RESUMEN

Beta-catenin is a multifunctional protein that is both an integral component of adherens junctions and a pivotal member of a signal transduction pathway. The cytoplasmic pool of beta-catenin, which participates in signal transduction, is highly regulated. Binding to the adenomatous polyposis coli tumor suppressor protein can stimulate the degradation of beta-catenin, whereas signaling initiated by the extracellular Wnt-1 oncoprotein or selected mutations in beta-catenin itself results in the accumulation of higher levels of beta-catenin in the cytoplasm. A variety of experiments from several model systems have converged to elucidate the mechanisms involved in this regulation as well as the downstream effectors of beta-catenin. These studies have recently been extended to demonstrate that deregulation of this pathway contributes to cancer in humans.

Asunto(s)

Cadherinas/fisiología , Adhesión Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Neoplasias/fisiopatología , Transducción de Señal/fisiología , Transactivadores , Animales , Humanos , beta Catenina

RESUMEN

Recent studies indicate that disruption of the E-cadherin-mediated cell-cell adhesion system is frequently associated with human cancers of epithelial origin. Reduced levels of both E-cadherin and the associated protein, alpha-catenin, have been reported in human tumors. This report describes the characterization of a human ovarian carcinoma-derived cell line (Ov2008) which expresses a novel mutant form of the alpha-catenin protein lacking the extreme N terminus of the wild-type protein. The altered form of alpha-catenin expressed in Ov2008 cells fails to bind efficiently to beta-catenin and is localized in the cytoplasm. Deletion mapping has localized the beta-catenin binding site on alpha-catenin between amino acids 46 and 149, which encompasses the same region of the protein that is deleted in the Ov2008 variant. Restoration of inducible expression of the wild-type alpha-catenin protein in these cells caused them to assume the morphology typical of an epithelial sheet and retarded their growth in vitro. Additionally, the induction of alpha-catenin expression in Ov2008 cells injected into nude mice attenuated the ability of these cells to form tumors. These observations support the classification of alpha-catenin as a growth-regulatory and candidate tumor suppressor gene.

Asunto(s)

Carcinoma/patología , Proteínas del Citoesqueleto/genética , Genes Supresores de Tumor , Neoplasias Ováricas/patología , Transactivadores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias de la Mama/química , Cadherinas , Carcinoma/química , Carcinoma/genética , Adhesión Celular , División Celular , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Células Epiteliales , Epitelio/química , Femenino , Expresión Génica , Genes , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias Ováricas/química , Neoplasias Ováricas/genética , Mutación Puntual/genética , Células Tumorales Cultivadas , alfa Catenina , beta Catenina

RESUMEN

The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the beta-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg2+, a Km of 0.8 mM and a kcat of 5.2 s-1 at 37 degrees C for dATP. dAMP is a weak competitive inhibitor with a Ki of approximately 4 mM, while PPi is a much stronger inhibitor with an apparent Ki of approximately 20 microM. The enzyme contains the highly conserved signature sequence GXVEX2ETX6REVXEEX2I designating the MutT family of proteins. However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT- mutator phenotype, and thus must serve a different biological role in the cell.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Monoéster Fosfórico Hidrolasas/genética , Pirofosfatasas/genética , Secuencia de Aminoácidos , Clonación Molecular , Genes Bacterianos , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pirofosfatasas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

The mutX gene of Streptococcus pneumoniae, a homologue of the Escherichia coli mutT mutator gene (Méjean, V., Salles, C., Bullions, L. C., Bessman, M. J., and Claverys, J.-P. (1993) Mol. Microbiol. 11, 323-330) has been cloned into an expression vector, and its gene product, the MutX protein, has been purified to apparent homogeneity. Like MutT, the pure MutX protein hydrolyzes all of the canonical nucleoside triphosphates at different rates with a preference for dGTP, yielding nucleoside monophosphates and inorganic pyrophosphate. Despite this similarity in enzymatic activity, the two proteins have notably dissimilar primary and quaternary structures. They share only a small region of amino acid homology, and under the same conditions in which MutT exists as a monomer in solution, MutX behaves as a trimer. The small region of conserved amino acid sequence most likely identifies a protein domain responsible for the novel nucleoside triphosphate pyrophosphohydrolase activity shared by the two enzymes, and by another protein of unknown function, the product of the E. coli orf17 gene (Takahagi, M., Iwasaki, H., Nakata, A., and Shinegawa, H. (1991) J. Bacteriol. 173, 5747-5753).

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/metabolismo , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Genes Bacterianos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Pirofosfatasas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/genética

RESUMEN

We show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin-resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are co-ordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17,870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside triphosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E. coli mutT mutator phenotype when introduced on a plasmid. Site-directed mutagenesis and analysis of nitrosoguanidine-induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus-encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Monoéster Fosfórico Hidrolasas/genética , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Secuencia Conservada , ADN Bacteriano/química , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Monoéster Fosfórico Hidrolasas/biosíntesis , Pirofosfatasas , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/efectos de los fármacos , Estreptomicina/toxicidad

RESUMEN

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.

Asunto(s)

Proteínas Bacterianas/metabolismo , Reparación del ADN , Nucleótidos de Desoxiguanina/metabolismo , Proteínas de Escherichia coli , Monoéster Fosfórico Hidrolasas , Difosfatos/metabolismo , Escherichia coli/metabolismo , Hidrólisis , Marcaje Isotópico/métodos , Espectroscopía de Resonancia Magnética/métodos , Isótopos de Oxígeno , Pirofosfatasas , Agua

RESUMEN

The mutT protein, which prevents A:T----C:G transversions during DNA replication, has the following enzymatic properties. Although it prefers dGTP as a substrate, it hydrolyzes all of the canonical nucleoside triphosphates to some extent. It has no activity in the absence of divalent cations, is maximally activated by magnesium ions, and has a pH optimum of 9.0. Nucleoside triphosphates are hydrolyzed according to the following equation. dGTP----dGMP + PPi Studies with nucleotide analogues suggest that the enzyme may prefer the syn rather than the anti conformation of the nucleoside triphosphates, which might explain the role it plays in preventing mutations.

Asunto(s)

Escherichia coli/enzimología , Monoéster Fosfórico Hidrolasas/genética , Secuencia de Bases , Unión Competitiva , Nucleótidos de Desoxiguanina/metabolismo , Escherichia coli/genética , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis , Nucleósido-Trifosfatasa , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Especificidad por Sustrato

RESUMEN

The mutT nucleoside triphosphatase, which prevents AT----CG transversions during DNA replication, has been crystallized from ammonium sulfate utilizing a novel technique involving vapor diffusion in capillaries. X-ray diffraction analysis has revealed that the crystals are monoclinic, space group P2(1), with cell constants a = 34.14, b = 72.54, c = 56.38, and beta = 98.90. The Vm value of 2.31 A3/Da is consistent with two molecules of enzyme per asymmetric unit. The crystals are reasonably stable in the x-ray beam, and a data set to 2.5 A resolution has been collected for native protein. There is evidence that the crystals diffract to at least 2.1 A.

Asunto(s)

Escherichia coli/enzimología , Monoéster Fosfórico Hidrolasas/ultraestructura , Pirofosfatasas/ultraestructura , Cristalografía , Nucleósido-Trifosfatasa , Conformación Proteica , Difracción de Rayos X

RESUMEN

With a probe constructed from the wild-type gene, a DNA fragment containing the entire mutT1 mutator gene was isolated and cloned into pUC18. Nucleotide sequence analysis revealed that the mutator defect was most likely due to an IS1 insertion into the wild-type gene.

Asunto(s)

Escherichia coli/genética , Genes Bacterianos , Mutación , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Mapeo Restrictivo