RESUMEN
A model of Sr metabolism was developed by using plasma and urinary Sr kinetic data obtained in groups of postmenopausal women who received four different oral doses of Sr and collected during the Sr administration period (25 days) and for 28 days after cessation of treatment. A nonlinear compartmental formalism that is appropriate for study of non-steady-state kinetics and allows dissociation of variables pertaining to Sr metabolism (system 1) from those indirectly operating on it (system 2) was used. At each stage of model development, the dose-dependent model response was fitted to the four sets of data considered simultaneously (1 set per dose). A seven-compartment model with internal Sr distribution and intestinal, urinary, and bone metabolic pathways was selected. It includes two kinds of nonlinearities: those accounting for saturable intestinal and bone processes, which behave as intrinsic nonlinearities because they are directly dependent on Sr, and extrinsic nonlinearities (dependent on system 2), which suggest the cooperative involvement of plasma Sr changes in modulating some intestinal and bone mineral metabolic pathways. With the set of identified parameter values, the initial steady-state model predictions are relevant to known physiology, and some peculiarities of model behavior for long-term Sr administration were simulated.
Asunto(s)
Modelos Biológicos , Dinámicas no Lineales , Estroncio/metabolismo , Femenino , Humanos , Cinética , Persona de Mediana EdadRESUMEN
We have studied the peculiarities of the nonlinear compartmental model for human Sr metabolism (Staub JF, Foos E, Courtin B, Jochemsen R, and Perault-Staub AM. Am J Physiol Regul Integr Comp Physiol 284: R819-R834, 2003), including its physiological reliability in the context of Sr-Ca similarity-dissimilarity. We found it to be relevant to Ca metabolism, except for discrimination against Sr relative to Ca at urinary and intestinal levels. The main findings are as follows: 1) the saturable part of intestinal absorption, shared by Sr and Ca, does not seem to be responsible for the discrimination of the transcellular pathway; 2) although there is little discrimination in bone, the physicochemical behaviors of Sr and Ca at the bone surface differ, at least quantitatively; and 3) Sr behaves as a "tracer" for Ca metabolic pathways and, under non-steady-state conditions, can also reveal self-regulatory processes. It is suggested that they depend on Ca2+ (cationic)-sensing receptors that are apparently more sensitive to Sr than to Ca. Acting on gastrointestinal and osteoblast lineage cells, these slow processes might contribute to adaptive, rather than homeostatic, regulation of Ca metabolism. Understanding these features could help clarify the pharmacological and therapeutic effects of oral Sr.
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Calcio/metabolismo , Modelos Biológicos , Dinámicas no Lineales , Estroncio/metabolismo , Femenino , Humanos , Persona de Mediana EdadRESUMEN
The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris. hTFF1 was secreted in large amounts in the extracellular medium of P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography. The resulting hTFF1 was found to be intact by Western blot analysis. Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide. Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells.
Asunto(s)
Proteínas/genética , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular/métodos , Escherichia coli , Fermentación , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Pichia/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Proteínas/química , Proteínas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
BACKGROUND & AIMS: This study looked for new proteins with expression restricted to the gastric epithelium that may provide insight to the differentiation and function of the gastric unit. METHODS: A novel complementary DNA was isolated and sequenced, and its expression was examined in mouse tissues at both messenger RNA and protein levels. Subcellular localization was studied using immunoelectron microscopy. The posttraductional processing of the protein was analyzed in vitro by protein microsequencing and in vivo by Western blotting. RESULTS: We identified a novel protein that is mainly expressed by the secretory granules of the stomach enteroendocrine cells. This protein has sequence similarity with prepromotilin, the precursor of the motilin hormone and the motilin-associated peptide. As for the prepromotilin, a posttraductional maturation leads to a secreted peptide that is further cleaved at a dibasic site and gives rise to the motilin-related peptide (MTLRP) and MTLRP-associated peptide. CONCLUSIONS: We have identified and characterized a novel gene encoding the preproMTLRP protein. MTLRP presents similarity to motilin and is specifically expressed by enteroendocrine cells of the stomach and therefore represents a novel hormone.
