RESUMEN
Using forward genetics, we revealed that the signal peptide peptidase (SPP) SppA, an aspartyl protease involved in regulated intramembrane proteolysis (RIP), is essential for hypoxia adaptation in Aspergillus nidulans, as well as hypoxia-sensitive mutant alleles of a sterol regulatory element-binding protein (SREBP) srbA and the Dsc ubiquitin E3 ligase complex dscA-E. Both null and dead activity [D337A] mutants of sppA failed to grow in hypoxia, and the growth defect of ΔsppA was complemented by nuclear SrbA-N381 expression. Additionally, SppA interacted with SrbA in the endoplasmic reticulum, where SppA localized in normoxia and hypoxia. Expression of the truncated SrbA-N414 covering the SrbA sequence prior to the second transmembrane region rescued the growth of ΔdscA but not of ΔsppA in hypoxia. Unlike ΔdscA and ΔdscA;ΔsppA double mutants, in which SrbA cleavage was blocked, the molecular weight of cleaved SrbA increased in ΔsppA compared to the control strain in immunoblot analyses. Overall, our data demonstrate the sequential cleavage of SrbA by Dsc-linked proteolysis followed by SppA, proposing a new model of RIP for SREBP cleavage in fungal hypoxia adaptation. Furthermore, the function of SppA in hypoxia adaptation was consistent in Aspergillus fumigatus, suggesting the potential roles of SppA in fungal pathogenesis.
Asunto(s)
Adaptación Fisiológica , Ácido Aspártico Endopeptidasas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Oxígeno/fisiología , Proteolisis , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Adaptación Fisiológica/genética , Alelos , Ácido Aspártico Endopeptidasas/genética , Aspergillus nidulans/enzimología , Aspergillus nidulans/crecimiento & desarrollo , Retículo Endoplásmico/metabolismo , Prueba de Complementación Genética , Mutación , Procesamiento Proteico-Postraduccional , Transducción de Señal , Proteínas de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Several Fusarium species produce the polyketide mycotoxin zearalenone (ZEA), a causative agent of hyperestrogenic syndrome in animals that is often found in F. graminearum-infected cereals in temperate regions. The ZEA biosynthetic cluster genes PKS4, PKS13, ZEB1 andâ ZEB2 encode a reducing polyketide synthase, a non-reducing polyketide synthase, an isoamyl alcohol oxidase and a transcription factor respectively. In this study, the production of two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum via an alternative promoter was characterized. ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggers the induction of both ZEB2L and ZEB2S transcription. ZEB2L and ZEB2S interact with each other to form a heterodimer that regulates ZEA production by reducing the binding affinity of ZEB2L for the ZEB2L gene promoter. Our study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production; this regulatory mechanism is similar to that observed in higher eukaryotes.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Zearalenona/biosíntesis , Grano Comestible/química , Retroalimentación Fisiológica , Proteínas Fúngicas/química , Fusarium/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Homeostasis , Leucina Zippers , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Isoformas de Proteínas , Multimerización de Proteína , Factores de Transcripción/química , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Zearalenona/farmacologíaRESUMEN
Fusarium graminearum, a prominent fungal pathogen that infects major cereal crops, primarily utilizes asexual spores to spread disease. To understand the molecular mechanisms underlying conidiogenesis in F. graminearum, we functionally characterized the F. graminearum ortholog of Aspergillus nidulans wetA, which has been shown to be involved in conidiogenesis and conidium maturation. Deletion of F. graminearum wetA did not alter mycelial growth, sexual development, or virulence, but the wetA deletion mutants produced longer conidia with fewer septa, and the conidia were sensitive to acute stresses, such as oxidative stress and heat stress. Furthermore, the survival rate of aged conidia from the F. graminearum wetA deletion mutants was reduced. The wetA deletion resulted in vigorous generation of single-celled conidia through autophagy-dependent microcycle conidiation, indicating that WetA functions to maintain conidial dormancy by suppressing microcycle conidiation in F. graminearum. Transcriptome analyses demonstrated that most of the putative conidiation-related genes are expressed constitutively and that only a few genes are specifically involved in F. graminearum conidiogenesis. The conserved and distinct roles identified for WetA in F. graminearum provide new insights into the genetics of conidiation in filamentous fungi.
