RESUMEN
Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1-4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9-12, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis13-15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17-20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNß after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.
Asunto(s)
Secuencias de Aminoácidos , Secuencia Conservada , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Células HEK293 , Humanos , Interferón beta/metabolismo , Proteínas de la Membrana/genética , Modelos Moleculares , Mutación , Nucleótidos Cíclicos/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved in Sgg pathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria and Staphylococcus aureus. Through genome analysis, we identified a locus in Sgg strain TX20005 that encodes a putative type VII secretion system (designated as SggT7SST05). We showed that core genes within the SggT7SST05 locus are expressed in vitro and in the colon of mice. Western blot analysis showed that SggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that this SggT7SST05 is functional. Deletion of SggT7SST05 (TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability of Sgg to stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest that SggT7SST05-secreted factors are responsible for the pro-proliferative activity of Sgg, whereas Sgg adherence to host cells requires both SggT7SST05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesx showed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesx did not. Examination of the Sgg load in the colon in the CRC model suggests that SggT7SST05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results reveal SggT7SST05 as a previously unrecognized pathogenicity determinant for Sgg colonization of the colon and promotion of colon tumors.
Asunto(s)
Proliferación Celular , Neoplasias del Colon/patología , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus gallolyticus subspecies gallolyticus/fisiología , Sistemas de Secreción Tipo VII/metabolismo , Animales , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/microbiología , Humanos , Ratones , Ratones Endogámicos A , Infecciones Estreptocócicas/metabolismoRESUMEN
The innate immune system is the first line of defense against bacterial and viral infections. The recognition of pathogen-associated molecular patterns by the RIG-I-like receptors, TLRs, and cGAS leads to the induction of IFN-I by activating the transcription factor IRF-3. Although the mechanism of IRF-3 activation has been extensively studied, the structural basis of IRF-3 activation upon phosphorylation is not fully understood. In this study, we determined the crystal structures of phosphorylated human and mouse IRF-3 bound to CREB-binding protein (CBP), which reveal that phosphorylated IRF-3 forms a dimer via pSer386 (pSer379 in mouse IRF-3) and a downstream pLxIS motif. Size-exclusion chromatography and cell-based studies show that mutations of key residues interacting with pSer386 severely impair IRF-3 activation and IFN-ß induction. By contrast, phosphorylation of Ser396 within the pLxIS motif of human IRF-3 only plays a moderate role in IRF-3 activation. The mouse IRF-3/CBP complex structure reveals that the mechanism of mouse IRF-3 activation is similar but distinct from human IRF-3. These structural and functional studies reveal the detailed mechanism of IRF-3 activation upon phosphorylation.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Animales , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Ratones , Mutagénesis Sitio-Dirigida , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Fosforilación , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Células Sf9 , Especificidad de la Especie , Spodoptera , Relación Estructura-ActividadRESUMEN
Deregulated Wnt signaling and altered lipid metabolism have been linked to obesity, diabetes, and various cancers, highlighting the importance of identifying inhibitors that can modulate Wnt signaling and aberrant lipid metabolism. We have established a Drosophila model with hyperactivated Wnt signaling caused by partial loss of axin, a key component of the Wnt cascade. The Axin mutant larvae are transparent and have severe adipocyte defects caused by up-regulation of ß-catenin transcriptional activities. We demonstrate pharmacologic mitigation of these phenotypes in Axin mutants by identifying bortezomib and additional peptide boronic acids. We show that the suppressive effect of peptide boronic acids on hyperactive Wnt signaling is dependent on α-catenin; the rescue effect is completely abolished with the depletion of α-catenin in adipocytes. These results indicate that rather than targeting the canonical Wnt signaling pathway directly, pharmacologic modulation of ß-catenin activity through α-catenin is a potentially attractive approach to attenuating Wnt signaling in vivo.
Asunto(s)
Adipocitos/efectos de los fármacos , Ácidos Borónicos/farmacología , Péptidos/farmacología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Animales , Proteína Axina/metabolismo , Drosophila/efectos de los fármacos , Drosophila/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, we have previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. Here we report that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. We observed that cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on lipid metabolism and developmental timing in Drosophila larvae.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/fisiología , Proteínas de Drosophila/fisiología , Larva/metabolismo , Necesidades Nutricionales/fisiología , Animales , Ciclina C/metabolismo , Ciclina C/fisiología , Quinasa 8 Dependiente de Ciclina/metabolismo , Dieta , Proteínas en la Dieta/metabolismo , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/metabolismo , Expresión Génica , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The serine/threonine kinase IKBKE is frequently overexpressed or activated in a variety of human cancers. Ectopic expression of IKBKE induces malignant transformation, cell migration, invasion and chemoresistance. Thus, IKBKE is an attractive target for anti-cancer drug development. METHODS: By screening of NCI Diversity Set and Clinical Collection I and II compound libraries using cell-based assay, we identified several candidates of IKBKE inhibitors, which directly inhibited IKBKE kinase activity in vitro and in vivo. One of them, malachite green oxalate (MCCK1), was further characterized. The mechanism was examined by western blot, immunoprecipitation (IP) and Immunofluorescence. We also evaluated in a mouse xenograft model. In vitro kinase assay and luciferase reporter assay were also performed in our experiments. RESULTS: MCCK1 inhibits IKBKE kinase as well as its downstream targets such as IκBα, p65 and IRF3. MCCK1 is a selective inhibitor for IKBKE, with moderate effect on TBK1, but does not inhibit the activation of IKKα/ß, STAT3, Erk-1/2, p38 or JNK. The inhibition of IKBKE by MCCK1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor aberrant expression of IKBKE. Furthermore, MCCK1 inhibits tumor growth in nude mice of human cancer cells in which IKBKE is elevated but not of those cancer cells in which it is not. CONCLUSION: These data indicate that MCCK1 is an IKBKE inhibitor with anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients with tumors over expressing IKBKE.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Quinasa I-kappa B/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Colorantes de Rosanilina/farmacología , Células A549 , Animales , Apoptosis/efectos de los fármacos , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Humanos , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Neoplasias/patología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-ß) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.
Asunto(s)
Factor 3 Regulador del Interferón/química , Rotavirus/química , Proteínas no Estructurales Virales/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Secuencias de Aminoácidos , Proteína de Unión a CREB/química , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/inmunología , Humanos , Evasión Inmune , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Dominios Proteicos , Rotavirus/genética , Rotavirus/inmunología , Infecciones por Rotavirus/genética , Infecciones por Rotavirus/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.
Asunto(s)
Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animales , Animales Modificados Genéticamente , Ciclina C/genética , Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisteroides/biosíntesis , Femenino , Privación de Alimentos , Regulación de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutación , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Ephrin type-B receptor 4 (EphB4) plays an important role in human carcinogenesis. This study investigated the effects of EphB4 expression in drug-resistance of acute myeloid leukemia cells to Adriamycin using myeloid leukemia cell lines with different degrees of differentiation, including an Adriamycin-resistant HL60 cell line as a model. The data showed that the EphB4 protein was differentially expressed in these myeloid leukemia cell lines, which expression was associated with sensitivity of myeloid leukemia cells to Adriamycin treatment in vitro. Furthermore, EphB4 protein stimulated by EphrinB2-Fc sensitized HL60/ADM cells to Adriamycin in a dose-dependent manner. Specifically, pre-incubation of HL60/ADM with 4 µg/ml EphrinB2-Fc protein for 30 min significantly sensitized tumor cell to Adriamycin treatment by reduction of tumor cell viability and induction of apoptosis (p<0.001), while there was no significant change in other groups (p>0.05). These data provided a proof-of-principle for further development of the EphB4-based strategy for treatment of drug-resistant leukemia.
Asunto(s)
Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Doxorrubicina , Humanos , Receptor EphB4RESUMEN
KEY MESSAGE: Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.
Asunto(s)
Hevea , Triploidía , Hevea/genética , Diploidia , Células Germinativas , Centrómero/genéticaRESUMEN
BACKGROUND: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells. METHODS: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFß-signaling. RESULTS: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFß responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFß target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOX(lo)/ANGPTL4(hi) cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent candidates for anti-TGFß targeted therapies. CONCLUSIONS: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFß signaling in advanced breast cancer.
Asunto(s)
Oxidorreductasas/fisiología , Proteína smad3/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas Supresoras de Tumor/fisiología , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/genética , Angiopoyetinas/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Células MCF-7 , Oxidorreductasas/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Activación Transcripcional , Transcriptoma , Neoplasias de la Mama Triple Negativas/genética , Proteínas Supresoras de Tumor/química , Oxidorreductasa que Contiene Dominios WWRESUMEN
Understanding the genetic basis of rubber tree (Hevea brasiliensis) domestication is crucial for further improving natural rubber production to meet its increasing demand worldwide. Here we provide a high-quality H. brasiliensis genome assembly (1.58 Gb, contig N50 of 11.21 megabases), present a map of genome variations by resequencing 335 accessions and reveal domestication-related molecular signals and a major domestication trait, the higher number of laticifer rings. We further show that HbPSK5, encoding the small-peptide hormone phytosulfokine (PSK), is a key domestication gene and closely correlated with the major domestication trait. The transcriptional activation of HbPSK5 by myelocytomatosis (MYC) members links PSK signaling to jasmonates in regulating the laticifer differentiation in rubber tree. Heterologous overexpression of HbPSK5 in Russian dandelion (Taraxacum kok-saghyz) can increase rubber content by promoting laticifer formation. Our results provide an insight into target genes for improving rubber tree and accelerating the domestication of other rubber-producing plants.
Asunto(s)
Hevea , Hevea/genética , Goma , Domesticación , Análisis de Secuencia de ADN , Genómica , Regulación de la Expresión Génica de las PlantasRESUMEN
Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumour syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP, suggesting that a GTPase might be the physiological target of Tsc2. Here we show that the small GTPase Rheb (Ras homologue enriched in brain) is a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupted its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. Our studies identify Rheb as a molecular target of the TSC tumour suppressors.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/genética , Neuropéptidos/genética , Proteínas Represoras/genética , Esclerosis Tuberosa/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Regulación Fúngica de la Expresión Génica , Genes Supresores , Glutatión Transferasa/metabolismo , Técnicas In Vitro , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Mutación Puntual , Proteínas Quinasas , Proteínas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
Insulin signalling is a potent stimulator of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin) [corrected]. Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity [corrected]. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, we identified Rheb (Ras homologue enriched in brain), a member of the Ras superfamily of GTP-binding proteins. Increased levels of Rheb in Drosophila melanogaster promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth.
Asunto(s)
Proteínas de Drosophila/metabolismo , Sustancias de Crecimiento/metabolismo , Insulina/metabolismo , Proteínas de Unión al GTP Monoméricas/fisiología , Neuropéptidos/fisiología , Transducción de Señal , Animales , División Celular/genética , División Celular/fisiología , Proteínas de Drosophila/genética , Femenino , Eliminación de Gen , Sustancias de Crecimiento/genética , Insulina/genética , Interfase , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fosfatidilinositol 3-Quinasas , Ploidias , Proteínas Quinasas , Proteínas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TOR , Transgenes , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor , Alas de Animales/crecimiento & desarrolloRESUMEN
Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Here, we show that tuberous sclerosis 1 (Tsc1) and Tsc2, tumour suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. We show that Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. We propose that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. Our studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumour suppressors.
Asunto(s)
Aminoácidos/metabolismo , Proteínas de Drosophila , Proteínas de Insectos/metabolismo , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Animales , Línea Celular , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Genes de Insecto , Genes Supresores de Tumor , Humanos , Proteínas de Insectos/genética , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas/genética , Proteínas Represoras/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
Tuberous sclerosis complex (TSC) is a human syndrome characterized by a widespread development of benign tumors. This disease is caused by mutations in the TSC1 or TSC2 tumor suppressor genes; the molecular mechanisms underlying the activity of these have long been elusive. Recent studies of Drosophila and mammalian cells demonstrate that the TSC1-TSC2 complex functions as GTPase activating protein against Rheb - a Ras-like small GTPase, which in turn regulates TOR signaling in nutrient-stimulated cell growth. These findings provide a new paradigm for how proteins involved in nutrient sensing could function as tumor suppressors and suggest novel therapeutic targets against TSC. Here, we review these exciting developments with an emphasis on Drosophila studies and discuss how Drosophila can be a powerful model system for an understanding of the molecular mechanisms of the activity of human disease genes.
Asunto(s)
Drosophila melanogaster/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Esclerosis Tuberosa/metabolismo , Animales , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Neuropéptidos/genética , Proteínas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Represoras/genética , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Addiction is proposed to arise from alterations in synaptic strength via mechanisms of long-term potentiation (LTP) and depression (LTD). However, the causality between these synaptic processes and addictive behaviors is difficult to demonstrate. Here we report that LTP and LTD induction altered operant alcohol self-administration, a motivated drug-seeking behavior. We first induced LTP by pairing presynaptic glutamatergic stimulation with optogenetic postsynaptic depolarization in the dorsomedial striatum, a brain region known to control goal-directed behavior. Blockade of this LTP by NMDA-receptor inhibition unmasked an endocannabinoid-dependent LTD. In vivo application of the LTP-inducing protocol caused a long-lasting increase in alcohol-seeking behavior, while the LTD protocol decreased this behavior. We further identified that optogenetic LTP and LTD induction at cortical inputs onto striatal dopamine D1 receptor-expressing neurons controlled these behavioral changes. Our results demonstrate a causal link between synaptic plasticity and alcohol-seeking behavior and suggest that modulation of this plasticity may inspire a therapeutic strategy for addiction.
Asunto(s)
Consumo de Bebidas Alcohólicas , Corteza Cerebral/fisiología , Comportamiento de Búsqueda de Drogas/fisiología , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Neostriado/fisiología , Animales , Potenciales Evocados/fisiología , Glutamatos/fisiología , Masculino , Optogenética , Ratas , Ratas Long-Evans , Receptores de Dopamina D1/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Receptores Presinapticos/fisiología , AutoadministraciónRESUMEN
Endogenous cyclic GMP-AMP (cGAMP) binds and activates STING to induce type I interferons. However, whether cGAMP plays any roles in regulating metabolic homeostasis remains unknown. Here we show that exogenous cGAMP ameliorates obesity-associated metabolic dysregulation and uniquely alters proinflammatory responses. In obese mice, treatment with cGAMP significantly decreases diet-induced proinflammatory responses in liver and adipose tissues and ameliorates metabolic dysregulation. Strikingly, cGAMP exerts cell-type-specific anti-inflammatory effects on macrophages, hepatocytes, and adipocytes, which is distinct from the effect of STING activation by DMXAA on enhancing proinflammatory responses. While enhancing insulin-stimulated Akt phosphorylation in hepatocytes and adipocytes, cGAMP weakens the effects of glucagon on stimulating hepatocyte gluconeogenic enzyme expression and glucose output and blunts palmitate-induced hepatocyte fat deposition in an Akt-dependent manner. Taken together, these results suggest an essential role for cGAMP in linking innate immunity and metabolic homeostasis, indicating potential applications of cGAMP in treating obesity-associated inflammatory and metabolic diseases.
Asunto(s)
Adipocitos/inmunología , Dieta Alta en Grasa/efectos adversos , Hepatocitos/inmunología , Nucleótidos Cíclicos/administración & dosificación , Obesidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adipocitos/efectos de los fármacos , Animales , Hepatocitos/efectos de los fármacos , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Nucleótidos Cíclicos/farmacología , Obesidad/inducido químicamente , Obesidad/inmunología , Fosforilación , Xantonas/administración & dosificación , Xantonas/farmacologíaRESUMEN
The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Ciclo Celular , Muerte Celular , División Celular , Núcleo Celular/metabolismo , Proliferación Celular , Separación Celular , Supervivencia Celular , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Activación Enzimática , Citometría de Flujo , Vectores Genéticos , Humanos , Microtúbulos/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Especificidad de la Especie , Huso Acromático , Serina-Treonina Quinasas TOR , TransgenesRESUMEN
In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments.