Asunto(s)
Proteínas Musculares/genética , Síndromes Miasténicos Congénitos/genética , Mutación Puntual , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Exones , Femenino , Genes Recesivos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Humanos , Lactante , Masculino , Proteínas Musculares/metabolismo , Linaje , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Fosforilasas/antagonistas & inhibidores , Piperidinas/farmacología , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Cafeína/farmacología , Proteínas de Unión a Calmodulina/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Flavonoides/antagonistas & inhibidores , Flavonoides/metabolismo , Glucógeno/metabolismo , Células HeLa , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Hígado/enzimología , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Proteínas del Tejido Nervioso/metabolismo , Neurogranina , Fragmentos de Péptidos/metabolismo , Fosforilasas/metabolismo , Fosforilación/efectos de los fármacos , Piperidinas/antagonistas & inhibidores , Piperidinas/metabolismo , Unión Proteica , Conejos , Células Tumorales CultivadasRESUMEN
Flavopiridol (L86-8275) ((-)-cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl] -4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC(50) = 15.5 microm. The inhibition is synergistic with glucose resulting in a reduction of IC(50) for flavopiridol to 2.3 microm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-A resolution, and refined to crystallographic R values of 0.216 (R(free) = 0.247), 0.189 (R(free) = 0.219), and 0.195 (R(free) = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe(285) and Tyr(613). Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes.
Asunto(s)
Flavonoides/metabolismo , Fosforilasas/antagonistas & inhibidores , Piperidinas/metabolismo , Animales , Sitios de Unión , Cafeína/química , Glucosa/metabolismo , Cinética , Modelos Moleculares , Músculo Esquelético/enzimología , Fosforilasas/química , Fosforilasas/metabolismo , Conformación Proteica , ConejosRESUMEN
7-Hydroxystaurosporine (UCN-01), a protein kinase inhibitor in clinical development, demonstrates potent antineoplastic activity. To determine whether specific genetic abnormalities would modulate the response to UCN-01, a model of human non-small cell lung carcinoma (NSCLC) cell lines with differential abnormalities of p16CDKN2, RB, and p53 was used for these studies. Cell growth was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and cell cycling was studied using flow cytometric analysis of DNA content. Changes in protein levels and phosphorylation were assessed by Western blotting. In cell lines expressing wild-type RB (A549 and Calul), UCN-01 treatment resulted in dose-dependent growth inhibition, arrest of cells in G1, and a reduction of cells in S phase. p16CDKN2-null cells showed similar growth inhibition to normal fetal lung fibroblasts. UCN-01-induced growth arrest was accompanied by induction of p21CDKN1 and a shift of Rb to the hypophosphorylated state in both p53 wild-type and mutant cell lines. In contrast, UCN-01 treatment of the RB-null cell line H596 resulted in less growth inhibition. To test the role of RB in response to UCN-01, effects of treatment were examined in two human isogenic models of RB expression: the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). In the Rb-expressing 5637 subline (RB5), UCN-01 treatment resulted in Rb hypophosphorylation and an accumulation in G1 in contrast to the parent line. Similarly, the wild-type Rb-expressing DU-145 sublines (DU1.1 and B5) showed increased G1 arrest compared with the parent cells. We conclude that UCN-01-induced G1 arrest can occur in cells null for p53 and p16CDKN2, and that RB status influences the ability of UCN-01 to induce a G1 arrest. These data suggest that the molecular profile of cell cycle regulating genes in individual tumors may predict responsiveness and provide insight into optimal therapeutic application of this new antineoplastic agent.
