RESUMEN
This study aims to identify the novel biomarkers of cold-dampness syndrome(RA-Cold) of rheumatoid arthritis(RA) by gene set enrichment analysis(GSEA), weighted gene correlation network analysis(WGCNA), and clinical validation. Firstly, transcriptome sequencing was carried out for the whole blood samples from RA-Cold patients, RA patients with other traditional Chinese medicine(TCM) syndromes, and healthy volunteers. The differentially expressed gene(DEG) sets of RA-Cold were screened by comparison with the RA patients with other TCM syndromes and healthy volunteers. Then, GSEA and WGCNA were carried out to screen the key DEGs as candidate biomarkers for RA-Cold. Experimentally, the expression levels of the candidate biomarkers were determined by RT-qPCR for an independent clinical cohort(not less than 10 cases/group), and the clinical efficacy of the candidates was assessed using the receiver operating characteristic(ROC) curve. The results showed that 3 601 DEGs associated with RA-Cold were obtained, including 106 up-regulated genes and 3 495 down-regulated genes. The DEGs of RA-Cold were mainly enriched in the pathways associated with inflammation-immunity regulation, hormone regulation, substance and energy metabolism, cell function regulation, and synovial pannus formation. GSEA and WGCNA showed that recombinant proteasome 26S subunit, ATPase 2(PSMC2), which ranked in the top 50% in terms of coefficient of variation, representativeness of pathway, and biological modules, was a candidate biomarker of RA-Cold. Furthermore, the validation results based on the clinical independent sample set showed that the F1 value, specificity, accuracy, and precision of PSMC2 for RA-Cold were 70.3%, 61.9%, 64.5%, and 81.3%, respectively, and the area under the curve(AUC) value was 0.96. In summary, this study employed the "GSEA-WGCNA-validation" integrated strategy to identify novel biomarkers of RA-Cold, which helped to improve the TCM clinical diagnosis and treatment of core syndromes in RA and provided an experimental basis for TCM syndrome differentiation.
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Artritis Reumatoide , Humanos , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/genética , Artritis Reumatoide/tratamiento farmacológico , Biomarcadores/metabolismo , Medicina Tradicional China , Perfilación de la Expresión Génica/métodos , Biología Computacional , Redes Reguladoras de Genes , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/uso terapéutico , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/uso terapéuticoRESUMEN
OBJECTIVE: To investigate the efficacy and mechanism of the Qingre Huayu Fang () on atherosclerotic vulnerable plaque in apolipoprotein E (ApoE) knockout mice through the ubiquitin proteasome pathway. METHODS: Sixty 8-week-old C57BL/6J ApoE knockout mice were fed a high-fat for 12 weeks and randomly divided into four treatment groups (n = 15 each): high-fat control, bortezomib (a proteasome inhibitor), bortezomib combined with Qingre Huayu Fang, and Qingre Huayu Fang alone. Aortic sections were examined for plaque development, inflammatory cell infiltration, type â /â ¢ collagen expression and immunohistochemical staining of CD40L, nuclear factor-kappa B (NF-κB)/P65 and ubiquitin. RESULTS: Mice in the high-fat control group had obvious atherosclerosis, with increased aortic plaque area. The degree of atherosclerosis of the atherosclerotic plaque was reduced in all of the treatment groups that received bortezomib and/or Duzhong (Cortex Eucommiae) Qingre Huayu. The expression of NF-?B, CD40L and ubiquitin were all reduced in the group that received combination bortezomib + Qingre Huayu Fang. CONCLUSION: The Qingre Huayu Fang inhibited aortic atherosclerosis in mice through a mechanism that may involve inhibition of the ubiquitin proteasome pathway.
