RESUMO
Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch3/antagonistas & inibidores , Receptor Notch3/genética , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Códon , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Conformação Proteica , Receptor Notch3/química , Receptor Notch3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Escherichia coli/genética , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.
Assuntos
Fígado/enzimologia , Fosforilases/química , Fosforilases/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Desenho de Fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Modelos Moleculares , Músculos/enzimologia , Fosforilases/genética , Conformação Proteica , Estrutura Secundária de Proteína , CoelhosRESUMO
Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.
Assuntos
Escherichia coli/metabolismo , Histidina/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Acilação , Sequência de Aminoácidos , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli/genética , Gluconatos/metabolismo , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteína-Tirosina Quinase ZAP-70 , Quinases de Receptores Adrenérgicos betaRESUMO
Ro 41-5253 is a RARalpha-selective antagonist that binds RARalpha but does not induce transcriptional activation and does not influence RAR/RXR heterodimerization and DNA binding. This retinoid inhibits proliferation and induces apoptosis in MCF-7 and ZR-75.1 estrogen-receptor-positive breast-carcinoma cells in a dose-dependent way. The anti-proliferative effect is more evident in ZR-75.1 cells than in MCF-7 cells and is probably mediated by anti-AP1 activity, a mechanism known to be implied in the action of several retinoids. In the induction of apoptosis also ZR-75.1 cells are more sensitive to treatment with Ro 41-5253 than MCF-7 cells. In ZR-75.1 cells an apoptotic/hypodiploid DNA peak is already evident after 2 days of incubation, whereas in MCF-7 cells it appears only after 4 days. The highest percentage of apoptotic cells, for both cell lines, is reached after 6 days of treatment. The apoptosis pathway is p53-independent and bcl-2 downregulation seems to be correlated with an increase in TGF-beta1 protein. The MDA-MB-231 estrogen-receptor-negative cell line is poorly responsive to Ro 41-5253 treatment, both in terms of proliferation inhibition and apoptosis induction. Ro 41-5253 has proliferation-inhibiting and apoptosis-inducing properties that are not mediated by transcriptional activation from retinoic-acid response elements. This retinoid antagonist seems to be a compound that exerts an anti-tumor activity but does not induce the toxic side effects of retinoids and might, therefore, be considered as a candidate for cancer therapy.
Assuntos
Apoptose , Benzoatos/farmacologia , Cromanos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Retinoides/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA , Feminino , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
In order to confirm and further explore the significance of the overexpression of the CRABP II (cellular retinoic acid binding protein type II) and psoriasin genes in psoriatic versus normal skin, we examined the mRNA expression levels of these two genes by in situ hybridization in skin samples from psoriatic plaques and in one case from the border between a psoriatic plaque and uninvolved skin. Both genes were markedly upregulated in lesional skin, with a shift from low to high expression in the transitional zone of the plaque. Expression of the cytokeratin 1 (K1) gene was, in contrast, high in normal skin and decreased in the transition from uninvolved skin to psoriatic plaque, Examination of mRNA levels of CRABP II and psoriasin in other hyperproliferative and inflammatory skin diseases showed high expression of psoriasin, and in some cases also of CRABP II, in atopic dermatitis, mycosis fungoides, Darier's disease and inflammatory lichen sclerosus et atrophicus. In atrophic lesions of lichen sclerosus et atrophicus that lacked an inflammatory infiltrate, these changes were only weakly expressed. These findings demonstrate that altered epidermal gene expression of K1, psoriasin and CRABP II is not disease-specific and may reflect instead an altered state of epidermal differentiation and/or may be linked to the inflammation and cellular infiltration common to all the conditions studied.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Queratinas/genética , Psoríase/metabolismo , RNA Mensageiro/biossíntese , Receptores do Ácido Retinoico/genética , Biópsia , Estudos de Casos e Controles , Humanos , Psoríase/patologia , Proteína A7 Ligante de Cálcio S100 , Proteínas S100RESUMO
The broad spectrum of physiological activities of retinoids is mediate d by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Though they have 9-cis retinoic acid as a common ligand, the amino acid sequence of their ligand binding domains is only distantly related (27%). This fact makes it probable that the ligand binding pockets of RARs and RXRs differ significantly with respect to their three dimensional structure. Therefore, one can expect that selective ligands for these receptor subclasses do exit. A clear example of a naturally existing RAR-selective retinoid is all-trans retinoic acid. Here we report on two synthetic retinoids which are very closely related to retinoic acid in structure yet show good receptor subclass selectivity. These compounds have a saturated double bond in the polyene side chain between either the 7, 8 or 9, 10 carbon atoms and are highly RAR or RXR selective, respectively (as shown by receptor binding, transactivation activity and the ability to induce RXR homodimer formation). In addition, we present compounds of the synthetic arotinoid class which are highly RAR selective. Interestingly the corresponding '9-cis analogs' are not able to bind or activate RXR alpha and show greatly reduced activity on the RARs.
