Cellular abundance of sodium phosphate cotransporter SLC20A1/PiT1 and phosphate uptake are controlled post-transcriptionally by ESCRT.

Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate cotransporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here, we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, whereas mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein downregulation in phosphate-replete cells. We show that SLC20A1 colocalizes with ESCRT and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes.

Characterization of the endogenous DAF-12 ligand and its use as an anthelmintic agent in Strongyloides stercoralis.

A prevalent feature of Strongyloides stercoralis is a life-long and potentially lethal infection that is due to the nematode parasite's ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasite's nuclear receptor, Ss-DAF-12, in governing infection. We identified Δ7-DA as the endogenous Ss-DAF-12 ligand and elucidated the hormone's biosynthetic pathway. Genetic loss of function of the ligand's rate-limiting enzyme demonstrated that Δ7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for the development of infectious larvae. Availability of the ligand permits Ss-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when Δ7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

Identification of a nuclear receptor/coactivator developmental signaling pathway in the nematode parasite Strongyloides stercoralis.

DAF-12 is nematode-specific nuclear receptor that has been proposed to govern development of the infectious stage of parasitic species, including Strongyloides stercoralis Here, we identified a parasite-specific coactivator, called DAF-12 interacting protein-1 (DIP-1), that is required for DAF-12 ligand-dependent transcriptional activity. DIP-1 is found only in Strongyloides spp. and selectively interacts with DAF-12 through an atypical receptor binding motif. Using CRISPR/Cas9-directed mutagenesis, we demonstrate that DAF-12 is required for the requisite developmental arrest and the ligand-dependent reactivation of infectious S. stercoralis infective third-stage larvae, and that these effects require the DIP-1 coactivator. These studies reveal the existence of a distinct nuclear receptor/coactivator signaling pathway that governs parasite development.

A novel functional cross-interaction between opioid and pheromone signaling may be involved in stress avoidance in Caenorhabditis elegans.

Upon sensing starvation stress, Caenorhabditis elegans larvae (L2d) elicit two seemingly opposing behaviors to escape from the stressful condition: food-seeking roaming mediated by the opioid peptide NLP-24 and dauer formation mediated by pheromones. Because opioid and pheromone signals both originate in ASI chemosensory neurons, we hypothesized that they might act sequentially or competitively to avoid starvation stress. Our data shows that NPR-17 opioid receptor signaling suppressed pheromone biosynthesis and the overexpression of opioid genes disturbed dauer formation. Likewise, DAF-37 pheromone receptor signaling negatively modulated nlp-24 expression in the ASI neurons. Under short-term starvation (STS, 3 h), both pheromone and opioid signaling were downregulated in gpa-3 mutants. Surprisingly, the gpa-3;nlp-24 double mutants exhibited much higher dauer formation than seen in either of the single mutants. Under long-term starvation (LTS, >24 h), the stress-activated SKN-1a downregulated opioid signaling and then enhanced dauer formation. Both insulin and serotonin stimulated opioid signaling, whereas NHR-69 suppressed opioid signaling. Thus, GPA-3 and SKN-1a are proposed to regulate cross-antagonistic interaction between opioids and pheromones in a cell-specific manner. These regulatory functions are suggested to be exerted via the selective interaction of GPA-3 with NPR-17 and site-specific SKN-1 binding to the promoter of nlp-24 to facilitate stress avoidance.

An opioid-like system regulating feeding behavior in C. elegans.

Neuropeptides are essential for the regulation of appetite. Here we show that neuropeptides could regulate feeding in mutants that lack neurotransmission from the motor neurons that stimulate feeding muscles. We identified nlp-24 by an RNAi screen of 115 neuropeptide genes, testing whether they affected growth. NLP-24 peptides have a conserved YGGXX sequence, similar to mammalian opioid neuropeptides. In addition, morphine and naloxone respectively stimulated and inhibited feeding in starved worms, but not in worms lacking NPR-17, which encodes a protein with sequence similarity to opioid receptors. Opioid agonists activated heterologously expressed NPR-17, as did at least one NLP-24 peptide. Worms lacking the ASI neurons, which express npr-17, did not response to naloxone. Thus, we suggest that Caenorhabditis elegans has an endogenous opioid system that acts through NPR-17, and that opioids regulate feeding via ASI neurons. Together, these results suggest C. elegans may be the first genetically tractable invertebrate opioid model.

NSBP-1 mediates the effects of cholesterol on insulin/IGF-1 signaling in Caenorhabditis elegans.

Nematode sterol-binding protein 1 (NSBP-1) is a homolog of nucleosome assembly protein 1 in mammals that is expressed widely in Caenorhabditis elegans. NSBP-1 mutants are biologically lethal, demonstrating the significance of the gene in growth and development. We investigated how cholesterol influences the insulin signaling pathway through this novel sterol-binding protein in C. elegans. Here we report that NSBP-1 influences many biological processes mediated by insulin signaling, such as longevity, dauer formation, fat storage, and resistance to oxidative stress. We found that NSBP-1 is phosphorylated by AKT-1 downstream of insulin signaling. In the absence of insulin signaling, NSBP-1 is translocated to the nucleus and binds to DAF-16, a FOXO transcription factor, in a cholesterol-dependent manner. Moreover, NSBP-1 and DAF-16 regulate a common set of genes that can directly modulate fat storage, longevity, and resistance to stress. Together, our results present a new steroid-binding molecule that can connect sterol signaling to insulin signaling through direct interaction with FOXO.

A potential biochemical mechanism underlying the influence of sterol deprivation stress on Caenorhabditis elegans longevity.

To investigate the biochemical mechanism underlying the effect of sterol deprivation on longevity in Caenorhabditis elegans, we treated parent worms (P0) with 25-azacoprostane (Aza), which inhibits sitosterol-to-cholesterol conversion, and measured mean lifespan (MLS) in F2 worms. At 25 µM (∼EC(50)), Aza reduced total body sterol by 82.5%, confirming sterol depletion. Aza (25 µM) treatment of wild-type (N2) C. elegans grown in sitosterol (5 µg/ml) reduced MLS by 35%. Similar results were obtained for the stress-related mutants daf-16(mu86) and gas-1(fc21). Unexpectedly, Aza had essentially no effect on MLS in the stress-resistant daf-2(e1370) or mitochondrial complex II mutant mev-1(kn1) strains, indicating that Aza may target both insulin/IGF-1 signaling (IIS) and mitochondrial complex II. Aza increased reactive oxygen species (ROS) levels 2.7-fold in N2 worms, but did not affect ROS production by mev-1(kn1), suggesting a direct link between Aza treatment and mitochondrial ROS production. Moreover, expression of the stress-response transcription factor SKN-1 was decreased in amphid neurons by Aza and that of DAF-28 was increased when DAF-6 was involved, contributing to lifespan reduction.