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Placa Aterosclerótica , Animales , Apolipoproteínas E/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/genética , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Ginsenoside 20(S)-Rg3 is identified as an active saponin monomer which derived from red ginseng and is demonstrated to play an anti-tumor role in diverse cancers. MicroRNAs (miRNAs) are important regulators in the progression of cancers, containing esophageal squamous cell carcinoma (ESCC). It was reported that microRNA 324-5p (miR-324-5p) exerted critical functions in some cancers; however, the detailed molecular mechanism of miR-324-5p mediated by 20(S)-Rg3 to suppress cell viability in ESCC has not been explored. Herein, we explored the function of 20(S)-Rg3 or miR-324-5p on ESCC cell viability by MTT assay, colony formation assay, flow cytometry analysis and western blot analysis. The binding of miR-324-5p to its target gene, proteasome activator subunit 3 (PSME3), was confirmed through RNA pull down and luciferase reporter assays. The results indicated that 20(S)-Rg3 significantly inhibited cell viability and the cell cycle and facilitated cell apoptosis. Furthermore, this effect was strengthened with the increased concentration of 20(S)-Rg3. Moreover, we found that miR-324-5p level was increased under 20(S)-Rg3 treatment. Additionally, overexpressed miR-324-5p inhibited ESCC cell viability, and downregulated miR-324-5p recovered inhibited cell viability caused by 20(S)-Rg3. Further exploration verified that miR-324-5p targeted PSME3, and PSME3 deficiency countervailed the effect of miR-324-5p inhibition on ESCC cell viability under 20(S)-Rg3 treatment. Conclusively, 20(S)-Rg3 suppresses cell viability in ESCC via mediating miR-324-5p-targeted PSME3.
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Antineoplásicos Fitogénicos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ginsenósidos/uso terapéutico , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Línea Celular Tumoral/efectos de los fármacos , Humanos , MicroARNs , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Yuanzhisan (YZS) is a classic type of Traditional Chinese Medicine, which has been reported to aid in the treatment of Alzheimer's disease (AD). The present study aimed to investigate the effects of YZS on tau protein aggregation, a hallmark of AD pathology, and its possible mechanisms. The results demonstrated that YZS improved learning and memory abilities, and decreased the severity of AD pathology in ßamyloid (Aß140)induced AD rats. Moreover, YZS administration inhibited the hyperphosphorylation of tau protein at Ser199 and Thr231 sites. Several vital enzymes in the ubiquitinproteasome system (UPS), including ubiquitinactivating enzyme E1a/b, ubiquitinconjugating enzyme E2a, carboxyl terminus of Hsc70interacting protein, ubiquitin C236 terminal hydrolase L1 and 26S proteasome, were all significantly downregulated in AD rats, which indicated an impaired enzymatic cascade in the UPS. In addition, it was identified that YZS treatment partly increased the expression levels of these enzymes in the brains of AD rats. In conclusion, the present results suggested that YZS could effectively suppress the hyperphosphorylation of tau proteins, which may be partially associated with its beneficial role in restoring functionality of the UPS.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/genética , Medicamentos Herbarios Chinos/farmacología , Fragmentos de Péptidos/genética , Agregado de Proteínas/efectos de los fármacos , Proteínas tau/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Humanos , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Ratas , Ubiquitina/genéticaRESUMEN
BACKGROUND: Curcumin functions as a proteasome inhibitor. However, the molecular mechanisms behind this action need more detailed explanations. PURPOSE: This study aimed to investigate the inhibitory effect of curcumin on 20S proteasome activity and to elucidate its exact mechanism in triple-negative breast cancer (TNBC) MDA-MB-231 cells. METHODS: Proteasomal peptidase activities were assayed using synthetic fluorogenic peptide substrates. Knockdown or overexpression of microRNA (miRNA or miR) or protein was used to investigate its functional effect on downstream cellular processes. BrdU (5bromo2'-deoxyuridine) assay was performed to identify cell proliferation. Western blot and quantitative real-time PCR(qRT-PCR) were carried out to determine protein abundance and miRNA expression, respectively. Correlations between protein expressions, miRNA levels, and proteasome activities were analyzed in TNBC tissues. Xenograft tumor model was performed to observe the in vivo effect of curcumin on 20S proteasome activity. RESULTS: Curcumin significantly reduced PSMB5 protein levels, accompanied with a reduction in the chymotrypsin-like (CT-l) activity of proteasome 20S core. Loss of PSMB5 markedly inhibited the CT-l activity of 20S proteasome. Furthermore, curcumin treatment significantly elevated miR-142-3p expression. PSMB5 was a direct target of miR-142-3p and its protein levels were negatively regulated by miR-142-3p. Moreover, histone acetyltransferase p300 suppressed miR-142-3p expression. Overexpression of p300 mitigated the promotive effect of curcumin on miR-142-3p expression. The correlations among p300 abundances, miR-142-3p levels, PSMB5 expressions, and the CT-l activities of 20S proteasome were evidenced in TNBC tissues. In addition, loss of p300 and PSMB5 reduced cell proliferation. Inhibition of miR-142-3p significantly attenuated the inhibitory impact of curcumin on cell proliferation. These curcumin-induced changes on p300, miR-142-3p, PSMB5, and 20S proteasome activity were further confirmed in in vivo solid tumor model. CONCLUSION: These findings demonstrated that curcumin suppressed p300/miR-142-3p/PSMB5 axis leading to the inhibition of the CT-l activity of 20S proteasome. These results provide a novel and alternative explanation for the inhibitory effect of curcumin on proteasome activity and also raised potential therapeutic targets for TNBC treatment.
