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1.
Plant Mol Biol ; 101(4-5): 499-506, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31621004

RESUMO

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different auxin response factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos Orgânicos , Proteínas de Plantas/metabolismo , Soja/metabolismo , Fatores de Transcrição/metabolismo , Fluorescência , Ligação Proteica , Sensibilidade e Especificidade
3.
J Cancer Res Clin Oncol ; 145(12): 3075-3087, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31595333

RESUMO

PURPOSE: Bladder cancer represents a major cause of malignancy-related morbidity and the most expensive per-patient-to-treat cancer, due to the lifelong surveillance of the patients. Accurate disease prognosis is essential in establishing personalized treatment decisions; yet optimum tools for precise risk stratification remain a competing task. In the present study, we have performed the complete evaluation of TP63 clinical significance in improving disease prognosis. METHODS: The levels of ΔNp63 and TAp63 transcripts of TP63 were quantified in 342 bladder tissue specimens of our screening cohort (n = 182). Hedegaard et al. (Cancer Cell 30:27-42. doi:10.1016/j.ccell.2016.05.004, 2016) (n = 476) and TCGA provisional (n = 413) were used as validation cohorts for NMIBC and MIBC, respectively. Survival analysis was performed using recurrence and progression for NMIBC or mortality for MIBC as endpoint events. Bootstrap analysis was performed for internal validation, while decision curve analysis was used for the evaluation of the clinical net benefit on disease prognosis. RESULTS: ΔNp63 was significantly expressed in bladder tissues, and was found to be over-expressed in bladder tumors. Interestingly, reduced ΔNp63 levels were correlated with muscle-invasive disease, high-grade tumors and high-EORTC-risk NMIBC patients. Moreover, ΔNp63 loss was independently associated with higher risk for NMIBC relapse (HR = 2.730; p = 0.007) and progression (HR = 7.757; p = 0.016). Hedegaard et al. and TCGA validation cohorts confirmed our findings. Finally, multivariate models combining ΔΝp63 loss with established prognostic markers led to a superior clinical benefit for NMIBC prognosis and risk stratification. CONCLUSIONS: ΔΝp63 loss is associated with adverse outcome of NMIBC resulting in superior prediction of NMIBC early relapse and progression.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Recidiva Local de Neoplasia/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Doença Crônica , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Invasividade Neoplásica/patologia , Recidiva Local de Neoplasia/patologia , Prognóstico , Recidiva , Neoplasias da Bexiga Urinária/patologia
4.
Anticancer Res ; 39(10): 5437-5448, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570438

RESUMO

BACKGROUND/AIM: Epithelial-mesenchymal transition (EMT) is a key multi-step process which enables cancer cells to detach from the epithelial primary tumor mass and allows them to metastasize to distant organs. We immunohistochemically analyzed the expression of the transcription factors (TWIST-1, SLUG, ZEB1, ZEB2) and components of the extracellular matrix (laminin-5, fibronectin) which influence the EMT. MATERIALS AND METHODS: Primary human breast (MDA-MB-231), colon (HT29, HCT116), ovarian (SKOV3, OVCAR3) and head and neck squamous cell carcinoma cell lines (UTSCC2, UTSCC24A) grown as xenografts were immunohistochemically analyzed in vitro and in vivo. RESULTS: A high SLUG expression was observed in every cancer entity both in vitro and in vivo. ZEB1 and ZEB2 showed a high in vivo expression especially in SKOV3 and in in vitro grown MDA-MB-231 cells. CONCLUSION: SLUG expression showed the highest expression in all cancer entities investigated. Hence, it presumably represents the master regulator of EMT in these metastatic tumor entities.


Assuntos
Biomarcadores Tumorais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Células HCT116 , Células HT29 , Humanos , Imuno-Histoquímica/métodos , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
5.
J Agric Food Chem ; 67(37): 10513-10520, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31475823

RESUMO

Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected ß-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of SREBP-1c gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.


Assuntos
Aminoácidos/metabolismo , Bovinos/metabolismo , Células Epiteliais/metabolismo , Gorduras/metabolismo , Glândulas Mamárias Animais/citologia , Leite/metabolismo , Fatores de Transcrição/metabolismo , Animais , Bovinos/genética , Feminino , Glândulas Mamárias Animais/metabolismo , Camundongos , Fosforilação , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética
6.
Chem Biol Interact ; 312: 108813, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494105

RESUMO

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival. YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus. The aim of this study is to evaluate the action of VP on RMS cell lines. This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment. In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.


Assuntos
Proliferação de Células/efeitos dos fármacos , Verteporfina/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Fatores de Transcrição/metabolismo
7.
J Agric Food Chem ; 67(42): 11815-11824, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31550160

RESUMO

Yan73 is a teinturier Vitis vinifera variety with red berry flesh, but the molecular mechanisms underlying its flesh coloration remain unclear. We analyzed the flavonoid metabolic and transcriptome profiles of Yan73 berry red and white flesh using HPLC-ESI-MS/MS and RNA-sequencing technologies. Anthocyanins are the main flavonoids responsible for Yan73 berry flesh color, and the coloration is coordinately regulated by the VvMYBA1 transcriptional activator and VvMYBC2-L1 transcriptional repressor. Furthermore, yeast one- and two-hybrid, dual luciferase, and bimolecular fluorescence complementation assays suggested that VvMYBA1 positively regulates Yan73 berry flesh color via interactions with VvWDR1 and the activation of the VvCHI3, VvOMT, and VvGST4 promoters, whereas VvMYBC2-L1 negatively regulates Yan73 berry flesh color, possibly by competing with the R2R3-MYB transcriptional activators for bHLH partners or by repressing VvOMT and VvGST4 expression. Our findings provide new insights into the molecular mechanisms regulating grape flesh color.


Assuntos
Frutas/química , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas Repressoras/genética , Vitis/genética , Cor , Frutas/genética , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vitis/química , Vitis/metabolismo
8.
Nat Cell Biol ; 21(10): 1191-1205, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548610

RESUMO

Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Morfogênese , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Repetição de Anquirina/genética , Células Cultivadas , Proteínas do Citoesqueleto/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Domínios Proteicos/genética , Ratos , Fatores de Transcrição/genética
9.
Genes Dev ; 33(17-18): 1117-1135, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31481536

RESUMO

T-cell development in mammals is a model for lineage choice and differentiation from multipotent stem cells. Although T-cell fate choice is promoted by signaling in the thymus through one dominant pathway, the Notch pathway, it entails a complex set of gene regulatory network and chromatin state changes even before the cells begin to express their signature feature, the clonal-specific T-cell receptors (TCRs) for antigen. This review distinguishes three developmental modules for T-cell development, which correspond to cell type specification, TCR expression and selection, and the assignment of cells to different effector types. The first is based on transcriptional regulatory network events, the second is dominated by somatic gene rearrangement and mutation and cell selection, and the third corresponds to establishing a poised state of latent regulator priming through an unknown mechanism. Interestingly, in different lineages, the third module can be deployed at variable times relative to the completion of the first two modules. This review focuses on the gene regulatory network and chromatin-based kinetic constraints that determine activities of transcription factors TCF1, GATA3, PU.1, Bcl11b, Runx1, and E proteins in the primary establishment of T-cell identity.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Linfócitos T/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Cromatina/metabolismo , Redes Reguladoras de Genes , Hematopoese , Linfócitos T/metabolismo , Timo/citologia , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
J Agric Food Chem ; 67(39): 10891-10903, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31505929

RESUMO

Jasmonates (JAs) play an important role in plant developmental processes and regulate the biosynthesis of various specialized metabolites, and transcription factors are crucial in mediating JA signaling to regulate these processes. Capsaicinoids (Caps) are intriguing specialized metabolites produced uniquely by Capsicum species that give their fruits a pungent flavor to defend against herbivory and pathogens. In this study, we identify a R2R3-MYB transcription factor CaMYB108 and demonstrate its roles in regulating the biosynthesis of Caps and stamen development. Transcriptional analysis indicated that CaMYB108 was preferentially expressed in the flower and fruit, while the subcellular localization of CaMYB108 was shown to be the nucleus. Virus-induced gene silencing of CaMYB108 led to the expression of capsaicinoid biosynthetic genes (CBGs), and the contents of Caps dramatically reduce. Moreover, the CaMYB108-silenced plants showed delayed anther dehiscence and reduced pollen viability. Transient overexpression of CaMYB108 caused the expression of CBGs to be upregulated, and the Caps content significantly increased. The results of dual-luciferase reporter assays showed that CaMYB108 targeted CBG promoters. In addition, the expression of CaMYB108 and CBGs was inducible by methyl jasmonate and was consistent with the increased content of Caps. Overall, our results indicate that CaMYB108 is involved in the regulation of Caps biosynthesis and stamen development.


Assuntos
Capsaicina/metabolismo , Capsicum/metabolismo , Ciclopentanos/metabolismo , Flores/crescimento & desenvolvimento , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Capsicum/genética , Capsicum/crescimento & desenvolvimento , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética
11.
Zhonghua Bing Li Xue Za Zhi ; 48(8): 590-595, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31422588

RESUMO

Objective: To investigate the expression of SMARCA4 (BRG1) and SMARCB1 (INI-1) protein in endometrial dedifferentiated carcinoma (DDC) and undifferentiated carcinoma (UDC), and their correlation with clinicopathologic features. Methods: Clinicopathological information was gathered for 26 cases of DDC and UDC and consulting hospitals from January, 2006 to December, 2018 in Fudan University Shanghai Cancer Center, including 10 cases of DDC and 16 cases of UDC. Morphologic features and diagnosis were reviewed by two pathologists. Immunohistochemistry for expression of BRG1 and INI1 protein was performed. The correlations with clinicopathologic features were analyzed. Results: BRG1 and INI1 loss were present in 14 of 26 cases of DDC/UDC, including 12 BRG1-deficient cases and 2 INI1-deficient cases, respectively. Six cases demonstrated variable amounts of rhabdoid cells in 14 BRG1/INI1-deficient cases, and only 1 case showed rhabdoid cells in the 12 intact expression cases. However, there was no significantly statistical difference (P=0.060). Age, invasive depth, lymph node status and FIGO stage were not associated with the expression of the BRG1 and INI1 (P=0.437, P=0.672, P=0.242, P=0.348). Remarkably, the BGR1/INI1-deficient patients had worse survival than those with intact expression (4.7 vs. 22.9, P=0.033). Conclusion: BRG1/INI1-deficient is observed in approximately half of DDC and UDC. Identification of these tumors is clinically relevant due to their more aggressive behavior and poor prognosis. Hence, BRG1 and INI1 immunohistochemical stains should be performed for DDC and UDC in order to help the pathologists to distinguish these tumors from other carcinomas, and to predict the clinical prognosis.


Assuntos
DNA Helicases/metabolismo , Neoplasias do Endométrio , Proteínas Nucleares/metabolismo , Proteína SMARCB1/metabolismo , Fatores de Transcrição/metabolismo , Biomarcadores Tumorais , China , Neoplasias do Endométrio/mortalidade , Feminino , Humanos , Imuno-Histoquímica
12.
Nat Methods ; 16(9): 866-869, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451765

RESUMO

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ácidos Indolacéticos/farmacologia , Proteólise/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Colesterol/metabolismo , Citoplasma/metabolismo , Células HEK293 , Humanos , Reguladores de Crescimento de Planta/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
13.
Gene ; 718: 144018, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31454543

RESUMO

Cytosine DNA methylation (5mC) is an epigenetic mark that regulates gene expression in plant responses to environmental stresses. Zinc-finger protein (ZFP) is the largest family of DNA-binding transcription factors that also plays an essential role in eukaryote. In plant we have already identified and characterized different useful ZFP-genes. While, the main objective of this research was to observe and identify more targeted stress responsive genes of ZFPs epigenetically throughout genome in rice for the first time. A comprehensive correlation analysis was performed through methylated DNA immunoprecipitation (MeDIP)-chip hybridization in rice under salt and osmotic stresses. High salinity and drought are two major abiotic hazards that are destroying the crop world-wide. As a result, Through-out genome 14 unique stress responsive transcription factors of ZFP-genes with varying level of methylation and expression under two conditions (control vs. stress) were isolated. All the identified genes were confirmed from different databases for their specific structure, cis-regulatory elements, phylogenetic analysis, and synteny analysis. Moreover, the tissue-specific expression patterns, and expression under abiotic and phytohormones stresses were also investigated. Phylogenetically all the genes were divided into 6 distinct subgroups with Arabidopsis and orthologous proteins were find-out through synteny analysis. Available RNA-seq data in response to various phytohormones provided hormone inducible gene expression profile. Through Reverse Transcriptase qPCR (RT-qPCR) analysis tissue-specific expression in shoot and root over various time points against salt and osmotic stresses exhibited the diverse expression patterns of identified genes. Overall, the present study providing a foundation for in-depth characterization of identified genes and to further understand the epigenetic role of DNA methylation for genes expression and environmental stresses regulation in higher plant.


Assuntos
Metilação de DNA/fisiologia , DNA de Plantas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza , Proteínas de Plantas , Estresse Fisiológico/fisiologia , Fatores de Transcrição , DNA de Plantas/genética , DNA de Plantas/metabolismo , Estudo de Associação Genômica Ampla , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Adv Exp Med Biol ; 1173: 21-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456203

RESUMO

Iron is an essential trace element in the human body, but excess iron is toxic as it contributes to oxidative damage. To keep iron concentration within the optimal physiologic range, iron metabolism at the cellular level and the whole systemic level are tightly regulated. Balance of iron homeostasis depends on the expression levels and activities of iron carriers, iron transporters, and iron regulatory and storage proteins. Divalent metal transporter 1 (DMT1) at the apical membrane of intestinal enterocyte brings in non-heme iron from the diet, whereas ferroportin 1 (FPN1) at the basal membrane exports iron into the circulation. Plasma transferrin (Tf) then carries iron to various tissues and cells. After binding to transferrin receptor 1 (TfR1), the complex is endocytosed into the cell, where iron enters the cytoplasm via DMT1 on the endosomal membrane. Free iron is either utilized in metabolic processes, such as synthesis of hemoglobin and Fe-S cluster, or sequestered in the cytosolic ferritin, serving as a cellular iron store. Excess iron can be exported from the cell via FPN1. The liver-derived peptide hepcidin plays a major regulatory role in controlling FPN1 level in the enterocyte, and thus controls the whole-body iron absorption. Inside the cells, iron regulatory proteins (IRPs) modulate the expressions of DMT1, TfR1, ferritin, and FPN1 via binding to the iron-responsive element (IRE) in their mRNAs. Both the release of hepcidin and the IRP-IRE interaction are coordinated with the fluctuation of the cellular iron level. Therefore, an adequate and steady iron supplement is warranted for the utilization of cells around the body. Investigations on the molecular mechanisms of cellular iron metabolism and regulation could advance the fields of iron physiology and pathophysiology.


Assuntos
Ferro/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Enterócitos/metabolismo , Ferritinas/metabolismo , Homeostase , Humanos , Sobrecarga de Ferro , Receptores da Transferrina/metabolismo , Fatores de Transcrição/metabolismo , Transferrina/metabolismo
15.
Hypertension ; 74(4): 833-842, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31446800

RESUMO

Heart failure (HF) is a major cause of morbidity and mortality in patients with various cardiovascular diseases. Restoration of cardiac function is critical in improving the clinical outcomes of patients with HF. Long noncoding RNAs are widely involved in the development of multiple cardiac diseases, whereas their role in regulating cardiac function remains unclear. In this study, we found that the expression of long noncoding RNA-DACH1 (dachshund homolog 1) was upregulated in the failing hearts of mice and human. We tested the hypothesis that the intronic long noncoding RNA of DACH1 (LncDACH1) can participate in the regulation of cardiac function and HF. Transgenic overexpression of LncDACH1 in the cardiac myocytes of mice led to impaired cardiac function, reduced calcium transient and cell shortening, and decreased SERCA2a (sarcoplasmic reticulum calcium ATPase 2a) protein expression. In contrast, conditional knockout of LncDACH1 in cardiac myocytes resulted in increased calcium transient, cell shortening, SERCA2a protein expression, and improved cardiac function of transverse aortic constriction induced HF mice. The same qualitative data were obtained by overexpression or knockdown of LncDACH1 with adenovirus carrying LncDACH1 or its siRNA. Moreover, therapeutic administration of adenovirus carrying LncDACH1 siRNA to transverse aortic constriction mice abolished the development of HF. Mechanistically, LncDACH1 directly binds to SERCA2a. Overexpression of LncDACH1 augments the ubiquitination of SERCA2a. LncDACH1 upregulation impairs cardiac function by promoting ubiquitination-related degradation of SERCA2a.


Assuntos
Proteínas do Olho/metabolismo , Insuficiência Cardíaca/metabolismo , Coração/fisiologia , RNA Longo não Codificante/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Humanos , Masculino , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fatores de Transcrição/genética
16.
J Cancer Res Clin Oncol ; 145(9): 2273-2283, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31428934

RESUMO

OBJECTIVES: Recent research has classified lung adenocarcinoma patients with KRAS mutation into three subtypes by co-occurring genetic events in TP53 (KP subgroup), STK11/LKB1 (KL subgroup) and CDKN2A/B inactivation plus TTF-1 low expression (KC subgroup). The aim of this study was to identify valuable biomarkers by searching the candidate molecules that contribute to lung adenocarcinoma pathogenesis, especially KC subtype. MATERIALS AND METHODS: We analyzed the publicly available database and identified the candidate REG4 using the E-GEOD-31210 dataset, and then confirmed by TCGA dataset. In addition, an independent cohort of 55 clinical samples was analyzed by quantitative real-time PCR analysis. Functional studies and RNA sequencing were performed after silencing the REG4 expression. RESULTS: REG4, an important regulator of gastro-intestinal carcinogenesis, was highly expressed in KRAS mutant lung adenocarcinoma with low expression of TTF-1 (KC subtype). The results were validated both by gene expression analysis and immunohistochemistry study in an independent 55 clinical samples from Fudan University Shanghai Cancer Center. Further in vitro and in vivo functional assays revealed silencing REG4 expression significantly reduces cancer cell proliferation and tumorigenesis. Moreover, RNA sequencing and GSEA analysis displayed that REG4 knockdown might induce cell cycle arrest by regulating G2/M checkpoint and E2F targets. CONCLUSION: Our results indicate that REG4 plays an important role in KRAS-driven lung cancer pathogenesis and is a novel biomarker of lung adenocarcinoma subtype. Future studies are required to clarify the underlying mechanisms of REG4 in the division and proliferation of KC tumors and its potential therapeutic value.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/diagnóstico , Proteínas Associadas a Pancreatite/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fatores de Transcrição/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/metabolismo
17.
J Sci Food Agric ; 99(15): 6850-6858, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31385316

RESUMO

BACKGROUND: Transparent Testa8 (TT8) and Homeobox12 (HB12) are two transcriptional factors in plant phenylpropanoid pathways and were reported to be positively related to lignin content. Alfalfa with silenced TT8 (TT8i) and HB12 (HB12i) was therefore generated using the RNA interference (RNAi) technique. Although lignin was found to be high in HB12i, such gene-silencing of alfalfa resulted in nutrient profiles that might be suitable for grazing. To extend the nutritional evaluation of transformed alfalfa, ground samples of 11 HB12i, 5 TT8i and 4 wild type (WT) were incubated in rumen fluid : buffer solution for 0, 2, 4, 8, 12, 24 and 48 h at 39 °C. Dry matter (DM) and neutral detergent fiber (NDF) degradations at each time point, and production of volatile fatty acids (VFA) at 4, 12, 24 and 48 h were analyzed, as well as degradation and production kinetics. The correlations and regressions between nutritive profiles and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectral parameters were determined. RESULTS: Both transformed genotypes had lower DM degradation and HB12i had lower VFA production compared with WT. Structural carbohydrate (STC) parameters were found to be negatively correlated with DM degradation and VFA production. The kinetics of DM degradation and VFA production were predicted from spectral parameters with good estimation power. CONCLUSION: Silencing of HB12 and TT8 affected fermentation characteristics of alfalfa and some fermentation characteristics were predictable from spectral parameters using ATR-FTIR spectroscopy. © 2019 Society of Chemical Industry.


Assuntos
Ácidos Graxos Voláteis/metabolismo , Inativação Gênica , Medicago sativa/genética , Medicago sativa/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Ração Animal/análise , Animais , Bovinos , Fibras na Dieta/metabolismo , Digestão , Proteínas de Plantas/metabolismo , Rúmen/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Transcrição/metabolismo
18.
Cancer Sci ; 110(10): 3110-3121, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385398

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the fourth leading cause of cancer-related death worldwide. Our previous study showed that EYA4 functioned by suppressing growth of HCC tumor cells, but its molecular mechanism is still not elucidated. Based on the results of gene microassay, EYA4 was inversely correlated with MYCBP and was verified in human HCC tissues by immunohistochemistry and western blot. Overexpressed and KO EYA4 in human HCC cell lines confirmed the negative correlation between EYA4 and MYCBP by qRT-PCR and western blot. Transfected siRNA of MYCBP in EYA4 overexpressed cells and overexpressed MYCBP in EYA4 KO cells could efficiently rescue the proliferation and G2/M arrest effects of EYA4 on HCC cells. Mechanistically, armed with serine/threonine-specific protein phosphatase activity, EYA4 reduced nuclear translocation of ß-catenin by dephosphorylating ß-catenin at Ser552, thereby suppressing the transcription of MYCBP which was induced by ß-catenin/LEF1 binding to the promoter of MYCBP. Clinically, HCC patients with highly expressed EYA4 and poorly expressed MYCBP had significantly longer disease-free survival and overall survival than HCC patients with poorly expressed EYA4 and highly expressed MYCBP. In conclusion, EYA4 suppressed HCC tumor cell growth by repressing MYCBP by dephosphorylating ß-catenin S552. EYA4 combined with MYCBP could be potential prognostic biomarkers in HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/genética , Neoplasias Hepáticas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , beta Catenina/metabolismo , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Serina/metabolismo , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Transcrição Genética , beta Catenina/química
19.
J Agric Food Chem ; 67(34): 9569-9578, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31385495

RESUMO

Acetyl-CoA synthetase (ACS) plays a key role in microalgal lipid biosynthesis and acetyl-CoA industrial production. In the present study, two ACSs were cloned and characterized from the oleaginous microalga Chromochloris zofingiensis. In vitro kinetic analysis showed that the Km values of CzACS1 and CzACS2 for potassium acetate were 0.99 and 0.81 mM, respectively. Moreover, CzACS1 and CzACS2 had outstanding catalytic efficiencies (kcat/Km), which were 70.67 and 79.98 s-1 mM-1, respectively, and these values were higher than that of other reported ACSs. CzACS1 and CzACS2 exhibited differential expression patterns at the transcriptional level under various conditions. Screening a recombinant library of 52 transcription factors (TFs) constructed in the present study via yeast one-hybrid assay pointed to seven TFs with potential involvement in the regulation of the two ACS genes. Expression correlation analysis implied that GATA20 was likely an important regulator of CzACS2 and that ERF9 could regulate two CzACSs simultaneously.


Assuntos
Acetato-CoA Ligase/metabolismo , Clorófitas/enzimologia , Regulação Enzimológica da Expressão Gênica , Microalgas/enzimologia , Acetato-CoA Ligase/química , Acetato-CoA Ligase/genética , Biocatálise , Clorófitas/química , Clorófitas/genética , Cinética , Metabolismo dos Lipídeos , Microalgas/química , Microalgas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Genes Dev ; 33(17-18): 1159-1174, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371436

RESUMO

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.


Assuntos
Nucleossomos/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo/metabolismo
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