RESUMEN
In summary, we show here that HCV infection is associated with an upregulation of ARF4, which promotes HCV replication. Upon HCV infection, CREB3 was redistributed to nucleus and activated ARF4 transcription. Our studies demonstrate a host factor ARF4 upregulated in HCV replication, which may provide new therapeutic targets for antiviral therapy.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Replicación Viral , Perfilación de la Expresión Génica , ARN Mensajero/biosíntesisRESUMEN
It is well established that Notch functions as a transcriptional activator through the formation of a ternary complex that comprises Notch, Maml, and CSL. This ternary complex then serves to recruit additional transcriptional cofactors that link to higher order transcriptional complexes. The mechanistic details of these events remain unclear. This report reveals that the Notch ternary complex can direct the formation of a repressor complex to terminate gene expression of select target genes. Herein, it is demonstrated that p19Arf and Klf4 are transcriptionally repressed in a Notch-dependent manner. Furthermore, results indicate that Notch recruits Polycomb Repressor Complex 2 (PRC2) and Lysine Demethylase 1 (KDM1A/LSD1) to these promoters, which leads to changes in the epigenetic landscape and repression of transcription. The demethylase activity of LSD1 is a prerequisite for Notch-mediated transcriptional repression. In addition, a stable Notch transcriptional repressor complex was identified containing LSD1, PRC2, and the Notch ternary complex. These findings demonstrate a novel function of Notch and provide further insight into the mechanisms of Notch-mediated tumorigenesis.Implications: This study provides rationale for the targeting of epigenetic enzymes to inhibit Notch activity or use in combinatorial therapy to provide a more profound therapeutic response. Mol Cancer Res; 15(9); 1173-83. ©2017 AACR.
Asunto(s)
Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Línea Celular Tumoral , Epigénesis Genética , Regulación de la Expresión Génica , Células HEK293 , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Linfoma/genética , Linfoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Empalme Alternativo/fisiología , Encéfalo/metabolismo , Variación Genética/fisiología , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/análisis , Factores de Ribosilacion-ADP/genética , Animales , Encéfalo/citología , Química Encefálica/fisiología , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/genética , Cobayas , Masculino , Ratones , Ratones Endogámicos C57BL , Conejos , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
The small GTPase, ADP-ribosylation factor-like 3 (ARL3), has been proposed to participate in the transport of proteins in photoreceptor cells. Moreover, it has been implicated in the pathogenesis associated with X-linked retinitis pigmentosa (XLRP) resulting from mutations in the ARL3 GTPase activating protein, retinitis pigmentosa 2 (RP2). To determine the importance of ARL3 in rod photoreceptor cells, we generated transgenic mice expressing a dominant active form of ARL3 (ARL3-Q71L) under a rod-specific promoter. ARL3-Q71L animals exhibited extensive rod cell death after post-natal day 30 (PN30) and degeneration was complete by PN70. Prior to the onset of cell death, rod photoresponse was significantly reduced along with a robust decrease in rod phosphodiesterase 6 (PDE6) and G-protein receptor kinase-1 (GRK1) levels. Furthermore, assembled phosphodiesterase-6 (PDE6) subunits, rod transducin and G-protein receptor kinase-1 (GRK1) accumulated on large punctate structures within the inner segment in ARL3-Q71L retina. Defective trafficking of prenylated proteins is likely due to sequestration of prenyl binding protein δ (PrBPδ) by ARL3-Q71L as we demonstrate a specific interaction between these proteins in the retina. Unexpectedly, our studies also revealed a novel role for ARL3 in the migration of photoreceptor nuclei. In conclusion, this study identifies ARL3 as a key player in prenylated protein trafficking in rod photoreceptor cells and establishes the potential role for ARL3 dysregulation in the pathogenesis of RP2-related forms of XLRP.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Pirofosfatasas/genética , Retinitis Pigmentosa/genética , Factores de Ribosilacion-ADP/biosíntesis , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/biosíntesis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/biosíntesis , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Proteínas de Unión al GTP , Regulación de la Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Prenilación de Proteína/genética , Retina/metabolismo , Retina/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/patología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/patologíaRESUMEN
There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.
