RESUMEN
Inherited mutations of the TFIIH helicase subunits xeroderma pigmentosum (XP) B or XPD yield overlapping DNA repair and transcription syndromes. The high risk of cancer in these patients is not fully explained by the repair defect. The transcription defect is subtle and has proven more difficult to evaluate. Here, XPB and XPD mutations are shown to block transcription activation by the FUSE Binding Protein (FBP), a regulator of c-myc expression, and repression by the FBP Interacting Repressor (FIR). Through TFIIH, FBP facilitates transcription until promoter escape, whereas after initiation, FIR uses TFIIH to delay promoter escape. Mutations in TFIIH that impair regulation by FBP and FIR affect proper regulation of c-myc expression and have implications in the development of malignancy.
Asunto(s)
Factores de Transcripción TFII , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xerodermia Pigmentosa/metabolismo , Western Blotting , Línea Celular , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Genes Dominantes , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Mutación , Neoplasias/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción TFIIH , Transcripción Genética , Transfección , Xerodermia Pigmentosa/genéticaRESUMEN
FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, possesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocked activator-dependent, but not basal, transcription through TFIIH. Recruited through FBP's nucleic acid-binding domain, FIR formed a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR contained an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targeted only TFIIH's p89/XPB helicase, required at several stages in transcription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Represoras/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Sitios de Unión , ADN Helicasas/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Células HeLa , Humanos , Sustancias Macromoleculares , Modelos Genéticos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Unión Proteica , Factores de Empalme de ARN , Proteínas de Unión al ARN , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción TFIIH , Técnicas del Sistema de Dos HíbridosRESUMEN
The K homology (KH) motif is one of the major classes of nucleic acid binding proteins. Some members of this family have been shown to interact with DNA while others have RNA targets. There have been no reports containing direct experimental evidence regarding the nature of KH module-DNA interaction. In this study, the interaction of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K (KH3) with its cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates that the first two helices, the conserved GxxG loop, beta 1, and beta 2, are the primary regions involved in DNA binding for KH3. The nature of the KH3-ssDNA interaction is further illuminated by a comparison of backbone 15N relaxation data for the bound and unbound KH3. Relaxation data are also used to confirm that the backbone of wild-type KH3 is structurally identical to that of the G26R mutant KH3, which was previously published. Amide proton exchange experiments indicate that the two helices involved in DNA binding are less stable than other regions of secondary structure and that a large portion of KH3 backbone amide hydrogens are protected in some manner upon ssDNA binding. The major backbone dynamics features of KH3 are similar to those of the structurally comparable human papillomavirus-31 E2 DNA binding domain. Secondary structure information for ssDNA-bound wild-type KH3 is also presented and shows that binding results in no global changes in the protein fold.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Homología de Secuencia de Aminoácido , Amidas , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteínas de Unión al ADN/genética , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Mapeo Peptídico , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/genética , Protones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleoproteínas/genéticaRESUMEN
Among it's many reported functions, heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transcription factor for the c- myc gene, a proto-oncogene critical for the regulation of cell growth and differentiation. We have determined the solution structure of the Gly26-->Arg mutant of the C-terminal K-homology (KH) domain of hnRNP K by NMR spectroscopy. This is the first structure investigation of hnRNP K. Backbone residual dipolar couplings, which provide information that is fundamentally different from the standard NOE-derived distance restraints, were employed to improve structure quality. An independent assessment of structure quality was achieved by comparing the backbone15N T1/T2ratios to the calculated structures. The C-terminal KH module of hnRNP K (KH3) is revealed to be a three-stranded beta-sheet stacked against three alpha-helices, two of which are nearly parallel to the strands of the beta-sheet. The Gly26-->Arg mutation abolishes single-stranded DNA binding without altering the overall fold of the protein. This provides a clue to possible nucleotide binding sites of KH3. It appears unlikely that the solvent-exposed side of the beta-sheet will be the site of protein-nucleic acid complex formation. This is in contrast to the earlier theme for protein-RNA complexes incorporating proteins structurally similar to KH3. We propose that the surface of KH3 that interacts with nucleic acid is comparable to the region of DNA interaction for the double-stranded DNA-binding domain of bovine papillomavirus-1 E2 that has a three-dimensional fold similar to that of KH3.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/genética , Ribonucleoproteínas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Bovinos , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Datos de Secuencia Molecular , Conformación Proteica , Proto-Oncogenes Mas , Ribonucleoproteínas/metabolismo , Soluciones , Factores de Transcripción/metabolismoRESUMEN
Genetic processes require direct interactions between proteins bound at nonadjacent cis elements. Because duplex DNA is rigid, either the protein-protein interactions are strong enough to deform the double helix or some feature of the intervening DNA must encourage juxtaposition of separated sites. For example, bent DNA can bring together only certain precisely positioned cis elements with the same helical phase. Interposing a DNA segment that both bends and twists easily to create a universal joint would provide an even more general mechanism to promote the association of separated sites regardless of position. A cis element of the human c-myc gene, known to be melted in vivo, and its associated single-strand DNA binding protein were examined and found to comprise just such a protein-DNA hinge.
Asunto(s)
ADN de Cadena Simple/metabolismo , Integrasas/metabolismo , Recombinación Genética/fisiología , Ribonucleoproteínas/metabolismo , Transcripción Genética/fisiología , Proteínas Virales , ADN de Cadena Simple/química , Proteínas Fúngicas/genética , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Integrasas/química , Conformación de Ácido Nucleico , Proteínas Proto-Oncogénicas c-myc/genética , Ribonucleoproteínas/química , TATA Box/genética , Transactivadores/genéticaRESUMEN
Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.
Asunto(s)
Anticuerpos Antibacterianos , Proteínas Opsoninas/inmunología , Infecciones Neumocócicas/diagnóstico , Infecciones Neumocócicas/inmunología , Adulto , Animales , Animales Recién Nacidos , Unión Competitiva/inmunología , Proteínas del Sistema Complemento/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Células HL-60/química , Células HL-60/inmunología , Células HL-60/microbiología , Humanos , Inmunoglobulina G , Inmunoglobulinas Intravenosas/administración & dosificación , Lactante , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Infecciones Neumocócicas/terapia , Conejos , Receptores de Superficie Celular/inmunologíaRESUMEN
A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.
Asunto(s)
Actividad Bactericida de la Sangre/inmunología , Neisseria meningitidis/inmunología , Adulto , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/inmunología , Preescolar , Proteínas del Sistema Complemento/inmunología , Humanos , Técnicas de Inmunoadsorción , Lactante , Laboratorios , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Persona de Mediana Edad , Neisseria meningitidis/clasificación , Estándares de Referencia , Reproducibilidad de los Resultados , Serotipificación , Especificidad de la EspecieRESUMEN
Two hundred twenty-one Gambian children vaccinated previously with one, two, or three doses of a meningococcal conjugate vaccine or two doses of polysaccharide vaccine before the age of 6 months were revaccinated at the age of 18-24 months with either meningococcal polysaccharide, conjugate, or inactivated polio vaccines. Children who had previously received one, two, or three doses of conjugate vaccine had significantly (P < .001) higher anti-group C meningococcal antibody levels following revaccination than did children vaccinated with a polysaccharide vaccine for the first time. Children vaccinated previously with two doses of polysaccharide vaccine had a lower group C antibody response than did control children. Group A antibody responses following revaccination of children who had previously received polysaccharide or conjugate vaccine were not significantly higher than those in control children. Thus, immunologic memory was probably induced by the group C but not by the group A component of the conjugate vaccine.