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1.
Nat Commun ; 11(1): 4627, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009389

RESUMO

Animals have evolved responses to low oxygen conditions to ensure their survival. Here, we have identified the C. elegans zinc finger transcription factor PQM-1 as a regulator of the hypoxic stress response. PQM-1 is required for the longevity of insulin signaling mutants, but surprisingly, loss of PQM-1 increases survival under hypoxic conditions. PQM-1 functions as a metabolic regulator by controlling oxygen consumption rates, suppressing hypoxic glycogen levels, and inhibiting the expression of the sorbitol dehydrogenase-1 SODH-1, a crucial sugar metabolism enzyme. PQM-1 promotes hypoxic fat metabolism by maintaining the expression of the stearoyl-CoA desaturase FAT-7, an oxygen consuming, rate-limiting enzyme in fatty acid biosynthesis. PQM-1 activity positively regulates fat transport to developing oocytes through vitellogenins under hypoxic conditions, thereby increasing survival rates of arrested progeny during hypoxia. Thus, while pqm-1 mutants increase survival of mothers, ultimately this loss is detrimental to progeny survival. Our data support a model in which PQM-1 controls a trade-off between lipid metabolic activity in the mother and her progeny to promote the survival of the species under hypoxic conditions.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Hipóxia/metabolismo , Metabolismo dos Lipídeos , Transativadores/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Glicogênio/metabolismo , Insulina/metabolismo , Larva/metabolismo , Mutação/genética , Consumo de Oxigênio , Transdução de Sinais , Estresse Fisiológico , Análise de Sobrevida , Transativadores/genética , Transcrição Genética , Vitelogeninas/metabolismo
2.
Nat Commun ; 11(1): 4496, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901024

RESUMO

Aging is characterized by the loss of homeostasis and the general decline of physiological functions, accompanied by various degenerative diseases and increased rates of mortality. Aging targeting small molecule screens have been performed many times, however, few have focused on endogenous metabolic intermediates-metabolites. Here, using C. elegans lifespan assays, we conducted a worm metabolite screen and identified an eukaryotes conserved metabolite, myo-inositol (MI), to extend lifespan, increase mobility and reduce fat content. Genetic analysis of enzymes in MI metabolic pathway suggest that MI alleviates aging through its derivative PI(4,5)P2. MI and PI(4,5)P2 are precursors of PI(3,4,5)P3, which is negatively related to longevity. The longevity effect of MI is dependent on the tumor suppressor gene, daf-18 (homologous to mouse Pten), independent of its classical pathway downstream genes, akt or daf-16. Furthermore, we found MI effects on aging and lifespan act through mitophagy regulator PTEN induced kinase-1 (pink-1) and mitophagy. MI's anti-aging effect is also conserved in mouse, indicating a conserved mechanism in mammals.


Assuntos
Envelhecimento/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Inositol/metabolismo , Longevidade/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Inositol/administração & dosagem , Locomoção/fisiologia , Longevidade/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Metabolômica , Camundongos , Mitofagia/fisiologia , Modelos Animais , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA-Seq
3.
PLoS Pathog ; 16(9): e1008826, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32970778

RESUMO

The nematode Caenorhabditis elegans has been extensively used as a model for the study of innate immune responses against bacterial pathogens. While it is well established that the worm mounts distinct transcriptional responses to different bacterial species, it is still unclear in how far it can fine-tune its response to different strains of a single pathogen species, especially if the strains vary in virulence and infection dynamics. To rectify this knowledge gap, we systematically analyzed the C. elegans response to two strains of Bacillus thuringiensis (Bt), MYBt18247 (Bt247) and MYBt18679 (Bt679), which produce different pore forming toxins (PFTs) and vary in infection dynamics. We combined host transcriptomics with cytopathological characterizations and identified both a common and also a differentiated response to the two strains, the latter comprising almost 10% of the infection responsive genes. Functional genetic analyses revealed that the AP-1 component gene jun-1 mediates the common response to both Bt strains. In contrast, the strain-specific response is mediated by the C. elegans GATA transcription factor ELT-2, a homolog of Drosophila SERPENT and vertebrate GATA4-6, and a known master regulator of intestinal responses in the nematode. elt-2 RNAi knockdown decreased resistance to Bt679, but remarkably, increased survival on Bt247. The elt-2 silencing-mediated increase in survival was characterized by reduced intestinal tissue damage despite a high pathogen burden and might thus involve increased tolerance. Additional functional genetic analyses confirmed the involvement of distinct signaling pathways in the C. elegans defense response: the p38-MAPK pathway acts either directly with or in parallel to elt-2 in mediating resistance to Bt679 infection but is not required for protection against Bt247. Our results further suggest that the elt-2 silencing-mediated increase in survival on Bt247 is multifactorial, influenced by the nuclear hormone receptors NHR-99 and NHR-193, and may further involve lipid metabolism and detoxification. Our study highlights that the nematode C. elegans with its comparatively simple immune defense system is capable of generating a differentiated response to distinct strains of the same pathogen species. Importantly, our study provides a molecular insight into the diversity of biological processes that are influenced by a single master regulator and jointly determine host survival after pathogen infection.


Assuntos
Bacillus thuringiensis/metabolismo , Infecções Bacterianas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fatores de Transcrição GATA/metabolismo , Sistema de Sinalização das MAP Quinases , Transcrição Genética , Animais , Bacillus thuringiensis/patogenicidade , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Fatores de Transcrição GATA/genética
4.
Nat Commun ; 11(1): 4869, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978394

RESUMO

Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.


Assuntos
Caenorhabditis elegans/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas , Glicosídeo Hidrolases/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional
5.
Nat Commun ; 11(1): 4865, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978396

RESUMO

The metabolic state of an organism instructs gene expression modalities, leading to changes in complex life history traits, such as longevity. Dietary restriction (DR), which positively affects health and life span across species, leads to metabolic reprogramming that enhances utilisation of fatty acids for energy generation. One direct consequence of this metabolic shift is the upregulation of cytoprotective (CyTP) genes categorized in the Gene Ontology (GO) term of "Xenobiotic Detoxification Program" (XDP). How an organism senses metabolic changes during nutritional stress to alter gene expression programs is less known. Here, using a genetic model of DR, we show that the levels of polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and eicosapentaenoic acid (EPA), are increased following DR and these PUFAs are able to activate the CyTP genes. This activation of CyTP genes is mediated by the conserved p38 mitogen-activated protein kinase (p38-MAPK) pathway. Consequently, genes of the PUFA biosynthesis and p38-MAPK pathway are required for multiple paradigms of DR-mediated longevity, suggesting conservation of mechanism. Thus, our study shows that PUFAs and p38-MAPK pathway function downstream of DR to help communicate the metabolic state of an organism to regulate expression of CyTP genes, ensuring extended life span.


Assuntos
Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Fenômenos Bioquímicos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Ácido Linoleico/metabolismo , Longevidade , Redes e Vias Metabólicas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
6.
Nat Commun ; 11(1): 4029, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788597

RESUMO

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5' AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.


Assuntos
Autofagia , Atrofia Óptica Autossômica Dominante/complicações , Transtornos da Visão/complicações , Adenilato Quinase/metabolismo , Animais , Autofagia/genética , Axônios/patologia , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , GTP Fosfo-Hidrolases/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitofagia , Mutação/genética , Fosforilação , Células Ganglionares da Retina/patologia
7.
PLoS Genet ; 16(8): e1008975, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750056

RESUMO

The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Receptores de Adiponectina/genética , Alelos , Animais , Caenorhabditis elegans/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Mutação com Ganho de Função/genética , Células HEK293 , Humanos , Fluidez de Membrana/genética , Fenótipo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo
8.
PLoS Genet ; 16(8): e1008984, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32857789

RESUMO

Mutations in human metabolic genes can lead to rare diseases known as inborn errors of human metabolism. For instance, patients with loss-of-function mutations in either subunit of propionyl-CoA carboxylase suffer from propionic acidemia because they cannot catabolize propionate, leading to its harmful accumulation. Both the penetrance and expressivity of metabolic disorders can be modulated by genetic background. However, modifiers of these diseases are difficult to identify because of the lack of statistical power for rare diseases in human genetics. Here, we use a model of propionic acidemia in the nematode Caenorhabditis elegans to identify genetic modifiers of propionate sensitivity. Using genome-wide association (GWA) mapping across wild strains, we identify several genomic regions correlated with reduced propionate sensitivity. We find that natural variation in the putative glucuronosyltransferase GLCT-3, a homolog of human B3GAT, partly explains differences in propionate sensitivity in one of these genomic intervals. We demonstrate that loss-of-function alleles in glct-3 render the animals less sensitive to propionate. Additionally, we find that C. elegans has an expansion of the glct gene family, suggesting that the number of members of this family could influence sensitivity to excess propionate. Our findings demonstrate that natural variation in genes that are not directly associated with propionate breakdown can modulate propionate sensitivity. Our study provides a framework for using C. elegans to characterize the contributions of genetic background in models of human inborn errors in metabolism.


Assuntos
Predisposição Genética para Doença , Glucuronosiltransferase/genética , Propionatos/farmacologia , Acidemia Propiônica/genética , Alelos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Glucuronosiltransferase/deficiência , Humanos , Mutação com Perda de Função/genética , Metabolismo/genética , Propionatos/metabolismo
9.
Nat Commun ; 11(1): 4242, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843637

RESUMO

Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/genética , Sítios de Ligação , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Mutação , Ligação Proteica , Proteômica , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética
10.
PLoS Biol ; 18(8): e3000817, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813728

RESUMO

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence/fluorescence in situ hybridization (immuno-FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.


Assuntos
Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Cromossomos/ultraestrutura , Meiose , Complexo Sinaptonêmico/ultraestrutura , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Análise Citogenética , Hibridização in Situ Fluorescente , Fase S/genética , Complexo Sinaptonêmico/metabolismo
11.
BMC Bioinformatics ; 21(1): 323, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32693790

RESUMO

BACKGROUND: Protein-protein interactions (PPIs) are central to many biological processes. Considering that the experimental methods for identifying PPIs are time-consuming and expensive, it is important to develop automated computational methods to better predict PPIs. Various machine learning methods have been proposed, including a deep learning technique which is sequence-based that has achieved promising results. However, it only focuses on sequence information while ignoring the structural information of PPI networks. Structural information of PPI networks such as their degree, position, and neighboring nodes in a graph has been proved to be informative in PPI prediction. RESULTS: Facing the challenge of representing graph information, we introduce an improved graph representation learning method. Our model can study PPI prediction based on both sequence information and graph structure. Moreover, our study takes advantage of a representation learning model and employs a graph-based deep learning method for PPI prediction, which shows superiority over existing sequence-based methods. Statistically, Our method achieves state-of-the-art accuracy of 99.15% on Human protein reference database (HPRD) dataset and also obtains best results on Database of Interacting Protein (DIP) Human, Drosophila, Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegan) datasets. CONCLUSION: Here, we introduce signed variational graph auto-encoder (S-VGAE), an improved graph representation learning method, to automatically learn to encode graph structure into low-dimensional embeddings. Experimental results demonstrate that our method outperforms other existing sequence-based methods on several datasets. We also prove the robustness of our model for very sparse networks and the generalization for a new dataset that consists of four datasets: HPRD, E.coli, C.elegan, and Drosophila.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Animais , Caenorhabditis elegans/metabolismo , Simulação por Computador , Bases de Dados de Proteínas , Drosophila/metabolismo , Escherichia coli/metabolismo , Humanos , Aprendizado de Máquina , Redes Neurais de Computação
12.
Proc Natl Acad Sci U S A ; 117(29): 17142-17150, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636256

RESUMO

Gut microbes play diverse roles in modulating host fitness, including longevity; however, the molecular mechanisms underlying their mediation of longevity remain poorly understood. We performed genome-wide screens using 3,792 Escherichia coli mutants and identified 44 E. coli mutants that modulated Caenorhabditis elegans longevity. Three of these mutants modulated C. elegans longevity via the bacterial metabolite methylglyoxal (MG). Importantly, we found that low MG-producing E. coli mutants, Δhns E. coli, extended the lifespan of C. elegans through activation of the DAF-16/FOXO family transcription factor and the mitochondrial unfolded protein response (UPRmt). Interestingly, the lifespan modulation by Δhns did not require insulin/insulin-like growth factor 1 signaling (IIS) but did require TORC2/SGK-1 signaling. Transcriptome analysis revealed that Δhns E. coli activated novel class 3 DAF-16 target genes that were distinct from those regulated by IIS. Taken together, our data suggest that bacteria-derived MG modulates host longevity through regulation of the host signaling pathways rather than through nonspecific damage on biomolecules known as advanced glycation end products. Finally, we demonstrate that MG enhances the phosphorylation of hSGK1 and accelerates cellular senescence in human dermal fibroblasts, suggesting the conserved role of MG in controlling longevity across species. Together, our studies demonstrate that bacteria-derived MG is a novel therapeutic target for aging and aging-associated pathophysiology.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Fatores de Transcrição Forkhead/metabolismo , Longevidade/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Aldeído Pirúvico , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Escherichia coli/metabolismo , Microbioma Gastrointestinal/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Modelos Biológicos , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genética
13.
Nature ; 584(7821): 410-414, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641833

RESUMO

In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1-5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6-9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.


Assuntos
Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Espaço Extracelular/metabolismo , Agregados Proteicos , Proteostase , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema de Sinalização das MAP Quinases , Agregação Patológica de Proteínas/prevenção & controle , Proteoma/genética , Proteoma/metabolismo , Interferência de RNA
14.
Chemosphere ; 260: 127627, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32673864

RESUMO

Nickel is the most prevailing metal allergen with the highest sensitization rate among the "TOP 25" contact allergens and can affect about 15% of the human population. It is an essential trace metal in plants, animals, and humans. However, the environmental levels of nickel are considerably higher than what is needed for human life. Exposure to high levels of nickel can lead to skin allergies, lung fibrosis, and carcinogenesis. Few existing studies have closely examined the toxicity of nickel, let alone investigated the effective detoxification pathways. Here, we developed a high-throughput screening platform to comprehensively evaluate the nickel toxicity in wild-type C. elegans and explore the underlying detoxification mechanisms in transgenic nematodes. We demonstrated that nickel exerted multiple toxic effects on growth, brood size, feeding, and locomotion in C. elegans. Of which, brood size is the most sensitive endpoint. Nickel was found to first bind to phytochelatin (PC) after entering the worms' body and this PC-Ni complex was further transported by the ABC transporter, CeHMT-1, into the coelomocytes for further detoxification. Our study also demonstrated that the high-throughput screening platform is a promising system for evaluation and investigation of the ecological risks of heavy metals.


Assuntos
Caenorhabditis elegans/fisiologia , Níquel/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans/metabolismo , Locomoção , Metais Pesados/toxicidade , Nematoides , Níquel/toxicidade , Fitoquelatinas/metabolismo
15.
Nature ; 582(7811): 283-288, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499657

RESUMO

Mobile genetic elements threaten genome integrity in all organisms. RDE-3 (also known as MUT-2) is a ribonucleotidyltransferase that is required for transposon silencing and RNA interference in Caenorhabditis elegans1-4. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (designated poly(UG) or pUG tails)5. Here we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to targets of RNA interference, as well as to transposon RNAs. RNA fragments attached to pUG tails with more than 16 perfectly alternating 3' U and G nucleotides become gene-silencing agents. pUG tails promote gene silencing by recruiting RNA-dependent RNA polymerases, which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed pUG RNA biogenesis underlie double-stranded-RNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA-siRNA silencing loop enables parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/parasitologia , Epigênese Genética/genética , Genoma/genética , Hereditariedade , Poli G/genética , Poli U/genética , RNA Mensageiro/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Nucleotidiltransferases/metabolismo , Interferência de RNA , RNA Replicase/metabolismo , RNA Interferente Pequeno/genética , Moldes Genéticos
16.
Proc Natl Acad Sci U S A ; 117(25): 14270-14279, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513699

RESUMO

Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in Caenorhabditis elegans, the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1-dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Movimento Celular/fisiologia , Neurônios/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Polaridade Celular/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Transdução de Sinais
17.
Ecotoxicol Environ Saf ; 201: 110857, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32534332

RESUMO

Caenorhabditis elegans is sensitive to toxicity of environmental pollutants. The alteration in expression of mir-794, a microRNA (miRNA) molecule, mediated a protective response to nanopolystyene (100 nm) at predicted environmental concentration (1 µg/L) in nematodes. However, the underlying molecular basis for mir-794 function in regulating the response to nanopolystyrene remains largely unclear. In this study, we found that intestinal overexpression of mir-794 caused the susceptibility to nanopolystyrene toxicity, suggesting that mir-794 acted in the intestine to regulate the response to nanopolystyrene. Intestinal overexpression of mir-794 further decreased the expressions of daf-16 encoding a FOXO transcriptional factor in insulin signaling pathway, skn-1 encoding a Nrf transcriptional factor in p38 MAPK signaling pathway, and mdt-15 encoding a lipid metabolic sensor acting downstream of SKN-1 in nanopolystyrene exposed nematodes. Meanwhile, intestinal overexpression of mir-794 could suppress the resistance of nematodes overexpressing intestinal daf-16, skn-1, or mdt-15 containing the corresponding 3' untranslated region (3' UTR) to nanopolystyrene toxicity. Therefore, DAF-16, SKN-1, and MDT-15 acted as the downstream targets of intestinal mir-794 to regulate the response to nanopolystyrene. In the intestine, DAF-16 functioned synergistically with SKN-1 or MDT-15 to regulate the response to nanopolystyrene. Our results suggested that the intestinal mir-794 provided an important epigenetic regulation mechanism to control the response to nanopolystyrene by linking insulin and p38 MAPK signaling pathways in nematodes.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Insulina/metabolismo , Intestinos/efeitos dos fármacos , MicroRNAs/metabolismo , Nanopartículas/toxicidade , Poliestirenos/toxicidade , Poluentes do Solo/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Lipídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/genética , Fatores de Transcrição/genética
18.
Nat Commun ; 11(1): 2983, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532993

RESUMO

Calorimetry has been widely used in metabolic studies, but direct measurements from individual small biological model organisms such as C. elegans or isolated single cells have been limited by poor sensitivity of existing techniques and difficulties in resolving very small heat outputs. Here, by careful thermal engineering, we developed a robust, highly sensitive and bio-compatible calorimetric platform that features a resolution of ~270 pW-more than a 500-fold improvement over the most sensitive calorimeter previously used for measuring the metabolic heat output of C. elegans. Using this calorimeter, we demonstrate time-resolved metabolic measurements of single C. elegans worms from larval to adult stages. Further, we show that the metabolic output is significantly lower in long-lived C. elegans daf-2 mutants. These demonstrations clearly highlight the broad potential of this tool for studying the role of metabolism in disease, development and aging of small model organisms and single cells.


Assuntos
Caenorhabditis elegans/metabolismo , Calorimetria/métodos , Análise de Célula Única/métodos , Temperatura , Animais , Metabolismo Basal/genética , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Calorimetria/instrumentação , Metabolismo Energético/genética , Humanos , Larva/citologia , Larva/genética , Larva/metabolismo , Longevidade/genética , Modelos Animais , Mutação , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única/instrumentação , Condutividade Térmica
19.
Proc Natl Acad Sci U S A ; 117(24): 13637-13646, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482879

RESUMO

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN in Caenorhabditis elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.


Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Redes Reguladoras de Genes , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510480

RESUMO

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico , Estresse Oxidativo , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Homeostase , Mitocôndrias/metabolismo
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