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Curcumina/farmacología , MicroARNs/genética , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos BALB C , MicroARNs/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Type B 8-keto-trichothecenes are muco-active mycotoxins that exist as inevitable contaminants in cereal-based foodstuffs. Gut-associated inflammation is an early frontline response during human and animal exposure to these mycotoxins. Despite various tools for chemical identification, optimized biomonitoring of sentinel response-associated biomarkers is required to assess the specific proinflammatory actions of 8-keto-trichothecenes in the gut epithelial barrier. In the present study, intoxication with 8-keto-trichothecenes in human intestinal epithelial cells was found to trigger early response gene 1 product (EGR-1) that plays crucial roles in proinflammatory chemokine induction. In contrast, epithelial exposure to 8-keto-trichothecenes resulted in downregulated expression of nuclear factor NF-kappa-B p65 protein, a key transcription factor, during general inflammatory responses in the gut. Based on the early molecular patterns of expression, the inflammation-inducing activity of 8-keto-trichothecenes was quantified using intestinal epithelial cells with dual reporters for EGR-1 and p65 proteins. EGR-1-responsive elements were linked to luciferase reporter while p65 promoter was bound to secretory alkaline phosphatase (SEAP) reporter. In response to conventional inflammagens such as endotoxins and cytokines such as TNF-α, both luciferase and SEAP activity were elevated in a dose-dependent manner. However, as expected from the mechanistic evaluation, 8-keto-trichothecene-exposed dual reporters of luciferase and SEAP displayed contrasting expression patterns. Furthermore, 8-keto-trichothecene-elevated EGR-1-responsive luciferase activity was improved by deficiency of PSMA3, an α-type subunit of the 20S proteasome core complex for ubiquitin-dependent EGR-1 degradation. This molecular event-based dual biomonitoring in epithelial cells is a promising supplementary tool for detecting typical molecular inflammatory pathways in response to 8-keto-trichothecenes in the food matrix.
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Bioensayo/métodos , Biomarcadores , Enterocitos/metabolismo , Mediadores de Inflamación/metabolismo , Tricotecenos Tipo B/efectos adversos , Animales , Línea Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Ratones , Micotoxinas , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
The ubiquitin (Ub)/26S proteasome system (UPS) plays a key role in plant growth, development, and survival by directing the turnover of numerous regulatory proteins. In the UPS, the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains function as hubs for ubiquitin-mediated protein degradation. Radiation sensitive 23 (RAD23), which has been identified as a UBL/UBA protein, contributes to the progression of the cell cycle, stress responses, ER proteolysis, and DNA repair. Here, we report that pollen development is arrested at the microspore stage in a rad23b null mutant. We demonstrate that RAD23B can directly interact with KIP-related protein 1 (KRP1) through its UBL-UBA domains. In addition, plants overexpressing KRP1 have defects in pollen development, which is a phenotype similar to the rad23b mutant. RAD23B promotes the degradation of KRP1 in vivo, which is accumulated following treatment with the proteasome inhibitor MG132. Our results indicate that RAD23B plays an important in pollen development by controlling the turnover of the key cell cycle protein, KRP1.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Polen/genética , Complejo de la Endopetidasa Proteasomal/genética , UbiquitinaRESUMEN
Alzheimer's disease (AD) is a common neurodegenerative disease which is partly characterized by the aggregation of hyperphosphorylated Tau proteins forming neurofibrillary tangles that promote AD pathogenesis. In this study, we investigated the effects of tanshinone IIA (Tan IIA) isolated from Salvia miltiorrhiza on Tau degradation in the treatment of AD. The results showed that Tan IIA reduced the Tau expression and attenuated Tau phosphorylation in N2a cells, Tau-overexpressing cells, and 3×Tg-AD mouse primary neuron cells. Moreover, Tan IIA increased polyubiquitinated Tau accumulation and induced proteasomal degradation of the Tau protein. Additionally, Tan IIA became bound to the Tau protein and inhibited the formation of heparin-induced Tau fibrils. In summary, Tan IIA can increase polyubiquitinated Tau accumulation and induce the proteasomal degradation of the Tau protein and the binding of Tan IIA to the Tau protein, inhibiting the formation of Tau fibrils. Tan IIA may be further explored as a potential candidate for AD treatment.
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Abietanos/farmacología , Enfermedad de Alzheimer/metabolismo , Medicamentos Herbarios Chinos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Salvia miltiorrhiza/química , Ubiquitinas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Animales , Línea Celular , Humanos , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Proteolisis/efectos de los fármacos , Proteínas tau/genéticaRESUMEN
Identifying genetic variation that increases crop yields is a primary objective in plant breeding. We used association analyses of oilseed rape/canola (Brassica napus) accessions to identify genetic variation that influences seed size, lipid content, and final crop yield. Variation in the promoter region of the HECT E3 ligase gene BnaUPL3 C03 made a major contribution to variation in seed weight per pod, with accessions exhibiting high seed weight per pod having lower levels of BnaUPL3 C03 expression. We defined a mechanism in which UPL3 mediated the proteasomal degradation of LEC2, a master transcriptional regulator of seed maturation. Accessions with reduced UPL3 expression had increased LEC2 protein levels, larger seeds, and prolonged expression of lipid biosynthetic genes during seed maturation. Natural variation in BnaUPL3 C03 expression appears not to have been exploited in current B napus breeding lines and could therefore be used as a new approach to maximize future yields in this important oil crop.
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Brassica napus/metabolismo , Productos Agrícolas/metabolismo , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassica napus/enzimología , Brassica napus/genética , Productos Agrícolas/química , Productos Agrícolas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Homeodominio/metabolismo , Ligasas/genética , Ligasas/metabolismo , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Mutación , Fenotipo , Mucílago de Planta/biosíntesis , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Aceite de Brassica napus/metabolismo , Semillas/química , Semillas/genética , Semillas/crecimiento & desarrollo , Factores de Transcripción/genética , Transcriptoma/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Proteasome inhibition represents an important anticancer strategy. Here, we studied the mechanisms at the basis of the pro-apoptotic activity of the standardized decoction of Hemidesmus indicus, a plant evoking a complex anticancer activity, and explored its inhibition of proteasome activity in human leukemia cells. Additionally, we preliminary tested the cytotoxicity of some H. indicus's phytochemicals on leukemia cells and their intestinal absorption on a human intestinal epithelium model consisting of a monolayer of differentiated Caco2 cells. We observed a potent antileukemic effect for H. indicus, imputable to the modulation of different critical targets at protein and mRNA levels and the reduction of the 26S proteasome expression. We found that some phytomarkers of H. indicus decoction passed through the enterocyte monolayer. Overall, our study supports the pharmacological potential of H. indicus, which can represent an interesting botanical drug in the oncological area.
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Antineoplásicos Fitogénicos/farmacología , Hemidesmus/química , Inhibidores de Proteasoma/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos Fitogénicos/química , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Absorción Intestinal , Células Jurkat , MicroARNs/genética , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/químicaRESUMEN
Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis. Abbreviations: AC: acinar cell; AMY: amylase; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATP6V1B2: ATPase, H+ transporting, lysosomal V1 subunit B2; ATP6V1D: ATPase, H+ transporting, lysosomal V1 subunit D; ATP6V1H: ATPase, H+ transporting, lysosomal V1 subunit H; AV: autophagic vacuole; CDE: choline-deficient, ethionine-supplemented; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EM: electron microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H & E: hematoxylin and eosin; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: normal donor; NEU: neutrophil; PPARGC1A/PGC1α: peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha; RIPA: radio-immunoprecipitation; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TM: tamoxifen; WT: wild-type; ZG: zymogen granule.
