Warnhoff K et al. (2019) Nat Chem Biol "Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification."

Ruvkun G, Warnhoff K

[Nat Chem Biol, 2019]

The kingdoms of life share many small molecule cofactors and coenzymes. Molybdenum cofactor (Moco) is synthesized by many archaea, bacteria, and eukaryotes, and is essential for human development. The genome of Caenorhabditis elegans contains all of the Moco biosynthesis genes, and surprisingly these genes are not essential if the animals are fed a bacterial diet that synthesizes Moco. C. elegans lacking both endogenous Moco synthesis and dietary Moco from bacteria arrest development, demonstrating interkingdom Moco transfer. Our screen of Escherichia coli mutants identifies genes necessary for synthesis of bacterial Moco or transfer to C. elegans. Developmental arrest of Moco-deficient C. elegans is caused by loss of sulfite oxidase, a Moco-requiring enzyme, and is suppressed by mutations in either C. elegans cystathionine gamma-lyase or cysteine dioxygenase, blocking toxic sulfite production from cystathionine. Thus, we define the genetic pathways for an interkingdom dialogue focused on sulfur homeostasis.

Warnhoff K et al. (2024) Elife "Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans."

Warnhoff K, Snoozy J, Ruvkun G, Breen PC, Bhattacharya S

[Elife, 2024]

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 (<i>cdo-1</i>) in the nematode <i>Caenorhabditis elegans,</i> we isolated loss-of-function mutations in <i>rhy-1</i> and <i>egl-9,</i> which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (<i>hif-1</i>). 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 <i>cdo-1</i> 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 <i>C. elegans cdo-1</i> is transcriptionally activated by high levels of cysteine and <i>hif-1. hif-1-</i>dependent activation of <i>cdo-1</i> occurs downstream of an H₂S-sensing pathway that includes <i>rhy-1, cysl-1,</i> and <i>egl-9. cdo-1</i> transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of <i>cdo-1</i> by <i>hif-1</i> reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H₂S signal. H₂S then acts through the <i>rhy-1/cysl-1/egl-9</i> signaling pathway to increase HIF-1-mediated transcription of <i>cdo-1,</i> promoting degradation of cysteine via CDO-1.

Warnhoff K et al. (2021) Genes Dev "Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans."

Ruvkun G, Mendel RR, Warnhoff K, Hercher TW

[Genes Dev, 2021]

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode <i>Caenorhabditis elegans</i> can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by <i>C. elegans</i> from its diet and is an effective dietary supplement, rescuing a <i>C</i><i>elegans</i> model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Warnhoff K et al. (2015) Worm "New links between protein N-terminal acetylation, dauer diapause, and the insulin/IGF-1 signaling pathway in Caenorhabditis elegans."

Kornfeld K, Warnhoff K

[Worm, 2015]

Protein N-terminal acetylation is a widespread posttranslational modification in eukaryotes that is catalyzed by N-terminal acetyltransferases (NATs). The biochemical activity of NATs has been characterized extensively, whereas the biological function of NATs is only beginning to be defined. Here we comment on recent progress in understanding the function of NAT activity in C. elegans based on the characterization of natc-1 by Warnhoff etal. (2014) and daf-31 by Chen etal. (2014).(1,2) natc-1 encodes an auxiliary subunit of the NatC complex and modulates stress tolerance, dauer entry, and adult lifespan. daf-31 encodes the catalytic subunit of the NatA complex and affects dauer entry, dauer formation, and adult lifespan. The analysis of these genes and genetic studies of NATs in other organisms suggests protein N-terminal acetylation plays an evolutionarily conserved role in promoting growth and development and inhibiting stress resistance. Furthermore, we propose that NATs may regulate growth and development in response to external cues such as nutrient deprivation and other physiologic stresses.

Warnhoff K et al. (2017) PLoS Biol "The Nuclear Receptor HIZR-1 Uses Zinc as a Ligand to Mediate Homeostasis in Response to High Zinc."

Kornfeld K, Schneider DL, Tan CH, Warnhoff K, Croswell D, Kocsisova Z, Morrison A, Roh HC

[PLoS Biol, 2017]

Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. Here we describe a novel Caenorhabditis elegans nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors.

Warnhoff K et al. (2014) PLoS Genet "The DAF-16 FOXO transcription factor regulates natc-1 to modulate stress resistance in Caenorhabditis elegans, linking insulin/IGF-1 signaling to protein N-terminal acetylation."

Robertson JD, Murphy JT, Guthrie J, Kumar S, Schneider DL, Kornfeld K, Peterson M, Warnhoff K, Hsu S

[PLoS Genet, 2014]

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.

Huang CY et al. (2012) PLoS One "C. elegans EIF-3.K promotes programmed cell death through CED-3 caspase."

Tan CH, Huang CY, Wu SC, Chen JY, Wu YF, Wu YC, Chen RH, Lu PJ, Tzeng RY

[PLoS One, 2012]

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.

Wojtovich AP et al. (2011) PLoS One "SLO-2 is cytoprotective and contributes to mitochondrial potassium transport."

Sherman TA, Nehrke K, Urciuoli WR, Wojtovich AP, Nadtochiy SM, Brookes PS

[PLoS One, 2011]

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance &quot;big&quot; K(+) channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K(+) transport and APC. The canonical Ca(2+)-activated BK (or &quot;maxi-K&quot;) channel SLO1 was dispensable for both mitochondrial K(+) transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K(+) channel SLO2 was required, and that SLO2-dependent mitochondrial K(+) transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K(+).

Johnson CK et al. (2020) J Neurophysiol "The Na+/K+-ATPase is needed in glia of touch receptors for responses to touch in C. elegans."

Wang Y, Bianchi L, Fernandez-Abascal J, Johnson CK, Wang L

[J Neurophysiol, 2020]

Four of the five types of mammalian mechanosensors are composed of nerve endings and accessory cells. In C. elegans we showed that glia supports the function of nose touch neurons via the activity of glial Na⁺ and K⁺channels. We show here that a third regulator of Na⁺ and K⁺, the Na⁺/K⁺-ATPase, is needed in glia of nose touch neurons for touch. Importantly, we show that the two Na⁺/K⁺-ATPase genes are needed for the function rather than structural integrity and that their ion transport activity is crucial for touch. Finally, when glial Na⁺/K⁺-ATPase genes are knocked-out, touch can be restored by activation of a third Na⁺/K⁺-ATPase. Taken together, these data show the requirement in glia of touch neurons of the function of the Na⁺/K⁺-ATPase. These data underscore the importance of the homeostasis of Na⁺ and K⁺, most likely in the space surrounding touch neurons, in touch sensation, a function that might be conserved across species.

Jospin M et al. (2002) J Physiol "Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans."

Jospin M, Allard B, Segelat L, Mariol MC, Segalat L

[J Physiol, 2002]

The properties of K+ channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K+ currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K+ channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mm external K+, respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca2+-activated K+ channels exhibiting an unusual sensitivity to internal Cl- and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca2+-activated K+ channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mm external K+). In providing a detailed description of native K+ currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K+ channels in this model organism that is highly amenable to molecular and classical genetics.