RESUMO
The epitopes important for receptor binding and activation of human interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta/IFNAR-2 complex formation, suggesting that steric hindrance of receptor binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction sites.
Assuntos
Alanina/genética , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Interferon beta/química , Interferon beta/imunologia , Receptores de Interferon/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Especificidade de Anticorpos , Ligação Competitiva , Relação Dose-Resposta a Droga , Mapeamento de Epitopos , Humanos , Interferon beta-1a , Interferon beta/genética , Interferon beta/metabolismo , Proteínas de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Receptor de Interferon alfa e beta , Alinhamento de SequênciaRESUMO
Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.
Assuntos
Displasia Ectodérmica/genética , Ligação Genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Fator de Necrose Tumoral alfa/química , Cromossomo X/genética , Processamento Alternativo , Sequência de Aminoácidos , Linhagem Celular , Cromatografia em Gel , Dimerização , Relação Dose-Resposta a Droga , Ectodisplasinas , Ensaio de Imunoadsorção Enzimática , Éxons , Furina , Glicosilação , Humanos , Íntrons , Ligantes , Dados de Sequência Molecular , Fenótipo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Subtilisinas/metabolismoRESUMO
HIPK2 (homeodomain-interacting protein kinase 2) is a CD95 binding partner in yeast. Its primary amino acid sequence is highly conserved between human and mouse. The highest HIPK2 mRNA expression is found in neuronal tissue. The HIPK2 gene is located on human chromosome 7q33-35 and the protein is mainly localized in the nucleus. HIPK2 has been described to play a role as a co-repressor for homeodomain transcription factors.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 7 , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Receptor fas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Códon de Iniciação , DNA Complementar , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.
Assuntos
Interferon beta/genética , Interferon beta/metabolismo , Receptores de Interferon/metabolismo , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Antivirais/metabolismo , Células COS , Análise Mutacional de DNA , Vetores Genéticos/metabolismo , Inibidores do Crescimento/fisiologia , Humanos , Interferon beta-1a , Interferon beta/biossíntese , Interferon beta/fisiologia , Proteínas de Membrana , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Ligação Proteica/genética , Estrutura Terciária de Proteína , Receptor de Interferon alfa e beta , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-beta-1a (IFN-beta-1a) were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-beta-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-beta-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-beta-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked species had an apparent Mr consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-beta-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-beta-1a/IFNAR2 and IFN-beta-1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-beta-1a and its receptor.
Assuntos
Interferon beta/química , Interferon beta/metabolismo , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Cromatografia em Gel , Dimerização , Histidina/química , Humanos , Interferon beta-1a , Interferon beta/isolamento & purificação , Substâncias Macromoleculares , Proteínas de Membrana , Receptor de Interferon alfa e beta , Receptores de Interferon/isolamento & purificação , SolubilidadeRESUMO
The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.
Assuntos
Anticorpos Monoclonais/análise , Mapeamento de Epitopos , Animais , Antivirais/metabolismo , Células COS , Humanos , Interferon-alfa/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologiaRESUMO
Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-beta (HuIFN-beta), in particular in relation to its activity. The recently determined crystal structure of HuIFN-beta provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-beta, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-beta.