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Células Acinares/metabolismo , Autofagosomas/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Lisosomas/metabolismo , Pancreatitis/metabolismo , Células Acinares/efectos de los fármacos , Células Acinares/enzimología , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/ultraestructura , Autofagia/efectos de los fármacos , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Núcleo Celular/metabolismo , Ceruletida/toxicidad , Modelos Animales de Enfermedad , Humanos , Inflamación/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Páncreas/efectos de los fármacos , Páncreas/enzimología , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/enzimología , Pancreatitis/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
A Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway.
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Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Proteasoma/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/parasitología , Ubiquitina/metabolismoRESUMEN
BACKGROUND: A low-protein diet supplemented with ketoacids (LPD + KA) maintains the nutritional status of patients with chronic kidney disease (CKD). Oxidative damage and mitochondrial dysfunction associated with the upregulation of p66SHC and FoxO3a have been shown to contribute to muscle atrophy. This study aimed to determine whether LPD + KA improves muscle atrophy and attenuates the oxidative stress and mitochondrial damage observed in CKD rats. METHODS: 5/6 nephrectomy rats were randomly divided into three groups and fed with either 22% protein (normal-protein diet; NPD), 6% protein (low-protein diets; LPD) or 5% protein plus 1% ketoacids (LPD + KA) for 24 weeks. Sham-operated rats with NPD intake were used as the control. RESULTS: KA supplementation improved muscle atrophy and function in CKD + LPD rats. It also reduced the upregulation of genes related to the ubiquitin-proteasome system and 26S proteasome activity, as well as protein and mitochondrial oxidative damage in the muscles of CKD + LPD rats. Moreover, KA supplementation prevented the drastic decrease in activities of mitochondrial electron transport chain complexes, mitochondrial respiration, and content in the muscles of CKD + LPD rats. Furthermore, KA supplementation reversed the elevation in p66Shc and FoxO3a expression in the muscles of CKD + LPD rats. CONCLUSIONS: Our results showed that KA supplementation to be beneficial to muscle atrophy in CKD + LPD, which might be associated with improvement of oxidative damage and mitochondrial dysfunction through suppression of p66Shc and FoxO3a.
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Suplementos Dietéticos , Cetoácidos/uso terapéutico , Mitocondrias Musculares/efectos de los fármacos , Atrofia Muscular/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Renal Crónica/complicaciones , Animales , Evaluación Preclínica de Medicamentos/métodos , Cetoácidos/farmacología , Riñón/fisiopatología , Masculino , Mitocondrias Musculares/fisiología , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatología , Nefrectomía , Complejo de la Endopetidasa Proteasomal/biosíntesis , Complejo de la Endopetidasa Proteasomal/genética , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Ubiquitina/biosíntesis , Ubiquitina/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
KEY MESSAGE: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2-haplotype and S 3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.
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Sistemas CRISPR-Cas , Proteínas F-Box/metabolismo , Petunia/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ribonucleasas/genética , Autoincompatibilidad en las Plantas con Flores/genética , Alelos , Proteínas F-Box/genética , Flores/enzimología , Flores/genética , Flores/fisiología , Técnicas de Inactivación de Genes , Petunia/enzimología , Petunia/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Polen/enzimología , Polen/genética , Polen/fisiología , Polinización , Complejo de la Endopetidasa Proteasomal/genética , Ribonucleasas/metabolismoRESUMEN
BACKGROUND: Although it has been reported to contain high polyphenols, the pharmacological studies of the calyx of Diospyros kaki Thunb (DKC) have not been elucidated in detail. In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells. METHODS: Anti-cell proliferative effect of 70% ethanol extracts from the calyx of Diospyros kaki (DKC-E70) was evaluated by MTT assay. The effect of DKC-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively. RESULTS: DKC-E70 suppressed the proliferation of human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29. Although DKC-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by DKC-E70 occurred at the earlier time than that of cyclin D1 mRNA, which indicates that DKC-E70-mediated downregulation of cyclin D1 protein may be a consequence of the induction of degradation and transcriptional inhibition of cyclin D1. In cyclin D1 degradation, we found that cyclin D1 downregulation by DKC-E70 was attenuated in presence of MG132. In addition, DKC-E70 phosphorylated threonine-286 (T286) of cyclin D1 and T286A abolished cyclin D1 downregulation by DKC-E70. We also observed that DKC-E70-mediated T286 phosphorylation and subsequent cyclin D1 degradation was blocked in presence of the inhibitors of ERK1/2, p38 or GSK3ß. In cyclin D1 transcriptional inhibition, DKC-E70 inhibited the expression of ß-catenin and TCF4, and ß-catenin/TCF-dependent luciferase activity. CONCLUSIONS: Our results suggest that DKC-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3ß, and cyclin D1 transcriptional inhibition through Wnt signaling. From these findings, DKC-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.
