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Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the cell migration and invasion assay data shown in Fig. 5C were strikingly similar to data appearing in different form in other articles by different authors at different research institutes, some of which have been retracted. Owing to the fact that the contentious data in the above article were already under consideration for publication, or had already been published, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 17: 4803­4810, 2018; DOI: 10.3892/mmr.2018.8417].

RESUMEN

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

RESUMEN

Chemotherapy-induced side effects affect the quality of life and efficacy of treatment of cancer patients. Current approaches for treating the side effects of chemotherapy are poorly effective and may cause numerous harmful side effects. Therefore, developing new and effective drugs derived from natural non-toxic compounds for the treatment of chemotherapy-induced side effects is necessary. Experiments in vivo and in vitro indicate that Panax ginseng (PG) and its ginsenosides are undoubtedly non-toxic and effective options for the treatment of chemotherapy-induced side effects, such as nephrotoxicity, hepatotoxicity, cardiotoxicity, immunotoxicity, and hematopoietic inhibition. The mechanism focus on anti-oxidation, anti-inflammation, and anti-apoptosis, as well as the modulation of signaling pathways, such as nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1), P62/keap1/Nrf2, c-jun N-terminal kinase (JNK)/P53/caspase 3, mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERK), AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase 4 (MKK4)/JNK, and phosphatidylinositol 3-kinase (PI3K)/AKT. Since a systemic review of the effect and mechanism of PG and its ginsenosides on chemotherapy-induced side effects has not yet been published, we provide a comprehensive summarization with this aim and shed light on the future research of PG.

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The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4.

Asunto(s)

Colorantes Fluorescentes/química , Hepatopatías/tratamiento farmacológico , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Vemurafenib/síntesis química , Carbolinas/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/química , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Vemurafenib/análogos & derivados , Vemurafenib/farmacología

RESUMEN

Mitogen-activated protein kinase kinase 4 (MAP2K4) is a tumor suppressor in many cancers. However, its roles and action mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Here, we analyzed MAP2K4 and its downstream kinases (c-Jun N-terminal kinase (JNK) and p38) using immunohistochemical staining and their prognostic significances using univariate and multivariate Cox proportional hazards regression analysis in our PDAC cohort. Then, we validated MAP2K4/JNK/p38 mRNA levels and prognostic significances using The Cancer Genome Atlas (TCGA) database. Finally, we evaluated the effects of MAP2K4 on the proliferation and invasion of PDAC cells. MAP2K4, JNK, and p38 proteins were expressed in 97.3 % (72/74), 95.6 % (65/68), and 88.6 % (62/70) of the samples, respectively, and their levels in tumor tissues were significantly higher than those in normal ducts. MAP2K4 protein expression was lower in male patients (p = 0.028). In our PDAC cohort, advanced TNM stage, low MAP2K4, and high JNK protein levels were significant prognostic factors for poor overall survival (OS) based on a univariate survival analysis (p = 0.006, p < 0.001, and p = 0.004, respectively). N stage and MAP2K4 and JNK protein levels were independent prognostic factors for OS based on multivariate analysis. We then built a prognosis prediction nomogram combining the standard TNM staging system with MAP2K4 and JNK expression that had a Harrell's C-index of 0.645. The new prognosis prediction model effectively stratified the resected patients with PDAC, from both our cohort and TCGA database, into low- and high-risk groups. Finally, MAP2K4 overexpression inhibited pancreatic cancer cell proliferation and migration in vitro. This study shows that reduced protein and mRNA levels of MAP2K4 found in PDAC patients, coupled to in vitro effects observed, support the tumor suppressor role of MAP2K4 in PDAC. Importantly, combining MAP2K4 and JNK expression with the TNM staging system results in a better prediction of postoperative survival of patients with PDAC.

Asunto(s)

Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/enzimología , Técnicas de Apoyo para la Decisión , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , MAP Quinasa Quinasa 4/análisis , Nomogramas , Neoplasias Pancreáticas/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/cirugía , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , MAP Quinasa Quinasa 4/genética , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Resultado del Tratamiento , Proteínas Quinasas p38 Activadas por Mitógenos/análisis