Asunto(s)
Infecciones por Clostridium/microbiología , Clostridium perfringens/genética , Enterotoxinas/genética , Enfermedades Transmitidas por los Alimentos/microbiología , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorocebus aethiops , Infecciones por Clostridium/epidemiología , Clostridium perfringens/aislamiento & purificación , Secuencia Conservada , Brotes de Enfermedades , Enterotoxinas/biosíntesis , Enterotoxinas/inmunología , Enfermedades Transmitidas por los Alimentos/epidemiología , Expresión Génica , Humanos , Íleon/microbiología , Masculino , Datos de Secuencia Molecular , NAD+ Nucleosidasa/biosíntesis , NAD+ Nucleosidasa/genética , NAD+ Nucleosidasa/inmunología , Conejos , Análisis de Secuencia de ADN , Tokio , Células VeroRESUMEN
Rapid and efficient growth is a major consideration and challenge for global mariculture. The differential growth rate of the sea cucumber, Apostichopus japonicus, has significantly hampered the total production of the industry. In the present study, forward and reverse suppression subtractive hybridization libraries were constructed and sequenced from a fast-growth group and a slow-growth group of the sea cucumber. A total of 142 differentially expressed sequence tags (ESTs) with insertions longer than 150 bp were identified and further analyzed. Fifty-seven of these ESTs (approximately 40%) were functionally annotated for cell structure, energy metabolism, immunity response, and growth factor categories. Six candidate genes, arginine kinase, cytochrome c oxidase subunit I, HSP70, ß-actin, ferritin, and the ADP-ribosylation factor, were further validated by quantitative PCR. Significant differences were found between the fast- and slow-growth groups (P < 0.05) for the expression levels of arginine kinase, cytochrome c oxidase, HSP70, the ADP-ribosylation factor, and ß-actin. However, no significant difference was observed for ferritin. Our results provide promising candidate gene markers for practical size screening, and also further promote marker-assisted selective breeding of this species.
Asunto(s)
Etiquetas de Secuencia Expresada , Regulación del Desarrollo de la Expresión Génica , Stichopus/genética , Factores de Ribosilacion-ADP/biosíntesis , Actinas/biosíntesis , Animales , Arginina Quinasa/biosíntesis , Complejo IV de Transporte de Electrones/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Stichopus/crecimiento & desarrolloRESUMEN
Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Biomarcadores de Tumor/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Neoplasias Pulmonares/patología , Proteínas de Unión al GTP ral/biosíntesis , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Expresión Génica/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Neoplasias Pulmonares/metabolismo , Metástasis de la Neoplasia , Estadificación de Neoplasias , Survivin , Proteínas de Unión al GTP ral/genéticaRESUMEN
Bardet-Biedl syndrome (BBS) is a syndromic form of retinal degeneration. Recently, homozygosity mapping with a consanguineous family with isolated retinitis pigmentosa identified a missense mutation in BBS3, a known BBS gene. The mutation in BBS3 encodes a single amino acid change at position 89 from alanine to valine. Since this amino acid is conserved in a wide range of vertebrates, we utilized the zebrafish model system to functionally characterize the BBS3 A89V mutation. Knockdown of bbs3 in zebrafish alters intracellular transport, a phenotype observed with knockdown of all BBS genes in the zebrafish, as well as visual impairment. Here, we find that BBS3 A89V is sufficient to rescue the transport delays induced by the loss of bbs3, indicating that this mutation does not affect the function of BBS3 as it relates to syndromic disease. BBS3L A89V, however, was unable to rescue vision impairment, highlighting a role for a specific amino acid within BBS3 that is necessary for visual function, but dispensable in other cell types. These data aid in our understanding of why patients with the BBS3 A89V missense mutation only present with isolated retinitis pigmentosa.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Síndrome de Bardet-Biedl/genética , Proteínas Recombinantes/genética , Retinitis Pigmentosa/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Factores de Ribosilacion-ADP/biosíntesis , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Silenciador del Gen , Melanosomas/metabolismo , Datos de Secuencia Molecular , Mutación Missense , Proteínas Recombinantes/biosíntesis , Reflejo de Sobresalto , Alineación de Secuencia , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesisRESUMEN
ADP-ribosylation factors (Arfs) that play an essential role in intracellular trafficking and organelle structure are small GTP-binding proteins, which have been identified recently to be involved in virus infection. However, little is known about the Arfs and their relationships with viral infection in the economically important crustaceans to date. In the present study, two novel members of the Arf family, designated as MjArf1 and MjArfn respectively, were cloned from the shrimp Marsupenaeus japonicus. Sequence and phylogenetic analysis showed that MjArf1 belongs to Class I Arf, which has very high homology in sequence to the known Arf 79F of insects and Arf1 of other animals (96-99%), whereas MjArfn is an unidentified Arf, which has only 62-66% identity to other known Arfs. In High Five cells, the distribution of MjArf1 was dependent on its GDP/GTP binding state but the distribution of MjArfn was not affected by that. Both Arfs were ubiquitously expressed in examined tissues. Further investigation with real-time quantitative PCR revealed that MjArf1 and MjArfn were significantly up-regulated after WSSV challenge. In virus-resistant shrimps, however, no distinct fluctuation of MjArf1 expression was found and MjArfn was even found to be notably repressed. These results suggested that MjArf1 and MjArfn might be involved in the shrimp innate immune response in WSSV infection and MjArfn might play a role in WSSV invasion. These studies may contribute to a better understanding of host defense and/or virus invasion interaction and for the control of marine crustacean diseases.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Penaeidae/metabolismo , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Infecciones por Virus ADN/virología , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Penaeidae/genética , Penaeidae/virología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Carga Viral , Virus del Síndrome de la Mancha Blanca 1RESUMEN
The INK4b-ARF-INK4a locus encodes for two cyclin-dependent kinase inhibitors, p15(INK4b) and p16(INK4a) and a regulator of the p53 pathway, ARF. In addition ANRIL, a non-coding RNA, is also transcribed from the locus. ARF, p15(INK4b) and p16(INK4a) are well-established tumor suppressors which function is frequently disabled in human cancers. Recent studies showed that single nucleotide polymorphisms mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including cardiovascular disease, ischemic stroke, type 2 diabetes, frailty and Alzheimer's disease. The INK4b-ARF-INK4a locus is regulated by Polycomb repressive complexes (PRCs), and its expression can be invoked by activating signals. Other epigenetic modifiers such as the histone demethylases JMJD3 and JHDM1B, the SWI/SNF chromatin remodeling complex and DNA methyltransferases regulate the locus interplaying with PRCs. In view of the intimate involvement of the INK4b-ARF-INK4a locus on disease, to understand its regulation is the first step for manipulate it to therapeutic benefit.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Enfermedad de Alzheimer/metabolismo , Enfermedades Cardiovasculares/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Diabetes Mellitus Tipo 2/metabolismo , Epigénesis Genética , Sitios Genéticos , Factores de Ribosilacion-ADP/genética , Enfermedad de Alzheimer/genética , Animales , Enfermedades Cardiovasculares/genética , Cromatina/genética , Cromatina/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilasas de Modificación del ADN , Diabetes Mellitus Tipo 2/genética , Fase G1/genética , Histona Demetilasas , Humanos , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN no Traducido/biosíntesis , ARN no Traducido/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In shrimp, small GTPases in the Ras superfamily can regulate hemolytic phagocytosis against WSSV infection. However, the ADP ribosylation factors (Arfs), also belonging to the regulatory GTP-binding proteins and playing a central role in membrane trafficking, have not been reported yet in shrimp and their relationship with WSSV infection is completely unknown to date. Here, a novel class II Arf (designated as MjArf4) was cloned and characterized from the shrimp Marsupenaeus japonicus. Like other Arfs, MjArf4 contains an N-terminal myristoylated site, a p loop, switch regions, as well as an interswitch region. In High Five cells, when MjArf4 was in its GDP-bound form, it dispersed into the whole cell, whereas in the GTP-bound form it promoted formation of a punctuate Golgi-like structure, indicating that the MjArf4 distribution was dependent on its GDP/GTP binding. After challenge with WSSV, the mRNA level of MjArf4 was up-regulated significantly as WSSV propagated. Thus, a member of the Arf family was characterized for the first time in shrimp and found to be involved in WSSV infection.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Penaeidae/inmunología , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Infecciones por Virus ADN/inmunología , Datos de Secuencia Molecular , Penaeidae/enzimología , Penaeidae/genética , Penaeidae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus del Síndrome de la Mancha Blanca 1/inmunologíaRESUMEN
This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Melanoma/patología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Pulmonares/secundario , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/secundario , Ratones , Fosfolipasa D/metabolismoRESUMEN
The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Anticonvulsivantes/farmacología , Proteínas Activadoras de GTPasa/fisiología , Neuritas/efectos de los fármacos , Ácido Valproico/farmacología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/biosíntesis , Animales , Línea Celular , Línea Celular Tumoral , Ratones , Neuritas/fisiología , Neurogénesis , Triazoles/farmacología , Regulación hacia ArribaRESUMEN
A common pathobiological feature of malignant gliomas is the insidious infiltration of single tumor cells into the brain parenchyma, rendering these deadly tumors virtually incurable with available therapies. In this study, we report that ADP-ribosylation factor 6 (ARF6), a Ras superfamily small GTPase, is abundantly expressed in invasive human glioma cells. Cellular depletion of ARF6 by small interfering RNA decreased Rac1 activation, impaired HGF-stimulated and serum-stimulated glioma cell migration in vitro, and markedly decreased the invasive capacity of invasive glioma in the brain. Furthermore, ectopic expression of ARF6 in glioma cells promoted cell migration via the activation of Rac1. Upon stimulation of glioma cells with HGF, we show that IQ-domain GTPase-activating protein 1 (IQGAP1) is recruited and overlaps with ARF6 at the leading edge of migrating cells. However, cellular depletion of ARF6 abrogated this recruitment of IQGAP1 and attenuated the formation of surface protrusions. ARF6 forms complexes with Rac1 and IQGAP1 in glioma cells upon HGF stimulation, and knockdown of IQGAP1 significantly inhibits ARF6-induced Rac1 activation and cell migration. Taken together, these data suggest that ARF6-mediated Rac1 activation is essential for glioma cell invasion via a signaling pathway that requires IQGAP1.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Neoplasias Encefálicas/patología , Glioma/patología , Proteína de Unión al GTP rac1/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/antagonistas & inhibidores , Factores de Ribosilacion-ADP/genética , Animales , Neoplasias Encefálicas/metabolismo , Movimiento Celular/fisiología , Factor de Crecimiento Epidérmico/farmacología , Glioma/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Agrobacterium-mediated transformation of leaf tissues of Atropa belladonna with an adenosine 5'-diphosphate (ADP)-ribosylation factor gene of carrot, arf-001, was performed employing pBCR82 as an expression vector. This vector co-expresses rol gene cluster together with arf-001, and thus, the transformed host cells were obtained as hairy roots. Two cell lines of the transformed belladonna were established as the liquid cultures of hairy root tissues, and expression of arf-001 and accumulation of its product in the cells were confirmed by RT-PCR and Western blot analyses, respectively. A marked increase in extracellular protein concentrations was observed in the transformed belladonna root cultures as compared with the controls transformed with an empty vector. However, the secretion of the proteins of the transformants was markedly reduced in the presence of a physiological concentration of monensin. These results suggest that over-expression of arf-001 in belladonna results in the enhancement of secretory activity in the transformed cells.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Atropa/química , Atropa/genética , Western Blotting , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Raíces de Plantas/química , ARN de Planta/biosíntesis , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhizobium/genéticaRESUMEN
ARLTS1 is a newly characterized tumor suppressor gene located at chromosome 13q14.3 and involved in the pathogenesis of various types of tumors: two single-nucleotide polymorphisms, one of them responsible for protein truncation, were found statistically associated with familial malignancies, whereas DNA hypermethylation and genomic deletions have been identified as a mechanism of ARLTS1 down-regulation in sporadic cancers. We found that in a large portion of lung carcinomas (37%) and in all analyzed lung cancer cell lines, ARLTS1 is strongly down-regulated due to DNA methylation in its promoter region. After its restoration by adenoviral transduction, ARLTS1-negative A549 and H1299 cells underwent apoptosis and inhibition of cell growth. Furthermore, ARLTS1 reexpression significantly reduced the ability of A549 and H1299 to form tumors in nude mice. Finally, we identified approximately 650 transcripts differentially expressed after restoration of ARLTS1 expression in A549 cells, suggesting that various pathways involved in cell survival, proliferation, signaling, and development mediate the effects of wild-type ARLTS1 in a lung cancer system.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Factores de Ribosilacion-ADP/biosíntesis , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Secuencia Conservada , Metilación de ADN , Regulación hacia Abajo , Terapia Genética/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Alineación de SecuenciaRESUMEN
