RESUMO
According to the hygiene hypothesis, the increasing incidence of autoimmune diseases in western countries may be explained by changes in early microbial exposure, leading to altered immune maturation. We followed gut microbiome development from birth until age three in 222 infants in Northern Europe, where early-onset autoimmune diseases are common in Finland and Estonia but are less prevalent in Russia. We found that Bacteroides species are lowly abundant in Russians but dominate in Finnish and Estonian infants. Therefore, their lipopolysaccharide (LPS) exposures arose primarily from Bacteroides rather than from Escherichia coli, which is a potent innate immune activator. We show that Bacteroides LPS is structurally distinct from E. coli LPS and inhibits innate immune signaling and endotoxin tolerance; furthermore, unlike LPS from E. coli, B. dorei LPS does not decrease incidence of autoimmune diabetes in non-obese diabetic mice. Early colonization by immunologically silencing microbiota may thus preclude aspects of immune education.
Assuntos
Bacteroides/imunologia , Diabetes Mellitus Tipo 1/imunologia , Microbioma Gastrointestinal , Lipopolissacarídeos/imunologia , Animais , Estônia , Fezes/microbiologia , Finlândia , Microbiologia de Alimentos , Humanos , Lactente , Camundongos , Camundongos Endogâmicos NOD , Leite Humano/imunologia , Federação RussaRESUMO
Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.
Assuntos
Proteínas ADAM/imunologia , Colite/imunologia , Matriz Extracelular/metabolismo , Fibroblastos/imunologia , Mucosa Intestinal/imunologia , Células-Tronco Mesenquimais/imunologia , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Animais , Diferenciação Celular , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/imunologia , Colo/patologia , Matriz Extracelular/imunologia , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Humanos , Inflamação , Interleucina-11/genética , Interleucina-11/imunologia , Mucosa Intestinal/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Análise de Célula Única , Dodecilsulfato de Sódio/administração & dosagem , Transcrição Gênica , Transcriptoma , Cicatrização/genética , Cicatrização/imunologiaRESUMO
Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.
Assuntos
Autofagia , Glutamina/metabolismo , Proteínas Quinases/metabolismo , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Drosophila melanogaster , Humanos , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismoRESUMO
Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Cromatografia de Afinidade , Proteômica , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Motivos de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Trombospondinas/químicaRESUMO
Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.
Assuntos
Antifúngicos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Antifúngicos/química , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450 , Farmacorresistência Fúngica/genética , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The endoplasmic reticulum (ER) is a membranous organelle that maintains proteostasis and cellular homeostasis, controlling the fine balance between health and disease. Dysregulation of the ER stress response has been implicated in intestinal inflammation associated with inflammatory bowel disease (IBD), a chronic condition characterized by changes to the mucosa and alteration of the gut microbiota. While the microbiota and microbially derived metabolites have also been implicated in ER stress, examples of this connection remain limited to a few observations from pathogenic bacteria. Furthermore, the mechanisms underlying the effects of bacterial metabolites on ER stress signaling have not been well established. RESULTS: Utilizing an XBP1s-GFP knock-in reporter colorectal epithelial cell line, we screened 399 microbiome-related metabolites for ER stress pathway modulation. We find both ER stress response inducers (acylated dipeptide aldehydes and bisindole methane derivatives) and suppressors (soraphen A) and characterize their activities on ER stress gene transcription and translation. We further demonstrate that these molecules modulate the ER stress pathway through protease inhibition or lipid metabolism interference. CONCLUSIONS: Our study identified novel links between classes of gut microbe-derived metabolites and the ER stress response, suggesting the potential for these metabolites to contribute to gut ER homeostasis and providing insight into the molecular mechanisms by which gut microbes impact intestinal epithelial cell homeostasis.