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Cardiovascular diseases prevail among modern societies and underdeveloped countries, and a high mortality rate has also been reported by the World Health Organization affecting millions of people worldwide. Hyperactive platelets are the major culprits in thrombotic disorders. A group of drugs is available to deal with such platelet-related disorders; however, sometimes, side effects and complications caused by these drugs outweigh their benefits. Ginseng and its nutraceuticals have been reported to reduce the impact of thrombotic conditions and improve cardiovascular health by antiplatelet mechanisms. This review provides (1) a comprehensive insight into the available pharmacological options from ginseng and ginsenosides (saponin and nonsaponin fractions) for platelet-originated cardiovascular disorders; (2) a discussion on the impact of specific functional groups on the modulation of platelet functions and how structural modifications among ginsenosides affect platelet activation, which may further provide a basis for drug design, optimization, and the development of ginsenoside scaffolds as pharmacological antiplatelet agents; (3) an insight into the synergistic effects of ginsenosides on platelet functions; and (4) a perspective on future research and the development of ginseng and ginsenosides as super nutraceuticals.

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The diagnosis and treatment of cervical squamous cell carcinoma is challenged by difficulties in the determination of tumor invasion. GATA binding protein 6 antisense (GATA6-AS) is a recently identified long non-coding RNA that exhibits critical functions in the growth of endothelial cells; however, to the best of our knowledge, its involvement in other physiological and pathological processes is unknown. By reverse transcription-quantitative polymerase chain reaction, the present study examined the expression of GATA6-AS in tumor tissues and adjacent healthy tissues, and the serum from patients with cervical squamous cell carcinoma and healthy controls. The diagnostic and prognostic potentials of GATA6-AS for cervical squamous cell carcinoma were analyzed by receiver operating characteristic curve analysis and survival curve analysis, respectively. Associations between the serum levels of GATA6-AS and clinical characteristics of patients with cervical squamous cell carcinoma were analyzed by a χ2 test. A GATA6-AS overexpression vector was transfected into cervical squamous cell carcinoma cells, and the effects on cell migration and invasion were investigated by Transwell migration and invasion assays, respectively. The expression of mitogen-activated protein kinase kinase 4 (MTK-1) following transfection with the GATA6-AS overexpression vector was detected by western blot analysis. It was identified that the expression levels of GATA6-AS were lower in tumor tissues compared with healthy tissues. In addition, serum levels of GATA6-AS were higher in patients compared with healthy controls. The serum levels of GATA6-AS were associated with tumor metastasis, and may serve as a potential diagnostic and prognostic marker for cervical squamous cell carcinoma. Furthermore, GATA6-AS overexpression inhibited cancer cell migration and invasion. The expression levels of MTK-1 were also reduced following GATA6-AS overexpression. Therefore, the present study proposed that downregulated GATA6-AS expression was associated with tumor metastasis in cervical squamous cell carcinoma, and that GATA6-AS expression may inhibit cancer cell migration and invasion by downregulating MTK-1.

RESUMEN

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers in China. Mitogen-activated protein kinase kinase 4 (MKK4) regulates tumorigenesis as a component of the MKK4 pathway. A number of studies have suggested a correlation between the MKK4 -1304T>G polymorphism and the risk of CRC. However, the results are still controversial. Therefore, we conducted a meta-analysis to obtain a more accurate assessment of the association between the MKK4 -1304T>G polymorphism and the risk of CRC. METHODS: Systematic literature searches were performed using PubMed, Embase, Cochrane Library, and CNKI. Four trials, including 1,255 cancer cases and 1,181 controls, were recruited in our study to assess the relationship of the MKK4 -1304T>G polymorphism with the risk of CRC. RESULTS: Four studies met our inclusion criteria and were finally included in the analysis, involving 1,255 cancer patients and 1,181 controls. Our meta-analysis revealed that the MKK4 -1304T>G polymorphism could reduce the risk of CRC (G vs. T: OR, 0.60, 95% CI: 0.48-0.76, P<0.0001; GG vs. TT: OR, 0.43, 95% CI: 0.29-0.62, P<0.0001; GG vs. TT + TG: OR, 0.50, 95% CI: 0.34-0.72, P=0.0003; TG + GG vs. TT: OR, 0.62, 95% CI: 0.53-0.73, P<0.0001; and TG vs. TT + GG: OR, 0.70, 95% CI: 0.59-0.82, P<0.0001). CONCLUSIONS: In conclusion, our meta-analysis showed that the MKK4 -1304T>G polymorphism was associated with the susceptibility to CRC. In the future, large and well-designed case-control studies are needed to validate our findings.

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) exerts a significant protective effect against neurotoxicity and mitochondrial damage in neurons. However, the mechanisms underlying PF-mediated rescue remain elusive. Therefore, we endeavored to further research the molecular mechanisms underlying PF-mediated inhibition of tributyltin chloride (TBTC)-induced apoptosis of neurons. AIM OF THE STUDY: To investigate the influence and possible mechanism of action of PF in TBTC-induced neurodegenerative disease. MATERIALS AND METHODS: First, primary hypothalamic neurons were treated with tributyltin chloride (150 µg/L) and PF (25, 50, and 100 µM). 17ß-estradiol (1 nM) was used as a positive control. Subsequently, CCK-8 assay was performed. The level of apoptosis was examined by flow cytometry and the function of mitochondria was reflected by MMP levels. The mRNA expression levels of B-cell lymphoma-2 (Bcl-2), together with Bax, were examined using qRT-PCR. The protein levels of mitogen-activated protein kinase kinase 4 (MKK4), c-Jun N-terminal kinase (JNK), Bcl-2, Bax, and Caspase-3 were examined using western blotting. Finally, pretreatment with JNK agonist, anisomycin, was done to observe the change in expressions of MKK4 and JNK. RESULTS: Paeoniflorin treatment reduced TBTC-induced damage and neuron loss in a dose-dependent manner. Decrease in mitogen-activated protein kinase (MAPK) as well as JNK levels were reversed by treatment with paeoniflorin via inhibition of JNK activation. Furthermore, ratio of levels of Bcl-2/Bax increased while the activation of caspase-3 was suppressed. In addition, pretreatment with JNK agonist, anisomycin effectively suppressed TBTC-induced cytotoxicity in hypothalamic neuron. CONCLUSIONS: PF can potentially be used to prevent and/or treat neurodegenerative diseases and neural injury by inhibiting MKK4-JNK signaling pathway.

Asunto(s)

Glucósidos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Monoterpenos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Compuestos de Trialquiltina/toxicidad , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Femenino , Hipotálamo/citología , Neuronas/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos

RESUMEN

Prostate cancer (PCa) is the second most common type of cancer and the 6th leading cause of cancer­associated mortality worldwide. Accumulated evidence suggests that PCa initiation and progression are controlled by microRNAs (miRNAs). Therefore, investigating PCa­associated miRNAs may provide novel biomarkers for the diagnosis and treatment of patients with PCa. In the present study it was demonstrated that miRNA­136 (miR­136) expression was significantly downregulated in PCa tissues and cell lines. The resumption of miR­136 expression suppressed cell proliferation and invasion in PCa cells. Bioinformatics analysis predicted that mitogen­activated protein kinase kinase 4 (MAP2K4) was a direct target of miR­136. This prediction was experimentally confirmed by a luciferase reporter assay, RT­qPCR and western blot analysis. MAP2K4 was highly expressed in PCa tissues and inversely correlated with the miR­136 expression level. Additionally, the restoration of MAP2K4 expression significantly blocked the inhibitory effects of miR­136 on cell proliferation and invasion in PCa cells. Therefore, miR­136 may suppress the proliferation and invasion of PCa cells by targeting MAP2K4 and may be a novel candidate target for cancer therapy against PCa.

Asunto(s)

MAP Quinasa Quinasa 4/metabolismo , MicroARNs/metabolismo , Neoplasias de la Próstata/patología , Regiones no Traducidas 3' , Antagomirs/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Humanos , MAP Quinasa Quinasa 4/química , MAP Quinasa Quinasa 4/genética , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Alineación de Secuencia

RESUMEN

The NFκB family of transcription factors is crucial for innate or adaptive immunity, inflammation, and diseases including cancer. The two NFκB signaling pathways (canonical and non-canonical) differ from each other in extracellular signals, membrane receptors, signaling adaptors, and dimer subunits. The p52 (NFκB2) subunit, which participates in the non-canonical pathway, is generated by ubiquitin-mediated processing of the p100 precursor. Here, we found that NFκB2 processing and activation were mediated by mitogen-activated protein kinase kinase-4 (MKK4) and its substrate c-Jun N-terminal kinase (JNK). In MKK4-null mouse embryonic fibroblasts (MEFs), serum- and lymphotoxin ß receptor (LTßR) antibody-induced processing of p100 and nuclear translocation of p52 were found to be defective. Serum and LTßR antibody activated the MKK4-JNK signaling pathway, and SP600125, a JNK inhibitor, blocked p100 processing. Cellular senescence, one of the responses regulated by the non-canonical NFκB pathway, was observed more frequently in MKK4-null MEFs than in wildtype cells. These results suggest that the MKK4/JNK-dependent pathway regulates NFκB2 processing/activation and, through this mechanism, MKK4 and NFκB2 control cellular growth and senescence.

Asunto(s)

Células Epiteliales/metabolismo , Fibroblastos/metabolismo , MAP Quinasa Quinasa 4/genética , Subunidad p52 de NF-kappa B/genética , Animales , Antracenos/farmacología , Bronquios/citología , Bronquios/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Células Epiteliales/citología , Fibroblastos/citología , Regulación de la Expresión Génica , Humanos , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/metabolismo , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/metabolismo , Ratones , Subunidad p52 de NF-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal

RESUMEN

Mitogen-activated protein kinase kinase 4 (MKK4) is a key mediator of Jun N-terminal kinase signaling and influences malignant metastasis. Here, we used immunohistochemistry to assess phosphorylated MMK4 (pMKK4) levels and examine their association with the clinicopathological features of a pilot set of patient samples consisting of normal colonic mucosa (NCM), colorectal adenoma (CA), and colorectal cancer (CRC) tissues. pMKK4 levels were also assessed in a validation set of CRC cases with accompanying follow-up data to confirm their clinicopathological and prognostic significance. pMKK4 levels, which were high in 79.17% of NCM samples, were downregulated in 33.33% of CA and 63.54% of CRC samples. pMKK4 downregulation was associated with metastasis, especially to the liver. In the validation set, pMKK4 downregulation was associated with increases in invasive depth, lymph node metastasis, distant metastasis, and TNM stage. Univariate analysis indicated that pMKK4 score, tumor differentiation, and TNM stage were correlated with disease-free survival and overall survival. Multivariate analysis indicated that decreased pMKK4 expression was an independent risk factor for disease-free survival in CRC patients. These results suggest that CRC patients with low pMKK4 immunochemistry scores should be monitored carefully for early detection of possible recurrences, especially liver metastasis.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Regulación hacia Abajo , MAP Quinasa Quinasa 4/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fosforilación , Proyectos Piloto , Pronóstico , Adulto Joven

RESUMEN

MicroRNAs (miRNAs or miRs) can function as tumor-suppressor or oncogenic genes. Upregulation of miRNA-141 has been frequently observed in colorectal cancer (CRC) samples. The experimentally observed targets of miR-141 include the tumor-suppressor gene mitogen-activated protein kinase kinase 4 (MAP2K4). The aim of the present study was to investigate the role of miR-141 in the proliferation of colonic cancer. Western blotting, immunohistochemistry and reverse transcription-quantitative polymerase chain reaction were used to detect the expression levels of miR-141 and MAP2K4 in colonic adenocarcinoma (CAC) and adjacent non-cancerous (NC) tissue samples, as well as in human CAC cell lines (HT29, T94 and LS174). MTT assay was used to investigate the proliferation and apoptosis of these three cell lines. The expression levels of miR-141 were significantly upregulated in clinical samples of CAC, compared with adjacent NC tissues. By contrast, MAP2K4 was downregulated in CAC. The in vitro assays demonstrated that overexpression of miR-141 resulted in cell proliferation of CAC by inhibiting MAP2K4 activity. Our study suggests that targeting the miR-141-MAP2K4 signaling pathway may represent a novel approach for the treatment of CRC.

RESUMEN

OBJECTIVE: Pregnancy is accompanied by dramatic physiological changes in maternal plasma proteins. Characterization of the maternal plasma proteome in normal pregnancy is an essential step for understanding changes to predict pregnancy outcome. The objective of this study was to describe maternal plasma proteins that change in abundance with advancing gestational age and determine biological processes that are perturbed in normal pregnancy. STUDY DESIGN: A longitudinal study included 43 normal pregnancies that had a term delivery of an infant who was appropriate for gestational age without maternal or neonatal complications. For each pregnancy, 3 to 6 maternal plasma samples (median, 5) were profiled to measure the abundance of 1125 proteins using multiplex assays. Linear mixed-effects models with polynomial splines were used to model protein abundance as a function of gestational age, and the significance of the association was inferred via likelihood ratio tests. Proteins considered to be significantly changed were defined as having the following: (1) >1.5-fold change between 8 and 40 weeks of gestation; and (2) a false discovery rate-adjusted value of P < .1. Gene ontology enrichment analysis was used to identify biological processes overrepresented among the proteins that changed with advancing gestation. RESULTS: The following results were found: (1) Ten percent (112 of 1125) of the profiled proteins changed in abundance as a function of gestational age; (2) of the 1125 proteins analyzed, glypican-3, sialic acid-binding immunoglobulin-type lectin-6, placental growth factor, C-C motif-28, carbonic anhydrase 6, prolactin, interleukin-1 receptor 4, dual-specificity mitogen-activated protein kinase 4, and pregnancy-associated plasma protein-A had more than a 5-fold change in abundance across gestation (these 9 proteins are known to be involved in a wide range of both physiological and pathological processes, such as growth regulation, embryogenesis, angiogenesis immunoregulation, inflammation etc); and (3) biological processes associated with protein changes in normal pregnancy included defense response, defense response to bacteria, proteolysis, and leukocyte migration (false discovery rate, 10%). CONCLUSION: The plasma proteome of normal pregnancy demonstrates dramatic changes in both the magnitude of changes and the fraction of the proteins involved. Such information is important to understand the physiology of pregnancy and the development of biomarkers to differentiate normal vs abnormal pregnancy and determine the response to interventions.

Asunto(s)

Proteínas Sanguíneas/análisis , Edad Gestacional , Resultado del Embarazo , Proteoma/análisis , Adulto , Biomarcadores/sangre , Femenino , Humanos , Estudios Longitudinales , Embarazo , Complicaciones del Embarazo/sangre , Estudios Prospectivos , Proteómica/métodos

RESUMEN

Apoptosis signal-regulating kinase I (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the downstream MAP kinase kinases (MKKs) from two MAP kinase cascades: c-Jun N-terminal kinase (JNK) and p38. The essential physiological functions of ASK1 have attracted extensive attention. However, our understanding of the molecular mechanisms of ASK1, including the activation mechanism of ASK1 and the catalytic mechanism of ASK1-mediated MKK phosphorylation, remain unclear. The lack of purified ASK1 protein has hindered the elucidation of ASK1-initiated signal transduction mechanisms. Here, we report a one-step chromatography method for the expression and purification of functional full-length ASK1 from Escherichia coli. The purified ASK1 demonstrates auto-phosphorylation activity. The kinase activity of auto-phosphorylated ASK1 (pASK1) was also evaluated on two MKK substrates, MKK4 and 7, from the JNK cascades. Our results show that MKK7 can be phosphorylated by pASK1 more effectively than MKK4. The steady-state kinetic analysis demonstrates that MKK7 is a better ASK1 substrate than MKK4. These observations are further confirmed by direct pull-down assays which shows ASK1 binds MKK7 significantly stronger than MKK4. Furthermore, robust phospho-tyrosine signal is observed in MKK4 phosphorylation by pASK1 in addition to the phospho-serine and phospho-threonine. This study provides novel mechanistic and kinetic insights into the ASK1-initiated MAPK signal transduction via highly controlled reconstructed protein systems.

Asunto(s)

Expresión Génica , MAP Quinasa Quinasa Quinasa 5 , Activación Enzimática , Escherichia coli , Humanos , MAP Quinasa Quinasa 4/química , MAP Quinasa Quinasa 7/química , MAP Quinasa Quinasa Quinasa 5/biosíntesis , MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

INTRODUCTION: c-Jun N-terminal kinases (JNKs) are involved in the emergence and progression of diverse pathologies such as neurodegenerative, cardiovascular and metabolic disorders as well as inflammation and cancer. In recent years, several highly selective pan-JNK inhibitors have been characterized and three chemical entities targeting JNKs have been investigated in clinical trials. AREAS COVERED: This review summarizes patents claiming inhibitors of all JNK isoforms published between 2010 and 2014. Although primarily focusing on the patent literature, relevant peer-reviewed publications related to the covered patents have also been included. Moreover, key patents claiming novel applications of previously published chemical entities are reviewed. The article highlights a total of 28 patents from nine pharmaceutical companies and academic research groups. EXPERT OPINION: Although some selective pan-JNK inhibitors with reasonable in vivo profiles are now available, little is known about the isoform selectivity required for each particular indication and the development of isoform-selective JNK inhibitors still represents a challenge in JNK drug discovery. Moreover, isoform-selective tool compounds are a prerequisite to a comprehensive understanding of the biology of each JNK isoform. Potential approaches towards such compounds include the design of type-II and type-I(1)/2 binders, which are absent in the current JNK inhibitor portfolios, as well as the design of novel allosteric inhibitors. Furthermore, covalent inhibition, which already led to the first high-quality probe for JNKs, might be further exploited for gaining selectivity and in vivo efficacy. With regard to a potential therapeutic application, the recently proposed concept of covalent reversible inhibitors is expected to be attractive.

Asunto(s)

Diseño de Fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Humanos , Isoenzimas , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida , Patentes como Asunto , Inhibidores de Proteínas Quinasas/farmacología

RESUMEN

The exocyst complex, also called the Sec6/8 complex, is important for targeting exocytic vesicles to specific docking sites on the plasma membrane in yeast and mammalian cells. In addition to these original findings, recent results of studies suggest that Sec8 is also involved in oncogenesis, although the functional implications of Sec8 in cancer cells are not well understood. c-Jun N-terminal kinase-interacting protein 4 (JIP4) is a scaffold protein that plays a crucial role in the regulation of mitogen-activated protein kinase (MAPK) signaling cascades. The present study examined how Sec8 is involved in JIP4-mediated MAPK signaling under apoptotic conditions. It was found that Sec8 binds to and regulates JIP4, and that knockdown of Sec8 enhances the binding of JIP4 to MAPK kinase 4, thereby decreasing the phosphorylation of MAPK kinase 4, JNK, and p38. These results raise the possibility that Sec8 serves as an important regulator of MAPK signaling cascades.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de Transporte Vesicular/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Células HEK293 , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Fosforilación , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

OBJECTlVE To explore the mechanisms of tau hyperphosphorylation induced by calyculin A ( CA) in neuroblastoma cells and the effect of melatonin. METHODS N2a cells were treated with CA 5 nmol·L-1 , or CA with melatonin 50 μmol·L-1 , or CA with vitamin E ( Vit E ) 50 μmol·L-1 for 12 h. The level of tau phosphorylation at Ser422 ( recognized by R145d antibody) site and the level of phosphorylated c-Jun N-terminal kinases ( p-JNK ) and phosphorylated mitogen-activated protein kinase kinase 4 ( p-MKK4 ) were detected with immunoblotting, the level of malondialdehyde ( MDA ) was assayed with fluorimetry, and the activity of p38-mitogen activated protein kinase ( p38MAPK ) was assayed by radioimmunobloting. RESULTS CA treatment increased the level of phosphorylated tau at Ser422 site (1.70±0.19, 1.0, P<0.01), and melatonin attenuated the effect of CA (0.98±0.12, 1.70± 0.19, P<0.01). ln addition, CA treatment increased the level of MDA (μmol·g-1 protein:0.241±0.006, 0.141±0.006, P<0.01) and melatonin antagonized the increase of MDA induced by CA (μmol·g-1 protein:0.172±0.004, 0.193±0.005, 0.241±0.006, P<0.01) . CA treatment increased the level of p-JNK (1.91±0.27, 1, P<0.01) and p-MKK4 (1.81±0.09, 1, P<0.01) and melatonin antagonized the effect of CA induced increase of p-JNK (1.11±0.15, 1.91±0.27, P<0.01) and p-MKK4 (1.14±0.06, 1.81±0.09, P<0.01) without changing the level or activity of p38MAPK. Both JNK inhibitor ( SP600125 ) and MKK4/JNK transduction pathway inhibitor antagonized CA induced tau phosphorylation at Ser422 site and JNK phosphorylation. CONCLUSlON lnhibiton of JNK phosphorylation is possibly involved in the protection of melatonin on CA-induced tau hyperphosphorylation at Ser422 site.

RESUMEN

Obesity is characterized as an excess accumulation of body fat resulting from a positive energy balance. It is the major risk factor for type 2 diabetes (T2D). The evidence for familial aggregation of obesity and its associated metabolic diseases is substantial. To date, about 150 genetic loci identified in genome-wide association studies (GWAS) are linked with obesity and T2D, each accounting for only a small proportion of the predicted heritability. However, the percentage of overall trait variance explained by these associated loci is modest (~5-10% for T2D, ~2% for BMI). The lack of powerful genetic associations suggests that heritability is not entirely attributable to gene variations. Some of the familial aggregation as well as many of the effects of environmental exposures, may reflect epigenetic processes. This review summarizes our current knowledge on the genetic basis to individual risk of obesity and T2D, and explores the potential role of epigenetic contribution.