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1.
Cells ; 10(3)2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33801464

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.


Asunto(s)
/metabolismo , Proteína 58 DEAD Box/metabolismo , Interferones/metabolismo , Receptores Inmunológicos/metabolismo , /metabolismo , Proteína 58 DEAD Box/genética , Interacciones Huésped-Patógeno/genética , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interferones/genética , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Fosfoproteínas/metabolismo , Poli C/farmacología , Poli I/farmacología , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Inmunológicos/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia Arriba , Virus Zika/genética , Virus Zika/metabolismo
2.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33804889

RESUMEN

In chronic kidney disease, hyperphosphatemia upregulates the Ca2+ channel ORAI and its activating Ca2+ sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca2+ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+. The present study explored whether sustained exposure to Mg2+ or Gd3+ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl2 or 50 µM GdCl3. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and SOCE from the increase in [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, Mg2+ and Gd3+ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg2+ and the CaSR agonist Gd3+ interfere with phosphate-induced dysregulation of [Ca2+]i in megakaryocytes.


Asunto(s)
Señalización del Calcio , Gadolinio/farmacología , Cloruro de Magnesio/farmacología , Megacariocitos/efectos de los fármacos , Proteína ORAI1/metabolismo , Células Cultivadas , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Megacariocitos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
3.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33803640

RESUMEN

The LATS1 kinase has been described as a tumor suppressor in various cancers. However, its role in melanoma has not been fully elucidated. There are several processes involved in tumorigenesis, including melanin production. Melanin content positively correlates with the level of reactive oxygen species (ROS) inside the cell. Accordingly, the purpose of the study was to assess the role of LATS1 in melanogenesis and oxidative stress and its influence on tumor growth. We have knocked down LATS1 in primary melanocytes and melanoma cells and found that its expression is crucial for melanin synthesis, ROS production, and oxidative stress response. We showed that LATS1 ablation significantly decreased the melanogenesis markers' expression and melanin synthesis in melanocyte and melanoma cell lines. Moreover, silencing LATS1 resulted in enhanced oxidative stress. Reduced melanin content in LATS1 knocked down tumors was associated with increased tumor growth, pointing to melanin's protective role in this process. The study demonstrated that LATS1 is highly engaged in melanogenesis and oxidative stress control and affects melanoma growth. Our results may find the implications in the diagnosis and treatment of pigmentation disorders, including melanoma.


Asunto(s)
Melaninas/biosíntesis , Melanoma/patología , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Cinética , Melanocitos/metabolismo , Melanoma/genética , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Hipoxia Tumoral/genética
4.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802742

RESUMEN

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.


Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/patología , Endorribonucleasas/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/patología , Pulmón/patología , Terapia Molecular Dirigida , Proteínas Serina-Treonina Quinasas/metabolismo , Células Cultivadas , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citocinas/biosíntesis , Endorribonucleasas/genética , Epitelio/efectos de los fármacos , Epitelio/patología , Humanos , Imidazoles/química , Imidazoles/farmacología , Inflamación/genética , Modelos Biológicos , Naftalenos/química , Naftalenos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Pirazinas/química , Pirazinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína 1 de Unión a la X-Box/metabolismo
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(3): 336-343, 2021 Mar 25.
Artículo en Chino | MEDLINE | ID: mdl-33849823

RESUMEN

OBJECTIVE: To explore the effect of estradiol (E2) binding to its receptor ERß on the proliferation and apoptosis of C28I2 cells. OBJECTIVE: We cloned the sequence of ESR2 into a recombinant adenovirus plasmid (pAd-ESR2) and packaged the plasmid in HEK293 cells. Normal human chondrocyte C28I2 cells were transfected with Ad-ESR2 or small interfering RNA targeting ESR2-siRNA (ESR2-siRNA), and the effects of treatment with DMSO or E2 on the expression of the proteins associated with endoplasmic reticulum (ER) stress and cell apoptosis were determined using Western blotting. qRT-PCR was used to detect the expressions of proliferation-related marker genes, and an EdU kit and flow cytometry were used to assess cell proliferation and apoptosis. We also tested the effects of U0126 (an ERK pathway inhibitor) and E2, alone or in combination, on ER stress, apoptosis and the ERK signaling pathway in C28I2 cells infected with Ad-ESR2 using Western blotting. OBJECTIVE: Overexpression of Ad-ESR2 in C28I2 cells significantly promoted the expressions of IRE1α, PERK, XBP1s, and cleaved caspase-12, inhibited proliferation related marker genes PCNA, cyclin B1, cyclin D1, and decreased the level of ERK phosphorylation following E2 treatment (all P < 0.05). Interference of ESR2 caused significant reduction in the expressions of ER stress-related proteins and apoptosis-related proteins, up-regulated the genes related to cell proliferation, and increased intracellular pERK/ERK ratio in C28I2 cells. The effect of E2 binding to ERß, which promoted the expressions of ER stress associated proteins and apoptosis related proteins, was obviously antagonized by treatment of the cells with U0126. OBJECTIVE: The binding of E2 to ERß promotes ER stress and apoptosis in human chondrocytes by activating ERK pathway phosphorylation inhibit cell proliferation.


Asunto(s)
Estradiol , Receptor beta de Estrógeno , Apoptosis , Proliferación Celular , Condrocitos/metabolismo , Endorribonucleasas/metabolismo , Estradiol/farmacología , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Células HEK293 , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo
6.
Nat Commun ; 12(1): 1924, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772006

RESUMEN

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Demencia Frontotemporal/genética , Proteínas Mitocondriales/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Demencia Frontotemporal/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Homología de Secuencia de Aminoácido
7.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670622

RESUMEN

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Glándulas Paratiroides/metabolismo , Neoplasias de las Paratiroides/genética , Receptores Sensibles al Calcio/genética , Factores de Transcripción/genética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Amidas/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Neoplasias de las Paratiroides/metabolismo , Fenetilaminas/farmacología , Propilaminas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Piridinas/farmacología , Interferencia de ARN , Receptores Sensibles al Calcio/agonistas , Receptores Sensibles al Calcio/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo
8.
Nature ; 592(7852): 105-109, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33692546

RESUMEN

The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)1-4. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades5,6. Here we show that Arabidopsis PRR and PRR co-receptor mutants-fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants-are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI.


Asunto(s)
Arabidopsis/inmunología , Proteínas NLR/inmunología , Inmunidad de la Planta/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , NADPH Oxidasas/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas Serina-Treonina Quinasas/metabolismo , Pseudomonas syringae/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología
9.
Front Immunol ; 12: 573078, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33692778

RESUMEN

Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-ß promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-ß production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.


Asunto(s)
Alphacoronavirus/metabolismo , Interferón beta/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Factor 3 Asociado a Receptor de TNF/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alphacoronavirus/inmunología , Animales , Línea Celular , Coronavirus/inmunología , Coronavirus/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Interferón beta/inmunología , Interferón beta/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Porcinos , Factor 3 Asociado a Receptor de TNF/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo
10.
Viruses ; 13(2)2021 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-33668405

RESUMEN

Porcine deltacoronavirus (PDCoV) is an emerging infectious disease of swine with zoonotic potential. Phylogenetic analysis suggests that PDCoV originated recently from a host-switching event between birds and mammals. Little is known about how PDCoV interacts with its differing hosts. Human-derived cell lines are susceptible to PDCoV infection. Herein, we compare the gene expression profiles of an established host swine cells to potential emerging host human cells after infection with PDCoV. Cell lines derived from intestinal lineages were used to reproduce the primary sites of viral infection in the host. Porcine intestinal epithelial cells (IPEC-J2) and human intestinal epithelial cells (HIEC) were infected with PDCoV. RNA-sequencing was performed on total RNA extracted from infected cells. Human cells exhibited a more pronounced response to PDCoV infection in comparison to porcine cells with more differentially expressed genes (DEGs) in human, 7486, in comparison to pig cells, 1134. On the transcriptional level, the adoptive host human cells exhibited more DEGs in response to PDCoV infection in comparison to the primary pig host cells, where different types of cytokines can control PDCoV replication and virus production. Key immune-associated DEGs and signaling pathways are shared between human and pig cells during PDCoV infection. These included genes related to the NF-kappa-B transcription factor family, the interferon (IFN) family, the protein-kinase family, and signaling pathways such as the apoptosis signaling pathway, JAK-STAT signaling pathway, inflammation/cytokine-cytokine receptor signaling pathway. MAP4K4 was unique in up-regulated DEGs in humans in the apoptosis signaling pathway. While similarities exist between human and pig cells in many pathways, our research suggests that the adaptation of PDCoV to the porcine host required the ability to down-regulate many response pathways including the interferon pathway. Our findings provide an important foundation that contributes to an understanding of the mechanisms of PDCoV infection across different hosts. To our knowledge, this is the first report of transcriptome analysis of human cells infected by PDCoV.


Asunto(s)
Infecciones por Coronavirus/metabolismo , Células Epiteliales/virología , Enfermedades de los Porcinos/metabolismo , Transcriptoma , Animales , Línea Celular , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Interferones/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Porcinos
13.
Nat Metab ; 3(3): 428-441, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33758424

RESUMEN

Obesity reduces adipocyte mitochondrial function, and expanding adipocyte oxidative capacity is an emerging strategy to improve systemic metabolism. Here, we report that serine/threonine-protein kinase 3 (STK3) and STK4 are key physiological suppressors of mitochondrial capacity in brown, beige and white adipose tissues. Levels of STK3 and STK4, kinases in the Hippo signalling pathway, are greater in white than brown adipose tissues, and levels in brown adipose tissue are suppressed by cold exposure and greatly elevated by surgical denervation. Genetic inactivation of Stk3 and Stk4 increases mitochondrial mass and function, stabilizes uncoupling protein 1 in beige adipose tissue and confers resistance to metabolic dysfunction induced by high-fat diet feeding. Mechanistically, STK3 and STK4 increase adipocyte mitophagy in part by regulating the phosphorylation and dimerization status of the mitophagy receptor BNIP3. STK3 and STK4 expression levels are elevated in human obesity, and pharmacological inhibition improves metabolic profiles in a mouse model of obesity, suggesting STK3 and STK4 as potential targets for treating obesity-related diseases.


Asunto(s)
Adipocitos/metabolismo , Metabolismo Energético , Mitofagia , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Línea Celular , Humanos , Ratones , Ratones Noqueados , Obesidad/prevención & control , Obesidad/terapia , Proteínas Serina-Treonina Quinasas/genética
14.
Nat Commun ; 12(1): 1899, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771996

RESUMEN

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.


Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis , Treonina/metabolismo , Secuencias de Aminoácidos/genética , Animales , Aurora Quinasa A/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Activación Enzimática , Femenino , Humanos , Oocitos/metabolismo , Fosforilación , Prolina/genética , Prolina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Serina/genética , Serina/metabolismo , Treonina/genética , Xenopus laevis
15.
Eur J Med Chem ; 216: 113318, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33730624

RESUMEN

Identifying a pharmacological agent that targets only one of more than 500 kinases present in humans is an important challenge. One potential solution to this problem is the development of bivalent kinase inhibitors, which consist of two connected fragments, each bind to a dissimilar binding site of the bisubstrate enzyme. The main advantage of bivalent (type V) kinase inhibitors is generating more interactions with target enzymes that can enhance the molecules' selectivity and affinity compared to single-site inhibitors. Earlier type V inhibitors were not suitable for the cellular environment and were mostly used in in vitro studies. However, recently developed bivalent compounds have high kinase affinity, high biological and chemical stability in vivo. This review summarized the hetero-bivalent kinase inhibitors described in the literature from 2014 to the present. We attempted to classify the molecules by serine/threonine and tyrosine kinase inhibitors, and then each target kinase and its hetero-bivalent inhibitor was assessed in depth. In addition, we discussed the analysis of advantages, limitations, and perspectives of bivalent kinase inhibitors compared with the monovalent kinase inhibitors.


Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor EphA1/antagonistas & inhibidores , Receptor EphA1/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo
16.
Int J Mol Sci ; 22(4)2021 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-33668437

RESUMEN

Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.


Asunto(s)
Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Fosforilación/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transactivadores/genética
17.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670800

RESUMEN

Copper (Cu) dyshomeostasis plays a pivotal role in several neuropathologies, such as Parkinson's disease (PD). Metal accumulation in the central nervous system (CNS) could result in loss-of-function of proteins involved in Cu metabolism and redox cycling, generating reactive oxygen species (ROS). Moreover, neurodegenerative disorders imply the presence of an excess of misfolded proteins known to lead to neuronal damage. In PD, Cu accumulates in the brain, binds α-synuclein, and initiates its aggregation. We assessed the correlation between neuronal differentiation, Cu homeostasis regulation, and α-synuclein phosphorylation. At this purpose, we used differentiated SHSY5Y neuroblastoma cells to reproduce some of the characteristics of the dopaminergic neurons. Here, we reported that differentiated cells expressed a significantly higher amount of a copper transporter protein 1 (CTR1), increasing the copper uptake. Cells also showed a significantly more phosphorylated form of α-synuclein, further increased by copper treatment, without modifications in α-synuclein levels. This effect depended on the upregulation of the polo-like kinase 2 (PLK2), whereas the levels of the relative protein phosphatase 2A (PP2A) remained unvaried. No changes in the oxidative state of the cells were identified. The Cu dependent alteration of α-synuclein phosphorylation pattern might potentially offer new opportunities for clinical intervention.


Asunto(s)
Cobre/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , alfa-Sinucleína/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cobre/farmacología , Proteínas Transportadoras de Cobre/genética , Proteínas Transportadoras de Cobre/metabolismo , ATPasas Transportadoras de Cobre/genética , ATPasas Transportadoras de Cobre/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
18.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673252

RESUMEN

Senescence is the final stage of plant development, affecting individual organs or the whole organism, and it can be induced by several environmental factors, including shading or darkness. Although inevitable, senescence is a complex and tightly regulated process, ensuring optimal remobilization of nutrients and cellular components from senescing organs. Photoreceptors such as phytochromes and cryptochromes are known to participate in the process of senescence, but the involvement of phototropins has not been studied to date. We investigated the role of these blue light photoreceptors in the senescence of individually darkened Arabidopsis thaliana leaves. We compared several physiological and molecular senescence markers in darkened leaves of wild-type plants and phototropin mutants (phot1, phot2, and phot1phot2). In general, all the symptoms of senescence (lower photochemical activity of photosystem II, photosynthetic pigment degradation, down-regulation of photosynthetic genes, and up-regulation of senescence-associated genes) were less pronounced in phot1phot2, as compared to the wild type, and some also in one of the single mutants, indicating delayed senescence. This points to different mechanisms of phototropin operation in the regulation of senescence-associated processes, either with both photoreceptors acting redundantly, or only one of them, phot1, playing a dominant role.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Complejo de Proteína del Fotosistema II/genética , Hojas de la Planta/genética , Proteínas Serina-Treonina Quinasas/genética
19.
Nat Commun ; 12(1): 1736, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741957

RESUMEN

Metastasis is the leading cause of cancer-related death. Despite the recent advancements in cancer treatment, there is currently no approved therapy for metastasis. The present study reveals a potent and selective activity of PRAK in the regulation of tumor metastasis. While showing no apparent effect on the growth of primary breast cancers or subcutaneously inoculated tumor lines, Prak deficiency abrogates lung metastases in PyMT mice or mice receiving intravenous injection of tumor cells. Consistently, PRAK expression is closely associated with metastatic risk in human cancers. Further analysis indicates that loss of function of PRAK leads to a pronounced inhibition of HIF-1α protein synthesis, possibly due to reduced mTORC1 activities. Notably, pharmacological inactivation of PRAK with a clinically relevant inhibitor recapitulates the anti-metastatic effect of Prak depletion, highlighting the therapeutic potential of targeting PRAK in the control of metastasis.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metástasis de la Neoplasia , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias de la Mama , Línea Celular Tumoral , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Neoplasias/terapia , Proteínas Serina-Treonina Quinasas/genética
20.
Nat Commun ; 12(1): 1731, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741962

RESUMEN

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.


Asunto(s)
Supervivencia Celular/fisiología , Cerebelo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transporte de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Exosomas/metabolismo , Femenino , Interneuronas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Mutación , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Canales de Potasio Shaw/genética , Transducción de Señal
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