Asunto(s)
Proteínas Fúngicas/metabolismo , Fusarium/genética , Secuencia de Aminoácidos , Autofagia , Proteínas Fúngicas/genética , Fusarium/metabolismo , Fusarium/fisiología , Genes Fúngicos , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Micelio/citología , Micelio/crecimiento & desarrollo , Estrés Oxidativo , Fenotipo , Eliminación de Secuencia , Esporas Fúngicas/citología , Esporas Fúngicas/crecimiento & desarrollo , Transcriptoma , Virulencia/genéticaRESUMEN
An acireductone dioxygenase (ARD) gene of potatoes was isolated from the expressed sequence tags (ESTs) of potato post-suberization cDNA libraries. The highest expression levels of the StARD gene and the protein appeared 36 h after suberization. An approximate 9-fold increase in ARD activity was detected at 36 h after wounding. Real-time reverse transcription-PCR (RT-PCR) analysis and immunolocalization studies revealed that StARD transcripts increase at the wound surface of potato tubers. The polyamine (PA) contents increased significantly after wounding at the wound surface. The increased PA content and ARD activity may play an important role in wound periderm formation.
Asunto(s)
Dioxigenasas/metabolismo , Tubérculos de la Planta/metabolismo , Poliaminas/metabolismo , Solanum tuberosum/enzimología , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Lípidos/biosíntesis , Metionina/metabolismo , Tubérculos de la Planta/genética , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solanum tuberosum/genéticaRESUMEN
uvsF201 was the first highly UV-sensitive repair-defective mutation isolated in Aspergillus nidulans. It showed epistasis only with postreplication repair mutations, but caused lethal interactions with many other repair-defective strains. Unexpectedly, closest homology of uvsF was found to the large subunit of human DNA replication factor RFC that is essential for DNA replication. Sequencing of the uvsF201 region identified changes at two close base pairs and the corresponding amino acids in the 5'-region of uvsF(RFC1). This viable mutant represents a novel and possibly important type. Additional sequencing of the uvsF region confirmed a mitochondrial ribosomal protein gene, mrpA(L16), closely adjacent, head-to-head with a 0.2kb joint promoter region. MMS-induced transcription of both the genes, but especially uvsF(RFC1), providing evidence for a function in DNA damage response.
Asunto(s)
Aspergillus nidulans/metabolismo , Reparación del ADN , Replicación del ADN , Proteínas Fúngicas/metabolismo , Metilmetanosulfonato/farmacología , Proteína de Replicación C/metabolismo , Regulación hacia Arriba , Secuencia de Aminoácidos , Aspergillus nidulans/química , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Daño del ADN , Replicación del ADN/efectos de los fármacos , Epistasis Genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Mutágenos/farmacología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteína de Replicación C/química , Proteína de Replicación C/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Transcripción Genética/efectos de los fármacosRESUMEN
The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.
Asunto(s)
Proteínas del Choque Térmico HSP40/farmacología , Chaperonas Moleculares , Tiorredoxinas/metabolismo , Zinc/metabolismo , Cisteína/química , Cisteína/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSP40/genética , Humanos , Peróxido de Hidrógeno/farmacología , Luciferasas/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Dedos de ZincRESUMEN
Apoptosis-inducing factor (AIF) is a ubiquitous FAD-binding flavoprotein comprised of 613 amino acids and plays an important role in caspase-independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two-hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N-terminus interacts with the C-terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull-down assay and also verified by the results of co-immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription-translation system, we found that mature AIF could inhibit newly-translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase-7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIF's cellular functions of the inhibition of protein synthesis during apoptosis.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Apoptosis/fisiología , Núcleo Celular/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Biosíntesis de Proteínas/fisiología , Transporte Activo de Núcleo Celular/fisiología , Factor Inductor de la Apoptosis/genética , Caspasa 7/genética , Caspasa 7/metabolismo , Núcleo Celular/genética , Sistema Libre de Células/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Células HeLa , Humanos , Células Jurkat , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/genéticaRESUMEN
Thioredoxin (Trx) is a cellular redox enzyme that plays multiple roles in regulating cell growth and apoptosis. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein 1 (AP-1) transcription and was also shown to promote degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Recently, Jab1 expression was associated with the progression and poor prognosis of pituitary, epithelial ovarian, and breast cancers, suggesting that it plays a role in oncogenesis. Here, we report that Trx specifically interacts with and modulates the function of Jab1. Fluorescence resonance energy transfer and co-immunoprecipitation studies revealed that Trx and Jab1 colocalize and directly interact with each other. Further, Trx negatively regulates two important Jab1-controlled signaling pathways, activation of AP-1 transcription and degradation of p27Kip1, probably through a direct interaction between Trx and C-terminal of Jab1. The negative effect of Trx on AP-1 activity is Jab1-dependent, as it disappears when Jab1 levels are suppressed by an antisense approach. In addition, Trx competes with p27Kip1 for Jab1 binding. Taken together, our results suggest that Trx may regulate cell cycle and growth through a novel modulation of Jab1-mediated proliferation signals, further indicating that Trx may have the ability to control tumor progression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Tiorredoxinas/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Sitios de Unión , Complejo del Señalosoma COP9 , Línea Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Cisteína/química , ADN Complementario/metabolismo , Progresión de la Enfermedad , Disulfuros , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Genes Reporteros , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Mutación , Neoplasias/metabolismo , Oligonucleótidos Antisentido/química , Oxidación-Reducción , Péptido Hidrolasas , Pronóstico , Unión Proteica , Proteínas Recombinantes/química , Transducción de Señal , Factores de Tiempo , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
Rice blast fungus, Magnaporthe oryzae, is the most destructive pathogen in the rice-growing area. This fungus has a biotrophic phase early in infection and later switches to a necrotrophic lifestyle. During the biotrophic phase, the fungus competes with its host for nutrients and oxygen. Continuous uptake of oxygen is essential for successful establishment of blast disease of this pathogen. Here, we report transcriptional responses of the fungus to oxygen limitation. Transcriptome analysis using RNA-Seq identified that 1,047 genes were up-regulated in response to hypoxia. Those genes are involved in mycelial development, sterol biosynthesis, and metal ion transport based on hierarchical GO terms, and are well-conserved among three fungal species. In addition, null mutants of two hypoxia-responsive genes were generated and their roles in fungal development and pathogenicity tested. The mutant for the sterol regulatory element-binding protein gene, MoSRE1, exhibited increased sensitivity to a hypoxia-mimicking agent, increased conidiation, and delayed invasive growth within host cells, which is suggestive of important roles in fungal development. However, such defects did not cause any significant decrease in disease severity. The other null mutant, for the alcohol dehydrogenase gene MoADH1, showed no defect in the hypoxia-mimicking condition (using cobalt chloride) and fungal development. Taken together, this comprehensive transcriptional profiling in response to a hypoxic condition with experimental validations would provide new insights into fungal development and pathogenicity in plant pathogenic fungi.
Asunto(s)
Hipoxia de la Célula/genética , Genes Fúngicos , Magnaporthe/genética , Oryza/microbiología , Alcohol Deshidrogenasa/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Estudio de Asociación del Genoma Completo , Magnaporthe/efectos de los fármacos , Magnaporthe/metabolismo , Magnaporthe/patogenicidad , Mutación , Estrés Oxidativo , Oxígeno/farmacología , ARN de Hongos/genética , ARN Mensajero/genética , Especificidad de la Especie , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Transcriptoma , Transformación Genética , Virulencia/genéticaRESUMEN
To investigate additional functions of the T cell adaptor, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD (SLP-76), we performed a yeast two-hybrid assay using the N-terminal region of SLP-76 fused with the kinase domain of Syk. By screening a human leukemia cDNA library, we identified the p85 subunit of phosphoinositide 3-kinase (PI3K) as one of the interacting molecules. Unlike the SH2 domain of Vav or Nck, tyrosine phosphorylation of SLP-76 at position 113 or 128 was sufficient for it to associate with the N-terminal SH2 of p85. Collectively, these data suggest that SLP-76 may play a role in PI3K signaling pathways.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Oncogénicas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/genética , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Quinasa Syk , Técnicas del Sistema de Dos HíbridosRESUMEN
Growth at the restrictive temperature (42 degrees C) of Aspergillus nidulans B120, carrying the conditional-lethal mutation sod(VI)C1, was partially improved by the addition of 1.0 M sorbitol to the medium. The mutant grown at 42 degrees C, with osmotic stabilizer, showed abnormal hyphal morphology, a decrease in beta-1,3-glucan synthase activity as well as cell wall sugar content, but an increase in chitin synthase activity and N-acetyl-glucosamine content. The mutation also affected the secretion of extracellular protease. The temperature-dependent osmo-sensitive phenotype of a Saccharomyces cerevisiae alpha-COP mutation can be rescued by the A. nidulans sod(VI)C(+) gene. These results indicate that the sod(VI)C1 mutation affects proper processing of secretory proteins destined for the surface of cells or beyond.
Asunto(s)
Aspergillus nidulans/crecimiento & desarrollo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes Fúngicos , beta-Glucanos , Aspergillus nidulans/metabolismo , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Quitina Sintasa/metabolismo , Proteína Coatómero/genética , Proteína Coatómero/metabolismo , Glucanos/biosíntesis , Mutación , Ósmosis , Sorbitol/farmacología , TemperaturaRESUMEN
The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1 approximately 447) and its four different derivatives, denoted as NS5A-A (aa 1 approximately 150), -B (aa 1 approximately 300), -C (aa 300 approximately 447) and D (aa 150 approximately 447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and centaurindelta 2 (CENTdelta2). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, CENTdelta2 and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and CENTdelta2 were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.
Asunto(s)
Hígado/química , Hígado/metabolismo , Proteínas no Estructurales Virales/metabolismo , Hepacivirus/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/genéticaRESUMEN
Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.
Asunto(s)
Fusarium/genética , Hifa/crecimiento & desarrollo , Esporas Fúngicas/crecimiento & desarrollo , Aspergillus nidulans/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/fisiología , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Hifa/genética , Reproducción Asexuada , Esporas Fúngicas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Fusarium graminearum (teleomorph Gibberella zeae) is a prominent pathogen that infects major cereal crops such as wheat, barley, and maize. Both sexual (ascospores) and asexual (conidia) spores are produced in F. graminearum. Since conidia are responsible for secondary infection in disease development, our objective of the present study was to reveal the molecular mechanisms underlying conidiogenesis in F. graminearum based on the framework previously described in Aspergillus nidulans. In this study, we firstly identified and functionally characterized the ortholog of AbaA, which is involved in differentiation from vegetative hyphae to conidia and known to be absent in F. graminearum. Deletion of abaA did not affect vegetative growth, sexual development, or virulence, but conidium production was completely abolished and thin hyphae grew from abnormally shaped phialides in abaA deletion mutants. Overexpression of abaA resulted in pleiotropic defects such as impaired sexual and asexual development, retarded conidium germination, and reduced trichothecene production. AbaA localized to the nuclei of phialides and terminal cells of mature conidia. Successful interspecies complementation using A. nidulans AbaA and the conserved AbaA-WetA pathway demonstrated that the molecular mechanisms responsible for AbaA activity are conserved in F. graminearum as they are in A. nidulans. Results from RNA-sequencing analysis suggest that AbaA plays a pivotal role in conidiation by regulating cell cycle pathways and other conidiation-related genes. Thus, the conserved roles of the AbaA ortholog in both A. nidulans and F. graminearum give new insight into the genetics of conidiation in filamentous fungi.
Asunto(s)
Proteínas Fúngicas/genética , Fusarium/genética , Esporas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Eliminación de Gen , Expresión Génica , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Orden Génico , Marcación de Gen , Genes Reporteros , Prueba de Complementación Genética , Sistemas de Lectura Abierta , Fenotipo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Transducción de Señal , Esporas Fúngicas/metabolismoRESUMEN
Eukaryotic translation initiation factor 3 is composed of 13 subunits (eIF3a through eIF3m) and plays an essential role in translation. During apoptosis, several caspases rapidly down-regulate protein synthesis by cleaving eIF4G, -4B, -3j, and -2α. In this study, we found that the activation of caspases by cisplatin in T24 cells induces the cleavage of subunit G of the eIF3 complex (eIF3g). The cleavage site (SLRD(220)G) was identified, and we found that the cleaved N-terminus was translocated to the nucleus, activating caspase-3, and that it also showed a strong DNase activity. These data demonstrate the important roles of eIF3g in the translation initiation machinery and in DNA degradation during apoptosis.
Asunto(s)
Caspasa 3/metabolismo , Caspasa 7/metabolismo , Desoxirribonucleasas/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Línea Celular Tumoral , Núcleo Celular/metabolismo , División del ADN , Activación Enzimática , Factor 3 de Iniciación Eucariótica/química , Humanos , Fragmentos de Péptidos/metabolismo , ProteolisisRESUMEN
NDRG2 is a member of the N-myc downstream regulated gene (NDRG) family, implicated in cell growth and differentiation. Investigation of NDRG2 molecular interactions by yeast two-hybrid screening identified prenylated Rab acceptor-1 (PRA1), involved in vesicle trafficking and protein transport, as binding partner. Binding of NDRG2 (and NDRG1-4) with PRA1 in vitro was confirmed by GST pull-down assay and immunoprecipitation, and colocalization was verified by confocal microscopy in HCT116 cells. Intracellular coexpression showed that NDRG2 and PRA1 synergistically downregulate T-cell factor (TCF) promoter activity and GSK3ß phosphorylation. Results suggest that NDRG2 and PRA1 might act synergistically to prevent signaling of TCF/ß-catenin.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Transducción de Señal , Factores de Transcripción TCF/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Transporte Vesicular/metabolismo , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Sitios de Unión/genética , Western Blotting , Núcleo Celular/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Humanos , Microscopía Confocal , Mutación , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Supresoras de Tumor/genética , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte Vesicular/genéticaRESUMEN
The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Ratones , Microscopía Confocal , Fosfoinositido Fosfolipasa C/antagonistas & inhibidores , Fosfoinositido Fosfolipasa C/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Timidina/metabolismo , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Phospholipase Cepsilon (PLCepsilon) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCepsilon is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCepsilon RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCepsilon, and the mutation of Lys-2186 of the PLCepsilon RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCepsilon upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCepsilon, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCepsilon. The EGF-dependent degradation of PLCepsilon was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCepsilon, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCepsilon-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCepsilon knockdown. Our results suggest that the Siah-dependent degradation of PLCepsilon plays a role in the regulation of growth factor-dependent cell growth.
Asunto(s)
Factor de Crecimiento Epidérmico/fisiología , Proteínas Nucleares/fisiología , Fosfoinositido Fosfolipasa C/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Embrión de Mamíferos , Fibroblastos/fisiología , Haplorrinos , Humanos , Riñón , Ratones , Proteínas Nucleares/genética , Fosforilación , Fosfotirosina/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The prenylated Rab acceptor 1 (PRA1) is a ubiquitously expressed 21 kDa protein containing two transmembrane domains that possibly induce its localization to the Golgi complex. It binds to prenylated Rab GTPases and VAMP2. In this study, we report that PRA1-overexpressing cells exhibited a significantly retarded growth rate as compared to that of the mock-transfected cells, and the transcriptional activity of TCF, as evaluated by TOPflash luciferase reporter assay, was profoundly reduced in the PRA1-overexpressed cells. These intracellular functions of PRA1 were verified by introducing deletion mutant or site-directed mutants, or small interfering RNA of PRA1. In addition, the translocation of beta-catenin from the cytosol to the nucleus was blocked to a significant degree in the PRA1-cells, and the interaction of PRA1 and beta-catenin was identified by confocal microscopy and immunoprecipitation analysis. Finally, we observed that the inhibition of TCF/beta-catenin signaling by PRA1 is associated with ERK1/2 dephosphorylation. Therefore, our data suggest that the in vivo modulation of PRA1 may be involved in TCF/beta-catenin signaling, as well as cellular proliferation and tumorigenesis.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Transporte Vesicular/fisiología , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al GTP/química , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/química , Modelos Biológicos , Mutación , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Transducción de Señal , Proteínas de Transporte Vesicular/químicaRESUMEN
Oxidative stress induces apoptosis in a variety of cell types by as yet unclear signaling mechanisms. The Daxx protein is reportedly involved in apoptosis through its interactions with Fas, transforming growth factor-beta receptor, and promyelocytic leukemia protein (PML). Here, we explored the possible roles of Daxx in oxidative stress-induced apoptosis. We found that both the mRNA and protein levels of Daxx markedly increased when cells underwent apoptosis after H2O2 treatment. Pretreatment with the cell-permeable antioxidant, N-acetyl cysteine, prevented cells from H2O2-induced Daxx upregulation and subsequent apoptosis, indicating that the endogenous oxidant regulated Daxx expression. Furthermore, suppression of endogenous Daxx expression by antisense oligonucleotide technology inhibited oxidative stress-induced apoptosis in HeLa cells. Taken together, these results suggest that Daxx acts as an intermediary messenger of pro-apoptotic signals triggered by oxidative stress.