Asunto(s)
Alcaloides/farmacología , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Genes de Retinoblastoma/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Genes p53/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Humanos , Neoplasias Pulmonares/metabolismo , Fosforilación , Proteína de Retinoblastoma/biosíntesis , Proteína de Retinoblastoma/genética , Estaurosporina/análogos & derivados , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The synthetic flavone flavopiridol can be cytostatic or cytotoxic to mammalian cells, depending on the concentration of the drug and the duration of exposure. It has been shown to inhibit the cyclin-dependent kinase (CDK) family of cell cycle regulatory enzymes. However, the existence of additional potential targets for drug action remains a matter of interest to define. To identify cellular targets, flavopiridol was immobilized. CDKs, particularly CDK 4, bound weakly to immobilized flavopiridol when ATP was absent but not in its presence. Two proteins with molecular weights of 40 kDa and 120 kDa had high affinities to the immobilized flavopiridol independent of the presence of ATP. They were present in all cell lines analyzed: cervical (HeLa), prostate and non-small cell lung carcinoma (NSCLC) cell lines. A 60-kDa protein, which was present only in NSCLC cells and bound similarly well to immobilized flavopiridol, was identified as cytosolic aldehyde dehydrogenase class 1 (ALDH-1). The level of this protein correlated with the resistance of NSCLC cell lines to cytotoxicity caused by 500 nM flavopiridol but not higher flavopiridol concentrations. Despite binding to ALDH-1, there was no inhibition of dehydrogenase activity by flavopiridol concentrations as high as 20 microM and flavopiridol was not metabolized by ALDH-1. The results suggest that high cellular levels of ALDH-1 may reduce cytotoxicity of flavopiridol and contribute to relative resistance to the drug. This is the first report that flavopiridol binds to proteins other than CDKs.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Citosol/enzimología , Flavonoides/metabolismo , Isoenzimas/metabolismo , Neoplasias Pulmonares/enzimología , Piperidinas/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Proteínas Portadoras , Cromatografía de Afinidad , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Retinal-Deshidrogenasa , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27kip1 but not p21cip1 was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27kip1 is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27kip1.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fase G1 , Proteínas Asociadas a Microtúbulos/metabolismo , Estaurosporina/farmacología , Proteínas Supresoras de Tumor , Células 3T3 , Animales , Línea Celular , Transformación Celular Neoplásica , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Fase G1/efectos de los fármacos , Células HeLa , Humanos , Ratones , Oncogenes , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Células Tumorales CultivadasRESUMEN
Previous studies have shown that heptachlor, a chlorinated hydrocarbon insecticide, is a liver tumor promoter in rats and mice and induces tumor promoting-like alterations in human myeloblastic leukemia cells. The nature of tumor promotion is multifaceted and has recently been shown to include suppression of programmed cell death (apoptosis) as a mechanism by which a tumor promoter can prolong cell viability. The ability of tumor promoters to suppress apoptosis prompted us to address the question of whether heptachlor is capable of effecting the expression of genes involved in lymphocyte apoptosis, in particular, the p53 tumor suppressor gene. Experiments with a CEM x 174 cell line, a hybrid of human T and B cells, revealed that heptachlor downregulated p53 gene expression at the post-transcriptional level without changing levels of mRNA in the cells. The heptachlor-induced reduction in the basal levels of expression of this gene was both in a concentration and time-dependent manner.
Asunto(s)
Apoptosis/efectos de los fármacos , Genes p53/efectos de los fármacos , Heptacloro/toxicidad , Insecticidas/toxicidad , Linfocitos/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Expresión Génica , Genes p53/genética , Humanos , Técnicas In VitroRESUMEN
The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.
Asunto(s)
Alcaloides/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fase G1/efectos de los fármacos , Proteínas Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Inhibidores Enzimáticos/farmacología , Estaurosporina , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Staurosporine (ST), a protein kinase inhibitor, at a concentration of 20 nM arrests normal diploid fibroblasts 3 h into G1 (H. A. Crissman et al., Proc. Natl. Acad. Sci. USA, 88: 7580-7584, 1991; K. Abe et al., Exp. Cell Res., 192: 122-127, 1991). ST (2 nM) induces a new G1 arrest point at 6 h into G1. Partial phosphorylation of the retinoblastoma protein was observed at the 2 nM ST arrest point, whereas the retinoblastoma protein was unphosphorylated or underphosphorylated at the 20 nM arrest point. This correlated with the activity of the cyclin-dependent kinase 2 (CDK2) and the phosphorylation of the Thr160 residue of p33CDK2. The cyclin E and cyclin D1/2 levels were reduced at the 20 nM ST arrest point. In HeLa cells that do not arrest in G1 in response to 2 or 20 nM ST, the retinoblastoma protein and CDK2 phosphorylations and CDK2 activity were not affected by ST. These results suggest that ST inhibits one or more G1-regulating protein kinases, which lie upstream of CDK2.
Asunto(s)
Alcaloides/farmacología , Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Fase G1/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Quinasa 2 Dependiente de la Ciclina , Ciclinas/metabolismo , Fibroblastos , Células HeLa , Humanos , Fosforilación , Estaurosporina , Factores de TiempoRESUMEN
The role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Tetrahidrofolato Deshidrogenasa/biosíntesis , Secuencia de Bases , Western Blotting , Línea Celular , Cromatografía Liquida , ADN/genética , Electroforesis en Gel de Poliacrilamida , Factor 2 Eucariótico de Iniciación/genética , Regulación Enzimológica de la Expresión Génica , Focalización Isoeléctrica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Plásmidos , Biosíntesis de Proteínas , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Serina/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Transfección , eIF-2 QuinasaRESUMEN
Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.
Asunto(s)
Genes Fúngicos , Lisina/análogos & derivados , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular/métodos , Codón/genética , Vectores Genéticos , Humanos , Cinética , Lisina/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Factores de Iniciación de Péptidos/fisiología , Fenotipo , Plásmidos , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Ácido Nucleico , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Bacillus stearothermophilus mutations which confer resistance to or dependence on a variety of ribosome-targeted antibiotics have been isolated. Many of these mutations produce ribosomal proteins with altered mobilities in a two-dimensional gel electrophoresis system. This collection of altered thermophilic ribosomal proteins will be useful in examining ribosomal structure and function.
Asunto(s)
Geobacillus stearothermophilus/genética , Proteínas Ribosómicas/genética , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana , Electroforesis en Gel Bidimensional , Mutación , Biosíntesis de ProteínasRESUMEN
Most naturally occurring mutants of the retinoblastoma (RB) protein contain large deletions or truncations. The small cell lung carcinoma cell line H209 contains a normal-sized but unphosphorylated RB protein (Hensel et al., Cancer Res., 50: 3067-3072, 1990), which fails to form a complex with SV40 T antigen, suggesting that the RB gene of H209 may contain a subtle mutation. To define this mutation, the RB complementary DNA and genomic DNA were sequenced, revealing a point mutation in exon 21 that changed a G to a T. This results in an amino acid substitution of a Phe for Cys706. The mutant RB complementary DNA was used as a template for in vitro transcription and translation to synthesize the mutated protein. The resulting protein failed to bind to SV40 T antigen, demonstrating that a single missense mutation of the RB gene led to the complete inactivation of the ability of the RB protein to bind T antigen.
Asunto(s)
Antígenos Transformadores de Poliomavirus/metabolismo , Proteína de Retinoblastoma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica/genética , Proteína de Retinoblastoma/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
To facilitate the study of the regulation of the rpsA gene, a translational fusion between the rpsA gene and the lacZ gene was constructed. Synthesis of the fusion protein was repressed about 10-fold when rpsA was supplied in trans on a multicopy plasmid. This repression is similar to the post-transcriptional regulation previously found for the wild type rpsA gene. Addition of purified protein S1 to a coupled in vitro transcription-translation system caused a specific reduction in the synthesis of the rpsA-lacZ fusion protein. Addition of various subdomain fragments of protein S1 to the coupled in vitro system showed that the N-terminal fragment, possessing the ribosome binding domain of protein S1, was able to repress the synthesis of the rpsA-lacZ fusion protein. In contrast, fragments from the C-terminal region, containing the nucleic acid binding domain of protein S1, were inactive in this repression. Induction of truncated rpsA genes, coding for either the N-terminal 101 or 329 amino acids caused a reduction in the synthesis of the chromosomally encoded protein S1, thus confirming in vivo that the N-terminal part of protein S1 represses rpsA expression.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/metabolismo , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/genética , Mapeo Restrictivo , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.
