RESUMEN
Lower selenium levels are observed in Alzheimer's disease (AD) brains, while supplementation shows multiple benefits. Selenoprotein W (SELENOW) is sensitive to selenium changes and binds to tau, reducing tau accumulation. However, whether restoration of SELENOW has any protective effect in AD models and its underlying mechanism remain unknown. Here, we confirm the association between SELENOW downregulation and tau pathology, revealing SELENOW's role in promoting tau degradation through the ubiquitinâproteasome system. SELENOW competes with Hsp70 to interact with tau, promoting its ubiquitination and inhibiting tau acetylation at K281. SELENOW deficiency leads to synaptic defects, tau dysregulation and impaired long-term potentiation, resulting in memory deficits in mice. Conversely, SELENOW overexpression in the triple transgenic AD mice ameliorates memory impairment and tau-related pathologies, featuring decreased 4-repeat tau isoform, phosphorylation at Ser396 and Ser404, neurofibrillary tangles and neuroinflammation. Thus, SELENOW contributes to the regulation of tau homeostasis and synaptic maintenance, implicating its potential role in AD.
Asunto(s)
Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Homeostasis , Ratones Transgénicos , Selenoproteína W , Proteínas tau , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Ratones , Selenoproteína W/metabolismo , Selenoproteína W/genética , Masculino , Fosforilación , Humanos , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Patients with multiple myeloma exhibit malignant osteolytic bone disease due to excessive osteoclast formation and function. We recently identified that osteoclastogenic stimulator selenoprotein W (SELENOW) is upregulated via ERK signaling and downregulated via p38 signaling during receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation. In the intrinsic physiological process, RANKL-induced downregulation of SELENOW maintains proper osteoclast differentiation; in contrast, forced overexpression of SELENOW leads to overactive osteoclast formation and function. METHODS AND RESULTS: We observed that SELENOW is highly expressed in multiple myeloma-derived peripheral blood mononuclear cells (PBMCs) and mature osteoclasts when compared to healthy controls. Also, the level of tumor necrosis factor alpha (TNFα), a pathological osteoclastogenic factor, is increased in the PBMCs and serum of patients with multiple myeloma. ERK activation by TNFα was more marked and sustained than that by RANKL, allowing SELENOW upregulation. Excessive expression of SELENOW in osteoclast progenitors and mature osteoclasts derived from multiple myeloma facilitated efficient nuclear translocation of osteoclastogenic transcription factors NF-κB and NFATc1, which are favorable for osteoclast formation. CONCLUSION: Our findings suggest a possibility that feedforward signaling of osteoclastogenic SELENOW by TNFα derived from multiple myeloma induces overactive osteoclast differentiation, leading to bone loss during multiple myeloma.
Asunto(s)
Diferenciación Celular , Mieloma Múltiple , Osteoclastos , Selenoproteína W , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Diferenciación Celular/genética , Leucocitos Mononucleares/metabolismo , Sistema de Señalización de MAP Quinasas , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Mieloma Múltiple/genética , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Selenoproteína W/metabolismo , Selenoproteína W/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.
Asunto(s)
Selenio , Selenoproteína W , Ratones , Animales , Selenoproteína W/genética , Selenoproteína W/metabolismo , Selenio/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Macrófagos/metabolismo , Oxidación-Reducción , Inflamación/genéticaRESUMEN
Selenium (Se) plays an essential role in the growth of fish and performs its physiological functions mainly through incorporation into selenoproteins. Our previous studies suggested that the selenoprotein W gene (selenow) is sensitive to changes in dietary Se in rainbow trout. However, the molecular characterization and tissue expression pattern of selenow are still unknown. Here, we revealed the molecular characterization, the tissue expression pattern of rainbow trout selenow and analyzed its response to dietary Se. The open reading frame (ORF) of the selenow gene was composed of 393 base pairs (bp) and encodes a 130-amino-acid protein. The 3' untranslated region (UTR) was 372 bp with a selenocysteine insertion sequence (SECIS) element. Remarkably, the rainbow trout selenow gene sequence was longer than those reported for mammals and most other fish. A ß1-α1-ß2-ß3-ß4-α2 pattern made up the secondary structure of SELENOW. Furthermore, multiple sequence alignment revealed that rainbow trout SELENOW showed a high level of identity with SELENOW from Salmo salar. In addition, the selenow gene was ubiquitously distributed in 13 tissues with various abundances and was predominantly expressed in muscle and brain. Interestingly, dietary Se significantly increased selenow mRNA expression in muscle. Our results highlight the vital role of selenow in rainbow trout muscle response to dietary Se levels and provide a theoretical basis for studies of selenow.
