4, we performed a forward genetic screen for suppressors of the drl-1 mutant phenotypes and identified mutations in flr-2 and fshr-1 , which encode the orthologues of follicle stimulating hormone and its putative G protein-coupled receptor, respectively. In the absence of DRL-1/FLR-4, neuronal FLR-2 acts through intestinal FSHR-1 and Protein Kinase A signaling to restrict growth. Furthermore, we show that opposing signaling through DRL-1 and FLR-2 coordinates TIR-1 phase transition, which modulates downstream p38/PMK-1 activity, lipid homeostasis, and development. Finally, we identify a surprising noncanonical role for the developmental transcription factor PHA-4/FOXA in the intestine where it restricts growth in response to impaired DRL-1 signaling. Our work uncovers a complex multi-tissue signaling network that converges on p38 signaling to maintain homeostasis during development.

The rate at which organisms grow is influenced by their biotic environment. The nematode Caenorhabditis elegans grows slower in the presence of Actinobacteria , but it is unknown why. Here, we show how perturbed levels of hydrogen sulfide and cysteine modulate the growth rate of C. elegans on Actinobacteria . Using an unbiased forward genetic screen of C. elegans we discovered alleles of the conserved cystathionine gamma-lyase ( cth-2/CTH) that improved animal growth rate on Actinobacteria . Conversely, null alleles of cth-2 cause developmental arrest of animals grown on Actinobacteria , which can be rescued by exogenous H2S. We also discovered a leucine rich repeat gene that regulates cysteine and H2S production, lrr-2/LRRC58. A wild isolate of C. elegans that naturally grows well on Actinobacteria has a mutant allele of lrr-2 , suggesting this sulfur metabolism pathway is important for the regulation of animal growth rate in its natural ecological context. We propose a model in which wild-type worms use sulfurous compounds to promote growth of their favored bacterial food sources by inhibiting Actinobacteria growth. This strategy becomes a liability when Actinobacteria are the sole food source but can be bypassed by mutations in sulfur metabolism. This study reveals how the homeostasis of sulfurous compounds controls the growth rate of animals in an ecological context.### Competing Interest StatementThe authors have declared no competing interest.

Abstract The amino acid cysteine is critical for many aspects of life, yet excess cysteine is toxic. Therefore, animals require pathways to maintain cysteine homeostasis. In mammals, high cysteine activates cysteine dioxygenase, a key enzyme in cysteine catabolism. The mechanism by which cysteine dioxygenase is regulated remains largely unknown. We discovered that C. elegans cysteine dioxygenase (cdo-1) is transcriptionally activated by high cysteine and the hypoxia inducible transcription factor (hif-1). hif-1- dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is sufficient to drive sulfur amino acid metabolism. EGL-9 and HIF-1 are core members of the cellular hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 functions largely independent of EGL-9 prolyl hydroxylation and the von Hippel-Lindau E3 ubiquitin ligase; classical hypoxia signaling pathway components. We propose that the intersection of hif-1 and cdo-1 reveals a negative feedback loop for maintaining cysteine homeostasis. High cysteine stimulates the production of an H2S signal. H2S then activates the rhy-1/cysl-1/egl-9 signaling pathway, increasing HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

Surveillance of Caenorhabditis elegans mitochondrial status is coupled to defense responses such as drug detoxification, immunity, antiviral RNA interference (RNAi), and regulation of life span. A cytochrome p540 detoxification gene, cyp-14A4 , is specifically activated by mitochondrial dysfunction. The nuclear hormone receptor NHR-45 and the transcriptional Mediator component MDT-15/MED15 are required for the transcriptional activation of cyp-14A4 by mitochondrial mutations, gene inactivations, or toxins. A genetic screen for mutations that fail to activate this cytochrome p450 gene upon drug or mutation-induced mitochondrial dysfunction identified a DNA helicase ARIP-4 that functions in concert with the NHR-45 transcriptional regulatory cascade. In response to mitochondrial dysfunction, ARIP-4 and NHR-45 protein interaction is enhanced, and they relocalize from the nuclear periphery to the interior of intestinal nuclei. NHR-45/ARIP-4 also regulates the transcriptional activation of the eol-1 gene that encodes a decapping enzyme required for enhanced RNAi and transgene silencing of mitochondrial mutants. In the absence of arip-4 , animals were more susceptible to the mitochondrial inhibitor antimycin. Thus, ARIP-4 serves as a transcriptional coactivator of NHR-45 to promote this defense response. A null mutation in arip-4 extends the life span and health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified. Thus, arip-4 acts in a pathway that couples mitochondrial surveillance to the activation of downstream immunity, detoxification, and RNAi responses.

Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.

Translation in eukaryotes is followed to detect toxins and virulence factors and coupled to the induction of defence pathways. Caenorhabditis elegans germline-specific mutations in translation components are detected by this system to induce detoxification and immune responses in distinct somatic cells. An RNA interference screen revealed gene inactivations that act at multiple steps in lipid biosynthetic and kinase pathways upstream of MAP kinase to mediate the systemic communication of translation defects to induce detoxification genes. Mammalian bile acids can rescue the defect in detoxification gene induction caused by C. elegans lipid biosynthetic gene inactivations. Extracts prepared from C. elegans with translation deficits but not from the wild type can also rescue detoxification gene induction in lipid-biosynthesis-defective strains. These eukaryotic antibacterial countermeasures are not ignored by bacteria: particular bacterial species suppress normal C. elegans detoxification responses to mutations in translation factors.

AbstractRNA interference (RNAi) is an antiviral pathway common to many eukaryotes that detects and cleaves foreign nucleic acids. In mammals, mitochondrially localized proteins such as MAVS, RIG-I, and MDA5 mediate antiviral responses. Here, we report that mitochondrial dysfunction inCaenorhabditis elegansactivates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. The induction of RNAi also requires the conserved RNA decapping enzyme EOL-1/DXO. The transcriptional induction ofeol-1requires DRH-1 as well as the mitochondrial unfolded protein response (UPRmt). Upon mitochondrial dysfunction, EOL-1 is concentrated into foci that depend on the transcription of mitochondrial RNAs that may form dsRNA, as has been observed in mammalian antiviral responses. The enhanced RNAi triggered by mitochondrial dysfunction contributes to the increase in longevity that is induced by mitochondrial dysfunction.

RNA interference (RNAi) is an antiviral pathway common to many eukaryotes that detects and cleaves foreign nucleic acids. In mammals, mitochondrially localized proteins such as mitochondrial antiviral signaling (MAVS), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) mediate antiviral responses. Here, we report that mitochondrial dysfunction in Caenorhabditis elegans activates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. The induction of RNAi also requires the conserved RNA decapping enzyme EOL-1/DXO. The transcriptional induction of eol-1 requires DRH-1 as well as the mitochondrial unfolded protein response (UPRmt). Upon mitochondrial dysfunction, EOL-1 is concentrated into foci that depend on the transcription of mitochondrial RNAs that may form double-stranded RNA (dsRNA), as has been observed in mammalian antiviral responses. Enhanced RNAi triggered by mitochondrial dysfunction is necessary for the increase in longevity that is induced by mitochondrial dysfunction.

RNA interference (RNAi) is an antiviral pathway common to many eukaryotes that detects and cleaves foreign nucleic acids. In mammals, mitochondrially localized proteins such as MAVS, RIG-I, and MDA5 mediate antiviral responses. Here, we report that mitochondrial dysfunction in Caenorhabditis elegans activates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. The induction of RNAi also requires the conserved RNA decapping enzyme EOL-1/DXO. The transcriptional induction of eol-1 requires DRH-1 as well as the mitochondrial unfolded protein response (UPRmt). Upon mitochondrial dysfunction, EOL-1 is concentrated into foci that depend on the transcription of mitochondrial RNAs that may form dsRNA, as has been observed in mammalian antiviral responses. The enhanced RNAi triggered by mitochondrial dysfunction contributes to the increase in longevity that is induced by mitochondrial dysfunction.