Vassilatis DK et al. (1997) J Biol Chem "Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans. Isolation and characterization."

Wilkinson HA, Schaeffer JM, Van der Ploeg LHT, Arena JP, Vassilatis DK, Plasterk RHA, Cully DF

[J Biol Chem, 1997]

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluClalpha and GluClbeta) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegans strain with a Tc1 transposable element insertion that functionally inactivated the GluClalpha gene (GluClalpha::Tc1). GluClalpha::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity. Xenopus oocytes expressing GluClalpha::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluClalpha::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced Bmax. These experiments suggest that GluClalpha is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluClalpha2) encoding a chloride channel that shares 75% amino acid identity with GluClalpha. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate. GluClalpha2 coassembles with GluClbeta to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluClalpha may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.

Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[International Worm Meeting, 2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

Ziogiene D et al. (2024) PLoS One "Dolichol kinases from yeast, nematode and human can replace each other and exchange their domains creating active chimeric enzymes in yeast."

Vasiliunaite E, Zinkeviciute R, Timinskas A, Gedvilaite A, Burdulis A, Norkiene M, Ziogiene D

[PLoS One, 2024]

Protein glycosylation is a fundamental modification crucial for numerous intra- and extracellular functions in all eukaryotes. The phosphorylated dolichol (Dol-P) is utilized in N-linked protein glycosylation and other glycosylation pathways. Dolichol kinase (DK) plays a key role in catalyzing the phosphorylation of dolichol. The glycosylation patterns in the Kluyveromyces lactis DK mutant revealed that the yeast well tolerated a minor deficiency in Dol-P by adjusting protein glycosylation. Comparative analysis of sequences of DK homologs from different species of eukaryotes, archaea and bacteria and AlphaFold3 structural model studies, allowed us to predict that DK is most likely composed of two structural/functional domains. The activity of predicted K. lactis DK C-terminal domain expressed from the single copy in the chromosome was not sufficient to keep protein glycosylation level necessary for survival of K. lactis. However, the glycosylation level was partially restored by additionally provided and overexpressed N- or C-terminal domain. Moreover, co-expression of the individual N-and C-terminal domains restored the glycosylation of vacuolar carboxypeptidase Y in both K. lactis and Saccharomyces cerevisiae. Despite the differences in length and non-homologous sequences of the N-terminal domains the human and nematode Caenorhabditis elegans DKs successfully complemented DK functions in both yeast species. Additionally, the N-terminal domains of K. lactis and C. elegans DK could functionally substitute for one another, creating active chimeric enzymes. Our results suggest that while the C-terminal domain remains crucial for DK activity, the N-terminal domain may serve not only as a structural domain but also as a possible regulator of DK activity.

Dent JA et al. (2000) Proc Natl Acad Sci U S A "The genetics of ivermectin resistance in Caenorhabditis elegans."

Dent JA, Avery L, Vassilatis DK, Smith M

[Proc Natl Acad Sci U S A, 2000]

The ability of organisms to evolve resistance threatens the effectiveness of every antibiotic drug. We show that in the nematode Caenorhabditis elegans, simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) alpha-type subunits confers high-level resistance to the antiparasitic drug ivermectin. In contrast, mutating any two channel genes confers modest or no resistance. We propose a model in which ivermectin sensitivity in C. elegans is mediated by genes affecting parallel genetic pathways defined by the family of GluCl genes. The sensitivity of these pathways is further modulated by unc-7, unc-9, and the Dyf (dye filling defective) genes, which alter the structure of the nervous system. Our results suggest that the evolution of drug resistance can be slowed by targeting antibiotic drugs to several members of a multigene family.

Olgun, A et al. (2005) J Gerontol A Biol Sci Med Sci "Functional analysis of MRG-1: the ortholog of human MRG15 in Caenorhabditis elegans."

Olgun, A, Pereira-Smith, OM, Vassilatis, DK, Aleksenko, T

[J Gerontol A Biol Sci Med Sci, 2005]

Mortality Factor on Chromosome 4 (MORF4) induces senescence in several immortal human cell lines. MORF-related gene on chromosome 15 (MRG15), another expressed family member, is highly conserved and expressed in yeast to humans. To determine the biological functions of human MRG15 (hMRG15) we used RNA-mediated interference (RNAi) to silence mrg-1, the Caenorhabditis elegans ortholog, and its closest homolog Y37D8A.11. Expression of mrg-1 RNAi resulted in sterility, body wall defects, vulval protrusion, and posterior developmental defects in worms. We expressed mrg-1 under its own and the cytomegalovirus promoter in human cells. Both constructs were expressed, indicating that C. elegans promoter elements are functional in mammalian cells. Overexpression from the cytomegalovirus promoter eventually resulted in cell death, possibly due to competition with hMRG15 in endogenous nucleoprotein complexes. Recent data indicate a role for yeast and human MRG15 in transcriptional regulation via chromatin remodeling. Here we demonstrate the importance of mrg-1 in development and reproduction in C. elegans and discuss its potential to impact the aging process.

Cully DF et al. (1996) Parasitology "Molecular biology and electrophysiology of glutamate-gated chloride channels of invertebrates."

Etter A, Wilkinson HA, Vassilatis DK, Arena JP, Cully DF

[Parasitology, 1996]

In this chapter we summarize the available data on a novel class of ligand-gated anion channels that are gated by the neurotransmitter glutamate. Glutamate is classically thought to be a stimulatory neurotransmitter, however, studies in invertebrates have proven that glutamate also functions as an inhibitory ligand. The bulk of studies conducted in vivo have been on insects and crustaceans, where glutamate was first postulated to act on H-receptors resulting from hyperpolarizing response to glutamate. Recently, glutamate-gated chloride channels have been cloned from several nematodes and Drosophila. The pharmacology and electrophysiological properties of these channels have been studied by expression in Xenopus oocytes. Studies on the cloned channels demonstrate that the invertebrate glutamate-gated chloride channels are the H-receptors and represent important targets for the antiparasitic avermectins.

Etter A et al. (1999) J Neurochem "Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore."

Arena JP, Schaeffer JM, Etter A, Vassilatis DK, Liu KK, Reiss B, Cully DF

[J Neurochem, 1999]

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Cully DF et al. (1994) Nature "Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans."

Schaeffer JM, Cully DF, Van der Ploeg LHT, Liu KK, Arena JP, Paress PS, Vassilatis DK

[Nature, 1994]

The avermectins are a family of macrocyclic lactones used in the control of nematode and arthropod parasites. Ivermectin (22,23-dihydroavermectin B-1a) is widely used as an anthelmintic in veterinary medicine and is used to treat onchocerciasis or river blindness in humans. Abamectin (avermectin B-1a) is a miticide and insecticide used in crop protection. Avermectins interact with vertebrate and invertebrate GABA receptors and invertebrate glutamate-gated chloride channels the soil nematode Caenorhabditis elegans has served as a useful model to study the mechanism of action of avermectins. A C. elegans messenger RNA expressed in Xenopus oocytes encodes an avermectin-sensitive glutamate-gated chloride channel. To elucidate the structure and properties of this channel, we used Xenopus oocytes for expression cloning of two functional complementary DNAs encoding an avermectin-sensitive glutamate-gated chloride channel. We find that the electrophysiological and structural properties of these proteins indicate that they are new

Lamunyon, Craig W. et al. (2011) International Worm Meeting "Y47D7A.16: a riboflavin tranporter in C. elegans and homolog of human Riboflavin Transporter 2."

Said, Hamid, LaMunyon, Craig W., Rothman, Jason, Biswas, Arundhati

[International Worm Meeting, 2011]

The C. elegans gene Y47D7A.16 is an ortholog of isoform 1 of the human Riboflavin Transporter 2 vitamin transporter. We became interested in this gene as the riboflavin transporter in C. elegans. Cloning of the cDNA revealed an exonic structure different from that predicted and an alternative splice in the first exon. Expression of the cDNA in human ARPE19 retinal cells showed enhanced riboflavin uptake, establishing the protein as a functional transporter of riboflavin (cRFT). Uptake by cRFT was pH dependent (uptake at buffer pH 5 > than uptake at pH 8), Na independent, and was inhibited by riboflavin structural analogues lumichrome and lumiflavin. Worms exposed to Y47D7A.16 RNAi laid defective eggs at a slowed pace and exhibited a slower defecation rate compared to controls. The protein is expressed primarily in the gut, with expression most pronounced in the distal gut, although expression also occurs in the excretory canal. We will discuss the effects of riboflavin supplementation on RNAi knockdown worms and on the expression of Y47D7A.16. [Supported by grants from the DVA and the NIH (DK-58057)].

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).