Asunto(s)
Oncorhynchus mykiss , Selenio , Animales , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , Selenoproteína W/genética , Selenoproteína W/metabolismo , Selenio/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Clonación Molecular , Mamíferos/genéticaRESUMEN
Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas. The U14C mutant of SelW1 was isolated and biophysically characterized. It contains an intramolecular disulfide bond and is thermally stable up to 70 °C. NMR resonance assignment of reduced and oxidized SelW1 showed that SelW1 adopts a thioredoxin fold. Interestingly, both forms show two additional sets of resonance for amino acid residues near the termini and have basically identical dynamic behavior. Since SelW1 from Chlamydomonas resembles the ancestor of mammalian selenoproteins in certain aspects, this study lays the basis for future characterization of SelW1 function and possible interaction partners.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Mutación , Selenoproteína W/química , Selenoproteína W/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Disulfuros/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Estabilidad Proteica , Estructura Secundaria de Proteína , Selenoproteína W/genética , TermodinámicaRESUMEN
Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodeling. However, their underlying mechanism of action is not fully understood. Herein, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signaling. RNA-sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhances osteoclastogenesis in vitro via nuclear translocation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 mediated by 14-3-3γ, whereas its deficiency suppresses osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibit high bone mass phenotype and osteoporosis, respectively. Ectopic SELENOW expression stimulates cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW regulates osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism.
Asunto(s)
Remodelación Ósea/genética , Osteoclastos/fisiología , Osteogénesis/genética , Osteoporosis/genética , Selenoproteína W/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/metabolismo , Osteoporosis/patología , Ligando RANK/metabolismo , RNA-Seq , Selenoproteína W/genética , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Selenoprotein W (SelW) is a selenium-containing protein with a redox motif found abundantly in the skeletal muscle of rodents. Previous in vitro studies suggest that SelW plays an antioxidant role; however, relatively few in vivo studies have addressed the antioxidant role of SelW. Since oxidative stress is a causative factor for the development of insulin resistance in obese subjects, we hypothesized that if SelW plays a role as an antioxidant, SelW deficiency could aggravate the oxidative stress and insulin resistance caused by a high-fat diet. SelW deficiency did not affect insulin sensitivity and H2O2 levels in the skeletal muscle of control diet-fed mice. SelW levels in the skeletal muscle were decreased by high-fat diet feeding for 12 wk. High-fat diet induced obesity and insulin resistance and increased the levels of H2O2 and oxidative stress makers, which were not affected by SelW deficiency. High-fat diet feeding increased the expression of antioxidant enzymes; however, SelW deficiency did not affect the expression levels of antioxidants. These results suggest that SelW does not play a protective role against oxidative stress and insulin resistance in the skeletal muscle of high-fat diet-fed obese mice.