RESUMO
Tumor necrosis factor receptor-1 (TNFR-1) and CD95 (also called Fas or APO-1) are cytokine receptors that engage the apoptosis pathway through a region of intracellular homology, designated the "death domain." Another death domain-containing member of the TNFR family, death receptor 3 (DR3), was identified and was shown to induce both apoptosis and activation of nuclear factor kappaB. Expression of DR3 appears to be restricted to tissues enriched in lymphocytes. DR3 signal transduction is mediated by a complex of intracellular signaling molecules including TRADD, TRAF2, FADD, and FLICE. Thus, DR3 likely plays a role in regulating lymphocyte homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Caspases , NF-kappa B/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Clonagem Molecular , Cisteína Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Fas , Biblioteca Gênica , Humanos , Linfócitos , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral , Alinhamento de Sequência , Fator 1 Associado a Receptor de TNF , Fator 2 Associado a Receptor de TNF , Transfecção , Células Tumorais Cultivadas , Receptor fas/química , Receptor fas/fisiologiaRESUMO
Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, Elongin (SIII), is identified as a functional target of the VHL protein. Elongin (SIII) is a heterotrimer consisting of a transcriptionally active subunit (A) and two regulatory subunits (B and C) that activate transcription elongation by RNA polymerase II. The VHL protein was shown to bind tightly and specifically to the Elongin B and C subunits and to inhibit Elongin (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.
Assuntos
Genes Supressores de Tumor , Ligases , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Doença de von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Elonguina , Regulação da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Fibroblast growth factor 7 (FGF-7) or keratinocyte growth factor (KGF), is a potent and specific mitogen for epithelial cells. We have recently identified a novel human FGF-7 homologue, named FGF-10. To study the expression of this new FGF family member and its regulation in wound repair, we cloned the mouse FGF-10 (mFGF-10) cDNA. The encoded protein is 92% identical to human FGF-10 and 91% identical to rat FGF-10. When expressed in mammalian 293 cells, the mFGF-10 protein was glycosylated but remained cell- or extracellular matrix-associated. Upon addition of heparin, mFGF-10 protein was released into the media. mRNA encoding mFGF-10 was relatively abundant in lung, skin, brain and heart. In the skin, both FGF-7 and mFGF-10 were expressed in the dermal, but not the epidermal compartment. In contrast to FGF-7, mFGF-10 expression was not induced during cutaneous wound repair. In cultured fibroblasts, expression of mFGF-10 was strongly repressed by transforming growth factor beta and tumor necrosis factor alpha, whereas epidermal growth factor and interleukin-1beta had no effect. These results demonstrate a differential regulation of mFGF-10 and FGF-7 expression in vitro and during the wound healing process.
Assuntos
DNA Complementar/genética , DNA Complementar/metabolismo , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , RNA Mensageiro/metabolismo , Células 3T3/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Células Cultivadas , Clonagem Molecular , Feminino , Fator 10 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/fisiologia , Glicosilação , Heparina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Ratos , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Cicatrização/fisiologiaRESUMO
cDNA clones having extensive sequence identity with the sea urchin fascin and the Drosophila singed gene products were isolated from a human teratocarcinoma cDNA library. The human homolog, termed hsn, is a single-copy gene that was localized to human chromosome 7p22 by fluorescence in situ hybridization and is predicted to encode a 493-amino-acid product with a molecular mass of approximately 55,000. This protein would be similar in size to the fascin and singed proteins, as well as a previously described 55-kD actin-bundling protein that was purified from HeLa cells. Monoclonal antibodies directed against the 55-kD HeLa protein were reactive against a bacterially expressed hsn fusion protein, indicating that the hsn gene probably encodes the 55-kD protein. The hsn mRNA was variably expressed in all human tissues analyzed and was highly expressed in actively growing renal carcinoma cell lines and in activated, but not in resting, lymphocytes, suggesting a functional role for hsn in proliferation. The fascin family lacks homology with other characterized actin-binding proteins, and the high degree of evolutionary conservation of these proteins indicates a functional importance of their actin-bundling properties.