Assuntos
Receptores do Ácido Retinoico/efeitos dos fármacos , Retinoides/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Animais , Núcleo Celular/metabolismo , Ligantes , Camundongos , Receptores X de Retinoides , Relação Estrutura-AtividadeRESUMO
Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator-activated receptor include polyunsaturated fatty acids and, for RXR, the 9-cis isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXR alpha, being able to activate a RXR-responsive promoter. We present evidence that phytanic acid binds to RXR alpha, promotes formation of an RXR alpha/RXR response element complex (as detected by gel retardation), and induces a RXR alpha conformational change similar to that induced by 9-cis-retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXR alpha in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism.
Assuntos
Ácido Fitânico/metabolismo , Receptores do Ácido Retinoico/agonistas , Fatores de Transcrição/agonistas , Sequência de Bases , Relação Dose-Resposta a Droga , Escherichia coli/genética , Humanos , Ligantes , Dados de Sequência Molecular , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes/metabolismo , Doença de Refsum/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Tretinoína/metabolismoRESUMO
Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes. This effect is mediated by RAR alpha, as has been demonstrated through the use of a selective RAR alpha antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7129-7133). Here, we show that conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of magnitude the pro-differentiating effects of all-trans retinoic acid and an RAR alpha-selective synthetic retinoid. We also present evidence that these RXR-selective ligands are able to bind to a DNA RXR.RAR heterodimer complex. This finding demonstrates that agonists for RARs and RXRs can synergistically promote HL-60 differentiation, which could be mediated through a heterodimer of these receptors.
Assuntos
Diferenciação Celular/fisiologia , Receptores do Ácido Retinoico/fisiologia , Retinoides/farmacologia , Fatores de Transcrição/fisiologia , Ativação Transcricional/efeitos dos fármacos , Ligação Competitiva , Diferenciação Celular/efeitos dos fármacos , DNA Complementar , Células HL-60 , Humanos , Cinética , Estrutura Molecular , Receptores do Ácido Retinoico/biossíntese , Receptores do Ácido Retinoico/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Retinoides/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/efeitos dos fármacos , TransfecçãoRESUMO
The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with 13-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundibular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets, but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients.
Assuntos
Acne Vulgar/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/metabolismo , Glândulas Sebáceas/metabolismo , Tretinoína/uso terapêutico , Acne Vulgar/tratamento farmacológico , Adolescente , Adulto , Feminino , Humanos , Hibridização In Situ , Masculino , EstereoisomerismoRESUMO
Excessive intake of retinol or of retinoic acid causes a syndrome of characteristic toxic effects known as hypervitaminosis A. To test the role of the nuclear retinoic acid receptor (RAR gamma) in this process we produced mice with a targeted disruption of the RAR gamma gene and examined toxic effects of repeated doses of retinoic acid and two other synthetic retinoids, Ro 15-1570 and Ro 40-6055. Surprisingly, homozygous mutant mice were resistant to fourfold higher doses of retinoic acid than wild-type mice as well as to elevated doses of the synthetic retinoids, indicating that RAR gamma may have a major role in mediating retinoid toxicity, a finding that possibly has practical implications for reducing the toxicity of synthetic retinoids in clinical use.
Assuntos
Mutação , Receptores do Ácido Retinoico/genética , Tretinoína/toxicidade , Animais , Sequência de Bases , Benzoatos/toxicidade , Resistência a Medicamentos/genética , Camundongos , Camundongos Mutantes , Sondas Moleculares/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Retinoides/toxicidade , Pele/efeitos dos fármacos , Tetra-Hidronaftalenos/toxicidade , Tretinoína/sangue , Receptor gama de Ácido RetinoicoRESUMO
The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.
Assuntos
Receptores Citoplasmáticos e Nucleares/química , Receptores do Ácido Retinoico/química , Fatores de Transcrição , Animais , Benzoatos/farmacologia , Cromanos/farmacologia , Clonagem Molecular , Endopeptidases , Humanos , Camundongos , Modelos Biológicos , Naftalenos/farmacologia , Mapeamento de Peptídeos , Conformação Proteica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Retinoides/farmacologia , Deleção de Sequência , Tretinoína/farmacologiaRESUMO
In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3' end of the human cytokeratin 1 (K1) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes were then applied in ISH to human skin sections made from routinely fixed and paraffin-embedded clinical biopsy material. Consistent with published results, we observed strong expression of K1 mRNA in the suprabasal cell layers of the epidermis but only weak to undetectable signals in the basal and cornified cell layers and in the dermis. With this experimental procedure we see no decrease in probe efficiency or quality compared to conventional methods. The use of PCR-derived riboprobe templates for ISH makes it possible to detect expression of any desired gene of known sequence rapidly and efficiently.
Assuntos
Hibridização In Situ/métodos , Queratinas/genética , Reação em Cadeia da Polimerase/métodos , Sondas RNA/química , RNA Mensageiro/análise , Sequência de Bases , Genes , Histocitoquímica , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , Pele/químicaRESUMO
The binding affinity of retinoic acid receptors (RARs) to their response elements is strongly enhanced in vitro by the formation of heterodimers with retinoid X receptors (RXRs) suggesting that heterodimerization with RXR may be a prerequisite for a RAR-mediated transcriptional response. We found that in Drosophila SL-3 cells that are devoid of endogenous RARs and RXRs the presence of RAR is sufficient to confer a response to all-trans retinoic acid (RA). The transfection of both RAR and RXR and stimulation with their respective ligands all-trans and 9-cis RA leads to a synergistic response. On point mutations of the RAR beta 2 gene promoter RA response element (RARE) the stimulation by RARs showed distinct differences in the absence and presence of RXR. The same differences in transcriptional activity are observed, if mammalian cells containing endogenous RARs and RXRs are stimulated with all-trans RA only or additionally with 9-cis RA. This establishes an RXR-independent and an RXR-dependent pathway of all-trans RA action in Drosophila SL-3 cells as well as in mammalian cells. The presence or absence of 9-cis RA determines by which of the two pathways a response to all-trans RA is mediated.
Assuntos
Proteínas de Transporte/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição , Ativação Transcricional , Tretinoína/metabolismo , Animais , Sequência de Bases , Linhagem Celular , DNA/metabolismo , Drosophila , Humanos , Dados de Sequência Molecular , Mutação , Receptores do Ácido Retinoico , Receptores X de Retinoides , Células Tumorais CultivadasRESUMO
Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.
Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Retinoides/farmacologia , Tretinoína/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Cinética , Camundongos , Plasmídeos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores do Ácido Retinoico , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Ativação Transcricional , Transfecção , Tretinoína/metabolismoRESUMO
The sequence requirements for distinguishing the functional specificities of two homeodomain proteins, Antennapedia and Sex combs reduced, involved in the specification of segmental identities in Drosophila, have been determined. A series of deletions and hybrid proteins was generated and assayed for their function in vivo after heat shock-induced ectopic expression during development. A distinct portion of each protein, including the residues within and adjacent to both ends of the homeodomain, has been found to almost entirely determine its functional specificity as measured by diagnostic cuticular transformations of embryonic and adult head structures. The remaining sequences contribute to the potency with which the proteins act in different cells and are to a limited extent functionally transferable from one protein to the other.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Genes Homeobox , Proteínas de Homeodomínio , Proteínas Nucleares , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Deleção Cromossômica , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Embrião não Mamífero/fisiologia , Temperatura Alta , Hormônios de Inseto/genética , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
The homeotic gene Sex Combs Reduced (Scr) of Drosophila is required during embryogenesis for labial and first thoracic segment development. We define the Scr gene structure, showing that the major embryonic transcript is proximal to the fushi tarazu gene, and report the sequence of the transcript, which encodes a 413-amino acid, homeodomain-containing protein. We describe Scr protein distribution throughout embryogenesis. Expression begins at gastrulation and is eventually apparent in three tissues, epidermis, nervous system and visceral mesoderm, though there are clear contrasts in the domains of expression in these three tissues.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes Homeobox , Hormônios de Inseto/genética , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Drosophila melanogaster/embriologia , Embrião não Mamífero/anatomia & histologia , Epiderme/análise , Regulação da Expressão Gênica , Hormônios de Inseto/fisiologia , Mesoderma/análise , Dados de Sequência Molecular , Sistema Nervoso/análise , Sistema Nervoso/embriologia , Proteínas Recombinantes de Fusão/imunologiaRESUMO
The ada gene of Escherichia coli K-12 encodes the regulatory protein for the adaptive response to alkylating agents. A set of plasmids carrying ordered deletions from the 3' end of the ada gene were isolated and characterized. These ada deletions encode fusion proteins that derive their amino termini from ada and their carboxyl termini from the downstream vector sequence that occurs before an in-frame stop codon. Several of these ada deletions encode Ada derivatives that constitutively activate ada transcription to very high levels. A second class of ada deletions encode Ada derivatives that are dominant inhibitors of the inducible transcription of ada but are inducible activators of alkA transcription. In addition, we found that two Ada derivatives containing the same ada sequences but fused to different vector-derived tails have strikingly different properties. One Ada derivative constitutively activates both ada and alkA expression to very high levels. In contrast, the other Ada derivative is an inducible activator of ada expression, like the wild-type Ada protein, but is not an inducible activator of alkA transcription. Our data suggest that the carboxyl terminus of the Ada protein plays a key role in modulating the ability of the Ada protein to function as a transcriptional activator.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Genes , Transcrição Gênica , Alelos , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/fisiologia , Vetores Genéticos , Metiltransferases/metabolismo , Dados de Sequência Molecular , O(6)-Metilguanina-DNA Metiltransferase , Plasmídeos , Regiões Promotoras Genéticas , Fatores de Transcrição , beta-Galactosidase/metabolismoRESUMO
The ada gene of Escherichia coli K-12, the regulatory locus for the adaptive response to methylating agents, coded for a 39,000-dalton protein. An adjacent gene coding for a 27,000-dalton protein was coregulated with ada. The Ada protein was strongly induced upon exposure of cells to methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine. An analysis of ada regulation with an ada-lacZ operon fusion showed that ada+ function was required for induction of ada transcription. Derivatives of the ada gene truncated from the 3' end produced proteins which were more potent in stimulating transcription than the product of the intact ada gene, indicating that the transcription-activating function of the Ada protein resided in its amino terminus. The sequence of the ada-regulatory region and the identification of the start site of ada transcription are also presented.
Assuntos
Alquilantes/toxicidade , Reparo do DNA , Escherichia coli/genética , Genes Bacterianos , Genes Reguladores , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Metilação , Metilnitronitrosoguanidina/toxicidadeRESUMO
Alkaline and neutral elution techniques were used to characterize the production of single- and double-strand DNA breaks in human diploid fibroblasts by incorporated radionuclides. 125I was incorporated in DNA as [125I]iododeoxyuridine, 3H as [3H]-thymidine, and 14C as [14C]thymidine. Under frozen conditions, 125I was 3 times as efficient as 3H per decay in inducing single-strand breaks and 6 times as efficient as 14C. For double-strand break production, however, 125I was 6 times as efficient per decay as 3H. It was calculated that, on the average, each 125I decay produces about one double-strand break in the frozen state. Under nonfrozen conditions, 125I and external X-rays were roughly 5-fold and 3H about 3-fold more efficient in double-strand break induction than under frozen conditions.