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Músculo Esquelético/metabolismo , Obesidad/genética , Estrés Oxidativo , Selenoproteína W/genética , Animales , Catalasa/genética , Catalasa/metabolismo , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Humanos , Peróxido de Hidrógeno/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/patología , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Selenoproteína W/deficiencia , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Mouse selenoprotein W (SELENOW) is a small protein containing a selenocysteine (Sec, U) and four cysteine (Cys, C) residues. The Sec residue in SELENOW is located within the conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin (Trx). It is known that glutathione (GSH) binds to SELENOW and that this binding is involved in protecting cells from oxidative stress. However, the regulatory mechanisms controlling the glutathionylation of SELENOW in oxidative stress are unclear. In this study, using purified recombinant SELENOW in which Sec13 was changed to Cys, we found that SELENOW was glutathionylated at Cys33 and that this S-glutathionylation was enhanced by oxidative stress. We also found that the S-glutathionylation of SELENOW at Cys33 in HEK293 cells was due to glutathione S-transferase Pi (GSTpi) and that this modification was reversed by glutaredoxin1 (Grx1). In addition to the disulfide bond between the Cys10 and Cys13 of SELENOW, a second disulfide bond was formed between Cys33 and Cys87 under oxidative stress conditions. The second disulfide bond was reduced by Trx1, but the disulfide bond between Cys10 and Cys13 was not. The second disulfide bond was also reduced by glutathione, but the disulfide bond in the CXXC motif was not. The second disulfide bond of the mutant SELENOW, in which Cys37 was replaced with Ser, was formed at a much lower concentration of hydrogen peroxide than the wild type. We also observed that Cys37 was required for S-glutathionylation, and that S-glutathionylated SELENOW containing Cys37 protected the cells from oxidative stress. Furthermore, the SELENOW (C33, 87S) mutant, which could not form the second disulfide bond, also showed antioxidant activity. Taken together, these results indicate that GSTpi-mediated S-glutathionylation of mouse SELENOW at Cys33 is required for the protection of cells in conditions of oxidative stress, through inhibition of the formation of the second disulfide bond.
Asunto(s)
Disulfuros/metabolismo , Gutatión-S-Transferasa pi/genética , Estrés Oxidativo/genética , Selenoproteína W/genética , Animales , Sitios de Unión/genética , Muerte Celular/genética , Cisteína/genética , Disulfuros/antagonistas & inhibidores , Glutarredoxinas/genética , Glutatión/genética , Glutatión/metabolismo , Células HEK293 , Humanos , Ratones , Oxidación-Reducción , Unión Proteica/genética , Selenocisteína/genética , Selenoproteína W/metabolismoRESUMEN
Selenoprotien W (SelW) plays a key role in brain development, although the exact biological function and mechanisms remain unclear. We performed a yeast two-hybrid screen on a human fetal brain cDNA library and identified FAM96B as a novel binding partner of SelW. FRET analyses confirmed the interaction between SelW' and FAM96B. The mutated SelW' construct was cloned and overexpressed in E. coli, and a pull-down assay verified a direct interaction between SelW' and FAM96B. Finally, Co-Immunoprecipitation on murine brain tissue proteins demonstrated an endogenous interaction between the two proteins in the brain. Taken together, our findings prove a direct interaction between SelW and FAM96B, which may provide new insights into the role of SelW in brain development and neurodegenerative diseases.
Asunto(s)
Encéfalo/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Selenoproteína W/metabolismo , Animales , Femenino , Feto/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Genes , Células HEK293 , Humanos , Metaloproteínas/genética , Ratones , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenoproteína W/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Breast cancer is one of the most common malignancies and the major cause of cancer-related death in women. Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has been increasingly recognized, few studies have been explored the functional mechanism of piRNAs in breast cancer development and progression. METHODS: We examined the top 20 highly expressed piRNAs based on the analysis of TCGA breast cancer data in two patient cohorts to test the roles of piRNAs in breast cancer. The effects of piRNA-36,712 on the malignant phenotypes and chemosensitivity of breast cancer cells were detected in vitro and in vivo. MS2-RIP and reporter gene assays were conducted to identify the interaction and regulation among piRNA-36,712, miRNAs and SEPW1P. Kaplan-Meier estimate with log-rank test was used to compare patient survival by different piRNA-36,712 expression levels. RESULTS: We found piRNA-36,712 level was significantly lower in breast cancer than in normal breast tissues and low level was correlated with poor clinical outcome in patients. Functional studies demonstrated that piRNA-36,712 interacts with RNAs produced by SEPW1P, a retroprocessed pseudogene of SEPW1, and subsequently inhibits SEPW1 expression through competition of SEPW1 mRNA with SEPW1P RNA for microRNA-7 and microRNA-324. We also found that higher SEPW1 expression due to downregulation of piRNA-36,712 in breast cancer may suppress P53, leading to the upregulated Slug but decreased P21 and E-cadherin levels, thus promoting cancer cell proliferation, invasion and migration. Furthermore, we found that piRNA-36,712 had synergistic anticancer effects with the paclitaxel and doxorubicin, two chemotherapeutic agents for breast cancer. CONCLUSIONS: These findings suggest that piRNA-36,712 is a novel tumor suppressor and may serve as a potential predictor for the prognosis of breast cancer patients.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , ARN Interferente Pequeño/genética , Selenoproteína W/genética , Animales , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Humanos , MicroARNs/genética , Paclitaxel/farmacología , Pronóstico , Seudogenes , ARN Mensajero/genética , ARN Interferente Pequeño/biosíntesis , Regulación hacia ArribaRESUMEN
Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in metastatic development, yet this "premetastatic" stage of the metastatic cascade, when primary tumor cells cross the blood-brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable "premetastatic" phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2 Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124-34. ©2018 AACR.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Metástasis de la Neoplasia/tratamiento farmacológico , Apomorfina/farmacología , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Cinesinas/genética , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Proteínas Serina-Treonina Quinasas/genética , Selenoproteína W/genéticaRESUMEN
Selenoprotein W (SelW) is an important member of the avian selenoprotein family. It is well known for its important role in protecting neurons from oxidative stress during neuronal development. d-Amino acid (d-serine), as a neurotransmitter in the central nervous system (CNS), can mediate neurotoxicity. d-Amino acid oxidase (DAAO) is responsible for regulating the d-serine levels in cells. However, the correlation between SelW and DAAO is not clear yet. To investigate the regulations between SelW and DAAO, chicken embryo monolayer neurons were treated with d-serine and/or Se. In this study, we predicted molecular binding between SelW and DAAO. These results showed that the 9-16, 18, 41-47 and 66 residues of SelW could combine with the DAAO, which suggested that chicken SelW might be the target of DAAO. We determined the DAAO activity and the mRNA expression of SelW in in vitro cultured chicken embryo primitive neuron cells. d-Serine influenced the activity of DAAO and, moreover, a significant increase in the mRNA expression of SelW was found in neurons treated with Se. Notably, we also observed changes in the expression of SelW and DAAO when neurons were treated with various concentrations of d-serine and Se. In conclusion, these data suggest that d-serine could regulate the mRNA expression of SelW by interfering with the activity of DAAO in chicken embryo neurons.
Asunto(s)
D-Aminoácido Oxidasa/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Selenoproteína W/metabolismo , Serina/farmacología , Animales , Supervivencia Celular , Células Cultivadas , Embrión de Pollo , Pollos , D-Aminoácido Oxidasa/genética , Proyección Neuronal , Neuronas/citología , Neuronas/efectos de los fármacos , Estrés Oxidativo , Selenio/farmacología , Selenoproteína W/genéticaRESUMEN
Micronutrient selenium (Se) plays a key role in redox regulation through its incorporation into selenoproteins as the 21st amino acid selenocysteine (Sec). Because Se deficiency appears to be a cofactor in the anemia associated with chronic inflammatory diseases, we reasoned that selenoproteins may contribute to erythropoietic recovery from anemia, referred to as stress erythropoiesis. Here, we report that loss of selenoproteins through Se deficiency or by mutation of the Sec tRNA (tRNA[Sec]) gene (Trsp) severely impairs stress erythropoiesis at 2 stages. Early stress erythroid progenitors failed to expand and properly differentiate into burst-forming unit-erythroid cells , whereas late-stage erythroid progenitors exhibited a maturation defect that affected the transition of proerythroblasts to basophilic erythroblasts. These defects were, in part, a result of the loss of selenoprotein W (SelenoW), whose expression was reduced at both transcript and protein levels in Se-deficient erythroblasts. Mutation of SelenoW in the bone marrow cells significantly decreased the expansion of stress burst-forming unit-erythroid cell colonies, which recapitulated the phenotypes induced by Se deficiency or mutation of Trsp Similarly, mutation of SelenoW in murine erythroblast (G1E) cell line led to defects in terminal differentiation. In addition to the erythroid defects, the spleens of Se-deficient mice contained fewer red pulp macrophages and exhibited impaired development of erythroblastic island macrophages, which make up the niche supporting erythroblast development. Taken together, these data reveal a critical role of selenoproteins in the expansion and development of stress erythroid progenitors, as well as the erythroid niche during acute anemia recovery.
Asunto(s)
Anemia/metabolismo , Células Precursoras Eritroides/citología , Eritropoyesis , Selenio/deficiencia , Selenoproteínas/metabolismo , Anemia/genética , Animales , Regulación hacia Abajo , Eritroblastos/citología , Eritroblastos/metabolismo , Células Precursoras Eritroides/metabolismo , Ratones Endogámicos C57BL , Mutación , Selenio/metabolismo , Selenoproteína W/genética , Selenoproteína W/metabolismo , Selenoproteínas/genética , Bazo/citología , Bazo/metabolismoRESUMEN
This study aims to evaluate the protective effects of selenomethionine (SeMet) on aflatoxin B1 (AFB1)-induced hepatotoxicity in primary chicken hepatocytes. Cell viability and lactic dehydrogenase activity assays revealed the dose dependence of AFB1 toxicity to chicken hepatocytes. AFB1 concentrations of >0.05 µg/mL significantly reduced glutathione and total superoxide dismutase levels and increased the malondialdehyde concentration and cytochrome P450 enzyme 1A5 (CYP450 1A5) mRNA levels (P < 0.05). AFB1, however, did not affect CYP450 3A37 mRNA levels. Supplementation with 2 µM SeMet protected against AFB1-induced changes and significantly increased selenoprotein W (SelW) mRNA levels (P < 0.05). Additionally, SelW knockdown attenuated the protective effect of SeMet on AFB1-induced damage and significantly increased the level of CYP450 1A5 expression (P < 0.05). Therefore, SeMet alleviates AFB1-induced damage in primary chicken hepatocytes by improving SelW expression, thus inhibiting CYP450 1A5 expression.
Asunto(s)
Aflatoxina B1/toxicidad , Proteínas Aviares/genética , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/genética , Hepatocitos/efectos de los fármacos , Selenometionina/farmacología , Selenoproteína W/genética , Animales , Proteínas Aviares/metabolismo , Pollos , Sistema Enzimático del Citocromo P-450/metabolismo , Glutatión/metabolismo , Hepatocitos/enzimología , Hepatocitos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Selenoproteína W/metabolismo , Superóxido Dismutasa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In this study, a selenoprotein W cDNA was cloned from topmouth culter (Erythroculter ilishaeformis), and it was designated as EISelW. The EISelW open reading frame was composed of 261 base pairs (bp), encoding 86-amino-acid protein. The 5' untranslated region (UTR) consisted of 104 bp, and the 3'-UTR was composed of 365 bp. A selenocysteine insertion sequence (SECIS) element was found in the 3'-UTR of EISelW mRNA. The SECIS element was classified as form II because of a small additional apical loop presented in SECIS element of EISelW mRNA. Bioinformatic approaches showed that the secondary structure of EISelW was a ß1-α1-ß2-ß3-ß4-α2 pattern from amino-terminal to carboxy-terminal. Real-time PCR analysis of EISelW mRNAs expression in 17 tissues showed that the EISelW mRNA was predominantly expressed in liver, ovary, pituitary, various regions of the brain, spinal cord and head kidney. Study of intraperitoneal injection showed that the levels of EISelW mRNA in brain, liver, ovary and spleen were regulated by somatostatin 14 (SS14), 17ß-estradiol (E2), cysteamine hydrochloride (CSH) and a binary mixture of E2 and CSH, dependent on the dosage. These results suggest that E2, SS14 and CSH status may affect tissues of selenium metabolism by regulating the expression of SelW mRNA, as SelW plays a central role in selenium metabolism.
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Cisteamina/farmacología , Estradiol/farmacología , Perciformes/genética , Selenoproteína W/genética , Somatostatina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario/genética , Interacciones Farmacológicas , Femenino , Hígado/metabolismo , Masculino , Ovario/metabolismo , Filogenia , ARN Mensajero/metabolismo , Bazo/metabolismoRESUMEN
Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein.
Asunto(s)
Proteínas 14-3-3/metabolismo , Estrés Oxidativo/fisiología , Selenoproteína W/metabolismo , Proteínas 14-3-3/genética , Secuencias de Aminoácidos , Ditiotreitol/farmacología , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Células MCF-7 , Mutación Missense , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Selenoproteína W/genéticaRESUMEN
Selenoprotein W (SelW) is a low molecular weight and selenocysteine containing protein with redox activity involved in the antioxidant response. In the present study, the full-length cDNA of goldfish (Carassius auratus) selenoprotein W (gfSelW) was successfully cloned from the liver tissue by rapid amplification of cDNA ends technique. The obtained gfSelW cDNA was 730 bp long with a 79 bp 5'-untranslated region (UTR), a 390 bp 3'-UTR containing the consensus polyadenylation signal AATAAA and a 261 bp open reading frame coding a protein of 86 amino acid residues. gfSelW mRNA was observed in all regions of brain and peripheral tissues by semi-quantitative RT-PCR, and the most abundant was detected in testis. After fasting for 1 week, gfSelW mRNA expression levels were significantly decreased compared to the fed group in hypothalamus and liver. After refeeding for 7 days, gfSelW mRNA expression levels were increased back. Furthermore, the mRNA expressions of gfSelW in hypothalamus and liver were varied in periprandial changes and significantly up-regulated after meal 2 h and 4 h, respectively. With cadmium exposure for 24 h, gfSelW mRNA expression levels in gill and leucocytes were significantly decreased at different cadmium concentrations changing from 0.5 ppm to 10 ppm. However, the gfSelW mRNA expression level was sharply increased in liver, relatively to the control about 4.98-fold at 0.5 ppm. The results in this study provide molecular characterization of SelW in goldfish and imply that SelW mRNA expression may be associated with metabolic status and oxidative stress and regulated by metabolic factors and cadmium in fish.
Asunto(s)
Cadmio/toxicidad , Clonación Molecular , Proteínas de Peces/metabolismo , Carpa Dorada/metabolismo , ARN Mensajero/metabolismo , Selenoproteína W/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Peces/genética , Carpa Dorada/genética , Datos de Secuencia Molecular , Especificidad de Órganos , Selenoproteína W/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Selenoprotein W (SelW) is mainly understood in terms of its antioxidant effects in the cellular defense system. Inflammation is an important indicator of animal tissue injury, and the inflammatory cells may trigger a sophisticated and well-orchestrated inflammatory cascade, resulting in exaggerated oxidative stress. To investigate the role of SelW in inflammatory injury in chicken immune tissues and cultured splenic lymphocyte, in this report, the effects of selenium (Se) on mRNA expressions of SelW and inflammatory factors (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in the chicken immune organs (spleen, thymus and bursa of Fabricius) and cultured splenic lymphocyte treated with sodium selenite and H2O2, or knocked down SelW with small interfering RNAs (siRNAs) were examined. The results showed that Se-deficient diets effectively decreased the mRNA expression of SelW (P < 0.05), and induced a significantly up-regulation of COX-2, iNOS, NF-κB, PTGEs and TNF-α mRNA levels (P < 0.05). The histopathological analysis showed that immune tissues were obviously injured in the low-Se groups. In vitro, H2O2 induced a significantly up-regulation of the mRNA levels of inflammation-related genes (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in cultured splenic lymphocyte (P < 0.05). When lymphocytes were pretreated with Se before treated with H2O2, the inflammation-related genes were significantly decreased (P < 0.05). Silencing of SelW significantly up-regulated the inflammation-related genes (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in cultured splenic lymphocyte (P < 0.05). The results suggested that the expression levels of inflammatory factors (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) and SelW can be influenced by Se in birds. SelW commonly played an important role in the protection of immune organs of birds from inflammatory injury by the regulations of inflammation-related genes.