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Antineoplásicos/administración & dosificación , Neoplasias Colorrectales/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Diospyros/química , Extractos Vegetales/administración & dosificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/fisiopatología , Células HCT116 , Células HT29 , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Proteolisis , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The rates of protein turnover are higher during the neonatal period than at any other time in postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system are key pathways regulating cellular protein turnover. The objectives of this study were (1) to elucidate the effect of feeding colostrum versus milk-based formula on the mRNA abundance of key components of the mTOR pathway and of the ubiquitin-proteasome system in skeletal muscle of neonatal calves and (2) to compare different muscles. German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) up to 4 d of life. The nutrient content in formula and colostrum was similar, but formula had lower concentrations of free branched-chain AA (BCAA) and free total AA, insulin, and insulin-like growth factor (IGF)-I than colostrum. Blood samples were taken from d 1 to 4 before morning feeding and before and 2 h after the last feeding on d 4. Muscle samples from M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM) were collected after slaughter on d 4 at 2 h after feeding. The preprandial concentrations of free total AA and BCAA, insulin, and IGF-I in plasma changed over time but did not differ between groups. Plasma free total AA and BCAA concentrations decreased in COL, whereas they increased in FOR after feeding, resulting in higher postprandial plasma total AA and BCAA concentrations in FOR than in COL. Plasma insulin concentrations increased after feeding in both groups but were higher in COL than in FOR. Plasma IGF-I concentrations decreased in COL, whereas they remained unchanged in FOR after feeding. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in 3 different skeletal muscles was greater in COL than in FOR, whereas that of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) was unaffected by diet. The mRNA abundance of ubiquitin activating enzyme (UBA1) and ubiquitin conjugating enzyme 1 (UBE2G1) enzymes was not affected by diet, whereas that of ubiquitin conjugating enzyme 2 (UBE2G2) was greater (MLD) or tended to be greater (MM) in COL than in FOR. The mRNA abundance of atrogin-1 in MLD and MST was lower in COL than in FOR, whereas that of muscle ring finger protein-1 (MuRF1) was greater (MST) or tended to be greater (MLD). The abundance of MuRF1 mRNA was highest in MST, followed by MLD, and was lowest in MM. The results indicate that colostrum feeding may stimulate protein turnover that may result in a high rate of protein deposition in a muscle type-specific manner. Such effects seem to be mediated by the postprandial increase in plasma insulin.
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Alimentación Animal/análisis , Calostro , Alimentos Formulados , Complejo de la Endopetidasa Proteasomal/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Animales Recién Nacidos , Bovinos , Dieta/veterinaria , Femenino , Insulina/sangre , Músculo Esquelético/metabolismo , Embarazo , Complejo de la Endopetidasa Proteasomal/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
Traditionally, the main mammalian models used in aging research have been mice and rats, i.e. short-lived species that obviously lack effective maintenance mechanisms to keep their soma in a functional state for prolonged periods of time. It is doubtful that life-extending mechanisms identified only in such short-lived species adequately reflect the diversity of longevity pathways that have naturally evolved in mammals, or that they have much relevance for long-lived species such as humans. Therefore, some complementary, long-lived mammalian models have been introduced to aging research in the past 15-20 years, particularly naked mole-rats (and to a lesser extent also other mole-rats) and bats. Here, I summarize and compare the most important results regarding various aspects of aging - oxidative stress, molecular homeostasis and repair, and endocrinology - that have been obtained from studies using these new mammalian models of high longevity. I argue that the inclusion of these models was an important step forward, because it drew researchers' attention to certain oversimplifications of existing aging theories and to several features that appear to be universal components of enhanced longevity in mammals. However, even among mammals with high longevity, considerable variation exists with respect to other candidate mechanisms that also must be taken into account if inadequate generalizations are to be avoided.
