RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Liujunzi formula has been used to treat liver cancer in China for many years, but its underlying mechanism remains unclear. We previously found that decreased expression of miR-122-3p was associated with liver cancer. In this study, we aimed to explore the target of miR-122-3p and the effect of the Liujunzi formula on miR-122-3p and its downstream events in liver cancer. MATERIAL AND METHODS: Bioinformatics pinpointed potential targets of miR-122-3p. The actual target was confirmed by miRNA mimic/inhibitor transfections and a dual-luciferase reporter assay. RNA-seq looked at downstream genes impacted by this target. Flow cytometry checked for changes in T cell apoptosis levels after exposing them to liver cancer cells. Gene expression was measured by RT-qPCR, western blotting, and immunofluorescence staining. RESULTS: Cell experiments found the Liujunzi extract (LJZ) upregulated miR-122-3p and in a dose-dependent manner. Bioinformatics analysis found UBE2I was a potential target of miR-122-3p, which was validated through experiments using miRNA mimics/inhibitors and a dual-luciferase reporter assay. RNA-seq data implicated the NF-κB pathway as being downstream of the miR-122-3p/UBE2I axis, further confirmed by forcing overexpression of UBE2I. Bioinformatic evidence suggested a link between UBE2I and T cell infiltration in liver cancer. Given that the NF-κB pathway drives PD-L1 expression, which can inhibit T cell infiltration, we investigated whether PD-L1 is a downstream effector of miR-122-3p/UBE2I. This was corroborated through mining public databases, UBE2I overexpression studies, and tumor-T cell co-culture assays. In addition, we also confirmed that LJZ downregulates UBE2I and NF-κB/PD-L1 pathways through miR-122-3p. LJZ also suppressed SUMOylation in liver cancer cells and protected PD-1+ T cells from apoptosis induced by co-culture with tumor cells. Strikingly, a miR-122-3p inhibitor abrogated LJZ's effects on UBE2I and PD-L1, and UBE2I overexpression rescued the LJZ-mediated effects on NF-κB and PD-L1. CONCLUSIONS: miR-122-3p targets UBE2I, thereby suppressing the NF-κB signaling cascade and downregulating PD-L1 expression, which potentiates anti-tumor immune responses. LJZ bolsters anti-tumor immunity by modulating the miR-122-3p/UBE2I/NF-κB/PD-L1 axis in liver cancer cells.
Assuntos
Medicamentos de Ervas Chinesas , Neoplasias Hepáticas , MicroRNAs , Enzimas de Conjugação de Ubiquitina , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Humanos , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Apoptose/efeitos dos fármacos , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Hep G2 , Tolerância Imunológica/efeitos dos fármacosRESUMO
Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.
Assuntos
Solanum tuberosum , Enzimas de Conjugação de Ubiquitina , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Filogenia , Sequência de Aminoácidos , Saccharomyces cerevisiae/metabolismoRESUMO
A collection of 9050 natural products, their derivatives, and mimetics, was virtually screened against the human Atg3-Atg8 (Atg - autophagy) binding scaffold. By blocking this interaction, the lipidation of Atg8 does not occur and the formation of autophagosomes is inhibited. Forty-three (43) potential ligands were tested using enhanced Green Fluorescent Protein (eGFP) tagged LC3, the human ortholog of Atg8, in MCF7 breast cancer cells. Three hits showed single digit µM IC50 values with AT110, an isoflavone derivative, being the best at 1.2 ± 0.6 µM. Molecular modelling against Atg8 in conjunction with structural activity relationship (SAR) strongly supports the binding to this target. Testing in a panel of cancer cell lines showed little cytotoxic effect as compared to chloroquine. However, same concentration of AT110 was shown to be toxic to young zebrafish embryos. This can be explained in terms of the autophagy process being very active in the zebrafish embryos rendering them susceptible to AT110 whereas in the cancer cells tested the autophagy is not usually active. Nevertheless, AT110 blocks autophagy flux in the zebrafish confirming that the ligand is modulating autophagy. A small molecule non-cytotoxic autophagy inhibitor would open the door for adjunct therapies to bolster many established anticancer drugs, reducing their efficacious concentration thus limiting undesirable site effects. In addition, since many cancer types rely on the autophagy mechanism to survive a therapeutic regime, recurrence can potentially be reduced. The discovery of AT110 is an important step in establishing such an adjunct therapy.