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Inflamación/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Selenoproteína W/metabolismo , Animales , Células Cultivadas , Pollos , Inflamación/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Selenoproteína W/genética , Bazo/citología , Bazo/metabolismoRESUMEN
Selenoprotein W (SelW) is expressed in various tissues, particularly in skeletal muscle. We have previously reported that SelW is up-regulated during C2C12 skeletal muscle differentiation and inhibits binding of 14-3-3 to its target proteins. 14-3-3 reduces myogenic differentiation by inhibiting nuclear translocation of transcriptional co-activator with PDZ-binding motif (TAZ). Phosphorylation of TAZ at Ser89 is required for binding to 14-3-3, leading to cytoplasmic retention of TAZ and a delay in myogenic differentiation. Here, we show that myogenic differentiation was delayed in SelW-knockdown C2C12 cells. Down-regulation of SelW also increased TAZ binding to 14-3-3, which eventually resulted in decreasing translocation of TAZ to the nucleus. However, phosphorylation of TAZ at Ser89 was not affected. Although phosphorylation of TAZ at Ser89 was sustained by the phosphatase inhibitor okadaic acid, nuclear translocation of TAZ was increased by ectopic expression of SelW. This result was due to decreased binding of TAZ to 14-3-3. We also found that the interaction between TAZ and MyoD was increased by ectopic expression of SelW. Taken together, these findings strongly demonstrate that SelW enhances C2C12 cell differentiation by inhibiting TAZ binding to 14-3-3.
Asunto(s)
Proteínas 14-3-3/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Selenoproteína W/metabolismo , Factores de Transcripción/metabolismo , Proteínas 14-3-3/genética , Aciltransferasas , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Ratones , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Ácido Ocadaico/farmacología , Fosforilación , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Selenoproteína W/antagonistas & inhibidores , Selenoproteína W/genética , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Selenoprotein W (SelW) is a selenocysteine containing protein with redox activity involved in the antioxidant response. In this study, a selenoprotein W was cloned from pearl mussel Cristaria plicata (designated as CpSelW), and the expression patterns were characterized in tissues after Aeromonas hydrophila challenged. The full-length cDNA of cpSelW was of 858bp, containing a 5' untranslated region (UTR) of 145bp, a 3' UTR of 455bp with a poly (A) tail, and an open reading frame (ORF) of 258bp encoding a polypeptide of 85 amino acids with the predicted molecular mass of 9.277kDa, which shared 61% identity with SelW from Gallus gallus. A tertiary structure model generated for the CpSelW displayed a ß-α-ß-ß-ß-α secondary structure pattern, which was similar to mouse SelW protein 3D structure. The mRNA of CpSelW was constitutively expressed in tested tissues of healthy mussel, including mantle, gill, hemocytes, muscle, and hepatopancreas, and it was highly expressed in hepatopancreas. After mussels were stimulated by A. hydrophila, the mRNA expression of CpSelW in hemocytes at 6, 12 and 24h, in gill at 12h and in hepatopancreas at 24h was significantly down-regulated.