The beta-amyloid peptide (Abeta) is a major component of Alzheimer disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase (BACE1) and gamma-secretase. BACE1 cleaves APP at the N terminus of the Abeta domain, generating a membrane-bound C-terminal fragment (CTF-beta) that can be subsequently cleaved by gamma-secretase within the transmembrane domain to release Abeta. Because BACE1 initiates Abeta generation, it represents a potential target molecule to interfere with Abeta production in therapeutic strategies for AD. BACE1 interacts with Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA) proteins that are involved in the subcellular trafficking of BACE1. Here, we show that GGA1 is preferentially expressed in neurons of the human brain. GGA1 was also detected in activated microglia surrounding amyloid plaques in AD brains. Functional analyses with cultured cells demonstrate that GGA1 is implicated in the proteolytic processing of APP. Overexpression of GGA1 or a dominant-negative variant reduced cleavage of APP by BACE1 as indicated by a decrease in CTF-beta generation. Importantly, overexpression of GGA1 reduced, whereas RNAi-mediated suppression of GGA1 increased the secretion of Abeta. The modulation of APP processing by GGA1 is independent of a direct interaction of both proteins. Because total cellular activity of BACE1 was not affected by GGA1 expression, our data indicate that changes in the subcellular trafficking of BACE1 or other GGA1-dependent proteins contribute to changes in APP processing and Abeta generation. Thus, GGA proteins might be involved in the pathogenesis of AD.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Péptidos beta-Amiloides/biosíntesis , Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Proteínas Adaptadoras del Transporte Vesicular/genética , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
A differential display was performed to analyze differential gene expression in the brains of mice in association with dietary high beef-tallow. Consumption of a high beef-tallow diet downregulated the expression of ADP-ribosylation factor-like protein 8B (Arl8B) mRNA in the brain. Arl8B mRNA was widely expressed in the mouse brain, including primary neuronal cells. The current study indicates that green fluorescent protein-fused Arl8B protein accumulated at the growth cones in primary neuronal cells, and that protrusions of human embryonic kidney 293 (HEK293) cells were significantly elongated by overexpression of Arl8B, suggesting an important role of Arl8B in neurite formation.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Encéfalo/metabolismo , Grasas de la Dieta/administración & dosificación , Animales , Células Cultivadas , Regulación hacia Abajo , Perfilación de la Expresión Génica , Conos de Crecimiento/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Caveolas/metabolismo , Microdominios de Membrana/metabolismo , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Sustitución de Aminoácidos , Animales , Caveolas/efectos de los fármacos , Caveolina 1 , Caveolinas/genética , Caveolinas/aislamiento & purificación , Caveolinas/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Fraccionamiento Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/enzimología , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática , Femenino , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/genética , Isquemia/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Mutación Puntual , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Superóxidos/metabolismo , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/aislamiento & purificación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína de Unión al GTP rac1/aislamiento & purificación , Proteína de Unión al GTP rac1/metabolismoRESUMEN
OBJECTIVE: To detect expression of mouse ARL-1 homologous proteins in mouse tissues, and analyze homology, genetic distance and phylogenetic relationship between human aldose reductase like-1 (ARL-1) and mouse homologous proteins. METHODS: Homology of mouse ARL-1 homologous proteins with human ARL-1 was analyzed by software Clustal X 1.8 using GenBank and Swiss-Prot database; genetic distance and phylogenetic relationship between mouse ARL-1 homologous proteins and human ARL-1 were analyzed by software Mega 2.0; mouse tissues were detected by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits. RESULTS: The amino acid sequence of human ARL-1 was 83%, 82%, 81%, 79%, 70%, 51%, 50% and 45% identical to that of the Chinese hamster ovary reductase (CHO-Red), the mouse fibroblast growth factor-regulated protein (FR-1), rat aldose reductase-like protein (rARLP), the mouse vas deferens protein (MVDP), rat lens aldose reductase (LeAR), delta4-3-ketosteroid-5beta-reductase (5beta-Red), rat aldo-keto reductase protein c (RaK-c) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Among all the mouse ARL-1 homologous proteins, the genetic distance between CHO-Red and human ARL was the shortest (18.0%, P = 0.023), next was FR-1 (19.1%, P=0.023) and rARLP (19.9%, P = 0.025). From the phylogenetic tree, the protein whose relationship with human ARL-1 was the closest with CHO-Red, next was mouse FR-1, rARLP, MVDP and LeAR. Homologous proteins were found in mouse tissues including vas deferens, testis, bladder and uterus by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits. CONCLUSIONS: CHO-Red has the highest homology, the shortest genetic distance and the closest relationship with human ARL-1, next is FR-1, rARLP, MVDP. The major distribution of mouse ARL-1 homologous proteins is in vas deferens, testis, bladder and uterus, deducing they might be CHO-Red, FR-1, rARLP or MVDP