Dataset Information

Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

ABSTRACT: Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1) of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ) triple mutant ("Cyb") with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors.

SUBMITTER: Tehseen M

PROVIDER: S-EPMC4240545 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

Tehseen Muhammad M Dumancic Mira M Briggs Lyndall L Wang Jian J Berna Amalia A Anderson Alisha A Trowell Stephen S

PloS one 20141121 11

Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands ...[more]

PMID: 25415379

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Project description:Parasitic nematodes constitute one of the major threats to human health, causing diseases of major socioeconomic importance worldwide. Recent estimates indicate that more than 1 billion people are infected with parasitic nematodes around the world. Current measures to combat parasitic nematode infections include anthelmintic drugs. However, heavy exposure to anthelmintics has selected populations of livestock parasitic nematodes that are no longer susceptible to the drugs, rendering several anthelmintics useless for parasitic nematode control in many areas of the world. The rapidity with which anthelmintic resistance developed in response to these drugs suggests that increasing the selective pressure on human parasitic nematodes will also rapidly generate resistant worm populations. Therefore, development of new anthelmintics is of major importance before resistance becomes widespread in human parasitic nematode populations. G-Protein Coupled Receptors (GPCRs) represent an important target for many pharmacological interventions due to their ubiquitous expression in various cell types. GPCRs contribute to numerous physiological processes, and their ligand binding sites located on cell surfaces make them accessible targets and attractive substrates in terms of druggability. In fact, ∼35 % of Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drugs target GPCRs and their associated proteins, with over 300 additional drugs targeting GPCRs at the clinical trial stage. Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent ideal substrates for target-based drug discovery. Here we set out to identify NemChRs that are transcriptionally active inside the host, and to use these NemChRs in a reverse pharmacological screen to impede parasitic development. Our data identified several NemChRs, and we focused on one that was expressed in neuronal cells and exhibited the highest fold change in transcription after host activation. Next, we performed homology modelling and molecular dynamics simulations of this NemChR in order to conduct a virtual screening campaign to identify candidate drug targets which were ranked and selected for experimental testing in bioassays. Taken together, our results identify and characterize a candidate NemChR drug target, and provide a chemogenomic pipeline for identifying nematicide substrates.

Project description:A MAPK cascade consists of three kinases, (MEKK, MEK and MAPK), that are sequentially activated in response to a stimulus and serve to transmit signals. In C. albicans and in yeast, an MAPK cascade is linked to the pheromone pathway through a scaffold protein (Cst5 and Ste5, respectively). Cst5 is much shorter and lacks key domains compared to Ste5, so in C. albicans, other elements, in particular the MEKK Ste11, play key roles in controlling the associations and localizations of network components.AbstractCandida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or β subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

Dataset Information

Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

Publications

Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

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