Assuntos
Antineoplásicos/farmacologia , Família da Proteína 8 Relacionada à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Isoflavonas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Animais , Antineoplásicos/química , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Desenvolvimento Embrionário/efeitos dos fármacos , Humanos , Isoflavonas/química , Estrutura Molecular , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/metabolismo , Peixe-Zebra/embriologiaRESUMO
A series of novel indole derivatives were synthesized and evaluated for their antiproliferative activity against three selected cancer cell lines (MGC803, EC-109 and PC-3). Among these analogues, 2-(5-methoxy-1H-indol-1-yl)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (V7) showed the best inhibitory activity against MGC803 cells with an IC50 value of 1.59 µM. Cellular mechanisms elucidated that V7 inhibited colony formation, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, indole analogue V7 inhibited NEDDylation pathway and MAPK pathway against MGC803 cells.
Assuntos
Antineoplásicos/farmacologia , Indóis/química , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Indóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Exposure to environmental toxicants such as Bisphenol A (BPA) has raised serious health issues globally particularly in developing countries. It is ubiquitously used in the manufacturing of canned food and feeding bottles. BPA generated reactive oxygen species can lead to several diseases including cardiotoxicity. However, the endpoints stimulated in BPA cardiotoxicity yet need to be investigated. The current study was aimed to investigate the underlying molecular pathways which may contribute in revealing the protective effects of Pistacia integerrima against BPA induced oxidative stress. The dose of 100 µg/kg BW of BPA, 200 mg/kg BW P. integerrima, and 4 mg/kg BW melatonin was administered to Sprague Dawley rats. Present results of western blotting and qRT-PCR showed the increased expression of p53, PUMA and Drp1, while downregulation of Ubc13 in heart tissues of BPA treated group whereas the levels were reversed upon treatment with P. integerrima. The role of BPA in heart tissue apoptosis was further confirmed by the increased level of P-p53, cytochrome C and disrupted cellular architecture whereas the P. integerrima has shown its ameliorative potential by mitigating the adverse effects of BPA. Moreover, the oxidant, antioxidant, lipid, and liver markers profile has also revealed the therapeutic potential of P. integerrima by maintaining the levels in the normal range. However, melatonin has also manifested the normalized expression of apoptotic markers, biochemical markers, and tissue architecture. Conclusively, the data suggest that P. integerrima may be a potential candidate for the treatment of BPA induced toxicity by neutralizing the oxidative stress through Ubc13/p53 pathway.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Benzidrílicos/toxicidade , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/toxicidade , Pistacia/química , Extratos Vegetais/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Compostos Benzidrílicos/administração & dosagem , Glicemia/efeitos dos fármacos , Citocromos c/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Feminino , Hipodermóclise , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Melatonina/administração & dosagem , Melatonina/farmacologia , Fenóis/administração & dosagem , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Tumores de Planta , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Enzimas de Conjugação de Ubiquitina/genética , Regulação para CimaRESUMO
The ubiquitin/proteasome system is a primary conduit for selective intracellular protein degradation. Since its discovery over 30 years ago, this highly regulated system continues to be an active research area for drug discovery that is exemplified by several approved drugs. Here we review compounds in preclinical testing, clinical trials, and approved drugs, with the aim of highlighting innovative discoveries and breakthrough therapies that target the ubiquitin system.