Asunto(s)
Lisina/análogos & derivados , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN , Células HeLa/metabolismo , Humanos , Cinética , Factores de Iniciación de Péptidos/genética , Puromicina/análogos & derivados , Puromicina/biosíntesis , ARN de Transferencia de Metionina/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Protein synthesis initiation factor eIF-4D is a relatively abundant protein in mammalian cells and possesses a unique amino acid residue, hypusine. The role of the hypusine modification in eIF-4D function was addressed by studying the function of eIF-4D variants lacking hypusine. The cloned human cDNA encoding eIF-4D was overexpressed in Escherichia coli and a precursor form lacking hypusine was purified. This protein fails to stimulate methionyl-puromycin synthesis in vitro, nor does it significantly inhibit the action of native eIF-4D. Mammalian expression vectors were constructed with the wild-type cDNA and a mutant form in which the codon for lysine-50 (the residue hypusinated) was altered by site-directed mutagenesis to that for arginine. Transient co-transfection of COS-1 cells with the eIF-4D vector and a vector expressing dihydrofolate reductase led to strong synthesis of both eIF-4D and dihydrofolate reductase. This indicates that normal cellular levels of eIF-4D are saturating in these cells and that excess levels of eIF-4D are not detrimental. Cotransfection with the eIF-4D arginine variant caused no effect on dihydrofolate reductase synthesis, in agreement with the in vitro experiments. The inability of the unhypusinated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vivo strongly suggests that the hypusine modification is required for eIF-4D activity and for its interaction with the 80 S initiation complex in protein synthesis.
Asunto(s)
Lisina/fisiología , Factores de Iniciación de Péptidos/fisiología , Proteínas de Unión al ARN , Arginina/genética , Codón , ADN/genética , Escherichia coli/genética , Expresión Génica , Humanos , Lisina/genética , Mutación , Hibridación de Ácido Nucleico , Factores de Iniciación de Péptidos/genética , Plásmidos , Relación Estructura-Actividad , Transfección , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
The assembly of ribosomes in bacterial cells is a complex process that remains poorly characterized. The in vitro assembly of active ribosomal subunits from purified RNA and protein components indicates that all of the information for proper assembly resides in the primary sequences of these macromolecules. On the other hand, the in vitro requirement of unphysiological heating steps suggests that this pathway may not accurately reflect the in vivo pathway, and that other proteins may be required. One approach to identify any additional proteins is to isolate second-site revertants of mutants defective in ribosome assembly. Ribosomal protein L24 is essential in the assembly of 50S subunits. We have identified an Escherichia coli gene, srmB, that, when expressed at high copy number, can suppress the effect of a temperature-sensitive lethal mutation in L24. The SrmB amino-acid sequence has sequence identity with mouse translation initiation factor eIF-4A and with the human nuclear protein, p68. The purified SrmB protein is a nucleic acid-dependent ATPase, like eIF-4A, but can also bind RNA in the absence of ATP and other auxiliary protein factors. The RNA dependent ATPase activity of SrmB suggests that like, eIF-4A, it could be involved in specific alterations of RNA secondary structure.
Asunto(s)
Escherichia coli/genética , Factores de Iniciación de Péptidos/genética , Ribosomas/metabolismo , Supresión Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Bacteriano/genética , Factor 4A Eucariótico de Iniciación , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Mutación , ARN Bacteriano/genética , ARN Ribosómico 23S/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.
Asunto(s)
Proteínas Bacterianas/genética , Clonación Molecular , Genes Bacterianos , Rhizobium/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosfoproteínas/metabolismo , PlásmidosRESUMEN
A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA. The degradation and the synthesis rates of individual ribosomal proteins were determined. Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation. Other ribosomal proteins were either weakly or not at all degraded in both mutants. Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid. Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts.