daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan.

The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.

Pheromone sensing regulates Caenorhabditis elegans lifespan and stress resistance via the deacetylase SIR-2.1.

Lifespan in Caenorhabditis elegans, Drosophila, and mice is regulated by conserved signaling networks, including the insulin/insulin-like growth factor 1 (IGF-1) signaling cascade and pathways depending on sirtuins, a family of NAD(+)-dependent deacetylases. Small molecules such as resveratrol are of great interest because they increase lifespan in many species in a sirtuin-dependent manner. However, no endogenous small molecules that regulate lifespan via sirtuins have been identified, and the mechanisms underlying sirtuin-dependent longevity are not well understood. Here, we show that in C. elegans, two endogenously produced small molecules, the dauer-inducing ascarosides ascr#2 and ascr#3, regulate lifespan and stress resistance through chemosensory pathways and the sirtuin SIR-2.1. Ascarosides extend adult lifespan and stress resistance without reducing fecundity or feeding rate, and these effects are reduced or abolished when nutrients are restricted. We found that ascaroside-mediated longevity is fully abolished by loss of SIR-2.1 and that the effect of ascr#2 requires expression of the G protein-coupled receptor DAF-37 in specific chemosensory neurons. In contrast to many other lifespan-modulating factors, ascaroside-mediated lifespan increases do not require insulin signaling via the FOXO homolog DAF-16 or the insulin/IGF-1-receptor homolog DAF-2. Our study demonstrates that C. elegans produces specific small molecules to control adult lifespan in a sirtuin-dependent manner, supporting the hypothesis that endogenous regulation of metazoan lifespan functions, in part, via sirtuins. These findings strengthen the link between chemosensory inputs and conserved mechanisms of lifespan regulation in metazoans and suggest a model for communal lifespan regulation in C. elegans.

Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans.

A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-ß, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein-coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein-coupled receptors such as DAF-37 with more promiscuous G-protein-coupled receptors such as DAF-38.

Repression of a potassium channel by nuclear hormone receptor and TGF-ß signaling modulates insulin signaling in Caenorhabditis elegans.

Transforming growth factor ß (TGF-ß) signaling acts through Smad proteins to play fundamental roles in cell proliferation, differentiation, apoptosis, and metabolism. The Receptor associated Smads (R-Smads) interact with DNA and other nuclear proteins to regulate target gene transcription. Here, we demonstrate that the Caenorhabditis elegans R-Smad DAF-8 partners with the nuclear hormone receptor NHR-69, a C. elegans ortholog of mammalian hepatocyte nuclear factor 4α HNF4α), to repress the exp-2 potassium channel gene and increase insulin secretion. We find that NHR-69 associates with DAF-8 both in vivo and in vitro. Functionally, daf-8 nhr-69 double mutants show defects in neuropeptide secretion and phenotypes consistent with reduced insulin signaling such as increased expression of the sod-3 and gst-10 genes and a longer life span. Expression of the exp-2 gene, encoding a voltage-gated potassium channel, is synergistically increased in daf-8 nhr-69 mutants compared to single mutants and wild-type worms. In turn, exp-2 acts selectively in the ASI neurons to repress the secretion of the insulin-like peptide DAF-28. Importantly, exp-2 mutation shortens the long life span of daf-8 nhr-69 double mutants, demonstrating that exp-2 is required downstream of DAF-8 and NHR-69. Finally, animals over-expressing NHR-69 specifically in DAF-28-secreting ASI neurons exhibit a lethargic, hypoglycemic phenotype that is rescued by exogenous glucose. We propose a model whereby DAF-8/R-Smad and NHR-69 negatively regulate the transcription of exp-2 to promote neuronal DAF-28 secretion, thus demonstrating a physiological crosstalk between TGF-ß and HNF4α-like signaling in C. elegans. NHR-69 and DAF-8 dependent regulation of exp-2 and DAF-28 also provides a novel molecular mechanism that contributes to the previously recognized link between insulin and TGF-ß signaling in C. elegans.

Increased longevity of some C. elegans mitochondrial mutants explained by activation of an alternative energy-producing pathway.

The Caenorhabditis elegans misc-1 gene encodes a mitochondrial carrier with a role in oxidative stress response. The knock-out mutant has no lifespan phenotype and fails to upregulate the gei-7-mediated glyoxylate shunt, an extra-mitochondrial pathway of energy production. We show that gei-7 is required for the longevity of the mitochondrial mutant clk-1. Our data suggest that only mitochondrial mutants that upregulate gei-7 can achieve longevity.

MISC-1/OGC links mitochondrial metabolism, apoptosis and insulin secretion.

We identified MISC-1 (Mitochondrial Solute Carrier) as the C. elegans orthologue of mammalian OGC (2-oxoglutarate carrier). OGC was originally identified for its ability to transfer α-ketoglutarate across the inner mitochondrial membrane. However, we found that MISC-1 and OGC are not solely involved in metabolic control. Our data show that these orthologous proteins participate in phylogenetically conserved cellular processes, like control of mitochondrial morphology and induction of apoptosis. We show that MISC-1/OGC is required for proper mitochondrial fusion and fission events in both C. elegans and human cells. Transmission electron microscopy reveals that loss of MISC-1 results in a decreased number of mitochondrial cristae, which have a blebbed appearance. Furthermore, our pull-down experiments show that MISC-1 and OGC interact with the anti-apoptotic proteins CED-9 and Bcl-x(L), respectively, and with the pro-apoptotic protein ANT. Knock-down of misc-1 in C. elegans and OGC in mouse cells induces apoptosis through the caspase cascade. Genetic analysis suggests that MISC-1 controls apoptosis through the physiological pathway mediated by the LIN-35/Rb-like protein. We provide genetic and molecular evidence that absence of MISC-1 increases insulin secretion and enhances germline stem cell proliferation in C. elegans. Our study suggests that the mitochondrial metabolic protein MISC-1/OGC integrates metabolic, apoptotic and insulin secretion functions. We propose a novel mechanism by which mitochondria integrate metabolic and cell survival signals. Our data suggest that MISC-1/OGC functions by sensing the metabolic status of mitochondria and directly activate the apoptotic program when required. Our results suggest that controlling MISC-1/OGC function allows regulation of mitochondrial morphology and cell survival decisions by the metabolic needs of the cell.

Quantitative proteomics identifies ferritin in the innate immune response of C. elegans.

When encountering a pathogen, all organisms evoke a protective response by inducing defense mechanisms to help fight off the invader. The invertebrate model organism Caenorhabditis elegans has proven to be valuable for studies of the host response and the small nematode mounts a substantial transcriptional response to numerous pathogens. Here, we use global quantitative proteomics to profile the response to infection with E. coli strain LF82 isolated from patients suffering from Crohn's disease, an inflammatory bowel disease. We show that LF82 infection induces more than one hundred proteins. The response share many functional categories with other innate immunity studies in C. elegans, but also identifies novel host immune effector proteins. We demonstrate functional relevance for four LF82 induced proteins, including a lysozyme and a C-type lectin. The ferritin homolog FTN-2 was shown to be necessary for the full protective response against the Gram-negative LF82 and the Gram-positive pathogen Staphylococcus aureus. This study is the first to demonstrate a role for ferritin in the innate immune response of C. elegans, and our results suggests that quantitative proteomics is an attractive approach for identifying additional components in the complex immune response of the nematode.

Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function.

Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism.

Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25.

In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-ß and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.

Regulation of metabolism in Caenorhabditis elegans longevity.

The nematode Caenorhabditis elegans is a favorite model for the study of aging. A wealth of genetic and genomic studies show that metabolic regulation is a hallmark of life-span modulation. A recent study in BMC Biology identifying metabolic signatures for longevity suggests that amino-acid pools may be important in longevity.