Asunto(s)
Mucosa Gástrica/química , Motilina/genética , Hormonas Peptídicas , Animales , Anticuerpos , Secuencia de Bases , Northern Blotting , Células COS , Clonación Molecular , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Glándulas Endocrinas/citología , Enterocitos/química , Enterocitos/fisiología , Mucosa Gástrica/citología , Expresión Génica/fisiología , Ghrelina , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Motilina/inmunología , Motilina/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/análisis , Conejos , Homología de Secuencia de Aminoácido , Transcripción Genética/fisiologíaRESUMEN
The hallmark of rheumatoid arthritis (RA) is specific destruction of the synovial joints. In a mouse line that spontaneously develops a disorder with many of the features of human RA, disease is initiated by T cell recognition of a ubiquitously expressed self-antigen; once initiated, pathology is driven almost entirely by immunoglobulins. In this study, the target of both the initiating T cells and pathogenic immunoglobulins was identified as glucose-6-phosphate isomerase, a glycolytic enzyme. Thus, some forms of RA or related arthritides may develop by a mechanism fundamentally different from the currently popular paradigm of a joint-specific T cell response.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Linfocitos B/inmunología , Glucosa-6-Fosfato Isomerasa/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Reacciones Cruzadas , Modelos Animales de Enfermedad , Glucosa-6-Fosfato Isomerasa/química , Humanos , Inmunoglobulinas/inmunología , Articulaciones/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The largest subunit of the human basal transcription factor TFIIFalpha (also called RAP74) was reported previously to be the target of some phospho/dephosphorylation process. We show that TFIIFalpha possesses a serine/threonine kinase activity, allowing an autophosphorylation of the two residues at position serine 385 and threonine 389. Mutation analysis strongly suggests that autophosphorylation of both sites regulates the transcription elongation process. Moreover we also evidence three additional phosphorylation sites located at positions 207-230, 271-283, and 335-344. These sites are phosphorylated by casein kinase II-like kinases and TAF(II)250, a component of TFIID.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Especificidad por Sustrato , Factor de Transcripción TFIID , Factores de Transcripción/genética , Factores de Transcripción TFII/metabolismoRESUMEN
Recently we identified a novel human (h) multiprotein complex, called TATA-binding protein (TBP)-free TAFII-containing complex (TFTC), which is able to nucleate RNA polymerase II transcription and can mediate transcriptional activation. Here we demonstrate that TFTC, similar to other TBP-free TAFII complexes (yeast SAGA, hSTAGA, and hPCAF) contains the acetyltransferase hGCN5 and is able to acetylate histones in both a free and a nucleosomal context. The recently described TRRAP cofactor for oncogenic transcription factor pathways was also characterized as a TFTC subunit. Furthermore, we identified four other previously uncharacterized subunits of TFTC: hADA3, hTAFII150, hSPT3, and hPAF65beta. Thus, the polypeptide composition of TFTC suggests that TFTC is recruited to chromatin templates by activators to acetylate histones and thus may potentiate initiation and activation of transcription.
Asunto(s)
Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transducción de Señal , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII/fisiología , Factores de Transcripción/fisiología , Acetilación , Acetiltransferasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas , Humanos , Sustancias Macromoleculares , Complejos Multiproteicos , ARN Polimerasa II/metabolismo , Proteína de Unión a TATA-Box , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBPRESUMEN
Human DNA helicase VIII (HDH VIII) was isolated in the course of a systematic study of the DNA unwinding enzymes present in human cells. From a HeLa cell nuclear extract a protein with an Mrof 68 kDa in SDS-PAGE was isolated, characterised and micro-sequenced. The enzyme shows ATP- and Mg2+-dependent activity is not stimulated by RPA, prefers partially unwound 3'-tailed substrates and moves along the bound strand in the 5' to 3' direction. HDH VIII can also unwind partial RNA/DNA and RNA/RNA duplexes. Microsequencing of the polypeptide showed that this enzyme corresponds to G3BP, an element of the Ras pathway which binds specifically to the GTPase-activating protein. HDH VIII/G3BP is analogous to the heterogeneous nuclear ribonucleoproteins and contains a sequence rich in RGG boxes similar to the C-terminal domain of HDH IV/nucleolin, another DNA and RNA helicase.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , GTP Fosfohidrolasas/metabolismo , Proteínas/metabolismo , Proteínas ras/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , ADN Helicasas/química , ADN Helicasas/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina) , Electroforesis en Gel de Poliacrilamida , Endodesoxirribonucleasas/aislamiento & purificación , Proteínas Activadoras de GTPasa , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Activadoras de ras GTPasa , NucleolinaRESUMEN
The gamma-hydroxybutyrate biosynthetic enzyme succinic semialdehyde reductase (SSR) was purified to homogeneity from rat brain. Peptides were generated by tryptic cleavage and sequenced. PCR primers were designed from the amino acid sequences of two of the peptides showing a similarity (75-85%) to a mitochondrial aldehyde dehydrogenase. A PCR-amplified DNA fragment was generated from recombinant plasmids prepared by a mass excision procedure from a rat hippocampal cDNA library and used as a probe to screen this cDNA library. One cDNA of 1341 bp had an open reading frame encoding a protein of 447 residues with a deduced molecular mass of 47967 Da. The enzyme was expressed in Escherichia coli. Immunoblotting analysis revealed the existence of a protein with the same electrophoretic mobility as the SSR purified from rat brain and with an estimated molecular mass of 45 kDa. Northern blot experiments showed that this enzyme was not expressed in the kidney or in the liver. In the brain tissue, a single but rather broad band was labelled under high stringency conditions, suggesting the presence of more than one messenger species coding for SSR. Hybridization in situ performed on brain tissue slices showed specific labelling of the hippocampus, the upper cortex layer, the thalamus, the substantia nigra, the cerebellum, the pons medulla and the olfactory tract. The recombinant enzyme showed catalytic properties similar to those of the SSR purified from rat brain, particularly in regard to its substrate affinities and Ki for inhibition by phthalaldehydic acid. Valproic acid did not inhibit the cloned SSR. This enzyme had 20-35% identity in highly conserved regions involved in NADPH binding with four other proteins belonging to the aldo-oxo reductase family.
Asunto(s)
Encéfalo/enzimología , Hidroxibutirato Deshidrogenasa/genética , Hidroxibutirato Deshidrogenasa/metabolismo , Oxibato de Sodio/metabolismo , Aldehído Reductasa/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Femenino , Biblioteca de Genes , Hipocampo/enzimología , Humanos , Hidroxibutirato Deshidrogenasa/química , Hidroxibutirato Deshidrogenasa/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fragmentos de Péptidos/química , Reacción en Cadena de la Polimerasa , Embarazo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
TFIID is the DNA binding component of the RNA polymerase II transcriptional machinery and is composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs). Here we report the characterization of a new human TAF, hTAFII100, which is the human homologue of Drosophila TAFII80 and yeast TAFII90. hTAFII100 interacts strongly with hTAFII250, hTAFII55 and hTAFII28, less with hTAFII20 and hTAFII18, weakly with TBP and not at all with delta NTAFII135 and hTAFII30. Deletion analysis revealed that the C-terminal half of hTAFII100, which contains six WD-40 repeats, is not required for incorporation into the TFIID complex. Our results suggest that hTAFII100 can be divided into two domains, the N-terminal region responsible for interactions within the TFIID complex and the C-terminal WD repeat-containing half responsible for interactions between hTAFII100 and other factors. An anti-hTAFII100 antibody, raised against a C-terminal epitope, selectively inhibited basal TFIID-dependent in vitro transcription and the specific interaction between hTAFII100 and the 30 kDa subunit of TFIIF (RAP30). We demonstrate that the hTAFII100-TFIIF interaction supports pre-initiation complex formation in the presence of TFIID. Thus, this is the first demonstration that a TAFII functionally interacts with a basal transcription factor in vitro.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , Cartilla de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Spodoptera/citología , Factor de Transcripción TFIID , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
A nonlinear two-variable reaction-diffusion model of bone mineral metabolism, built from an overall self-oscillatory compartmental model of calcium metabolism in vivo, has been studied for its ability to generate spatial and spatio-temporal self-organizations in a two-dimensional space. Analytical and numerical results confirm the theoretical properties previously described for this kind of model. In particular, it is shown that, for a given set of reactional parameter values and certain values of the ratio of the two diffusion coefficients, there exists a set of unstable wavenumbers leading spontaneously to the development, from the homogeneous steady state, of either different types of stationary spatial patterns (hexagonal, striped and re-entrant hexagonal patterns) or more or less complex spatio-temporal expressions. We discuss the relevance of analogies established between some spatial or spatio-temporal structures predicted by the model and some peculiar features of the primary bone trabecular architecture which appear during embryonic ossification.
Asunto(s)
Densidad Ósea/fisiología , Huesos/anatomía & histología , Modelos Lineales , Animales , Calcio/metabolismo , Difusión , Procesamiento de Imagen Asistido por Computador , Osteogénesis/fisiología , RatasRESUMEN
Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling processes, including those associated with cancer progression. Stromelysin-3, which is expressed in most invasive human carcinomas, is a matrix metalloproteinase with unusual functional properties. In particular, its mature form does not cleave any of the major extracellular matrix components. To define critical structural determinants involved in controlling stromelysin-3 proteolytic activity, we have used site-directed mutagenesis. We show that the deletion of at least 175 C-terminal amino-acids is sufficient to endow mouse stromelysin-3 with activities against casein, laminin, and type IV collagen. In the case of the human enzyme, however, a further and single Ala-235-->Pro substitution is necessary to observe similar activities. Ala-235, which characterizes human stromelysin-3 among matrixins, is located immediately after the C terminus of the "Met-turn," which forms a hydrophobic basis for the catalytic zinc atom in the metzincin family. We conclude that human stromelysin-3 has gained specific functional properties during evolution by amino acid substitution in the catalytic zinc environment, and that it represents an attractive target for specific inhibitors that may be used to prevent cancer progression.
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Metaloendopeptidasas/metabolismo , Alanina , Animales , Secuencia de Bases , Caseínas/metabolismo , Clonación Molecular , Colágeno/metabolismo , Cartilla de ADN , ADN Complementario , Humanos , Cinética , Laminina/metabolismo , Metaloproteinasa 11 de la Matriz , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/química , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Prolina , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
The phosphorylation of retinoic acid receptor-alpha 1 (RAR alpha 1) by PKA was investigated both in vitro and in vivo. We show that bacterially expressed RAR alpha 1 is phosphorylated in vitro by protein kinase A (PKA) at the unique serine residue 369 located in the C-terminal end of the E region. We also show that RAR alpha 1 overexpressed in COS-1 cells is phosphorylated on multiple serine residues and that phosphorylation at serine 369 occurs only when COS-1 cells are cotransfected with PKA or treated with forskolin. RAR alpha 1 mutants were constructed in which serine 369 was replaced by an alanine (S369A) or a glutamic acid (S369E) residue. Comparison of the tryptic phosphopeptide patterns of wild type and mutated RAR alpha 1 overexpressed in COS-1 cells allowed us to confirm that serine 369 is the unique phosphorylation site for PKA in cultured cells. The DNA-binding efficiency of RAR alpha/retinoid X receptor-alpha (RXR alpha) heterodimers was enhanced in vitro by the S369E mutation. However, in transfected RAC65 cells, the same S369E mutation did not affect the ligand-dependent transcriptional activation by RAR alpha 1 of reporter genes containing a retinoic acid (RA)-response element. In contrast, the S369A mutation slightly decreased both DNA binding and the efficiency of PKA to enhance RA-induced transactivation by RAR alpha 1. Finally, we show that endogenous RAR alpha is also phosphorylated in vivo at serine 369 in forskolin-treated F9 cells, supporting the idea that phosphorylation of RARs at this site is involved in the modulation of the RA-induced differentiation of F9 cells by (Bu)2cAMP.
Asunto(s)
Proteína Quinasa C/metabolismo , Receptores de Ácido Retinoico/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Técnicas de Transferencia de Gen , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Receptores de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Activación TranscripcionalAsunto(s)
Niño Hospitalizado/psicología , Niño , Humanos , Recién Nacido , Relaciones Interpersonales , Alojamiento ConjuntoRESUMEN
We have cloned cDNAs encoding three novel TAFIIs [TATA-binding protein (TBP)-associated factors] from the human (h) HeLa cell TFIID complexes hTAFII28, hTAFII20 and hTAFII18. hTAFII28 is a core hTAFII present in both of the previously described hTFIID species which either lack or contain hTAFII30 (hTFIID alpha and hTFIID beta respectively), and is the homologue of Drosophila (d)TAFII30 beta. hTAFII18 is a novel hTAFII which shows homology to the N-terminal region of the yeast TAFIISPT3, but has no known Drosophila counterpart. In contrast to hTAFII28, hTAFII18 is a TFIID beta-specific hTAFII. hTAFII20 is the homologue of p22, an alternatively spliced form of dTAFII30 alpha (p32). Using a combination of protein affinity chromatography and cotransfection and immunoprecipitation assays, we have identified a series of in vitro and intracellular interactions among the novel hTAFIIs and between the novel hTAFIIs and hTAFII30 or TBP. We show that hTAFII28 interacts with hTAFII18 both in vitro and intracellularly; in contrast to its Drosophila homologue, hTAFII28 also interacts directly with TBP. Deletion analysis indicates that TBP and hTAFII18 bind to distinct domains of hTAFII28. hTAFII18 also interacts with TBP, but it interacts more strongly with hTAFII28 and hTAFII30. The binding of hTAFII28 and hTAFII30 requires distinct domains of hTAFII18. As observed with the homologous Drosophila proteins, hTAFII20 interacts directly with TBP; however, additional interactions between hTAFII20 and hTAFII28 or hTAFII30 were detected. These results reveal differences not only in subunit composition, but also in the organization of dTFIID and hTFIID complexes.
Asunto(s)
Factores de Transcripción/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Humanos , Sustancias Macromoleculares , Modelos Estructurales , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TATA Box , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , TransfecciónRESUMEN
During the course of lytic infection, the adenovirus major late promoter (MLP) is induced to high levels after replication of viral DNA has started. We had previously shown that sequence elements located downstream of the MLP start site were implicated in this late-specific transcriptional activation (DE1, between +85 and +98; DE2, between +100 and +120). Two positive transcription factors involved in this activation have been detected. DEF-A, which specifically binds to DE1 and also to the 3' portion of DE2 (DE2a), and DEF-B, which interacts with the 5' part of DE2 (DE2b). When present together, these two proteins cooperatively assemble onto the DE2 element. We now report the purification of DEF-B and show that it is identical to the product of the adenovirus IVa2 gene product. This conclusion is based on microsequence analysis of DEF-B as well as on the inhibitory effect of antibodies against IVa2 on the DNA-binding activity of DEF-B and also on DE-dependent in vitro transcription. In addition, we show that bacterially synthesized IVa2 protein binds to the DE sequences with the same specificity as DEF-B. Finally, in transfected cells, a recombinant IVa2 protein stimulates MLP activity in a DE-dependent fashion. The physiological implications of these findings are discussed.
Asunto(s)
Adenoviridae/genética , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Anticuerpos/inmunología , Secuencia de Bases , Sitios de Unión , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , ADN Viral/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
ERCC2 is involved in the DNA repair syndrome xeroderma pigmentosum (XP) group D and was found to copurify with the RNA polymerase II (B) transcription factor BTF2/TFIIH that possesses a bidirectional helicase activity. Antibodies directed towards the 89 kDa (ERCC3) or the p62 subunit of BTF2 are able to either immunoprecipitate ERCC2 or shift the polypeptide in a glycerol gradient. Conversely, an antibody directed towards ERCC2 also retains or shifts BTF2. ERCC2 could be resolved from the other characterized components of BTF2 upon salt treatment, while its readdition enhanced BTF2 transcription activity. ERCC2, ERCC3 and p44 are three repair proteins found in association with BTF2. Two of them, ERCC2 and ERCC3, are responsible for atypical forms of XP disorders which confer a high predisposition to skin cancer. This includes clinical features that lack an adequate rationalization on the basis of nucleotide excision repair (NER) deficiency but which may now be explained better in terms of a partial transcription deficiency.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN , Proteínas/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Proteínas/inmunología , Factor de Transcripción TFIIH , Proteína de la Xerodermia Pigmentosa del Grupo DRESUMEN
An inhibitor of RNA polymerase II transcription in vitro has been purified from HeLa cell nuclear extracts. Partial amino acid sequences derived from the purified protein revealed that the inhibitor of transcription corresponded to human topoisomerase II. Order of addition experiments provided evidence indicating that topoisomerase II inhibited transcription by binding over the core promoter and blocking preinitiation complex formation. Topoisomerase II-mediated repression could be relieved by sequence-specific transcriptional activators, having different activating and/or DNA binding domains, but antirepression required a transcriptional activation function in addition to a DNA binding domain. Moreover, transcription by RNA polymerase I was also inhibited by topoisomerase II and this inhibition could be relieved by the RNA polymerase I transactivator UBF. These observations suggest that topoisomerase II may participate in a general repression of transcription which can be counteracted by transcriptional activators.