RESUMO
Ca(2+)-activated Cl(-) channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.
Assuntos
Caenorhabditis elegans/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Filogenia , Animais , Transporte Biológico/genética , Caenorhabditis elegans/genética , Cálcio/metabolismo , Canais de Cloreto/genética , Proteínas de Membrana/genética , Neurônios Aferentes/metabolismo , Transdução de Sinais/genéticaRESUMO
Nematodes parasitize an alarming number of people and agricultural animals globally and cause debilitating morbidity and mortality. Anthelmintics have been the primary tools used to control parasitic nematodes for the past several decades, but drug resistance is becoming a major obstacle. Xenobiotic detoxification pathways defend against drugs and other foreign chemicals in diverse organisms, and evidence is accumulating that they play a role in mediating resistance to anthelmintics in nematodes. Related antioxidation pathways may also provide filarial parasites with protection against host free-radical-mediated immune responses. Upstream regulatory pathways have received almost no attention in nematode parasites, despite their potential to coregulate multiple detoxification and antioxidation genes. The nuclear eurythroid 2-related factor 2 (NRF2) transcription factor mediates inducible detoxification and antioxidation defenses in mammals, and recent studies have demonstrated that it promotes multidrug resistance in some human tumors. Recent studies in the free-living model nematode, Caenorhabditis elegans, have defined the homologous transcription factor, SKN-1, as a master regulator of detoxification and antioxidation genes. Despite similar functions, SKN-1 and NRF2 have important differences in structure and regulatory pathways. Protein alignment and phylogenetic analyses indicate that these differences are shared among many nematodes, making SKN-1 a candidate for specifically targeting nematode detoxification and antioxidation.
Assuntos
Anti-Helmínticos/farmacologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistência a Medicamentos , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Anti-Helmínticos/farmacocinética , Proteínas de Caenorhabditis elegans/química , Proteínas de Ligação a DNA/química , Resistência a Múltiplos Medicamentos , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Humanos , Inativação Metabólica , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Nematoides/efeitos dos fármacos , Nematoides/metabolismo , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/químicaRESUMO
Nematodes parasitize â¼1/3 of humans worldwide, and effective treatment via administration of anthelmintics is threatened by growing resistance to current therapies. The nematode transcription factor SKN-1 is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics, which can promote resistance. Distinct differences in regulation and DNA binding relative to mammalian Nrf2 make SKN-1 a promising and selective target for the development of anthelmintics with a novel mode of action that targets stress resistance and drug detoxification. We report 17 (ML358), a first in class small molecule inhibitor of the SKN-1 pathway. Compound 17 resulted from a vanillamine-derived hit identified by high throughput screening that was advanced through analog synthesis and structure-activity studies. Compound 17 is a potent (IC50 = 0.24 µM, Emax = 100%) and selective inhibitor of the SKN-1 pathway and sensitizes the model nematode C. elegans to oxidants and anthelmintics. Compound 17 is inactive against Nrf2, the homologous mammalian detoxification pathway, and is not toxic to C. elegans (LC50 > 64 µM) and Fa2N-4 immortalized human hepatocytes (LC50 > 5.0 µM). In addition, 17 exhibits good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Therefore, 17 is a valuable probe to study regulation and function of SKN-1 in vivo. By selective targeting of the SKN-1 pathway, 17 could potentially lead to drug candidates that may be used as adjuvants to increase the efficacy and useful life of current anthelmintics.
Assuntos
Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Caenorhabditis elegans/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Helmintíase/tratamento farmacológico , Helmintíase/parasitologia , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a ß-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , DNA de Protozoário/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Larva/metabolismo , Longevidade/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Repressoras/genética , Estresse Fisiológico/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
Negative-feedback loops between transcription factors and repressors in responses to xenobiotics, oxidants, heat, hypoxia, DNA damage, and infection have been described. Although common, the function of feedback is largely unstudied. Here, we define a negative-feedback loop between the Caenorhabditis elegans detoxification/antioxidant response factor SKN-1/Nrf and its repressor wdr-23 and investigate its function in vivo. Although SKN-1 promotes stress resistance and longevity, we find that tight regulation by WDR-23 is essential for growth and reproduction. By disabling SKN-1 transactivation of wdr-23, we reveal that feedback is required to set the balance between growth/reproduction and stress resistance/longevity. We also find that feedback is required to set the sensitivity of a core SKN-1 target gene to an electrophile. Interestingly, the effect of feedback on target gene induction is greatly reduced when the stress response is strongly activated, presumably to ensure maximum activation of cytoprotective genes during potentially fatal conditions. Our work provides a framework for understanding the function of negative feedback in inducible stress responses and demonstrates that manipulation of feedback alone can shift the balance of competing animal processes toward cell protection, health, and longevity.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Citoproteção , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Longevidade , Proteínas Repressoras/genética , Reprodução , Estresse Fisiológico , Fatores de Transcrição/genéticaRESUMO
High-throughput screening (HTS) is a powerful approach to drug discovery, but many lead compounds are found to be unsuitable for use in vivo after initial screening. Screening in small animals like C. elegans can help avoid these problems, but this system has been limited to screens with low-throughput or no specific molecular target. We report the first in vivo 1536-well plate assay for a specific genetic pathway in C. elegans. Our assay measures induction of a gene regulated by SKN-1, a master regulator of detoxification genes. SKN-1 inhibitors will be used to study and potentially reverse multidrug resistance in parasitic nematodes. Screens of two small commercial libraries and the full Molecular Libraries Small Molecule Repository (MLSMR) of â¼364,000 compounds validate our platform for ultra HTS. Our platform overcomes current limitations of many whole-animal screens and can be widely adopted for other inducible genetic pathways in nematodes and humans.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Acrilamida/farmacologia , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The nucleolus has recently emerged as a major coordinator of cellular stress responses by regulating the tumor suppressor p53. However, it is not known if the nucleolus regulates the cap 'n' collar (CnC) transcription factors SKN-1 and Nrf2, which activate conserved antioxidant and detoxification responses in C. elegans and mammals, respectively. A screen for negative regulators of detoxification genes in C. elegans identified the conserved WD40 repeat containing protein WDR-46. This protein is highly conserved with yeast UTP7, which functions in 18S rRNA processing and assembly of the 40S small ribosomal subunit. WDR-46 is expressed in the nucleoli of multiple tissues in C. elegans and is required for rRNA processing. Mutation or silencing of WDR-46 activates the single C. elegans CnC homologue SKN-1 and increases expression of its target genes. Depletion of wdr-46 reduces lifespan and stress resistance and SKN-1 partially compensates. Lastly, the C. elegans p53 homologue CEP-1 is partially required for activation of gst-4 when wdr-46 or other ribosome processing genes are silenced but not when translation initiation genes are silenced suggesting that disruptions to nucleolar function can activate SKN-1 by a mechanism that involves p53/cep-1 and is independent of protein translation.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Metabólica/genética , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Xenobióticos/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Nucléolo Celular/metabolismo , Fatores de Transcrição Forkhead , Expressão Gênica , Regulação da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Expectativa de Vida , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Estresse FisiológicoRESUMO
High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo(1-2). Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development. C. elegans is a multicellular model organism well suited for HTS. It is small (<1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action(3). We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them(4). When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap 'n' collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress(5-10). Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target(11-13).Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-4(14), which encodes a glutathione-s transferase(6). The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury(15-16).