9 Papers found (0.009 seconds)

Knox, Jessica et al. (2013) International Worm Meeting "Genetic analysis of pry-1/Axin regulation and Wnt signaling in nematode vulva development."

Cumbo, Philip, Knox, Jessica, Gupta, Bhagwati P

[

International Worm Meeting,

2013]

The Wnt pathway is a conserved signaling pathway vital for such cellular processes as polarity, migration and fate specification. In Caenorhabditis elegans, the Wnt pathway converges with the Notch and Ras pathways to induce 3 of 6 Vulval Precursor Cells (VPCs) to divide and form the mature vulva. The robust patterning of the vulva serves as a paradigm for the study of signal transduction and the complex integration of conserved signaling pathways controlling organ formation. Using the vulva model in C. elegans and the related species C. briggsae, we can analyze Wnt signaling function and conservation during development in an attempt to tease apart these pathway interactions. To this end, a genetic approach is being taken to dissect the regulation and function of the Axin homolog pry-1, a potent negative regulator of Wnt signaling. We have isolated Cbr-pry-1 mutants that, along with their C. elegans counterparts, display a Multivulva phenotype in which loss of PRY-1 results in activated Wnt signaling and formation of ectopic pseudo-vulvae. Using cell fate markers, cell ablation and genetic interaction techniques it was found that these ectopically induced VPCs adopt a 2 deg fate in both species, independent of inductive and lateral signals. A crucial interaction between the Wnt and Notch pathways in promoting 2 deg VPC fate has been uncovered in these pry-1 mutants, in which we observe a P7.p induction failure, caused by altered regulation of LIN-12 signaling and the resulting persistent expression of its transcriptional target lip-1. We have further embarked upon a dissection of pry-1 regulation throughout development. Using qRT-PCR and transcriptional GFP reporter fusions, we have found that pry-1 expression is responsive to Wnt signaling activity. In both species, pry-1 mutants exhibit a significant increase in pry-1 transcript level at all stages. The opposite trend is observed when key components of the Wnt pathway are knocked down (bar-1/b-catenin and pop-1/TCF/LEF). An analysis of pry-1 upstream and intronic UTRs indicates the presence of conserved TCF/LEF binding sites. These results together suggest that pry-1 is itself being positively regulated by Wnt pathway activity.

Knox, Jessica et al. (2021) International Worm Meeting "A Survey of the Kinome Pharmacopeia Reveals Multiple Scaffolds and Targets for the Development of Novel Anthelmintics"

Knox, Jessica, Carmona-Negron, Jose A., Roy, Peter J., Zuercher, William, Linossi, Edmond M., Joly, Nicolas, Pintard, Lionel, Jura, Natalia

[

International Worm Meeting,

2021]

Over one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintic drugs. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinase orthologs have diverged from vertebrates in key residues within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent important targets for the development of new anthelmintic medicines.

Burglin TR (1997) Nucleic Acids Res "Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals."

Burglin TR

[

Nucleic Acids Res,

1997]

A new Caenorhabditis elegans homeobox gene, ceh-25, is described that belongs to the TALE superclass of atypical homeodomains, which are characterized by three extra residues between helix 1 and helix 2. ORF and PCR analysis revealed a novel type of alternative splicing within the homeobox. The alternative splicing occurs such that two different homeodomains can be generated, which differ in their first 25 amino acids. ceh-25 is an orthologue of the vertebrate Meis genes and it shares a new conserved domain of 130 amino acids with them. A thorough analysis of all TALE homeobox genes was performed and a new classification is presented. Four TALE classes are identified in animals: PBC, MEIS, TGIF and IRO (Iroquois); two types in fungi: the mating type genes (M-ATYP) and the CUP genes; and two types in plants: KNOX and BEL. The IRO class has a new conserved motif downstream of the homeodomain. For the KNOX class, a conserved domain, the KNOX domain, was defined upstream of the homeodomain. Comparison of the KNOX domain and the MEIS domain shows significant sequence similarity revealing the existence of an archetypal group of homeobox genes that encode two associated conserved domains. Thus TALE homeobox genes were already present in the common ancestor of plants, fungi and animals and represent a branch distinct from the typical homeobox genes.

