RESUMO
A via Hippo consiste em uma cascata de serina-treonina quinases que desempenha um papel central na transdução de sinais mecânicos. Em mamíferos, o eixo canônico da via consiste na ativação das quinases MST1 e MST2 (codificadas pelos genes STK4 e STK3, respectivamente) e LATS1 e LATS2. A ativação dos dois últimos culmina na fosforilação, retenção citoplasmática e inativação dos coativadores transcricionais YAP e TAZ. A inativação de Hippo resulta na localização nuclear de YAP/TAZ, aumento da proliferação e contribui para a transformação maligna em células epiteliais. No presente trabalho, identificamos que o exon 7, que codifica um segmento do domínio quinase de MST2, estava ausente em células malignas da glândula mamária humana, T4-2, mas não na linhagem não maligna, S1. A exclusão do exon 7 compromete a interação de MST2 com MOB1, um dos principais substratos de MST2. Ao contrário da proteína completa, a superexpressão de MST2 sem o exon 7 não resultou em aumento da morte celular, bem como, não diminuiu a proliferação celular. Esta nova variante de STK3/MST2, a qual denominamos STK3Δ7/MST2Δ7 é produto de um exon skipping e foi encontrada em amostras de tumores de pacientes, mas pouco predominante em amostras de tecidos normais. Além disso, em pacientes com câncer pancreático, a expressão STK3Δ7 resultou em menor sobrevida específica da doença. A retenção do exon 7 foi menor em tumores mais agressivos e com alto grau histológico. Em ensaio 3D, células não malignas com expressão ectópica de MST2Δ7 não respondem aos sinais inibitórios da membrana basal reconstituída e formam estruturas tumor-like. Esta nova variante perde sua atividade quinase e pode perturbar a homeostase tecidual pela incapacidade de ativar morte e inibir a proliferação celular, mesmo em microambientes repressores desses processos em células normais, como na presença membrana basal. Esses achados podem avançar o nosso conhecimento sobre progressão tumoral com possível relevância clínica
The Hippo pathway consists of a cascade of serine-threonine kinases that plays a central role in the transduction of mechanical signals. In mammals, the canonical axis of the pathway consists of the activation of the kinases MST1 and MST2 (encoded by the genes STK4 and STK3, respectively) and LATS1 and LATS2 and their activation culminates in the phosphorylation, cytoplasmic retention and inactivation of the transcriptional coactivators YAP and TAZ. Hippo inactivation results in nuclear localization of YAP/TAZ, increased cell proliferation, and contributes to malignant transformation in epithelial cells. In the present work, we identified that exon 7, which encodes a segment of the kinase domain of MST2, was absent in malignant cells of the human mammary gland, T4-2, but not in the non-malignant S1 cell line. Exclusion of exon 7 compromises the interaction of MST2 with one of its main substrates, MOB1. Unlike the full-length protein, overexpression of MST2 without exon 7 did not result in increased cell death, nor decreased cell proliferation. This new variant of STK3/MST2, which we named STK3Δ7/MST2Δ7, is the product of an exon skipping and was found in tumor samples, but seldomly found in samples of normal tissues. Furthermore, in patients with pancreatic cancer, STK3Δ7 expression resulted in lower disease-specific survival. Exon 7 retention was reduced in aggressive tumors with a high histological grade. In a 3D assay, non-malignant cells with ectopic expression of MST2Δ7, even at low concentrations, do not respond to inhibitory signals from a reconstituted basement membrane and form tumor-like structures. This new variant loses its kinase activity and may disturb the tissue homeostasis due to its inability to activate death and to inhibit cell proliferation, even in microenvironments that repress these processes in normal cells, such as the basement membrane. These findings may advance our knowledge about tumor progression and might be clinically relevant
Assuntos
Neoplasias Pancreáticas/patologia , Proliferação de Células , Via de Sinalização Hippo , Células/classificação , Morte Celular/imunologia , Proteínas Serina-Treonina Quinases , Células Epiteliais/classificação , Expressão Ectópica do GeneRESUMO
The RPW8s (Resistance to Powdery Mildew 8) are atypical broad-spectrum resistance genes that provide resistance to the powdery mildew fungi. Powdery mildew of rubber tree is one of the serious fungal diseases that affect tree growth and latex production. However, the RPW8 homologs in rubber tree and their role of resistance to powdery mildew remain unclear. In this study, four RPW8 genes, HbRPW8-a, b, c, d, were identified in rubber tree, and phylogenetic analysis showed that HbRPW8-a was clustered with AtRPW8.1 and AtRPW8.2 of Arabidopsis. The HbRPW8-a protein was localized on the plasma membrane and its expression in rubber tree was significantly induced upon powdery mildew infection. Transient expression of HbRPW8-a in tobacco leaves induced plant immune responses, including the accumulation of reactive oxygen species and the deposition of callose in plant cells, which was similar to that induced by AtRPW8.2. Consistently, overexpression of HbRPW8-a in Arabidopsis thaliana enhanced plant resistance to Erysiphe cichoracearum UCSC1 and Pseudomonas syringae pv. tomato DC30000 (PstDC3000). Moreover, such HbRPW8-a mediated resistance to powdery mildew was in a salicylic acid (SA) dependent manner. Taken together, we demonstrated a new RPW8 member in rubber tree, HbRPW8-a, which could potentially contribute the resistance to powdery mildew.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ascomicetos , Hevea , Arabidopsis/metabolismo , Hevea/genética , Hevea/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Expressão Ectópica do Gene , Filogenia , Espécies Reativas de Oxigênio/metabolismo , Látex/metabolismo , Doenças das Plantas/microbiologia , Ascomicetos/fisiologia , Erysiphe , Ácido Salicílico/metabolismo , Nicotiana/metabolismo , Resistência à Doença/genéticaRESUMO
Wheat is the most widely grown crop globally, providing 20% of all human calories and protein. Achieving step changes in genetic yield potential is crucial to ensure food security, but efforts are thwarted by an apparent trade-off between grain size and number. Expansins are proteins that play important roles in plant growth by enhancing stress relaxation in the cell wall, which constrains cell expansion. Here, we describe how targeted overexpression of an α-expansin in early developing wheat seeds leads to a significant increase in grain size without a negative effect on grain number, resulting in a yield boost under field conditions. The best-performing transgenic line yielded 12.3% higher average grain weight than the control, and this translated to an increase in grain yield of 11.3% in field experiments using an agronomically appropriate plant density. This targeted transgenic approach provides an opportunity to overcome a common bottleneck to yield improvement across many crops.
Assuntos
Expressão Ectópica do Gene , Triticum , Produtos Agrícolas/metabolismo , Grão Comestível/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/genética , Sementes/metabolismo , Triticum/genética , Triticum/metabolismoRESUMO
BACKGROUND: APETALA3 (AP3) has significant roles in petal and stamen development in accordance with the classical ABC model. RESULTS: The AP3 homolog, CDM19, from Chrysanthemum morifolium cv. Jinba was cloned and sequenced. Sequence and phylogenetic analyses revealed that CDM19 is of DEF/AP3 lineage possessing the characteristic MIKC-type II structure. Expression analysis showed that CDM19 was transcribed in petals and stamens of ray and disc florets with weak expression in the carpels. Ectopic expression of CDM19 in Arabidopsis wild-type background altered carpel development resulting in multi-carpel siliques. CDM19 could only partially rescue the Arabidopsis ap33 mutant. CONCLUSIONS: Our results suggest that CDM19 may partially be involved in petal and stamen development in addition to having novel function in carpel development.
Assuntos
Proteínas de Plantas/fisiologia , Proteínas de Plantas/genética , Arabidopsis/crescimento & desenvolvimento , Chrysanthemum , Flores/crescimento & desenvolvimento , Expressão Ectópica do GeneRESUMO
Background: Flavonoids are a kind of important secondary metabolite and are commonly considered to provide protection to plants against stress and UV-B for a long time. Anthocyanidin synthase (ANS), which encodes a dioxygenase in the flavonoid pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanidins, but there is no direct evidence indicating that it provides tolerance to stress in plants. Results: To investigate whether ANS can increase tolerance to abiotic stress, MaANS was isolated from mulberry fruits and transformed into tobacco. Our results suggested that the bacterially expressed MaANS protein can convert dihydroquercetin to quercetin. Overexpression of MaANS remarkably increased the accumulation of total flavonoids in transgenic lines and anthocyanins in corollas of flowers. Transgenic lines showed higher tolerance to NaCl and mannitol stress. Conclusions: These results indicated that MaANS participates in various dioxygenase activities, and it can protect plants against abiotic stress by improving the ROS-scavenging ability. Thus, this alternative approach in crop breeding can be considered in the improvement of stress tolerance by enriching flavonoid production in plants
Assuntos
Oxigenases/metabolismo , Nicotiana , Morus/enzimologia , Oxigenases/genética , Quercetina , Estresse Fisiológico , Bactérias , Flavonoides/metabolismo , Plantas Geneticamente Modificadas , Dioxigenases/metabolismo , Expressão Ectópica do GeneRESUMO
Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes1. In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV2-5. However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.
Assuntos
Aedes/metabolismo , Proteínas de Transporte/metabolismo , Vírus da Dengue/metabolismo , Expressão Ectópica do Gene , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Aedes/genética , Aedes/virologia , Animais , Animais Geneticamente Modificados , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Transporte/genética , Replicação do DNA , Dengue/virologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Proteínas de Drosophila , Feminino , Trato Gastrointestinal/virologia , Inativação Gênica , Interações Hospedeiro-Patógeno , Mosquitos Vetores/virologia , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/farmacologia , Replicação Viral , Zika virus/metabolismoRESUMO
Somatic embryogenesis receptor-like kinase 1 (SERK1) is a membrane receptor that might serve as common co-regulator of plant cell differentiation processes by forming heterodimers with specific receptor-like kinases. The Coffea canephora SERK1 homolog (CcSERK1) was cloned in this work, and its early function in the transcription of embryogenesis master genes and of genes encoding proteins involved in auxin metabolism was investigated by externally manipulating its expression in embryogenic leaf explants, before the appearance of embryogenic structures. Overexpression of CcSERK1 early during embryogenesis caused an increase in the number of somatic embryos when the 55-day process was completed. Suppression of CcSERK1 expression by RNA interference almost abolished somatic embryogenesis. Real time-PCR experiments revealed that the transcription of the CcAGL15, CcWUS, CcBBM, CcPKL, CcYUC1, CcPIN1 and CcPIN4 homologs was modified in direct proportion to the expression of CcSERK1 and that only CcLEC1 was inversely affected by the expression levels of CcSERK1. The expression of the CcYUC4 homolog was induced to more than 80-fold under CcSERK1 overexpression conditions, but it was also induced when CcSERK1 expression was silenced. The level of CcTIR1 was not affected by CcSERK1 overexpression but was almost abolished during CcSERK1 silencing. These results suggest that CcSERK1 co-regulates the induction of somatic embryogenesis in Coffea canephora by early activation of YUC-dependent auxin biosynthesis, auxin transport mediated by PIN1 and PIN4, and probably auxin perception by the TIR1 receptor, leading to the induction of early-stage homeotic genes (CcAGL15, CcWUS, CcPKL and CcBBM) and repression of late-stage homeotic genes (CcLec1).
Assuntos
Coffea/genética , Coffea/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas Quinases/genética , Sementes/genética , Clonagem Molecular , Expressão Ectópica do Gene , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Sementes/crescimento & desenvolvimento , Transcrição GênicaRESUMO
The ability to noninvasively diagnose acute cellular rejection (ACR) with high specificity and sensitivity would significantly advance personalized liver transplant recipient care and management of immunosuppression. We performed microRNA (miRNA) profiling in 318 serum samples from 69 liver transplant recipients enrolled in the Immune Tolerance Network immunosuppression withdrawal (ITN030ST) and Clinical Trials in Organ Transplantation (CTOT-03) studies. We quantified serum miRNA at clinically indicated and/or protocol biopsy events (n = 130). The trajectory of ACR diagnostic miRNAs during immunosuppression withdrawal were also evaluated in sera taken at predetermined intervals during immunosuppression minimization before and at clinically indicated liver biopsy (n = 119). Levels of 31 miRNAs were significantly associated with ACR diagnosis with two miRNAs differentiating ACR from non-ACR (area under the receiver operating characteristic curve = 90%, 95% confidence interval = 82%-96%) and predicted ACR events up to 40 days before biopsy-proven rejection. The most differentially expressed miRNAs were low or absent in the blood of healthy individuals but highly expressed in liver tissue, indicating an ectopic origin from the liver allograft. Pathway analyses of rejection-associated miRNAs and their target messenger RNAs (mRNAs) showed induction of proinflammatory and cell death-related pathways. Integration of differentially expressed serum miRNA with concordant liver biopsy mRNA demonstrates interaction between molecules with a known role in transplant rejection. CONCLUSION: Distinct miRNA levels profiled from sera at the time of clinical allograft dysfunction can be used to noninvasively diagnose ACR. Predictive trajectories of the same profile during supervised immunosuppression minimization diagnosed rejection up to 40 days prior to clinical expression. The rejection-associated miRNAs in sera appear to be ectopically expressed liver and specific immune cell miRNAs that are biologically related, and the consequences of immune-mediated damage to the allograft. (Hepatology 2017;65:269-280).
Assuntos
Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Transplante de Fígado , MicroRNAs/sangue , Expressão Ectópica do Gene , Feminino , Rejeição de Enxerto/genética , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , Transcriptoma , Transplante HomólogoRESUMO
Drought and salt stresses are the two major factors influencing the yield and quality of crops worldwide. Na(+)(K(+))/H(+) antiporters (NHXs) are ubiquitous membrane proteins that play important roles in maintaining the cellular pH and Na(+)(K(+)) homeostasis. The model plant Arabidopsis potentially encodes six NHX genes, namely AtNHX1 to 6. In the present study, AtNHX5, a comparatively less well-studied NHX, was cloned and transferred into a soybean variety, Dongnong-50, via Agrobacterium-mediated cotyledonary node transformation to assess its role in improving salt tolerance of the transgenic plants. The transgenic soybean plants were tolerant to the presence of 300 mM NaCl whereas the non-transgenic plants were not. Furthermore, after NaCl treatment, the transgenic plants had a higher content of free proline but lower content of malondialdehyde compared to the non-transgenic plants. Our results revealed that that AtNHX5 possibly functioned by efficiently transporting Na(+) and K(+) ions from the roots to the leaves. Overall, the results obtained in this study suggest that soybean salt tolerance could be improved through the over expression of Arabidopsis AtNHX5.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Glycine max/fisiologia , Tolerância ao Sal/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/metabolismo , Secas , Expressão Ectópica do Gene , Regulação da Expressão Gênica de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Glycine max/genética , Glycine max/metabolismoRESUMO
Escherichia coli cells have dozens of two-component systems to sense and respond to various stimuli, and thereby cope with changing environments. BaeS/BaeR is one such two-component system, and it deals with a variety of envelope stresses. Interestingly, the ArcA/ArcB and TorS/TorR two-component systems are known to be associated with initiation of DNA replication; however, the effects of BaeS/BaeR on initiation of DNA replication remain unknown. Flow cytometry analysis revealed that the average number of replication origins (oriCs) per cell in ΔbaeR mutants was approximately 30% higher than that in wild-type cells. So was the growth rate of ΔbaeR cells. Ectopic expression of BaeR from the pbaeR plasmid reversed the ΔbaeR mutant phenotypes. The results indicate that absence of BaeR leads to the early initiation of DNA replication. Further, deletion of BaeR caused an increase in the amount of DnaA per cell, but did not change concentration of DnaA, which is the initiator protein.The average number of oriCs per cell in Δspy mutants was the same as that found in the wild-type cells although spy gene expression is controlled by BaeR. These results suggest that BaeR may indirectly affect initiation of replication by controlling expression of the dnaA gene.