Assuntos
Inibidores Enzimáticos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Huntingtin interaction protein 2 (HIP2) is an E2 ubiquitin-conjugating enzyme associated with neurodegenerative diseases, and HIP2 mRNA has been implicated as a potential blood biomarker for Parkinson's disease (PD). However, it is unclear whether the alteration of HIP2 expression may contribute to the development of PD, and whether the change of HIP2 in blood could reflect its expression in the brain or motor functions in PD patients. In this study, we established a mouse line with HIP2 haploinsufficiency. The reduction of the HIP2 expression led to spontaneous motor function impairment and dopaminergic neuronal loss. Furthermore, HIP2 haploinsufficiency increased the susceptibility of mice to 6-hydroxydopamine (6-OHDA) and caused severe loss of dopaminergic neurons. Interestingly, in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model for PD, we observed concurrent, highly correlated decrease of HIP2 expression in the brain and in the blood. Using blood samples from more than 300 patients, we validated the decreased HIP2 mRNA in PD patients, including de novo patients. Finally, in a 1-year, 20-patient study, we observed reversed blood HIP2 mRNA levels accompanying improved motor and overall daily functions in 75% of the PD patients with instructed Tai Chi training. Therefore, our in vivo studies have indicated HIP2 insufficiency as a contributing factor for PD, and functionally validated blood HIP2 as a useful and reversible biomarker for PD.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Atividade Motora/fisiologia , Doença de Parkinson/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Oxidopamina/farmacologiaRESUMO
Nine crossbred finishing barrows (body weight 94.4 ± 6.7 kg) randomly assigned to three dietary treatments were used to investigate the effects of dietary lysine on muscle growth related metabolic and signaling pathways. Muscle samples were collected from the longissimus dorsi of individual pigs after feeding the lysine-deficient (4.30 g/kg), lysine-adequate (7.10 g/kg), or lysine-excess (9.80 g/kg) diet for five weeks, and the total RNA was extracted afterwards. Affymetrix Porcine Gene 1.0 ST Array was used to quantify the expression levels of 19,211 genes. Statistical ANOVA analysis of the microarray data showed that 674 transcripts were differentially expressed (at p ≤ 0.05 level); 60 out of 131 transcripts (at p ≤ 0.01 level) were annotated in the NetAffx database. Ingenuity pathway analysis showed that dietary lysine deficiency may lead to: (1) increased muscle protein degradation via the ubiquitination pathway as indicated by the up-regulated DNAJA1, HSP90AB1 and UBE2B mRNA; (2) reduced muscle protein synthesis via the up-regulated RND3 and ZIC1 mRNA; (3) increased serine and glycine synthesis via the up-regulated PHGDH and PSPH mRNA; and (4) increased lipid accumulation via the up-regulated ME1, SCD, and CIDEC mRNA. Dietary lysine excess may lead to: (1) decreased muscle protein degradation via the down-regulated DNAJA1, HSP90AA1, HSPH1, and UBE2D3 mRNA; and (2) reduced lipid biosynthesis via the down-regulated CFD and ME1 mRNA. Collectively, dietary lysine may function as a signaling molecule to regulate protein turnover and lipid metabolism in the skeletal muscle of finishing pigs.
Assuntos
Perfilação da Expressão Gênica/métodos , Lisina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Animais , Análise por Conglomerados , Bases de Dados Genéticas , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Músculo Esquelético/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
We previously reported that the dietary flavonoids, luteolin and quercetin, might inhibit the invasiveness of cervical cancer by reversing epithelial-mesenchymal transition (EMT) signaling. However, the regulatory mechanism exerted by luteolin and quercetin is still unclear. This study analyzed the invasiveness activation by ubiquitin E2S ligase (UBE2S) through EMT signaling and inhibition by luteolin and quercetin. We found that UBE2S expression was significantly higher in highly invasive A431 subgroup III (A431-III) than A431-parental (A431-P) cells. UBE2S small interfering (si)RNA knockdown and overexpression experiments showed that UBE2S increased the migratory and invasive abilities of cancer cells through EMT signaling. Luteolin and quercetin significantly inhibited UBE2S expression. UBE2S showed a negative correlation with von Hippel-Lindau (VHL) and a positive correlation with hypoxia-induced factor (Hif)-1α. Our findings suggest that high UBE2S in malignant cancers contributes to cell motility through EMT signaling and is reversed by luteolin and quercetin. UBE2S might contribute to Hif-1α signaling in cervical cancer. These results show the metastatic inhibition of cervical cancer by luteolin and quercetin through reducing UBE2S expression, and provide a functional role for UBE2S in the motility of cervical cancer. UBE2S could be a potential therapeutic target in cervical cancer.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Luteolina/farmacologia , Quercetina/farmacologia , Neoplasias do Colo do Útero/fisiopatologia , Linhagem Celular Tumoral , Suplementos Nutricionais/análise , Feminino , Humanos , Invasividade Neoplásica , Transdução de Sinais/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
Chronic cadmium (Cd) exposure can induce renal toxicity. In Cd renal toxicity, p53 is thought to be involved. Our previous studies showed that Cd down-regulated gene expression of the UBE2D (ubiquitin-conjugating enzyme E2D) family members. Here, we aimed to define the association between UBE2D family members and p53-dependent apoptosis in human proximal tubular cells (HK-2 cells) treated with Cd. Cd increased intracellular p53 protein levels and decreased UBE2D2 and UBE2D4 gene expression via inhibition of YY1 and FOXF1 transcription factor activities. Double knockdown of UBE2D2 and UBE2D4 caused an increase in p53 protein levels, and knockdown of p53 attenuated not only Cd-induced apoptosis, but also Cd-induced apoptosis-related gene expression (BAX and PUMA). Additionally, the mice exposed to Cd for 6 months resulted in increased levels of p53 and induction of apoptosis in proximal tubular cells. These findings suggest that down-regulation of UBE2D family genes followed by accumulation of p53 in proximal tubular cells is an important mechanism for Cd-induced renal toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Regulação para Baixo/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Feminino , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Previous studies have demonstrated that dysregulation of micro (mi)RNAs is associated with the etiology of various neuropsychiatric disorders, including depression and schizophrenia. Cerebralcare Granule® (CG) is a Chinese herbal medicine, which has been reported to have an ameliorative effect on brain injury by attenuating bloodbrain barrier disruption and improving hippocampal neural function. The present study aimed to evaluate the cognitive behavior of rats continuously overexpressing miRNA30e (lentimiRNA30e), prior to and following the administration of CG. In addition, the mechanisms underlying the ameliorative effects of CG were investigated. The cognitive ability of the rats was assessed using an openfield test and a Morris water maze spatial reference/working memory test. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to detect neuronal apoptosis in the dentate gyrus of the hippocampus. Immunohistochemical analysis and western blotting were conducted to detect the expression levels of Bcell lymphoma 2 (BCL2) and ubiquitinconjugating enzyme 9 (UBC9), in order to examine neuronal apoptosis. The lentimiRNA30e rats exhibited increased signs of anxiety, depression, hyperactivity and schizophrenia, which resulted in a severe impairment in cognitive ability. Furthermore, in the dentate gyrus of these rats, the expression levels of BCL2 and UBC9 were reduced and apoptosis was increased. The administration of CG alleviated cognitive impairment, enhanced the expression levels of BCL2 and UBC9, and reduced apoptosis in the dentate gyrus in the lentimiRNA30e rats. No significant differences were detected in behavioral indicators between the lentimiRNA30e rats treated with CG and the normal controls. These findings suggested that CG exerts a potent therapeutic effect, conferred by its ability to enhance the expression levels of BCL2 and UBC9, which inhibits the apoptotic process in neuronal cells. Therefore, CG may be considered a potential therapeutic strategy for the treatment of cognitive impairment in mental disorders.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , MicroRNAs/metabolismo , Animais , Apoptose , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Giro Denteado/metabolismo , Giro Denteado/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-κB inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-κB inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Produtos Biológicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Inflamação/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Endotoxemia/tratamento farmacológico , Endotoxemia/etiologia , Endotoxemia/metabolismo , Inflamação/tratamento farmacológico , Inflamação/etiologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/imunologia , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B , Óxido Nítrico/metabolismo , Ligação Proteica , Proteólise , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer.
Assuntos
Autofagia/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Humanos , Proteínas de Membrana/metabolismo , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.
Assuntos
Estresse Oxidativo/fisiologia , Ubiquitinação/fisiologia , Endopeptidases/metabolismo , Lisina/química , Lisina/metabolismo , Poliubiquitina/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Massive changes in gene expression occur when plants are subjected to phosphorus (P) limitation, but the breadth of metabolic changes in these conditions and their regulation is barely investigated. Nearly 350 primary and secondary metabolites were profiled in shoots and roots of P-replete and P-deprived Arabidopsis thaliana wild type and mutants of the central P-signalling components PHR1 and PHO2, and microRNA399 overexpresser. In the wild type, the levels of 87 primary metabolites, including phosphorylated metabolites but not 3-phosphoglycerate, decreased, whereas the concentrations of most organic acids, amino acids, nitrogenous compounds, polyhydroxy acids and sugars increased. Furthermore, the levels of 35 secondary metabolites, including glucosinolates, benzoides, phenylpropanoids and flavonoids, were altered during P limitation. Observed changes indicated P-saving strategies, increased photorespiration and crosstalk between P limitation and sulphur and nitrogen metabolism. The phr1 mutation had a remarkably pronounced effect on the metabolic P-limitation response, providing evidence that PHR1 is a key factor for metabolic reprogramming during P limitation. The effects of pho2 or microRNA399 overexpression were comparatively minor. In addition, positive correlations between metabolites and gene transcripts encoding pathway enzymes were revealed. This study provides an unprecedented metabolic phenotype during P limitation in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Fósforo/metabolismo , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Expressão Gênica , Ácidos Glicéricos/metabolismo , Redes e Vias Metabólicas , Metaboloma , MicroRNAs/genética , Mutação , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , RNA de Plantas/genética , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
UbcH5 is the key ubiquitin-conjugating enzyme catalyzing ubiquitination during TNF-α-triggered NF-κB activation. Here, we identified an herb-derived sesquiterpene lactone compound IJ-5 as a preferential inhibitor of UbcH5 and explored its therapeutic value in inflammatory and autoimmune disease models. IJ-5 suppresses TNF-α-induced NF-κB activation and inflammatory gene transcription by inhibiting the ubiquitination of receptor-interacting protein 1 and NF-κB essential modifier, which is essential to IκB kinase activation. Mechanistic investigations revealed that IJ-5 preferentially binds to and inactivates UbcH5 by forming a covalent adduct with its active site cysteine and thereby preventing ubiquitin conjugation to UbcH5. In preclinical models, pretreatment of IJ-5 exhibited potent anti-inflammatory activity against TNF-α- and D-galactosamine-induced hepatitis and collagen-induced arthritis. These findings highlight the potential of UbcH5 as a therapeutic target for anti-TNF-α interventions and provide an interesting lead compound for the development of new anti-inflammation agents.
Assuntos
Lactonas/farmacologia , Plantas Medicinais/química , Sesquiterpenos/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Hepatite/tratamento farmacológico , Hepatite/patologia , Humanos , Quinase I-kappa B/metabolismo , Lactonas/química , Lactonas/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Plantas Medicinais/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Psoriasis is a chronic inflammatory skin disorder mediated by cross-talk occurring between epidermal keratinocytes, dermal vascular cells and immunocytes. Literature reveals that Signal transducer and activator of transcription 3 (STAT3), a protein involved in transmitting extracellular signals to the nucleus, is a possible important link between keratinocytes and immunocytes and is crucial to the development of psoriasis. Although photochemotherapy using UV in combination with 8 methoxypsoralen is one of the most effective therapy for moderate to severe plaque psoriasis, its mechanism of action is largely unknown. Herein, we studied the change in miRNA profiles of cultured human keratinocytes (HaCaT cells) before and after in vitro PUVA treatment by 8 methoxypsoralen and found significant up regulation of hsa-miR-4516. We for the first time demonstrate that ectopic expression of hsa-miR-4516 directly targets STAT3 protein by binding to its 3'UTR in HaCaT cells as confirmed by Luciferase reporter assays and Western blot analysis. We further show that overexpression of hsa-miR-4516 downregulates STAT3, p-STAT3, CDK6, and UBE2N proteins that are consistently upregulated in psoriasis and induces apoptosis in HaCaT cells. We also observed that anti-miR-4516 treatment was able to partially inhibit PUVA-induced apoptosis, suggesting that miR-4516 is involved in PUVA-induced apoptosis. Taken together, these results not only indicate the mechanistic involvement of hsa-miR-4516 in PUVA mediated effects by down-regulating STAT3 in HaCaT keratinocytes, but also highlight the potential of hsa-miR-4516 in development of novel therapeutic strategies. J. Cell. Physiol. 229: 1630-1638, 2014. © 2014 Wiley Periodicals, Inc.
Assuntos
Apoptose/genética , Quinase 6 Dependente de Ciclina/genética , Regulação para Baixo/genética , Queratinócitos/metabolismo , MicroRNAs/metabolismo , Terapia PUVA , Fator de Transcrição STAT3/genética , Enzimas de Conjugação de Ubiquitina/genética , Regiões 3' não Traduzidas/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Quinase 6 Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Perfilação da Expressão Gênica , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Metoxaleno/farmacologia , Metoxaleno/uso terapêutico , MicroRNAs/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/efeitos da radiação , Psoríase/tratamento farmacológico , Psoríase/genética , Psoríase/patologia , Reprodutibilidade dos Testes , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Enzimas de Conjugação de Ubiquitina/metabolismo , Raios UltravioletaRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Fibroblast-like synoviocytes (FLS) in the synovial tissues play critical roles in joint destruction. Recent studies implicate the sumoylation in the regulation of the inflammation and arthritis. Thus, we explored whether SUMO-conjugating enzyme UBC9 is involved in the progression of RA using a mouse collagen-induced arthritis (CIA) model. The effects of UBC9 siRNA on cell invasion and migration in human RA-FLS were also assessed in vitro. Treatment with siRNA against UBC9 for 3 weeks reduced the arthritis score and joint destruction. The expression of SUMO-1 and UBC9 protein in CIA joints was inhibited by UBC9 knockdown. Serum levels of anti-collagen (CII) antibodies, vascular endothelial growth factor A (VEGF-A), matrix metalloproteinases (MMP)-3, and MMP-9 were also decreased in CIA mice. In vitro, UBC9 silencing inhibited the secretion of VEGF-A, MMP-3, and MMP-9 from TNF-α-stimulated human RA-FLS. TNF-α-induced RA-FLS proliferation and migration were significantly attenuated by UBC9 knockdown. These findings indicate that SUMO-conjugating enzyme UBC9 promotes proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis. Inhibition of UBC9 activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating FLS proliferation, migration, and invasion.
Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/citologia , Membrana Sinovial/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Artrite/metabolismo , Artrite Reumatoide/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Inativação Gênica , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , RNA Interferente Pequeno/metabolismoRESUMO
Two new ß-carboline alkaloids, variabines A (1) and B (2), were isolated from the Indonesian marine sponge Luffariella variabilis. Their structures were elucidated from spectral data, and 1 was found to be a sulfonated derivative of 2. Although numerous ß-carboline alkaloids have been isolated from natural sources to date, 1 is the first ß-carboline derivative containing a sulfate group. Compound 2 inhibited chymotrypsin-like activity of the proteasome and Ubc13 (E2)-Uev1A interaction with IC50 values of 4 and 5 µg/mL, respectively, whereas 1 had little effect on the activity or interaction.
Assuntos
Alcaloides/isolamento & purificação , Carbolinas/isolamento & purificação , Poríferos/química , Inibidores de Proteassoma/farmacologia , Alcaloides/farmacologia , Animais , Carbolinas/farmacologia , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Estrutura Molecular , Inibidores de Proteassoma/isolamento & purificação , Ratos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.