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1.
Mol Genet Genomics ; 295(5): 1253-1262, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32556999

RESUMO

Monogenic diabetes is a rare type of diabetes resulting from mutations in a single gene. To date, most cases remain genetically unexplained, posing a challenge for accurate diabetes treatment, which leads to on a molecular diagnosis. Therefore, a trio exome scan was performed in a lean, nonsyndromic Caucasian girl with diabetes onset at 2½ years who was negative for autoantibodies. The lean father had diabetes from age 11 years. A novel heterozygous mutation in EDEM2, c.1271G > A; p.Arg424His, was found in the proband and father. Downregulation of Edem2 in rat RIN-m ß-cells resulted in a decrease in insulin genes Ins1 to 67.9% (p = 0.006) and Ins2 to 16.8% (p < 0.001) and reduced insulin secretion by 60.4% (p = 0.0003). Real-time PCR revealed a major disruption of endocrine pancreas-specific genes, including Glut2 and Pxd1, with mRNA suppression to 54% (p < 0.001) and 85.7% (p = 0.01), respectively. No other expression changes related to stress or apoptotic genes were observed. Extended clinical follow-up involving ten family members showed that two healthy individuals carried the same mutation with no sign of diabetes in the clinical screen except for a slight increase in IA-2 antibody in one of them, suggesting incomplete penetrance. In conclusion, we describe EDEM2 as a likely/potential novel diabetes gene, in which inhibition in vitro reduces the expression of ß-cell genes involved in the glucose-stimulated insulin secretion (GSIS) pathway, leading to an overall suppression of insulin secretion but not apoptosis.


Assuntos
Diabetes Mellitus/genética , Regulação para Baixo , Transportador de Glucose Tipo 2/genética , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Mutação Puntual , Transativadores/genética , alfa-Manosidase/genética , Idade de Início , Idoso , Animais , Linhagem Celular , Diabetes Mellitus/metabolismo , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Inativação Gênica , Humanos , Insulina/genética , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Linhagem , Ratos , Sequenciamento Completo do Exoma , Adulto Jovem
2.
Anticancer Res ; 40(6): 3255-3264, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487620

RESUMO

BACKGROUND/AIM: Rta, a transactivator of Epstein-Barr virus, is associated with progression of nasopharyngel carcinoma (NPC); however, its mechanism of contribution to the pathogenesis of NPC remains unclear. Interleukin-6 (IL-6), a tumor promoter, is detected in NPC. This in vitro study examined whether and how Rta promotes NPC progression by up-regulating IL-6. MATERIALS AND METHODS: Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, ELISA, immunoblotting assays, reporter gene assays, and transwell migration assays were performed. RESULTS: In NPC cells, Rta up-regulated IL-6 expression at the mRNA and protein levels, and the Rta's C-terminus was essential for promoter activation and expression of IL-6. The induction of IL-6 by Rta also required activation of extracellular signal-regulated kinase 1/2 and activator protein-1. Furthermore, IL-6 secreted from Rta-expressing NPC cells promoted migration of Rta-negative NPC cells by activating IL-6 receptor/Janus kinase/signal transducer and activator of transcription 3 pathway. CONCLUSION: Rta contributes to progression of NPC cells through induction of IL-6 in vitro.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Neoplasias/metabolismo , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transativadores/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/virologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologia , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia , Transativadores/genética , Fator de Transcrição AP-1/metabolismo , Transfecção , Regulação para Cima
3.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461317

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The nonstructural protein nsp5, also called 3C-like protease, is responsible for processing viral polyprotein precursors in coronavirus (CoV) replication. Previous studies have shown that PDCoV nsp5 cleaves the NF-κB essential modulator and the signal transducer and activator of transcription 2 to disrupt interferon (IFN) production and signaling, respectively. Whether PDCoV nsp5 also cleaves IFN-stimulated genes (ISGs), IFN-induced antiviral effector molecules, remains unclear. In this study, we screened 14 classical ISGs and found that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a (pDCP1A) through its protease activity. Similar cleavage of endogenous pDCP1A was also observed in PDCoV-infected cells. PDCoV nsp5 cleaved pDCP1A at glutamine 343 (Q343), and the cleaved pDCP1A fragments, pDCP1A1-343 and pDCP1A344-580, were unable to inhibit PDCoV infection. Mutant pDCP1A-Q343A, which resists nsp5-mediated cleavage, exhibited a stronger ability to inhibit PDCoV infection than wild-type pDCP1A. Interestingly, the Q343 cleavage site is highly conserved in DCP1A homologs from other mammalian species. Further analyses demonstrated that nsp5 encoded by seven tested CoVs that can infect human or pig also cleaved pDCP1A and human DCP1A, suggesting that DCP1A may be the common target for cleavage by nsp5 of mammalian CoVs.IMPORTANCE Interferon (IFN)-stimulated gene (ISG) induction through IFN signaling is important to create an antiviral state and usually directly inhibits virus infection. The present study first demonstrated that PDCoV nsp5 can cleave mRNA-decapping enzyme 1a (DCP1A) to attenuate its antiviral activity. Furthermore, cleaving DCP1A is a common characteristic of nsp5 proteins from different coronaviruses (CoVs), which represents a common immune evasion mechanism of CoVs. Previous evidence showed that CoV nsp5 cleaves the NF-κB essential modulator and signal transducer and activator of transcription 2. Taken together, CoV nsp5 is a potent IFN antagonist because it can simultaneously target different aspects of the host IFN system, including IFN production and signaling and effector molecules.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Coronavirus/metabolismo , Cisteína Endopeptidases/metabolismo , Endorribonucleases/metabolismo , Transativadores/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Exorribonucleases/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Suínos , Doenças dos Suínos/virologia
4.
PLoS Genet ; 16(5): e1008818, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469866

RESUMO

The Hippo signalling pathway and its central effector YAP regulate proliferation of cardiomyocytes and growth of the heart. Using genetic models in mice we show that the increased proliferation of embryonal and postnatal cardiomyocytes due to loss of the Hippo-signaling component SAV1 depends on the Myb-MuvB (MMB) complex. Similarly, proliferation of postnatal cardiomyocytes induced by constitutive active YAP requires MMB. Genome studies revealed that YAP and MMB regulate an overlapping set of cell cycle genes in cardiomyocytes. Protein-protein interaction studies in cell lines and with recombinant proteins showed that YAP binds directly to B-MYB, a subunit of MMB, in a manner dependent on the YAP WW domains and a PPXY motif in B-MYB. Disruption of the interaction by overexpression of the YAP binding domain of B-MYB strongly inhibits the proliferation of cardiomyocytes. Our results point to MMB as a critical downstream effector of YAP in the control of cardiomyocyte proliferation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/genética , Miócitos Cardíacos/citologia , Transativadores/química , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Miócitos Cardíacos/química , Regiões Promotoras Genéticas , Ratos
5.
PLoS Genet ; 16(5): e1008722, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392214

RESUMO

To survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins, depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA's cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Salmonella enterica/genética , Salmonella enterica/metabolismo , Transativadores/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo
6.
PLoS Negl Trop Dis ; 14(5): e0008274, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357189

RESUMO

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management.


Assuntos
Adesinas de Escherichia coli/genética , Bacteriemia/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/classificação , Fímbrias Bacterianas/genética , Genótipo , Transativadores/genética , Adolescente , Bacteriemia/epidemiologia , Criança , Pré-Escolar , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Moçambique/epidemiologia , Reação em Cadeia da Polimerase , Sorogrupo , Sequenciamento Completo do Genoma
7.
PLoS One ; 15(5): e0233394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453735

RESUMO

Chromodomain helicase DNA-binding (CHD) chromatin remodelers regulate transcription and DNA repair. They govern cell-fate decisions during embryonic development and are often deregulated in human pathologies. Chd1-8 show upon germline disruption pronounced, often developmental lethal phenotypes. Here we show that contrary to Chd1-8 disruption, Chd9-/-animals are viable, fertile and display no developmental defects or disease predisposition. Germline deletion of Chd9 only moderately affects gene expression in tissues and derived cells, whereas acute depletion in human cancer cells elicits more robust changes suggesting that CHD9 is a highly context-dependent chromatin regulator that, surprisingly, is dispensable for mouse development.


Assuntos
DNA Helicases/genética , Transativadores/genética , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa , Humanos , Células K562 , Camundongos , Células-Tronco Embrionárias Murinas/citologia
8.
Environ Pollut ; 264: 114778, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32417585

RESUMO

The hexafluoropropylene-oxide-dimer-acid (GenX) is a short-chain perfluoroalkyl substance that was recently introduced following the phase out of PFOA, as an alternative for the process of polymerization. GenX was detected at high concentrations in rivers, drinking water and in sera of exposed workers and recent findings suggested its potential dangerousness for human health. Aim of the study was to assess the consequences of GenX exposure on in vitro thyroid cells with particular attention to the effects on cell-viability, proliferation, DNA-damage and in the thyroid-related genes expression. FRTL-5 rat-thyroid cell line were incubated with increasing concentrations of GenX for 24 h, 48 h and 72 h to assess cell viability by WST-1. DNA-damage was assessed by comet assay and further confirmed by micronucleus assay. The proliferation of survived cells was measured by staining with crystal violet and evaluation of its optical density after incubation with SDS. Changes in TTF-1, Pax8, Tg, TSH-R, NIS and TPO genes expression were evaluated by RT-PCR. GenX exposure reduced FRTL-5 viability in a time and dose-dependent manner (24 h: ANOVA F = 22.286; p < 0.001; 48 h: F = 43.253, p < 0.001; 72 h: F = 49.708, p < 0.001). Moreover, GenX exerted a genotoxic effect, as assessed by comet assay (significant increase in tail-length, olive-tail-moment and percentage of tail-DNA) and micronucleus assay, both at cytotoxic and non-cytotoxic concentrations. Exposure to GenX at concentrations non-cytotoxic exerted a significant lowering of the expression of the regulatory gene TTF-1 (p < 0.05 versus untreated) and higher expression of Pax-8 (p < 0.05 versus untreated) and a down-regulation of NIS (p < 0.05 versus untreated). In addition, cells survived to GenX exposure showed a reduced re-proliferation ability (24 h: ANOVA F = 11,941; p < 0,001; 48 h: F = 93.11; p < 0.001; 72 h F = 21.65; p < 0.001). The exposure to GenX produces several toxic effects on thyroid cells in vitro. GenX is able to promote DNA-damage and to affect the expression of thyroid transcription-factor genes.


Assuntos
Proteínas Nucleares , Glândula Tireoide , Animais , Sobrevivência Celular , DNA , Humanos , Fatores de Transcrição Box Pareados , Ratos , Transativadores
9.
Gene ; 752: 144788, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32439375

RESUMO

Primulina genus is an ideal wild ornamental flower and emerging model for studying biosynthesis, diversity, and evolution of flower pigment. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation in Primulina remains unknown. Here, changes in anthocyanin content and the expression profiles of anthocyanin biosynthetic structural genes were examined in developing Primulina swinglei flowers and three other organs. Seventy-three R2R3-MYB transcription factor genes were identified from transcriptome of P. swinglei flowers, two of which, PsMYB1 and PsMYB2, are candidate regulators of anthocyanin biosynthesis according to clustering analysis. Furthermore, transient over-expression studies using tobacco leaves showed distinct pigment accumulation following co-infection with PsMYB1 and MrbHLH1 (a previously confirmed anthocyanin regulator from Morella rubra). Additionally, dual luciferase assays showed that PsMYB1 trans-activated the PsANS promoter, with the addition of MrbHLH1 resulting in a 5-fold increase in the intensity of this interaction. PsMYB1 did not, however, have any effect on the PsF3H promoter. The expression profile and dual luciferase assays showed that PsMYB2 plays no roles in anthocyanin regulation. Therefore, PsMYB1 is proposed to be the transcription factor gene regulating anthocyanin biosynthesis in P. swinglei.


Assuntos
Antocianinas/biossíntese , Antocianinas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos/genética , Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Lamiales/genética , Magnoliopsida/genética , Pigmentação/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Transativadores/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética
10.
PLoS One ; 15(5): e0232356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357159

RESUMO

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-ß (TGF-ß), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-ß2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-ß2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-ß2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-ß and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.


Assuntos
Ativinas/metabolismo , Células Endoteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/fisiologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Vasos Linfáticos/citologia , Proteína Smad2/fisiologia , Transativadores/fisiologia , Quinases Associadas a rho/metabolismo
11.
PLoS One ; 15(5): e0226453, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379825

RESUMO

Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic "on-off" switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate.


Assuntos
Adaptação Fisiológica/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Genes de Troca , Modelos Biológicos , Sítios de Ligação , Transporte Biológico/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ribonucleosídeos/metabolismo , Processos Estocásticos , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética
12.
Phys Chem Chem Phys ; 22(15): 8118-8127, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32242581

RESUMO

Many intrinsically disordered proteins (IDPs) are involved in complex signalling networks inside the cell. Their particular binding modes elicit different types of responses that can be subtly regulated. Here we study the binding of two disordered transactivation domains from proteins HIF-1α and CITED2, whose binding to the TAZ1 domain of CBP is critical for the hypoxic response. Experiments have shown that both IDPs compete for their shared partner, and that this competition is mediated by the formation of a ternary intermediate state. Here we use computer simulations with a coarse-grained model to provide a detailed molecular description of this intermediate. We find that the conserved LP(Q/E)L motif may have a critical role in the displacement of HIF-1α by CITED2 and show a possible mechanism for the transition from the intermediate to the bound state. We also explore the role of TAZ1 dynamics in the binding. The results of our simulations are consistent with many of the experimental observations and provide a detailed view of the emergent properties in the complex binding of these IDPs.


Assuntos
Simulação por Computador , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Modelos Moleculares , Domínios Proteicos , Proteínas Repressoras/química , Transativadores/química , Motivos de Aminoácidos , Ligação Proteica , Estrutura Quaternária de Proteína
13.
PLoS One ; 15(4): e0231265, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267872

RESUMO

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in the regulation of cell proliferation and differentiation. TAZ activity changes in response to the cellular environment such as mechanic and nutritional stimuli, osmolarity, and hypoxia. To understand the physiological roles of TAZ, chemical compounds that activate TAZ in cells are useful as experimental reagents. Kaempferol, TM-25659, and ethacridine are reported as TAZ activators. However, as each TAZ activator has a distinct property in cellular functions, additional TAZ activators are awaiting. We screened for TAZ activators and previously reported IB008738 as a TAZ activator that promotes myogenesis in C2C12 cells. In this study, we have characterized IBS004735 that was obtained in the same screening. IBS004735 also promotes myogenesis in C2C12 cells, but is not similar to IBS008738 in the structure. IBS004735 activates TAZ via Akt and has no effect on TAZ phosphorylation, which is the well-described key modification to regulate TAZ activity. Thus, we introduce IBS004735 as a novel TAZ activator that regulates TAZ in a yet unidentified mechanism.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Imidazóis/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologia , Transativadores/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Mioblastos Esqueléticos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Transativadores/genética , Transfecção
14.
Plant Physiol Biochem ; 151: 535-544, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32305820

RESUMO

NAC transcription factors play a pivotal role in plant growth, development and response to abiotic stress. However, their biological functions in desert trees are largely unknown. In this work, the NAC transcription factor HaNAC1 from Haloxylon ammodendron, a typical wooden plant normally grown in desert, was isolated, and its possible role in plant growth and resistance to drought stress was investigated. HaNAC1 encodes an ATAF subfamily transcription factor containing one NAC domain with five conserved regions. Quantitative real time PCR analyses revealed that HaNAC1 was ubiquitously expressed in various tissues and organs such as roots, stems, leaves and seeds, with a predominant expression in stems. Further studies demonstrated that expression of HaNAC1 was significantly induced by osmotic stress in Haloxylon ammodendron seedlings, and subcellular localization analysis indicated that GFP-HaNAC1 fusion protein was localized to the nucleus in Arabidopsis leaf protoplast. Ectopic expression of HaNAC1 led to promoted growth and drought tolerance in transgenic Arabidopsis, accompanied with up-regulated expression of stress-inducible marker genes, and increased accumulation of proline, IAA and ABA under both normal and drought stress conditions. In addition, co-immunoprecipitation and Bi-molecular fluorescence complementation assays illustrated that HaNAC1 directly interacted with AtNAC32. All these results suggest that HaNAC1 is involved in both the growth and drought resistance of Haloxylon ammodendron, and could be used as a promising candidate gene for the breeding of crops with augmented tolerance to drought stress.


Assuntos
Arabidopsis , Chenopodiaceae , Proteínas de Plantas , Estresse Fisiológico , Transativadores , Arabidopsis/genética , Chenopodiaceae/genética , Secas , Expressão Ectópica do Gene , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Estresse Fisiológico/genética , Transativadores/genética , Fatores de Transcrição/genética
15.
Nat Commun ; 11(1): 1794, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286285

RESUMO

Although group 3 innate lymphoid cells (ILC3s) are efficient inducers of T cell responses in the spleen, they fail to induce CD4+ T cell proliferation in the gut. The signals regulating ILC3-T cell responses remain unknown. Here, we show that transcripts associated with MHC II antigen presentation are down-modulated in intestinal natural cytotoxicity receptor (NCR)- ILC3s. Further data implicate microbiota-induced IL-23 as a crucial signal for reversible silencing of MHC II in ILC3s, thereby reducing the capacity of ILC3s to present antigen to T cells in the intestinal mucosa. Moreover, IL-23-mediated MHC II suppression is dependent on mTORC1 and STAT3 phosphorylation in NCR- ILC3s. By contrast, splenic interferon-γ induces MHC II expression and CD4+ T cell stimulation by NCR- ILC3s. Our results thus identify biological circuits for tissue-specific regulation of ILC3-dependent T cell responses. These pathways may have implications for inducing or silencing T cell responses in human diseases.


Assuntos
Apresentação do Antígeno/imunologia , Imunidade Inata , Linfócitos/imunologia , Microbiota , Baço/citologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/metabolismo , Polaridade Celular , Regulação para Baixo , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/metabolismo , Interleucina-23/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Microbiota/genética , Microbiota/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Fosforilação , Análise de Componente Principal , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T/imunologia , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética
16.
PLoS Pathog ; 16(4): e1008402, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251483

RESUMO

Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, late in infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection using dTag methodology and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that in late infection the IE2 proteins target a distinct subset of HCMV early-late and late promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/metabolismo , Replicação do DNA , Proteínas Imediatamente Precoces/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transativadores/metabolismo , Proteínas Virais/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , DNA Viral/genética , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , RNA Polimerase II/genética , Transativadores/genética , Iniciação da Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral
17.
Gene ; 749: 144679, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32330536

RESUMO

BACKGROUND: Hepatocellular carcinoma is one of the most common cancers worldwide. HBV-related HCC has characteristics of faster progression and worse prognosis. Previous studies have confirmed that HBx protein plays numbers of important roles in development of HBV-HCC. However, the molecular mechanism of carcinogenicity of HBx is still not well documented. METHODS: Firstly, a HCC cell line over-expressing HBx was established and its function was verified. Subsequently, the differentially expressed genes were detected by transcriptome sequencing technology and use the Western Blot technology to detect the up-regulated genes in HBx overexpressed cells, and the functional correlation of the genes was analyzed. Finally, tissue microarray was used to correlate up-regulated gene with clinical follow-up data to verify correlation with clinical prognosis. RESULTS: Over-expression of HBx could promote cell proliferation, and over-expression of HBx could up-regulate the expression of S100A4 protein. ShRNA experiments showed that HBx promoted cell proliferation by upregulating the expression of S100A4. IFN-α2b can down-regulate the expression of S100A4 and inhibit the proliferation of HCC cells. The expression of S100A4 in cancer was significantly up-regulated compared with adjacent tissues, and was also significantly associated with tumors volume, the expression of PD-L1 and the survival time of patients with HCC. CONCLUSION: In general, S100A4 may be an effective therapeutic target for HBV-HCC. And the connection between S100A4 and HBV are not clear yet. This study may play a guiding role in the future clinical treatment of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Transativadores/metabolismo , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Proliferação de Células , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína A4 de Ligação a Cálcio da Família S100/antagonistas & inibidores , Regulação para Cima
18.
PLoS Genet ; 16(4): e1008693, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324833

RESUMO

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.


Assuntos
Ciclo Celular , Proliferação de Células , Desenvolvimento Fetal , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico , Transativadores/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Ciclina B1/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Transativadores/genética
19.
Nat Commun ; 11(1): 1618, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238808

RESUMO

In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2-4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ70-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.


Assuntos
Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/genética , Proteínas de Escherichia coli , Genes Bacterianos/genética , Regiões Promotoras Genéticas , RNA Guia/genética , Transativadores , Ativação Transcricional
20.
Nat Commun ; 11(1): 1846, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296056

RESUMO

Seed size is a key agronomic trait that greatly determines plant yield. Elucidating the molecular mechanism underlying seed size regulation is also an important question in developmental biology. Here, we show that the KIX-PPD-MYC-GIF1 pathway plays a crucial role in seed size control in Arabidopsis thaliana. Disruption of KIX8/9 and PPD1/2 causes large seeds due to increased cell proliferation and cell elongation in the integuments. KIX8/9 and PPD1/2 interact with transcription factors MYC3/4 to form the KIX-PPD-MYC complex in Arabidopsis. The KIX-PPD-MYC complex associates with the typical G-box sequence in the promoter of GRF-INTERACTING FACTOR 1 (GIF1), which promotes seed growth, and represses its expression. Genetic analyses support that KIX8/9, PPD1/2, MYC3/4, and GIF1 function in a common pathway to control seed size. Thus, our results reveal a genetic and molecular mechanism by which the transcription factors MYC3/4 recruit KIX8/9 and PPD1/2 to the promoter of GIF1 and repress its expression, thereby determining seed size in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Transativadores/genética , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Pirofosfatases/genética , Pirofosfatases/metabolismo
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