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Quirópteros/genética , Reparación del ADN , Homeostasis/genética , Longevidad/genética , Ratas Topo/genética , Animales , Antioxidantes/metabolismo , Autofagia/genética , Quirópteros/crecimiento & desarrollo , Quirópteros/metabolismo , Regulación de la Expresión Génica , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratas Topo/crecimiento & desarrollo , Ratas Topo/metabolismo , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especificidad de la Especie , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Vitamina D/metabolismoRESUMEN
BACKGROUND: Carnosic acid (CA) is a diterpenoid found in Rosmarinus officinalis L. and Salvia officinalis L. as well as in many other Lamiaceae. This compound is reported to have antioxidant and antimicrobial properties. In addition, a number of reports showed that CA has a cytotoxic activity toward several cancer cell lines. PURPOSE: The aim of this study was to establish whether CA has any specific antiproliferative effect toward human glioblastoma (GBM) cells and to analyze the molecular mechanisms involved. METHODS: We evaluated cell survival by MTT assay, apoptosis and DNA content by flow cytometry, protein expression and phosphorylation by immunoblot analyses. RESULTS: Our results showed that CA inhibited cell survival on both normal astrocytes and GBM cells. In GBM cells, in particular, CA caused an early G2 block, a reduction in the percentage of cells expressing Ki67, an enhanced expression of p21(WAF) and induced apoptosis. Furthermore, we showed that CA promoted proteasomal degradation of several substrate proteins, including Cyclin B1, retinoblastoma (RB), SOX2, and glial fibrillary acid protein (GFAP), whereas MYC levels were not modified. In addition, CA dramatically reduced the activity of CDKs. CONCLUSION: In conclusion, our findings strongly suggest that CA promotes a profound deregulation of cell cycle control and reduces the survival of GBM cells via proteasome-mediated degradation of Cyclin B1, RB and SOX2.
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Abietanos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Ciclina B1/efectos de los fármacos , Glioblastoma/patología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Proteína de Retinoblastoma/efectos de los fármacos , Factores de Transcripción SOXB1/efectos de los fármacos , Astrocitos/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Ciclina B1/genética , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Relación Dosis-Respuesta a Droga , Fase G2/efectos de los fármacos , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Proteína de Retinoblastoma/genética , Factores de Transcripción SOXB1/genéticaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Existing evidences suggest that Radix Astragali and its polysaccharides composition (APS) can improve muscle mass, but the mechanisms need more research. AIM OF THE STUDY: In this study, we aimed to examine the effects of APS on muscle wasting at molecular level in 5/6 nephrectomised rats. MATERIALS AND METHODS: We performed 5/6 nephrectomy or sham operation in 160 6-week-old Sprague-Dawley rats, and feed animals with or without 2% APS for 155 days. After treatment, we compared the change of weight, muscle fibre, protein metabolism, pro-inflammatory factors (TNF-α, IL-15, CRP) and oxidative factors (MDA, SOD) among each group. In addition, we detected the Akt/mTOR, ubiquitin proteasome, autophagy signalling and AA transporters in vivo and in vitro. RESULTS: Data in vivo show 2% APS could alleviate weight loss and improve protein metabolism in nephrectomised rats. The levels of serum pro-inflammatory factors and oxidative factors were restored by APS treatment. In molecular levels, APS restored Akt/mTOR, MAFbx, MuRF1, Atg7, LC3B-II/LC3B-I and SLC38A2 which changed in nephrectomised rats. Data in vitro show the optimal dose of APS is 0.2mg/mL, and SLC38A2 siRNA attenuated the effects of 0.2mg/mL APS on atrophy and autophagy. CONCLUSIONS: Our results suggested APS could improve muscle wasting through Akt/mTOR, ubiquitin proteasome and autophagy signalling, and SLC38A2 may be one of potential targets.
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Planta del Astrágalo/química , Nefrectomía , Polisacáridos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina/metabolismo , Animales , Autofagia/fisiología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Masculino , Músculo Esquelético/patología , Mioblastos/efectos de los fármacos , Polisacáridos/química , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/genética , Ubiquitina/genéticaRESUMEN
Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy.