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1.
Braz J Med Biol Res ; 53(4): e9175, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32267308

RESUMEN

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.


Asunto(s)
Medicamentos Herbarios Chinos/uso terapéutico , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Agonistas de Aminoácidos Excitadores/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores AMPA/metabolismo , Adolescente , Adulto , Animales , Niño , Epilepsia del Lóbulo Temporal/inducido químicamente , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pentilenotetrazol , Ratas Sprague-Dawley , Adulto Joven
2.
Gene ; 744: 144608, 2020 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-32234541

RESUMEN

Prostate cancer (PCa) is the third most common malignancy worldwide. Novel and effective therapeutic targets are needed for PCa. The purpose of this study was to discover novel therapeutic targets for PCa by performing advanced analysis on PCa RNA sequencing (RNAseq) data from The Cancer Genome Atlas (TCGA). Weighted correlation-network analysis (WGCNA) was performed on the RNAseq data of tumor samples, and the module most relevant to the Gleason score was identified. Combining differential gene-expression analysis and survival analysis, we narrowed down potential therapeutic target genes and found that PKMYT1 might be one. Subsequently, functional studies (i.e., cell-proliferation assays, cell cycle analysis, and colony-formation assays) demonstrated that knockdown of PKMYT1 significantly inhibited the growth of PCa cells. Further investigation illustrated that PKMYT1 promoted the growth of PCa cells through targeting CCNB1 and CCNE1 expression. In addition, fostamatinib, an inhibitor of PKMYT1, effectively inhibited the proliferation of PCa cells. Taken together, our results suggest that PKMYT1 is a gene associated with malignancy of PCa and is a novel therapeutic target.


Asunto(s)
Neoplasias de la Próstata/enzimología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Humanos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Oxazinas/uso terapéutico , Pronóstico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Piridinas/uso terapéutico
3.
Gene ; 744: 144635, 2020 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-32244053

RESUMEN

Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Espermatogénesis , Proteínas Quinasas Activadas por AMP/metabolismo , Actinas/metabolismo , Animales , Humanos , Masculino , Ratones , Proteínas de Microfilamentos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal
4.
Tumour Biol ; 42(4): 1010428320914475, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32252611

RESUMEN

Hepatocellular carcinoma is a major cause of cancer mortality worldwide. The outcome of hepatocellular carcinoma depends mainly on its early diagnosis. To date, the performance of traditional biomarkers is unsatisfactory. Polo-like kinase 1 is a serine/threonine kinase that plays essential roles in cell cycle progression and deoxyribonucleic acid damage. Moreover, polo-like kinase 1 knockdown decreases the survival of hepatocellular carcinoma cells; therefore, polo-like kinase 1 is an attractive target for anticancer treatments. Nobiletin, a natural polymethoxy flavonoid, exhibits a potential antiproliferative effect against a wide variety of cancers. This study targets to identify a reliable diagnostic biomarker for hepatocellular carcinoma and provide a potential therapeutic target for its treatment. Polo-like kinase 1 levels were analyzed in 44 hepatocellular carcinoma patients, 33 non-hepatocellular carcinoma liver cirrhosis patients and 15 healthy controls using the enzyme-linked immunosorbent assay method. Receiver operating characteristics curve analysis was used to establish a predictive model for polo-like kinase 1 relative to α-fetoprotein in hepatocellular carcinoma diagnosis. Furthermore, in the in vitro study, gene expressions were assessed by quantitative polymerase chain reaction in two human hepatocellular carcinoma cell lines after treatment with doxorubicin and polo-like kinase 1 inhibitor volasertib (Vola) either alone or in combination with nobiletin. Cell viability was also determined using the crystal violet assay.: Serum polo-like kinase 1 levels in hepatocellular carcinoma patients were significantly higher than liver cirrhosis and control groups (p < 0.0001). Polo-like kinase 1 showed a reasonable sensitivity, specificity, positive predictive value, and negative predictive value in hepatocellular carcinoma diagnosis. Moreover, nobiletin improved inhibition of cell growth induced by Vola and doxorubicin. Regarding reverse transcription polymerase chain reaction results, nobiletin suppressed expressions of polo-like kinase 1 and proliferating cell nuclear antigen and elevated expressions of P53, poly (ADPribose) polymerase 1, and caspase-3. Nobiletin/doxorubicin and nobiletin/Vola showed a significant increase in caspase-3 activity indicating cell apoptosis. Polo-like kinase 1 may be a potential biomarker for hepatocellular carcinoma diagnosis and follow-up during treatment with chemotherapies. In addition, nobiletin synergistically potentiates the doxorubicin and Vola-mediated anticancer effect that may be attributed partly to suppression of polo-like kinase 1 and proliferating cell nuclear antigen expression and enhancement of chemotherapy-induced apoptosis.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Neoplasias Hepáticas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Caspasa 3/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Doxorrubicina/farmacología , Flavonas/farmacología , Células Hep G2 , Humanos , Cirrosis Hepática/patología , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pteridinas/farmacología
5.
PLoS One ; 15(3): e0224344, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32176701

RESUMEN

A key event in the development of both major forms of diabetes is the loss of functional pancreatic islet ß-cell mass. Strategies aimed at enhancing ß-cell regeneration have long been pursued, but methods for reliably inducing human ß-cell proliferation with full retention of key functions such as glucose-stimulated insulin secretion (GSIS) are still very limited. We have previously reported that overexpression of the homeobox transcription factor NKX6.1 stimulates ß-cell proliferation, while also enhancing GSIS and providing protection against ß-cell cytotoxicity through induction of the VGF prohormone. We developed an NKX6.1 pathway screen by stably transfecting 832/13 rat insulinoma cells with a VGF promoter-luciferase reporter construct, using the resultant cell line to screen a 630,000 compound chemical library. We isolated three compounds with consistent effects to stimulate human islet cell proliferation, but not expression of NKX6.1 or VGF, suggesting an alternative mechanism of action. Further studies of the most potent of these compounds, GNF-9228, revealed that it selectively activates human ß-cell relative to α-cell proliferation and has no effect on δ-cell replication. In addition, pre-treatment, but not short term exposure of human islets to GNF-9228 enhances GSIS. GNF-9228 also protects 832/13 insulinoma cells against ER stress- and inflammatory cytokine-induced cytotoxicity. GNF-9228 stimulates proliferation via a mechanism distinct from recently emergent DYRK1A inhibitors, as it is unaffected by DYRK1A overexpression and does not activate NFAT translocation. In conclusion, we have identified a small molecule with pleiotropic positive effects on islet biology, including stimulation of human ß-cell proliferation and insulin secretion, and protection against multiple agents of cytotoxic stress.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular Tumoral , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Glucagón/patología , Glucosa/farmacología , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/patología , Insulinoma/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ratas
6.
Nat Cell Biol ; 22(4): 412-424, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32203415

RESUMEN

Although the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a non-traditional role for Cu as a modulator of kinase signalling is emerging. Here, we found that Cu is required for the activity of the autophagic kinases ULK1 and ULK2 (ULK1/2) through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt the binding of Cu reduced ULK1/2-dependent signalling and the formation of autophagosome complexes. Increased levels of intracellular Cu are associated with starvation-induced autophagy and are sufficient to enhance ULK1 kinase activity and, in turn, autophagic flux. The growth and survival of lung tumours driven by KRASG12D is diminished in the absence of Ctr1, is dependent on ULK1 Cu binding and is associated with reduced levels of autophagy and signalling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to prevent autophagy signalling to limit proliferation and improve patient survival in cancer.


Asunto(s)
Adenocarcinoma del Pulmón/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Autofagia/genética , Cobre/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinasas/genética , Adenocarcinoma del Pulmón/enzimología , Adenocarcinoma del Pulmón/patología , Secuencia de Aminoácidos , Animales , Autofagosomas/enzimología , Proteína 5 Relacionada con la Autofagia/deficiencia , Proteína 5 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Transportador de Cobre 1/deficiencia , Transportador de Cobre 1/genética , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/deficiencia , Proteínas Proto-Oncogénicas p21(ras)/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Nat Cell Biol ; 22(4): 453-464, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32203417

RESUMEN

TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liquid-liquid phase separation to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1, and the transcription elongation factor CDK9 for transcription. TAZ forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signalling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain prevents the phase separation of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate that pathway-specific transcription factors also engage the phase-separation mechanism for efficient and specific transcriptional activation.


Asunto(s)
Proteínas de Ciclo Celular/genética , Quinasa 9 Dependiente de la Ciclina/genética , Proteínas de Unión al ADN/genética , Subunidad 1 del Complejo Mediador/genética , Proteínas Musculares/genética , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Compartimento Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Subunidad 1 del Complejo Mediador/metabolismo , Proteínas Musculares/metabolismo , Fosforilación , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo
8.
Life Sci ; 251: 117424, 2020 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-32057900

RESUMEN

AIMS: Dysfunction of the Hippo-Yes-Associated Protein (YAP) signaling pathway is known to be associated with hepatocellular carcinoma (HCC). Evodiamine (Evo), a plant-derived bioactive alkaloid, exerts inhibitory effects on cancer. However, the precise influence of Evo on HCC and its potential effects on Hippo-YAP signaling have yet to be ascertained. Here, the effects of Evo on cell proliferation and apoptosis were evaluated using HCC cell lines (HepG2 and Bel-7402) and nude mice with xenograft tumors. We further investigated whether Evo exerts anti-HCC activity through effects on Hippo-YAP signaling in vitro with the aid of XMU-MP-1, an inhibitor of the key component of this pathway, mammalian sterile 20-like kinase 1/2. MAIN METHODS: Cell proliferation and apoptosis were assessed using 5-ethynyl-2'-deoxyuridine staining, colony formation, flow cytometry, hematoxylin-eosin and dUTP nick-end labeling experiments. Bioinformatics and real-time quantitative polymerase chain reaction (RT-qPCR) arrays were performed to determine the associations among Evo, HCC progression and the Hippo-YAP pathway. The expression patterns of components of Hippo-YAP signaling and apoptotic genes were further examined via RT-qPCR and immunoblotting. KEY FINDINGS: Evo inhibited proliferation and promoted apoptosis of HCC cell lines in vitro, and attenuated xenograft tumor formation in nude mice in vivo. Mechanistically, Evo treatment stimulated the Hippo-YAP signaling pathway. In vitro, the effects of Evo on HCC cell proliferation and apoptosis were alleviated by XMU-MP-1. SIGNIFICANCE: Our collective results revealed that the anti-HCC effects of Evo were correlated with the Hippo-YAP signaling pathway.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Quinazolinas/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Nat Commun ; 11(1): 988, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-32080171

RESUMEN

Increasing grain yield of maize (Zea mays L.) is required to meet the rapidly expanding demands for maize-derived food, feed, and fuel. Breeders have enhanced grain productivity of maize hybrids by pyramiding desirable characteristics for larger ears. However, loci selected for improving grain productivity remain largely unclear. Here, we show that a serine/threonine protein kinase encoding gene KERNEL NUMBER PER ROW6 (KNR6) determines pistillate floret number and ear length. Overexpression of KNR6 or introgression of alleles lacking the insertions of two transposable elements in the regulatory region of KNR6 can significantly enhance grain yield. Further in vitro evidences indicate that KNR6 can interact with an Arf GTPase-activating protein (AGAP) and its phosphorylation by KNR6 may affect ear length and kernel number. This finding provides knowledge basis to enhance maize hybrids grain yield.


Asunto(s)
Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinasas/genética , Zea mays/genética , Mapeo Cromosómico , Grano Comestible/enzimología , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Proteínas Activadoras de GTPasa/metabolismo , Genes de Plantas , Fenotipo , Fosforilación , Fitomejoramiento , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sitios de Carácter Cuantitativo , Zea mays/enzimología , Zea mays/crecimiento & desarrollo
10.
Nat Commun ; 11(1): 735, 2020 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-32024846

RESUMEN

Multi-omics datasets represent distinct aspects of the central dogma of molecular biology. Such high-dimensional molecular profiles pose challenges to data interpretation and hypothesis generation. ActivePathways is an integrative method that discovers significantly enriched pathways across multiple datasets using statistical data fusion, rationalizes contributing evidence and highlights associated genes. As part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumor types, we integrated genes with coding and non-coding mutations and revealed frequently mutated pathways and additional cancer genes with infrequent mutations. We also analyzed prognostic molecular pathways by integrating genomic and transcriptomic features of 1780 breast cancers and highlighted associations with immune response and anti-apoptotic signaling. Integration of ChIP-seq and RNA-seq data for master regulators of the Hippo pathway across normal human tissues identified processes of tissue regeneration and stem cell regulation. ActivePathways is a versatile method that improves systems-level understanding of cellular organization in health and disease through integration of multiple molecular datasets and pathway annotations.


Asunto(s)
Biología Computacional/métodos , Redes y Vías Metabólicas/genética , Neoplasias/genética , Neoplasias/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Inmunoprecipitación de Cromatina , Bases de Datos Factuales , Femenino , Dosificación de Gen , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Genómica/métodos , Humanos , Mutación , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Análisis de Secuencia de ARN
11.
Nat Commun ; 11(1): 770, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-32034138

RESUMEN

Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca2+ levels to activate TBK1 for xenophagy via the Ca2+-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca2+-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca2+ chelator impairs TBC1D9-ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca2+-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca2+-dependent ubiquitin-recognition.


Asunto(s)
Autofagia/fisiología , Señalización del Calcio/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Infecciones Estreptocócicas/metabolismo , Sitios de Unión , Citosol/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Macroautofagia/fisiología , Mitocondrias/metabolismo , Mitocondrias/microbiología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Streptococcus pyogenes/patogenicidad , Ubiquitina/metabolismo
12.
Mol Carcinog ; 59(4): 425-438, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32064687

RESUMEN

Colorectal cancer (CRC) is a kind of malignant cancer with high morbidity and mortality. The purpose of this study was to explore potential regulated key genes involved in CRC through bioinformatics analysis and experimental verification. The gene expression profile data were downloaded from the Gene Expression Omnibus, and the differential expression genes were detected in cancerous and paracancerous samples of CRC patients, respectively. Then functional enrichment analysis, such as the Kyoto Encyclopedia of Genes and Genomes pathway analysis as well as the protein-protein interaction network were constructed, and the highly related genes were clustered by Molecular COmplex DEtection algorithm to find out the core interaction in different genes' crosstalk. The genes affecting CRC prognosis were screened by the Human Protein Atlas database. In addition, the expression level of core genes was detected by GEPIA database, and the core genes' changes in large-scale cancer genome data set were directly analyzed by cBioPortal database. The expression of the predicted hub genes DSN1, AHCY, and ERCC6L was verified by reverse-transcription quantitative polymerase chain reaction in CRC cells. The gene function of DSN1 was analyzed by wound healing and colony formation assays. The results showed that silencing of DSN1 could significantly reduce the migration and proliferation of CRC cells. Further, BUB1B, the potential interacting protein of DSN1, was also predicted via bioinformatics analysis. Above all, this study shows that bioinformatics analysis combined with experimental method verification provide more potential vital genes for the prevention and therapy of CRC.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ontología de Genes , Humanos , Pronóstico , Mapas de Interacción de Proteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
13.
Medicine (Baltimore) ; 99(5): e18864, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32000390

RESUMEN

Nuclear factor-κB-inducing kinase (NIK) is a new regulator of nuclear factor-κB signaling, which plays an important role in tumorigenesis. This study aimed to examine the expression of NIK in gastric cancer and investigate its clinical significance.Tumor issues were collected from 80 gastric cancer patients who received surgery and the diagnosis was confirmed by postoperative pathological analysis. The expression of NIK in gastric cancer tissues and adjacent normal mucosa was detected by immunohistochemical analysis. The associations between NIK expression and clinicopathological features of the patients were further analyzed.NIK expression was significantly higher in gastric cancer tissues than in adjacent normal tissues (P < .05). Furthermore, NIK expression showed significant association with UICC stage, T status, and differentiation, but not with age and gender of gastric cancer patients.NIK is overexpressed in gastric cancer and is a potential diagnostic marker of gastric cancer.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Gástricas/enzimología , Anciano , Biomarcadores de Tumor/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía
14.
Nat Commun ; 11(1): 613, 2020 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-32001690

RESUMEN

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Presión Osmótica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Estrés Fisiológico , Quinasas raf/metabolismo , Análisis Mutacional de ADN , Mutación/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica
15.
Nat Cell Biol ; 22(2): 246-256, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32015438

RESUMEN

The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/genética , Proteína Reguladora Asociada a mTOR/genética , Proteínas Supresoras de Tumor/genética , Animales , Sistemas CRISPR-Cas , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Edición Génica , Células HCT116 , Células HEK293 , Células HeLa , Xenoinjertos , Humanos , Hígado/anomalías , Hígado/metabolismo , Células MCF-7 , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Desnudos , Ratones Transgénicos , Miocardio/metabolismo , Miocardio/patología , Neurofibromina 2/deficiencia , Neurofibromina 2/genética , Tamaño de los Órganos , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia
16.
Am J Pathol ; 190(4): 844-861, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32035058

RESUMEN

Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1-/- knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase-Hippo-TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation.


Asunto(s)
Inflamación/patología , Enfermedades Neurodegenerativas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Interferón alfa y beta/fisiología , Replicación Viral , Infección por el Virus Zika/complicaciones , Virus Zika/aislamiento & purificación , Animales , Inflamación/virología , Masculino , Ratones , Ratones Noqueados , Enfermedades Neurodegenerativas/virología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Infección por el Virus Zika/virología
17.
PLoS Pathog ; 16(1): e1008138, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31961913

RESUMEN

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Phytophthora/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Esporas/crecimiento & desarrollo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/genética , Phytophthora/genética , Phytophthora/crecimiento & desarrollo , Phytophthora/patogenicidad , Enfermedades de las Plantas/parasitología , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Soja/parasitología , Esporas/enzimología , Esporas/genética , Esporas/metabolismo , Virulencia
18.
Cell Physiol Biochem ; 54(1): 1-14, 2020 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-31916733

RESUMEN

BACKGROUND/AIMS: Deubiquitinating enzymes (DUBs) are crucially involved in controlling signal transductions, and reverse ubiquitination by removing the ubiquitin from protein substrates. The Hippo signaling has an important role in tissue growth, cell proliferation, differentiation, and apoptosis. Since disruption of the Hippo signaling is associated with a number of diseases, it is imperative to investigate the molecular mechanism of the Hippo signaling. METHODS: DUB screening was performed using the kidney of the mouse unilateral ureteric obstruction (UUO) model to identify the cellular mechanism of the DUB-regulated Hippo signaling. In addition, kidney cells were used to confirm cell proliferation and protein levels in the Hippo signaling pathway. Densitometric analysis was conducted to compare the expression level of proteins using Image J. RESULTS: We found that YOD1, also known as OTU1, is downregulated in the mouse UUO model. We also demonstrated that YOD1 binds to and deubiquitinates neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4). Furthermore, we observed that YOD1 suppresses NEDD4-induced cell proliferation. CONCLUSION: YOD1 regulates the Hippo signaling pathway through NEDD4, and the K63-linked polyubiquitin chain of NEDD4 plays an important role. Also, our results indicate that YOD1 plays an important role in kidney diseases.


Asunto(s)
Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Transducción de Señal , Tioléster Hidrolasas/metabolismo , Animales , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Mutagénesis , Ubiquitina-Proteína Ligasas Nedd4/química , Ubiquitina-Proteína Ligasas Nedd4/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Tioléster Hidrolasas/química , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinación
19.
Proc Natl Acad Sci U S A ; 117(4): 2004-2013, 2020 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-31932432

RESUMEN

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.


Asunto(s)
Carcinogénesis/patología , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Glucosa/farmacología , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Acetilglucosamina/metabolismo , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/metabolismo , Proteínas Portadoras/genética , Proliferación Celular , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Células HEK293 , Humanos , Células MCF-7 , Ratones , N-Acetilglucosaminiltransferasas/genética , Fosforilación , Proteínas Quinasas/genética , Estabilidad Proteica , Proteínas Serina-Treonina Quinasas/genética , Edulcorantes/farmacología
20.
Nat Commun ; 11(1): 78, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31911626

RESUMEN

The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.


Asunto(s)
Encéfalo/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Hidrocarburos Clorados/administración & dosificación , Nitrilos/administración & dosificación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Humanos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo
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