Assuntos
Proteínas de Transporte/genética , Hormônios de Inseto/genética , Proteínas dos Microfilamentos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Drosophila , Células HeLa , Humanos , Linfócitos/metabolismo , Dados de Sequência Molecular , Ouriços-do-Mar , Homologia de Sequência de AminoácidosAssuntos
Clonagem Molecular , Genes Supressores de Tumor , Neoplasias Renais/genética , Ligases , Proteínas/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Doença de von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas/química , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
We recently isolated an RNA polymerase II elongation factor from rat liver nuclei and found it to be homologous to the product of the human ELL gene, a frequent target for translocations in acute myeloid leukemia. To further our understanding of the possible role(s) of ELL in transcriptional regulation and human disease, we initiated a search for ELL-related proteins. In this report we describe molecular cloning, expression, and characterization of human ELL2, a novel RNA polymerase II elongation factor 49% identical and 66% similar to ELL. Mechanistic studies indicate that ELL2 and ELL possess similar transcriptional activities. Structure-function studies localize the ELL2 elongation activation domain to an ELL2 N-terminal region that is highly homologous to ELL. Finally, Northern blot analysis reveals that the ELL2 and ELL genes are transcribed in many of the same tissues, but that the ratio of their transcripts exhibits tissue-to-tissue variation, raising the possibility that ELL2 and ELL may not perform completely general functions, but, instead, may perform gene- or tissue-specific functions.
Assuntos
Proteínas de Ligação a DNA/genética , Genoma Humano , Proteínas de Neoplasias , Fatores de Alongamento de Peptídeos , Fatores Genéricos de Transcrição , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Mutação , Ratos , Alinhamento de SequênciaRESUMO
Fibroblast growth factors (FGFs) transmit their signals through four transmembrane receptors that are designated FGFR1-4. Alternative splicing in the extracellular region of FGFR1-3 generates receptor variants with different ligand binding affinities. Thus two types of transmembrane receptors (IIIb and IIIc isoforms) have been identified for FGFR2 and FGFR3, and the existence of analogous variants has been postulated for FGFR1 based on its genomic structure. However, only a single full-length transmembrane FGFR1 variant (FGFR1-IIIc) has been identified so far. Here we describe the cloning of a full-length cDNA encoding FGFR1-IIIb from a mouse skin wound cDNA library. This receptor isoform was expressed at the highest levels in a subset of sebaceous glands of the skin and in neurons of the hippocampus and the cerebellum. FGFR1-IIIb was expressed in L6 rat skeletal muscle myoblasts and used in cross-linking and receptor binding studies. FGF-1 was found to bind the receptor with high affinity, whereas FGF-2, -10, and -7 bound with significantly lower affinities. Despite their apparently similar but low affinities, FGF-10 but not FGF-7 induced the activation of p44/42 mitogen-activated protein kinase in FGFR1-IIIb-expressing L6 myoblasts and stimulated mitogenesis in these cells, demonstrating that this new receptor variant is a functional transmembrane receptor for FGF-10.
Assuntos
Encéfalo/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Pele/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Reagentes de Ligações Cruzadas/metabolismo , DNA/biossíntese , Ativação Enzimática/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Glicosilação , Hibridização In Situ , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Homologia de Sequência de AminoácidosRESUMO
The human VHL tumor suppressor gene has been implicated in the inherited disorder von Hippel-Lindau disease and in sporadic renal carcinoma. The homologous rat gene encodes a 185-amino acid protein that is 88% sequence identical to the aligned 213-amino acid human VHL gene product. When expressed in COS-7 cells, both the human and the rat VHL proteins showed predominant nuclear, nuclear and cytosolic, or predominant cytosolic VHL staining by immunofluorescence. A complicated pattern of cellular proteins was seen that could be specifically coimmunoprecipitated with the introduced VHL protein. A complex containing VHL and proteins of apparent molecular masses 16 and 9 kDa was the most consistently observed. Certain naturally occurring VHL missense mutations demonstrated either complete or partial loss of the p16-p9 complex. Thus, the VHL tumor suppressor gene product is a nuclear protein, perhaps capable of specifically translocating between the nucleus and the cytosol. It is likely that VHL executes its functions via formation of specific multiprotein complexes. Identification of these VHL-associated proteins will likely clarify the physiology of this tumor suppressor gene.
Assuntos
Genes Supressores de Tumor , Ligases , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Doença de von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , DNA Complementar/química , Células HeLa , Humanos , Rim , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Frações Subcelulares/metabolismo , Transfecção , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.
Assuntos
Mapeamento Cromossômico , DNA Complementar/isolamento & purificação , Dimetilaliltranstransferase/genética , Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Cricetinae , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Feminino , Células HeLa , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Ovário , RNA Mensageiro/análise , Proteínas Recombinantes/metabolismo , Testículo/enzimologiaRESUMO
A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.