RESUMO
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with various clinical manifestations and serologic markers. In this study, we analyzed nine polyamine (PA) profiles of plasma from patients with SLE and healthy controls (HCs), and the relationship between the PA profiles and disease activity. PA alterations in plasma of 44 patients with SLE and fever were investigated using gas chromatography mass spectrometry (GC-MS) in selected ion monitoring mode using N-ethoxycarbonyl/ N-pentafluoropropionyl derivatives, and compared with those of 43 HCs. Patients with SLE and HCs showed differences in five of nine PA profiles. Among five changed PA levels, four PAs, namely N1-acetylcadaverine, spermidine, N1-acetylspermidine, and spermine, were dramatically decreased. However, the level of cadaverine was increased in patients with SLE. In the partial correlation with PA profiles and disease activity markers of SLE, several disease activity markers and nutritional markers were correlated with cadaverine, spermidine, and N 8-acetylspermidine. Thus, our results provide a comprehensive understanding of the relationship between PA metabolomics and disease activity markers in patients with SLE.
Assuntos
Febre/sangue , Lúpus Eritematoso Sistêmico/sangue , Poliaminas/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Febre/diagnóstico , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Estado Nutricional , Estudos Prospectivos , Índice de Gravidade de Doença , Adulto JovemRESUMO
Entry into anaphase and proteolysis of B-type cyclins depend on a complex containing the tetratricopeptide repeat proteins Cdc16p, Cdc23p, and Cdc27p. This particle, called the anaphase-promoting complex (APC) or cyclosome, functions as a cell cycle-regulated ubiquitin-protein ligase. Two additional subunits of the budding yeast APC were identified: The largest subunit, encoded by the APC1 gene, is conserved between fungi and vertebrates and shows similarity to BIMEp from Aspergillus nidulans. A small heat-inducible subunit is encoded by the CDC26 gene. The yeast APC is a 36S particle that contains at least seven different proteins.
Assuntos
Anáfase , Proteínas de Ciclo Celular/química , Proteínas Fúngicas/química , Ligases/química , Mitose , Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Centrifugação com Gradiente de Concentração , Ciclinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G1 , Genes Fúngicos , Ligases/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismoRESUMO
Physical and chemical agents can damage the genome. Part of the protective response to this damage is the increased expression of p53. p53, a transcription factor, controls the expression of genes, leading to cell cycle arrest and apoptosis. Another protective mechanism is the proliferative response required to replace the damaged cells. This proliferation is likely to be signaled by growth factors. In this communication, we show that the transforming growth factor alpha (TGF-alpha) gene is a direct target for p53-mediated transcriptional activation. In a stable cell line containing an inducible p53 construct, p53 induction leads to a threefold accumulation of the native TGF-alpha mRNA. IN cotransfection assays using a TGF-alpha promoter reporter construct, we show that expression of wild-type but not mutant p53 increases transcriptional activity of the TGF-alpha promoter by approximately 2.5-fold. In vitro, wild-type p53 binds to a consensus binding site found in the proximal portion of the promoter, and this sequence is necessary for the p53 transcriptional response. Furthermore, this element confers p53 induction to the otherwise nonresponsive adenovirus major late promoter. In addition to these results, we found that the TGF-alpha promoter contains a nonconsensus but functional TATA box-binding protein-binding site approximately 30 bp upstream of the transcription start site. Although p53 can repress transcription from promoters containing a TATA box, the nonconsensus TGF-alpha TATA motif is resistant to this effect. On the basis of these results, we propose that p53 may play a dual role, which includes both the elimination of irreparably genetically damage cells and the proliferative response necessary for their replacement, in the response to physical-chemical damage.
Assuntos
Regiões Promotoras Genéticas/genética , Transcrição Gênica , Fator de Crescimento Transformador alfa/genética , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/genética , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular , Dano ao DNA/genética , Análise Mutacional de DNA , Imunofluorescência , Regulação da Expressão Gênica , Glioma/metabolismo , Humanos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Deleção de Sequência , TATA Box/genética , Transfecção , Fator de Crescimento Transformador alfa/biossíntese , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.
