RESUMEN
Rapamycin, a natural product inhibitor of the Raptor-mammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN- and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2-/- cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.
Asunto(s)
Indazoles/farmacología , Indoles/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Diana Mecanicista del Complejo 1 de la Rapamicina , Morfolinas/farmacología , Complejos Multiproteicos , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Serina/metabolismo , Serina-Treonina Quinasas TOR , Factores de Tiempo , Factores de Transcripción/genética , Transfección , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.
Asunto(s)
Amidinas/química , Inhibidores de Serina Proteinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Amidinas/síntesis química , Amilorida/síntesis química , Secuencia de Aminoácidos , Animales , Antineoplásicos/síntesis química , Sitios de Unión , Proteínas Sanguíneas , Carcinoma Pulmonar de Lewis , Cristalografía por Rayos X , Humanos , Ratones , Datos de Secuencia Molecular , Naftalenos/síntesis química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/síntesis química , Especificidad de la Especie , Tiofenos/síntesis química , Células Tumorales CultivadasRESUMEN
Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.
Asunto(s)
Rastreo Diferencial de Calorimetría/métodos , Proteínas Fúngicas/química , Factores de Elongación de Péptidos/química , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Datos de Secuencia Molecular , Factores de Elongación de Péptidos/metabolismo , Fragmentos de Péptidos/química , Proteínas de Saccharomyces cerevisiae , Tripsina/química , Ultracentrifugación/métodosRESUMEN
Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nM). K(m) values for ATP were similar, but the Kcat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/K(m) values for EF-3A were about two-fold higher; however, the difference in Kcat/K(m) values between the two factors was small for basal ATPase activity.
Asunto(s)
Genes Fúngicos , Factores de Elongación de Péptidos/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Bases , Northern Blotting , Western Blotting , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Factores de Elongación de Péptidos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the Saccharomyces cerevisiae SOR1 gene, which codes for the sorbitol dehydrogenase protein. We cloned the coding sequence of the Saccharomyces YEF3 gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.
Asunto(s)
Proteínas Fúngicas , Vectores Genéticos , L-Iditol 2-Deshidrogenasa/genética , Factores de Elongación de Péptidos/biosíntesis , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiaeAsunto(s)
Proteínas Fúngicas , Factores de Elongación de Péptidos/metabolismo , Espectrofotometría/métodos , Adenosina Trifosfato/metabolismo , Alcohol Deshidrogenasa/genética , Western Blotting , Clonación Molecular , Cinética , Factores de Elongación de Péptidos/genética , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMEN
A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans, sequenced, and expressed in Saccharomyces cerevisiae. The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae, indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.
Asunto(s)
Candida albicans/enzimología , Candida albicans/genética , ADN-Topoisomerasas de Tipo I/genética , Genes Fúngicos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Secuencia Conservada , Cartilla de ADN/genética , ADN de Hongos/genética , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The construction of a human DNA library in a centromere-based circular yeast plasmid is described. The vector contains the yeast CEN3 sequence, the URA3 gene for propagation in yeast and a hygromycin-resistance gene (HyR) for selection in mammalian cells. The library consists of 64,000 members with an average insert size of 150 kb, with some members containing inserts of > 1 Mb. We calculate that the library contains three human genome equivalents of DNA. Clones can be identified by a PCR-based screening of DNA pools from individual colonies that have been stored in microtiter wells.
Asunto(s)
Biblioteca de Genes , Plásmidos/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Centrómero/genética , Cromosomas Artificiales de Levadura/genética , Vectores Genéticos , Humanos , Datos de Secuencia MolecularRESUMEN
The gene coding for the major outer capsid protein (VP7) of simian rotavirus SA-11 has been expressed in a baculovirus-insect cell system. The resulting protein is 35 kDa and is primarily associated with the endoplasmic reticulum. Neutralizing SA-11 polyclonal antiserum and VP7 monospecific antiserum reacted specifically with the expressed gene product. Antiserum derived against the recombinant VP7 protein neutralized SA-11 rotavirus infectivity in a fluorescent focus assay.
Asunto(s)
Baculoviridae/genética , Proteínas de la Cápside , Cápside/genética , Genes Virales , Vectores Genéticos , Rotavirus/genética , Proteínas Estructurales Virales/genética , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/inmunología , Secuencia de Bases , Cápside/inmunología , Clonación Molecular , ADN Viral/química , Cobayas , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Pruebas de Neutralización , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Mapeo Restrictivo , Rotavirus/inmunología , Integración ViralRESUMEN
The salivary protein gene complex consists of a series of loci coding for related but distinct proline-rich proteins (PRPs) found chiefly in saliva. We have screened a library of human genomic DNA fragments in bacteriophage lambda Charon 4A with a PRP cDNA synthesized and cloned from rat parotid gland mRNA. Two phages (PRP1 and PRP2) hybridizing to the rat probe under moderately stringent conditions contain related but not identical DNAs. Preliminary nucleotide sequence data indicate that both DNAs include regions comprised of nearly identical tandemly repeated sequences, each able to code for about 21 amino acids. The decoded consensus repeat sequence is homologous to the repeating amino acid units found by others in human PRPs. This and other features demonstrate that these two clones are members of the PRP gene family. Polymorphic differences between the DNAs of different individuals were observed after probing digests of human genomic DNA with a HinfI fragment from PRP1. These DNA polymorphisms reflect size differences, possibly caused by frequent unequal crossing-over between the repeated units in the PRP genes.