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[
Sci Rep,
2016]
G-protein signalling is an evolutionary conserved concept highlighting its fundamental impact on developmental and functional processes. Studies on the effects of G protein signals on tissues as well as an entire organism are often conducted in Caenorhabditis elegans. To understand and control dynamics and kinetics of the processes involved, pharmacological modulation of specific G protein pathways would be advantageous, but is difficult due to a lack in accessibility and regulation. To provide this option, we designed G protein-coupled receptor-based designer receptors (DREADDs) for C. elegans. Initially described in mammalian systems, these modified muscarinic acetylcholine receptors are activated by the inert drug clozapine-N-oxide, but not by their endogenous agonists. We report a novel C. elegans-specific DREADD, functionally expressed and specifically activating Gq-protein signalling in vitro and in vivo which we used for modulating mating behaviour. Therefore, this novel designer receptor demonstrates the possibility to pharmacologically control physiological functions in C. elegans.
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Schoneberg T, Promel S, Staunton D, Russ AP, Woollard A, Langenhan T, Vakonakis I, Hughes S, Schnabel R, Mestek L, Frickenhaus M
[
Cell Rep,
2012]
Adhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions.
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[
Worm,
2016]
Although several signaling pathways in oriented cell division have been well characterized such as delta/notch inductions or wnt/frizzled-based anterior-posterior polarity, there is strong evidence for additional signal pathways controlling early anterior-posterior polarity decisions. The homolog of the adhesion G protein-coupled receptor latrophilin, LAT-1 has been identified as a receptor essential for oriented cell division in an anterior-posterior direction of specific blastomeres in the early C. elegans embryo. We recently conducted a study aiming at clarifying the signals involved in LAT-1 function. We identified a Gs protein/adenylyl cyclase/cAMP pathway in vitro and demonstrated its physiological relevance in oriented cell division. By interaction with a Gs protein LAT-1 elevates cAMP levels. These data indicate that G-protein signaling in oriented cell division is not solely GPCR-independent. This commentary will discuss our findings in the context of the current knowledge of mechanisms controlling oriented cell division and anterior-posterior polarity. Further, we identify open questions which need to be addressed in the future.
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[
J Dev Biol,
2018]
Many vital processes during C. elegans development, especially the establishment and maintenance of cell polarity in embryogenesis, are controlled by complex signaling pathways. G protein-coupled receptors (GPCRs), such as the four Frizzled family Wnt receptors, are linchpins in regulating and orchestrating several of these mechanisms. However, despite being GPCRs, which usually couple to G proteins, these receptors do not seem to activate classical heterotrimeric G protein-mediated signaling cascades. The view on signaling during embryogenesis is further complicated by the fact that heterotrimeric G proteins do play essential roles in cell polarity during embryogenesis, but their activity is modulated in a predominantly GPCR-independent manner via G protein regulators such as GEFs GAPs and GDIs. Further, the triggered downstream effectors are not typical. Only very few GPCR-dependent and G protein-mediated signaling pathways have been unambiguously defined in this context. This unusual and highly intriguing concept of separating GPCR function and G-protein activity, which is not restricted to embryogenesis in <i>C. elegans</i> but can also be found in other organisms, allows for essential and multi-faceted ways of regulating cellular communication and response. Although its relevance cannot be debated, its impact is still poorly discussed, and <i>C.</i><i>elegans</i> is an ideal model to understand the underlying principles.
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[
J Biol Chem,
2007]
The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.
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Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G
[
Mol Cell,
2018]
In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
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[
PLoS One,
2017]
In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.
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[
Cell Commun Signal,
2019]
BACKGROUND: The neuropeptide Y system affects various processes, among others food intake, and is frequently discussed in the context of targeting obesity. Studies in model organisms are indispensable to enable molecular studies in a physiological context. Although the NPY system is evolutionarily conserved in all bilaterians, in the widely used model Caenorhabditis elegans there is controversy on the existence of NPY orthologous molecules. While the FMRFamide-like peptide (FLP)/Neuropeptide receptor-Resemblance (NPR) system in the nematode was initially suggested to be orthologous to the mammalian NPY system, later global phylogenetic studies indicate that FLP/NPR is protostome-specific. METHODS: We performed a comprehensive pharmacological study of the FLP/NPR system in transfected cells in vitro, and tested for functional substitution in C. elegans knockout strains. Further, we phenotypically compared different flp loss-of-function strains. Differences between groups were compared by ANOVA and post-hoc testing (Dunnett, Bonferroni). RESULTS: Our pharmacological analysis of the FLP/NPR system including formerly functionally uncharacterized NPY-like peptides from C. elegans demonstrates that G protein-coupling and ligand requirements for receptor activation are similar to the human NPY system. In vitro and in vivo analyses show cross-reactivity of NPY with the FLP/NPR system manifesting in the ability of the human GPCRs to functionally substitute FLP/NPR signaling in vivo. The high pharmacological/functional similarities enabled us to identify C. elegans FLP-14 as a key molecule in avoidance behavior. CONCLUSIONS: Our data demonstrate the pharmacological and functional similarities of human NPY and C. elegans NPR systems. This adds a novel perspective to current phylogenetic reconstructions of the neuropeptide Y system. NPY and NPR receptors are pharmacologically so similar that the human receptors can functionally compensate for the C. elegans ones, suggesting orthologous relationships. This is also underlined by the presence of NPY-like peptides and parallels in peptide requirements for receptor activation. Further, the results presented here highlight the potential of this knowledge for physiological as well as molecular studies on neuropeptide GPCRs such as the NPY system in the future.
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[
Pathog Dis,
2014]
Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.
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Haass C, Hegermann J, Giese A, Eimer S, Kamp F, Lutz AK, Nuscher B, Wender N, Brunner B, Winklhofer KF, Exner N, Beyer K, Bartels T
[
EMBO J,
2010]
Aggregation of -synuclein (S) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of S is largely unknown. We demonstrate with in vitro vesicle fusion experiments that S has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, S binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous S. In contrast, siRNA-mediated downregulation of S results in elongated mitochondria in cell culture. S can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, S prevents fusion of differently labelled mitochondrial populations. Thus, S inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of S is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin 1-79 or by DJ-1 C106A.