Assuntos
Azacitidina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Fator de Crescimento Transformador alfa/biossíntese , Sequência de Bases , DNA/metabolismo , Humanos , Cinética , Melanoma , Metilação , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Transfecção , Fator de Crescimento Transformador alfa/genética , Células Tumorais CultivadasRESUMO
Eukaryotic transcription requires promoter proximal elements. For class II promoters, two such elements are the TATA box and the initiator. The promoter for the human transforming growth factor-alpha (TGF alpha) gene has been shown to lack a TATA box, yet initiate transcription at a unique site. We have identified an approximately 13-basepair sequence between -5 and +8 as a new promoter element. We call this element the TGF alpha initiator based on the following observations: 1) it is located at the transcription initiation site; 2) the promoter activity is largely reduced by either deletion or mutation of the element; and 3) mutations result in initiation upstream of the authentic start site; the TGF alpha initiator directs site-specific initiation. An electrophoretic mobility shift assay demonstrated that a protein(s) in nuclear extracts forms complexes with the TGF alpha initiator. This interaction is sequence specific and depends on nucleotides that are critical for the promoter activity in vivo. Two polypeptides, 105 and 95 kilodaltons, were detected by Southwestern blot analysis on the basis that they specifically interact with the TGF alpha initiator. The larger polypeptide, TIBP-1, was subsequently purified by a matrix-immobilized TGF alpha initiator sequence and was shown to possess the TGF alpha initiator-specific mobility shift activity and an ability to interact with the initiator when immobilized on a membrane. In summary, we identified and characterized the TGF alpha initiator, a proximal element that is important for the accurate transcription of the TGF alpha gene, and the 105-kilodalton protein that interacts with this element.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fator de Crescimento Transformador alfa/análise , Sequência de Bases , Cromatografia de Afinidade , DNA/análise , DNA/genética , Deleção de Genes , Humanos , Immunoblotting , Dados de Sequência Molecular , Mutação , Nucleotídeos/análise , Nucleotídeos/fisiologia , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador alfa/genéticaRESUMO
We and others have previously reported that transforming growth factor-alpha (TGF alpha) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5'-flanking region of the TGF alpha gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormone-stimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGF alpha 5'-flanking region (-373 to -59 relative to the TGF alpha translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGF alpha gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGF alpha-luciferase reporter construct, indicating it is the TGF alpha promoter. A 1.1-kilobase segment of the TGF alpha 5'-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5'-flanking region to -3500 results in marked suppression of reporter gene expression. These results indicate that the TGF alpha gene 5'-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Transcrição Gênica , Fator de Crescimento Transformador alfa/biossíntese , Sequência de Bases , Neoplasias da Mama/metabolismo , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/biossíntese , Receptores ErbB/genética , Humanos , Técnicas In Vitro , Luciferases/biossíntese , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fator de Crescimento Transformador alfa/genética , Tretinoína/farmacologia , Tri-Iodotironina/farmacologia , Células Tumorais CultivadasRESUMO
The epidermal growth factor receptor is vital for normal development and plays a role in oncogenesis. The level of activation of this receptor by transforming growth factor-alpha (TGF-alpha) is controlled, in part, by the rate of transcription of the TGF-alpha gene. In the characterization of the proximal TGF-alpha promoter by DNase I footprinting, a 43-base pair element (-88 to -130 relative to the transcription start site), designated TalphaRE I, was found that was specifically protected by nuclear proteins from human mammary carcinoma MDA468 cells. TalphaRE I was essential for the maximal expression of the TGF-alpha gene as indicated by deletion and mutagenesis analyses. TalphaRE I consists of two cis-acting elements, a proximal regulatory element (PRE, -89 to -103) and a distal regulatory element (DRE, -121 to -128). Both elements were able to form specific complexes with protein from MDA468 cell nuclear extracts and are necessary for the full activity of the entire 1.1-kilobase pair TGF-alpha promoter. Competition and antibody studies determined that the DRE contains a binding site for the transcription factor AP-2, while the protein that binds to the PRE has yet to be identified. When linked upstream to the heterologous herpes simplex thymidine kinase promoter, the TalphaRE I enhanced transcription up to 11-fold in MDA468 cells. Cotransfection of an AP-2 expression vector was able to activate transcription from the TalphaREI-TK construct in a DRE-dependent manner. These results further our understanding of how TGF-alpha transcription is regulated.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Crescimento Transformador alfa/genética , Sequência de Bases , Feminino , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fator de Transcrição AP-2 , Células Tumorais CultivadasRESUMO
Cell division in haploid yeast gives rise to a "mother" cell capable of mating-type switching and a "daughter" cell that is not. Switching is initiated by the HO endonuclease, whose gene is only transcribed in cells that have previously given birth to a bud (mother cells). HO expression depends on a minimyosin, She1p/Myo4p, which accumulates preferentially in growing buds. We describe a gene, ASH1, that is necessary to repress HO in daughters. ASH1 encodes a zinc finger protein whose preferential accumulation in daughter cell nuclei at the end of anaphase depends on She1p/Myo4p. The greater abundance of Ash1p in daughter cells is responsible for restricting HO expression to mother cells.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Desoxirribonucleases de Sítio Específico do Tipo II/biossíntese , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Cadeias Pesadas de Miosina , Miosina Tipo V , Miosinas/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/biossíntese , Sequência de Aminoácidos , Anáfase , Sequência de Bases , Clonagem Molecular , Cruzamentos Genéticos , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Epitopos/análise , Proteínas Fúngicas/biossíntese , Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Miosinas/biossíntese , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , Dedos de ZincoRESUMO
In C. elegans, a Wnt/WG-like signaling pathway down-regulates the TCF/LEF-related protein, POP-1, to specify posterior cell fates. Effectors of this signaling pathway include a beta-catenin homolog, WRM-1, and a conserved protein kinase, LIT-1. WRM-1 and LIT-1 form a kinase complex that can directly phosphorylate POP-1, but how signaling activates WRM-1/LIT-1 kinase is not yet known. Here we show that mom-4, a genetically defined effector of polarity signaling, encodes a MAP kinase kinase kinase-related protein that stimulates the WRM-1/LIT-1-dependent phosphorylation of POP-1. LIT-1 kinase activity requires a conserved residue analogous to an activating phosphorylation site in other kinases, including MAP kinases. These findings suggest that anterior/posterior polarity signaling in C. elegans may involve a MAP kinase-like signaling mechanism.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal , Sequência Conservada , Proteínas de Ligação a DNA/química , Endoderma/fisiologia , Ativação Enzimática , Proteínas de Helminto/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Membrana/química , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. We show here that lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. We demonstrate that the WRM-1 protein binds to LIT-1 in vivo and that WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. Our findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas do Citoesqueleto/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Transdução de Sinais , Transativadores , Sequência de Aminoácidos , Animais , Células COS , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Proteínas de Helminto/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , beta CateninaRESUMO
In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.