Assuntos
Interferon beta/química , Cristalografia por Raios X , Dimerização , Glicosilação , Humanos , Interferon beta/uso terapêutico , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Receptores de Interferon/químicaRESUMO
Type I interferon (IFN) subtypes alpha and beta share a common multicomponent, cell surface receptor and elicit a similar range of biological responses, including antiviral, antiproliferative, and immunomodulatory activities. However, alpha and beta IFNs exhibit key differences in several biological properties. For example, IFN-beta, but not IFN-alpha, induces the association of tyrosine-phosphorylated receptor components ifnar1 and ifnar2, and has activity in cells lacking the IFN receptor-associated, Janus kinase tyk2. To define the structural basis for these functional differences we produced human IFN-beta with point mutations and compared them to wild-type IFN-beta in assays that distinguish alpha and beta IFN subtypes. IFN-beta mutants with charged residues (N86K, N86E, or Y92D) introduced at two positions in the C helix lost the ability to induce the association of tyrosine-phosphorylated receptor chains and had reduced activity on tyk2-deficient cells. The combination of negatively charged residues N86E and Y92D (homologous with IFN-alpha8) increased the cross-species activity of the mutant IFN-betas on bovine cells to a level comparable to that of human IFN-alphas. In contrast, point mutations in the AB loop and D helix had no significant effect on these subtype-specific activities. A subset of these latter mutations did, however, reduce activity in a manner analogous to IFN-alpha mutations. The effects of these mutations on IFN-beta activity are discussed in the context of a family of related ligands acting through a common receptor and signaling pathway.
Assuntos
Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Análise Mutacional de DNA , Humanos , Interferon-alfa/química , Interferon beta/química , Dados de Sequência Molecular , Mutação Puntual , Relação Estrutura-AtividadeRESUMO
PURPOSE: Two recombinant IFN-beta products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-beta-1a) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-beta-1b). The structural basis for activity differences between IFN-beta-1a and IFN-beta-1b, is determined. METHODS: In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-beta products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. RESULTS: IFN-beta-1a was 10 times more active than IFN-beta-1b with specific activities in a standard antiviral assay of 20 x 10(7) IU/mg for IFN-beta-1a and 2 x 10(7) IU/mg for IFN-beta-1b. Of the known structural differences between IFN-beta-1a and IFN-beta-1b, only glycosylation affected in vitro activity. Deglycosylation of IFN-beta-1a produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-beta-1b revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 x 10(7) IU/mg). Crystallographic data for IFN-beta-1a revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. CONCLUSIONS: Together these results suggest that the greater biological activity of IFN-beta-1a is due to a stabilizing effect of the carbohydrate on structure.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antivirais/farmacologia , Interferon beta/farmacologia , Proteínas Recombinantes/farmacologia , Adjuvantes Imunológicos/química , Sequência de Bases , Infecções por Cardiovirus/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cisteína/química , Vírus da Encefalomiocardite/efeitos dos fármacos , Glicosilação , Humanos , Interferon beta-1a , Interferon beta-1b , Interferon beta/química , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/química , Serina/químicaRESUMO
Interferons (IFNs) generally have been characterized as antiproliferative cytokines. The cell cycle arrest in G1/G0 phase induced by type I IFNs, especially IFN-alpha, was recognized as a manifestation of their antiproliferative effects. In this article, we report that the cell cycle block in G1/G0 is observed mainly in certain cell types, such as Daudi Burkitt's lymphoma cells. In a variety of human transformed cells, but not nontransformed primary cells, IFN-beta and IFN-alpha induced a significant increase in the S phase population. The increase appeared to be due to a continued S phase entry and subsequently a failure of S phase cells to transit efficiently into G2 and M phases. The ability of tumor cells to exhibit the S phase effect correlated with proper IFN signaling and loss or inactivation of the normal G1 checkpoint conferred by the retinoblastoma protein (pRB). Overriding the G1 checkpoint switched human nontransformed primary cells from nonresponsive to sensitive to the IFN-induced effect. Therefore, the cell cycle regulatory machinery could function, at least in part, as a determining factor that affects the IFN-induced cell cycle effect. The IFN effect in transformed cells may suggest intriguing prospects for combinatorial therapies for cancer.
Assuntos
Interferon beta/farmacologia , Fase S/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Fase G1/efeitos dos fármacos , Humanos , Proteína do Retinoblastoma/efeitos dos fármacos , Proteína do Retinoblastoma/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transativadores/efeitos dos fármacos , Transativadores/fisiologia , Células Tumorais CultivadasRESUMO
Apoptosis occurs in the normal liver and in various forms of liver disease. The CD95 (APO-1/Fas) (CD95) receptor mediates apoptosis, and liver cells in animal models are acutely sensitive to apoptosis initiated by this receptor. We have used primary human hepatocytes as a model system to investigate CD95-mediated apoptotic liver damage. Treatment of fresh human hepatocytes with low concentrations of agonistic antibodies against CD95 resulted in apoptosis of > 95% of the cultured liver cells within 4 and 7.5 h. Immunohistology of a panel of explanted liver tissues revealed that hepatocytes in normal livers (n = 5) and in alcoholic cirrhosis (n = 13) expressed low constitutive levels of CD95. CD95 receptor expression was highly elevated in hepatocytes in hepatitis B virus-related cirrhosis (n = 9) and in acute liver failure (n = 8). By in situ hybridization CD95 ligand messenger RNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of hepatitis B virus-related cirrhosis and acute hepatic failure, ligand expression was found primarily in areas with lymphocytic infiltration. In contrast, in patients with alcoholic liver damage, high CD95 ligand messenger RNA expression was found in hepatocytes. These findings suggest that liver destruction in hepatitis B may primarily involve killing of hepatocytes by T lymphocytes using the CD95 receptor-ligand system. In alcoholic liver damage, death of hepatocytes might occur by fratricide and paracrine or autocrine mechanisms mediated by the hepatocytes themselves.
Assuntos
Apoptose/fisiologia , Fígado/patologia , Glicoproteínas de Membrana/fisiologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Proteína Ligante Fas , Hepatite Viral Humana/complicações , Hepatite Viral Humana/patologia , Humanos , Hibridização in Situ Fluorescente , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Cirrose Hepática Alcoólica/patologia , Falência Hepática/patologia , Ativação Linfocitária , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , RNA Mensageiro/análise , Receptor fas/imunologiaRESUMO
Two-hybrid protein interaction screening in yeast was used to identify proteins that interact with the HBx nonstructural protein of hepatitis B virus (HBV). A new human member of the proteasome alpha-subunit family was obtained. Its protein sequence closely resembles the 28 kD subunits from other organisms. The interaction with HBx was abolished by a two amino-acid insertion behind position 128 in HBx, in a region previously found to be essential for its transcriptional transactivation function. These data support a model of HBx acting indirectly on transcriptional processes. By binding to a specific proteasome alpha-subunit, HBx might interfere with degradative processes, thereby enhancing the half-life of different transcription factors and other nuclear regulatory proteins. Interaction with the Hu 28k proteasome subunit could thus provide a unifying explanation for the markedly pleiotropic effects of HBx.
Assuntos
Cisteína Endopeptidases/metabolismo , Antígenos da Hepatite B/metabolismo , Complexos Multienzimáticos/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
During hepatitis B virus (HBV) infection, distinct host-virus interactions may establish the patterns of viral clearance and persistence and the extent of virus-associated pathology. It is generally thought that HBV-specific class I-restricted cytotoxic T lymphocytes (CTLs) play a critical role in this process by destroying infected hepatocytes. This cytopathic mechanism, however, could be lethal if most of the hepatocytes are infected. In the current study, we demonstrate that class I-restricted HBV-specific CTLs profoundly suppress hepatocellular HBV gene expression in HBV transgenic mice by a noncytolytic process, the strength of which greatly exceeds the cytopathic effect of the CTLs in magnitude and duration. We also show that the regulatory effect of the CTLs is initially mediated by interferon gamma and tumor necrosis factor alpha, is delayed in onset, and becomes independent of these cytokines shortly after it begins. The data indicate that the anti-viral CTL response activates a complex regulatory cascade that inhibits hepatocellular HBV gene expression without killing the cell. The extent to which this mechanism contributes to viral clearance or viral persistence during HBV infection remains to be determined.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Hepatopatias/imunologia , Linfócitos T Citotóxicos/microbiologia , Animais , Citocinas/genética , Citotoxicidade Imunológica , Regulação Viral da Expressão Gênica , Interferon gama/fisiologia , Hepatopatias/microbiologia , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Viral/genética , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
A panel of mutants of the hepatitis B virus X gene (HBx) was constructed by oligonucleotide-directed insertion of two codons (Arg Pro) at 10- or 20-amino-acid intervals along the entire gene. These mutants were tested in transiently transfected HepG2 cells for their effects on HBx trans-activation of an AP1-CAT reporter plasmid. The effects of HBx mutations on the mRNA and protein stability were also determined. Our results reveal two separate internal domains of the HBx, one around amino acid residue 68 and the other between residues 110 and 139, that are necessary for its activity. Mutations in the amino terminus (to position 45), between the two necessary domains and in the extreme carboxyl terminal portion of HBx, have little or no effect on its activity.
Assuntos
Regulação Viral da Expressão Gênica , Antígenos da Hepatite B/genética , Mutação , Transativadores/genética , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Códon , Análise Mutacional de DNA , Genes Reporter , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/biossíntese , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais Reguladoras e AcessóriasRESUMO
Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Análise Mutacional de DNA , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-fos , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
A sequence of 86 bp within the 5' region of the adult chicken beta-globin gene was found to undergo a DNA conformational transition at elevated levels of negative superhelical stress (- sigma = 0.068). In vitro chemical DNA modification studies which detect purine hyperreactivity (HR) to the alkylating agent diethyl pyrocarbonate (DEP) have identified this 86 bp long DEP-HR element. The DEP-HR element is composed of small, tandem segments with imperfect purine-pyrimidine alternations. Methylation of cytosines within GCGC sequences of the DEP-HR element facilitates this structural change. The binding of a monoclonal anti-Z-DNA antibody to the element has been revealed by chemical footprinting with DEP. These data suggest that the DEP-HR sequence can undergo a conformational transition to Z-DNA. It is unknown whether the conformational flexibility observed here occurs in vivo.
Assuntos
DNA/genética , Genes , Globinas/genética , Animais , Anticorpos , Sequência de Bases , Galinhas , Deleção Cromossômica , DNA/sangue , Eritrócitos/metabolismo , Conformação de Ácido Nucleico , PlasmídeosRESUMO
Diethylpyrocarbonate (DEPC) carbethoxylates Z-DNA to an increased extent because the reactive N-7 atoms of purine residues appear structurally more accessible on Z-DNA as opposed to B-DNA. This chemical probe was used in DEPC footprinting experiments, which confirm the specificity of binding of anti-Z-DNA monoclonal antibodies and which probe regions of close contact in this DNA-protein complex. Antibody binding to segments of Z-DNA existing in supercoiled plasmids resulted in specific protection from DEPC hyper-reactivity within the Z-DNA segment and induction of hyper-reactivity in purines lying adjacent to the Z-segment. Two different monoclonal immunoglobulin preparations, Z22 and Z44, are shown to generate specific and distinct footprint patterns when bound to the Z-helix. Binding of these antibodies was also found to affect DNA conformation within the Z-DNA segment by influencing the equilibrium between the B- and Z-helical conformations.
Assuntos
Anticorpos Antinucleares/imunologia , DNA/imunologia , Dietil Pirocarbonato/farmacologia , Formiatos/farmacologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Computadores , DNA/efeitos dos fármacos , DNA Super-Helicoidal , Epitopos , Conformação de Ácido NucleicoRESUMO
We determined the DNA sequence of the D-serine deaminase promoter region and of the N-terminal region of the structural gene. There are possibilities in the promoter for secondary structure and for initiation recognition sequences, and there is an open reading frame. The N-terminal sequence for the structural gene confirms that part of the amino acid sequence previously determined by E. Schlitz and W. Schmitt (FEBS Lett. 134:57-62, 1981), including the active site of the enzyme, and spans the two regions unresolved by their work.