Murareanu, Brandon et al. (2021) International Worm Meeting "High-Throughput Drug Screen Reveals Novel Inhibitors of Microsporidia Infection in C. elegans"

Roy, Peter, Knox, Jessica, Murareanu, Brandon, Reinke, Aaron

[

International Worm Meeting,

2021]

Microsporidia are a large and diverse group of fungal-related spore-forming obligate intracellular parasites known to infect hosts in most major animal phyla. Despite the emerging threat that microsporidia have become, few reliable treatment options exist. To address this, we developed a high-throughput drug screening protocol using C. elegans and the microsporidia species Nematocida parisii in order to identify novel chemical inhibitors of microsporidia infection. Using this protocol, we screened through the Spectrum Collection of 2,560 FDA-approved compounds and natural products. We developed a semi-automated method for quantifying progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that 6 of these compounds, including the known serine protease inhibitor ZPCK, are likely able to prevent microsporidia infection by inhibiting spore firing. We also show that 1 compound, Dexrazoxane, along with the known microsporidia inhibitor Fumagilin, are able to slow infection progression. Given the established role of Dexrazoxane as an iron chelator, it may act by depriving microsporidia of its essential iron resource, consequently putting the brakes on replication within the host. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors in the future.

Maaike C. W. van den Berg et al. (2006) European Worm Meeting "Sex-Dependent Resistance to the Fungal Pathogen Cryptococcus neoformans"

Maaike C. W. van den Berg, Jessica Z. Woerlee, Robin C. May, Hansong Ma

[

European Worm Meeting,

2006]

Maaike C. W. van den Berg1,2, Jessica Z. Woerlee2, Hansong Ma1,2 & Robin C. May1. Cryptococcus neoformans is the causative agent of cryptococcosis, a fatal fungal disease of immunocompromised patients. Our group and others have made use of C. elegans as an alternative host for Cryptococcus, in order to investigate the molecular basis of host-pathogen interactions. Using this system, we now report that male C. elegans show greater resistance to killing by Cryptococcus than hermaphrodite animals and that this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioural changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to Cryptococcus neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results suggest that many of the molecular determinants of longevity and immunity are the same and that differential regulation of these determinants may underlie much sex-dependent and species-dependent variation.

Kamal, Muntasir et al. (2021) International Worm Meeting "The Alimentary Cuticle of C. elegans Plays Multiple Roles in Mediating Xenobiotic Sensitivity"

Cummins, Carolyn, Kamal, Muntasir, Nguyen, Ken, Yip, Christopher, Knox, Jessica, Yeo, May, Roy, Peter, Szlapa, Karolina, Moshiri, Houtan, Won, Amy, Magomedova, Lilia, Hall, David, Bagg, Rachel, Han, Duhyun

[

International Worm Meeting,

2021]

Forward genetic screens with small molecules (SMs) in C. elegans carried out in our and other laboratories have repeatedly shown that some SMs aggregate in the anterior pharynx of worms as dark objects. Our group first identified the accumulation as at least partly, if not wholly composed of SM itself manifest as birefringent crystals and/or globular spherical objects. The crystallizing molecules are all lethal to worms. Our data indicates that the process of SM crystallization begins in the pharyngeal cuticle of the anterior pharynx, and over time, extends to the plasma membrane, ultimately causing extensive cellular damage. The crystallizing molecules can be grouped into 24 distinct structurally similar families (called scaffolds), and tend to be generally more hydrophobic and polar than molecules that do not aggregate. To understand the molecular basis of crystal accumulation, we screened 1.3 million randomly mutagenized wild-type F2 genomes and isolated 46 mutants that resist the lethal effects of a crystallizing SM, wact-190. This uncovered loss or reduction-of-function mutations in genes encoding components of a putative sphingomyelin synthesis pathway, SMS-5, TTM-5 and SPTL-2. Additionally, loss or reduction-of-function mutations in the ABC transporter PGP-14 also confer robust resistance to all crystallizing SMs. sms-5 and pgp-14 are moderately and highly expressed, respectively, in pharyngeal cells that line the pharynx cuticle. Both sms-5 and pgp-14 mutants show reduced accumulation of the hydrophobic, crystallizing SMs. Interestingly, both mutants show higher accumulation and sensitivity to a different set of lethal SMs with different physical properties relative to the wildtype. Together, these results indicate that (1) the anterior pharyngeal cuticle may be an important route of SM entry and a "sink" for many hydrophobic molecules, and (2) loss of SMS-5 or PGP-14 activity leads to altered barrier properties. Remarkably, sms-5 mutants are hypersensitive to cholesterol-deprived conditions, suggesting sms-5 is specifically required for accumulation of hydrophobic nutrients and other SMs in the pharynx cuticle and in turn into pharyngeal cells. PGP-14 maybe required for transporting a hydrophobic molecule related to SMS-5-catalyzed polar lipid synthesis into the developing cuticle. Taken together, our data suggest that SMS-5 and PGP-14 contribute to a lipid barrier in the anterior pharynx cuticle to limit access of small molecules to the animal.

Gupta, Pratyush et al. (2013) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Leahul, Lindsay, Gupta, Pratyush, Hansen, David, Jasper, Katie

[

International Worm Meeting,

2013]

We previously demonstrated that the proteasome inhibits the proliferative fate in the C. elegans germ line. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald, Knox et al. 2008). Presumably, the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To identify these PPPs, we first sought to identify the substrate recognition subunits (SRS) of E3 ubiquitin ligases that may target the PPPs for degradation by the proteasome. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five that enhance over-proliferation. One of these, RFP-1, specifically enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. Large-scale 2-hybrid screens identified potential binding partners for RFP-1 (Crowe and Candido 2004; Zhong and Sternberg 2006), which could be PPPs that are targeted for proteasomal degradation by RFP-1. One of these, mrg-1, partially suppresses the over-proliferation phenotype of glp-1(ar202gf); rfp-1(ok572), suggesting that it may function as a PPP. We found that MRG-1 levels do increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition. Therefore, MRG-1 protein levels are likely regulated through proteasomal-mediated degradation. We are currently determining how mrg-1 may participate in controlling the proliferative fate in the germline.

Burglin TR et al. (2016) Chromosoma "Homeodomain proteins: an update."

Affolter M, Burglin TR

[

Chromosoma,

2016]

Here, we provide an update of our review on homeobox genes that we wrote together with Walter Gehring in 1994. Since then, comprehensive surveys of homeobox genes have become possible due to genome sequencing projects. Using the 103 Drosophila homeobox genes as example, we present an updated classification. In animals, there are 16 major classes, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX/SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. In plants, there are 11 major classes, i.e., HD-ZIP (with four subclasses: I to IV), WOX, NDX, PHD, PLINC, LD, DDT, SAWADEE, PINTOX, and the two TALE classes KNOX and BEL. Most of these classes encode additional domains apart from the homeodomain. Numerous insights have been obtained in the last two decades into how homeodomain proteins bind to DNA and increase their specificity by interacting with other proteins to regulate cell- and tissue-specific gene expression. Not only protein-DNA base pair contacts are important for proper target selection; recent experiments also reveal that the shape of the DNA plays a role in specificity. Using selected examples, we highlight different mechanisms of homeodomain protein-DNA interaction. The PRD class of homeobox genes was of special interest to Walter Gehring in the last two decades. The PRD class comprises six families in Bilateria, and tinkers with four different motifs, i.e., the PAIRED domain, the Groucho-interacting motif EH1 (aka Octapeptide or TN), the homeodomain, and the OAR motif. Homologs of the co-repressor protein Groucho are also present in plants (TOPLESS), where they have been shown to interact with small amphipathic motives (EAR), and in yeast (TUP1), where we find an EH1-like motif in MAT2.

Roy, Peter et al. (2021) International Worm Meeting "A C. elegans Motor-Centric Screening Pipeline Yields Novel and Selective Nematicidal Scaffolds"

[

International Worm Meeting,

2021]

Nematode parasites of humans, livestock and crops pose a significant burden to human health and welfare. Alarmingly, our arsenal of effective nematocidal compounds is being depleted. Parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides used for crop protection have been restricted or banned because of poor phylum-selectivity. Here, we present our C. elegans-based discovery pipeline focused on lethal molecules that also induce motor defects. This pipeline yielded multiple new and selective nematicidal small molecule scaffolds (i.e., structurally-related families of molecules). We show that one of these scaffolds, tentatively called the APPs, stimulates neurotransmitter release and immobilizes larvae of multiple nematode parasites of plants and mammals in vitro. At similar concentrations, APP-1 does little to model flies, fish, plants or human cells. Forward genetic screens of 100,000s of mutant genomes failed to yield resistant animals that resist the lethal effects of APP-1, suggesting that resistance to the APPs in the field may not be easily generated. Hence, the APPs represent a novel, selective and potentially useful addition to our nematocidal armament.