Assuntos
Bactérias/metabolismo , Estresse do Retículo Endoplasmático , Microbioma Gastrointestinal , Resposta a Proteínas não Dobradas , Aldeídos/farmacologia , Apoptose , Dipeptídeos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HT29 , Humanos , Indóis/farmacologia , Macrolídeos/farmacologia , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Bacterial ADP-ribosyltransferases (ADPRTs) have been described as toxins involved in pathogenesis through the modification of host proteins. Here, we report that ADPRTs are not pathogen restricted but widely prevalent in the human gut microbiome and often associated with phage elements. We validated their biochemical activity in a large clinical isolate collection and further examined Bxa, a highly abundant ADPRT in Bacteroides. Bxa is expressed, secreted, and enzymatically active in Bacteroides and can ADP-ribosylate non-muscle myosin II proteins. Addition of Bxa to epithelial cells remodeled the actin cytoskeleton and induced secretion of inosine. Bxa-encoding B. stercoris can use inosine as a carbon source and colonizes the gut to significantly greater numbers than a bxa-deleted strain in germ-free and altered Schaedler flora (ASF) mice. Colonization correlated with increased inosine concentrations in the feces and tissues. Altogether, our results show that ADPRTs are abundant in the microbiome and act as bacterial fitness factors.
Assuntos
ADP Ribose Transferases/metabolismo , Citoesqueleto de Actina/metabolismo , Bacteroides thetaiotaomicron/metabolismo , Bacteroides/metabolismo , Células Epiteliais/metabolismo , Inosina/metabolismo , ADP Ribose Transferases/genética , Animais , Bacteriófagos/genética , Células CACO-2 , Linhagem Celular Tumoral , Fezes/química , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Vida Livre de Germes , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Pesadas de Miosina/metabolismoRESUMO
Inflammatory bowel diseases (IBD) are associated with alterations in gut microbial abundances and lumenal metabolite concentrations, but the effects of specific metabolites on the gut microbiota in health and disease remain largely unknown. Here, we analysed the influences of metabolites that are differentially abundant in IBD on the growth and physiology of gut bacteria that are also differentially abundant in IBD. We found that N-acylethanolamines (NAEs), a class of endogenously produced signalling lipids elevated in the stool of IBD patients and a T-cell transfer model of colitis, stimulated growth of species over-represented in IBD and inhibited that of species depleted in IBD in vitro. Using metagenomic sequencing, we recapitulated the effects of NAEs in complex microbial communities ex vivo, with Proteobacteria blooming and Bacteroidetes declining in the presence of NAEs. Metatranscriptomic analysis of the same communities identified components of the respiratory chain as important for the metabolism of NAEs, and this was verified using a mutant deficient for respiratory complex I. In this study, we identified NAEs as a class of metabolites that are elevated in IBD and have the potential to shift gut microbiota towards an IBD-like composition.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Etanolaminas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Animais , Bactérias/genética , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/isolamento & purificação , Estudos de Coortes , Modelos Animais de Doenças , Disbiose , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Perfilação da Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Masculino , Metagenoma , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Proteobactérias/efeitos dos fármacos , Proteobactérias/isolamento & purificação , Espectrometria de Massas em Tandem , Sequenciamento Completo do GenomaRESUMO
Sphingolipids are structural membrane components and important eukaryotic signaling molecules. Sphingolipids regulate inflammation and immunity and were recently identified as the most differentially abundant metabolite in stool from inflammatory bowel disease (IBD) patients. Commensal bacteria from the Bacteroidetes phylum also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To determine whether bacterial sphingolipids modulate intestinal health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids resulted in intestinal inflammation and altered host ceramide pools in mice. Using lipidomic analysis, we described a sphingolipid biosynthesis pathway and revealed a variety of Bacteroides-derived sphingolipids including ceramide phosphoinositol and deoxy-sphingolipids. Annotating Bacteroides sphingolipids in an IBD metabolomic dataset revealed lower abundances in IBD and negative correlations with inflammation and host sphingolipid production. These data highlight the role of bacterial sphingolipids in maintaining homeostasis and symbiosis in the gut.
Assuntos
Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Bacteroides thetaiotaomicron/metabolismo , Interações entre Hospedeiro e Microrganismos , Intestinos/microbiologia , Intestinos/fisiologia , Esfingolipídeos/metabolismo , Simbiose/efeitos dos fármacos , Animais , Vida Livre de Germes , Homeostase/efeitos dos fármacos , Doenças Inflamatórias Intestinais/prevenção & controle , Intestinos/efeitos dos fármacos , CamundongosRESUMO
In the Supplementary Tables 2, 4 and 6 originally published with this Article, the authors mistakenly included sample identifiers in the form of UMCGs rather than UMCG IBDs in the validation cohort; this has now been amended.
RESUMO
In the version of this Article originally published, in the first sentence of the second paragraph of the Discussion section, the word "operingrationally" should have read "operationally". This has now been amended in all versions of the Article.
RESUMO
The inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial chronic conditions of the gastrointestinal tract. While IBD has been associated with dramatic changes in the gut microbiota, changes in the gut metabolome-the molecular interface between host and microbiota-are less well understood. To address this gap, we performed untargeted metabolomic and shotgun metagenomic profiling of cross-sectional stool samples from discovery (n = 155) and validation (n = 65) cohorts of CD, UC and non-IBD control patients. Metabolomic and metagenomic profiles were broadly correlated with faecal calprotectin levels (a measure of gut inflammation). Across >8,000 measured metabolite features, we identified chemicals and chemical classes that were differentially abundant in IBD, including enrichments for sphingolipids and bile acids, and depletions for triacylglycerols and tetrapyrroles. While > 50% of differentially abundant metabolite features were uncharacterized, many could be assigned putative roles through metabolomic 'guilt by association' (covariation with known metabolites). Differentially abundant species and functions from the metagenomic profiles reflected adaptation to oxidative stress in the IBD gut, and were individually consistent with previous findings. Integrating these data, however, we identified 122 robust associations between differentially abundant species and well-characterized differentially abundant metabolites, indicating possible mechanistic relationships that are perturbed in IBD. Finally, we found that metabolome- and metagenome-based classifiers of IBD status were highly accurate and, like the vast majority of individual trends, generalized well to the independent validation cohort. Our findings thus provide an improved understanding of perturbations of the microbiome-metabolome interface in IBD, including identification of many potential diagnostic and therapeutic targets.
Assuntos
Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Biodiversidade , Biomarcadores/metabolismo , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/microbiologia , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Fezes/química , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/imunologia , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Doenças Inflamatórias Intestinais/imunologia , Complexo Antígeno L1 Leucocitário/análise , Metaboloma , MetagenomaRESUMO
The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.
Assuntos
Adaptação Fisiológica , Microbioma Gastrointestinal/genética , Variação Genética , Genoma Bacteriano , Fatores Etários , Bacteriófagos/genética , Bacteroides/genética , Bacteroides/virologia , Bifidobacterium bifidum/genética , Bifidobacterium longum/genética , Desenvolvimento Infantil , Pré-Escolar , Estônia , Fezes/microbiologia , Feminino , Finlândia , Humanos , Lactente , Estudos Longitudinais , Masculino , Redes e Vias Metabólicas , Metagenômica , Polimorfismo de Nucleotídeo Único , Probióticos , Federação RussaRESUMO
Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits.
Assuntos
Indóis/metabolismo , Indóis/farmacologia , Inflamação/metabolismo , Mucosa Intestinal/microbiologia , Peptostreptococcus/metabolismo , Simbiose , Animais , Anti-Inflamatórios/farmacologia , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Clostridiales/genética , Clostridiales/metabolismo , Colo/microbiologia , Colo/patologia , Citocinas/metabolismo , Disbiose/metabolismo , Humanos , Doenças Inflamatórias Intestinais , Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Camundongos , Mucina-2/genética , Mucina-2/metabolismo , Mucinas/genética , Mucinas/metabolismo , OrganoidesRESUMO
SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56ß, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.
Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Proteínas do Tecido Nervoso/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Transtorno do Espectro Autista/enzimologia , Transtorno do Espectro Autista/genética , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 22/genética , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Neurônios/enzimologia , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death. This study demonstrates that the selectivity profile for N-BICs is through conversion by SULT1A1 from an inactive prodrug to an active species that induces cell death and tumor suppression.
Assuntos
Arilsulfotransferase/metabolismo , Compostos de Benzil/farmacologia , Indóis/farmacologia , Animais , Compostos de Benzil/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HCT116 , Humanos , Indóis/farmacocinética , Camundongos , Camundongos Nus , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Vias Biossintéticas , Farmacorresistência Fúngica , Regulação Fúngica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.