RESUMEN
INTRODUCTION: Vorinostat is a small molecule inhibitor of class I and II histone deacetylases with preclinical activity in melanoma. METHODS: We evaluated 32 patients with advanced primary cutaneous or ocular melanoma in a multi-institutional setting (PMH Phase II Consortium) with continuous daily oral vorinostat 400 mg. The primary endpoint was response rate by RECIST, with time to progression as a secondary endpoint. The study was designed to distinguish a response rate of 20 % from a RR of 5 % and to distinguish a 2 month median progression-free survival (PFS), from one of 3.1 months. The study proceeded to stage 2 following 2 of 16 responses.. We also assessed VEGF, FGF levels, P52 polymorphisms and chromatin-associated proteins as potential biomarkers. RESULTS: Therapy was associated with significant side effects, including fatigue, nausea, lymphopenia, and hyperglycemia. Eleven patients experienced at least one grade 3 or higher adverse event. There were two confirmed PRs in patients with cutaneous melanoma. Sixteen patients had stable disease and 14 patients had progressive disease for best response. In addition, two patients with cutaneous melanoma scored as stable disease had early unconfirmed partial responses with subsequent progression. Patients with stable disease or partial response (n = 18) had a median progression free survival of 5 months. (range 2-12 months). CONCLUSIONS: Vorinostat demonstrated some early responses and a high proportion of patients with stable disease, but did not meet its primary endpoint of response. Different schedules of this agent with BRAF mutation status and markers of histone acetylation could be explored in melanoma.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Melanoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Biomarcadores/sangre , Supervivencia sin Enfermedad , Femenino , Factores de Crecimiento de Fibroblastos/sangre , Inhibidores de Histona Desacetilasas/efectos adversos , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/efectos adversos , Ácidos Hidroxámicos/farmacología , Masculino , Melanoma/genética , Melanoma/metabolismo , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Neoplasias Cutáneas , Proteína p53 Supresora de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/sangre , Vorinostat , Melanoma Cutáneo MalignoRESUMEN
A fast and robust method for determining the parameters for a flat (mask-based) bulk-solvent model and overall scaling in macromolecular crystallographic structure refinement and other related calculations is described. This method uses analytical expressions for the determination of optimal values for various scale factors. The new approach was tested using nearly all entries in the PDB for which experimental structure factors are available. In general, the resulting R factors are improved compared with previously implemented approaches. In addition, the new procedure is two orders of magnitude faster, which has a significant impact on the overall runtime of refinement and other applications. An alternative function is also proposed for scaling the bulk-solvent model and it is shown that it outperforms the conventional exponential function. Similarly, alternative methods are presented for anisotropic scaling and their performance is analyzed. All methods are implemented in the Computational Crystallography Toolbox (cctbx) and are used in PHENIX programs.
Asunto(s)
Bioingeniería/métodos , Biología Computacional/métodos , Sustancias Macromoleculares/química , Modelos Moleculares , Algoritmos , Anisotropía , Bioingeniería/tendencias , Biología Computacional/tendencias , Cristalografía por Rayos X , Sustancias Macromoleculares/metabolismo , Distribución Normal , Solventes , Factores de Tiempo , Difracción de Rayos X/métodos , Difracción de Rayos X/tendenciasRESUMEN
Equations in Sections 2.3 and 2.4 of the article by Afonine et al. [Acta Cryst. (2013). D69, 625-634] are corrected.
RESUMEN
Many cancers show an increase in incidence with age, and age is the biggest single risk factor for many cancers. However, the molecular basis of this relationship is poorly understood. Through a collection of review articles, our thematic issue discusses the link between aging and cancer in aspects including somatic mutations, proteostasis, mitochondria, metabolism, senescence, epigenetic regulation, immune regulation, DNA damage, and telomere function.
Asunto(s)
Epigénesis Genética , Neoplasias , Envejecimiento/genética , Envejecimiento/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Telómero/genéticaRESUMEN
The retinoblastoma protein and p53 are both cell-cycle regulators and are, directly or indirectly, inactivated in the majority of human tumors. Recent studies have provided new mechanistic insights into how these proteins regulate cell growth in response to various intracellular and extracellular signals.
Asunto(s)
Ciclo Celular/fisiología , Neoplasias/genética , Proteína de Retinoblastoma/fisiología , Proteína p53 Supresora de Tumor/fisiología , Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-microm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm(-1), differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.
Asunto(s)
Pared Celular/metabolismo , Lignina/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Populus/metabolismo , Plantas Modificadas Genéticamente/citología , Populus/citología , Espectrometría RamanRESUMEN
Deregulated AKT kinase activity due to PTEN deficiency in cancer cells contributes to oncogenesis by incompletely understood mechanisms. Here, we show that PTEN deletion in HCT116 and DLD1 colon carcinoma cells leads to suppression of CHK1 and CHK2 activation in response to irradiation, impaired G2 checkpoint proficiency and radiosensitization. These defects are associated with reduced expression of MRE11, RAD50 and NBS1, components of the apical MRE11/RAD50/NBS1 (MRN) DNA damage response complex. Consistent with reduced MRN complex function, PTEN-deficient cells fail to resect DNA double-strand breaks efficiently after irradiation and show greatly diminished proficiency for DNA repair via the error-free homologous recombination (HR) repair pathway. MRE11 is highly unstable in PTEN-deficient cells but stability can be significantly restored by inhibiting mTORC1 or p70S6 kinase (p70S6K), downstream kinases whose activities are stimulated by AKT, or by mutating a residue in MRE11 that we show is phosphorylated by p70S6K in vitro. In primary human fibroblasts, activated AKT suppresses MRN complex expression to escalate RAS-induced DNA damage and thereby reinforce oncogene-induced senescence. Taken together, our data demonstrate that deregulation of the PI3K-AKT/ mTORC1/ p70S6K pathways, an event frequently observed in cancer, exert profound effects on genome stability via MRE11 with potential implications for tumour initiation and therapy.
Asunto(s)
Inestabilidad Genómica/genética , Proteína Homóloga de MRE11/genética , Neoplasias/genética , Fosfohidrolasa PTEN/deficiencia , Reparación del ADN por Recombinación/genética , Daño del ADN/efectos de la radiación , Regulación hacia Abajo , Fibroblastos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Células HCT116 , Humanos , Proteína Homóloga de MRE11/antagonistas & inhibidores , Proteína Homóloga de MRE11/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias/radioterapia , Fosfohidrolasa PTEN/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinonas/farmacología , ARN Interferente Pequeño/metabolismo , Tolerancia a Radiación/genética , Reparación del ADN por Recombinación/efectos de la radiación , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/genética , Tionas/farmacología , Rayos X/efectos adversosRESUMEN
Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Especificidad por Sustrato/genéticaRESUMEN
Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteína de Retinoblastoma/química , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Quinasas Ciclina-Dependientes/química , Ciclinas/química , Cartilla de ADN/genética , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/genética , Especificidad por SustratoRESUMEN
Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates. Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1). Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity. EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle. Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2. A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells. Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Ciclo Celular/fisiología , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteína de Unión a CREB , Proteínas de Ciclo Celular , Diferenciación Celular , Clonación Molecular , Regulación hacia Abajo , Histona Acetiltransferasas , Datos de Secuencia Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Represoras , Proteína de Retinoblastoma/metabolismo , Transactivadores/antagonistas & inhibidores , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Ubiquitinas/metabolismoRESUMEN
Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1). Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/química , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Western Blotting , Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Separación Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Citometría de Flujo , Glutatión Transferasa/metabolismo , Chaperonas de Histonas , Humanos , Espectrometría de Masas , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Péptidos/química , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Fase S , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Treonina/química , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The automation of macromolecular structure determination by X-ray crystallography has long been a goal for many researchers. Recently, there have been improvements in the underlying algorithms, some of which have been implemented in software packages that deal with multiple stages of the structure determination process. These first steps towards complete automation have made X-ray crystallography more efficient.
Asunto(s)
Sistemas de Administración de Bases de Datos , Estructura Molecular , Automatización , Cristalografía por Rayos XRESUMEN
Macromolecular crystallographic refinement has recently been made more efficient by the use of cross-validated maximum likelihood targets and torsion-angle molecular dynamics simulated annealing. In combination with automated model building methods, the amount of manual intervention required to complete and refine a structure is greatly reduced.
Asunto(s)
Cristalografía por Rayos X/métodos , Sustancias Macromoleculares , Modelos Químicos , Modelos Moleculares , Funciones de VerosimilitudRESUMEN
Cellular senescence is a state of stable cell cycle arrest triggered by diverse stresses. Establishment of senescence occurs in conjunction with a multitude of chromatin changes, which are just beginning to be studied. These chromatin changes are hypothesized to be causative for senescence. Currently, a preferred method to study such changes is chromatin immunoprecipitation followed by sequencing (ChIP-Seq). This is usually done by cross-linking the cells with formaldehyde and then generating chromatin fragments between 150 and 300bp by sonication. The DNA replication-independent histone chaperone HIRA plays an important role in control of chromatin in nonproliferating senescent cells. While investigating the role of HIRA in senescence, we found conventional ChIP protocols to be problematic, routinely yielding too low amounts of DNA for sequencing. To overcome these problems we adapted and optimized an alternative ChIP method that does not rely on cross-linking and sonication for chromatin fragmentation, and is able to easily isolate chromatin from senescent cells ready for immunoprecipitation. This method uses Benzonase endonuclease for solubilization of uncross-linked chromatin by digestion of DNA and RNA, in the absence of proteolytic activity. Using this protocol, we were easily able to immunoprecipitate HIRA with sufficient DNA for Illumina sequencing.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Endodesoxirribonucleasas/metabolismo , Endorribonucleasas/metabolismo , Epigenómica/métodos , Serratia marcescens/enzimología , Animales , Puntos de Control del Ciclo Celular , Senescencia Celular , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Humanos , Análisis de Secuencia de ADN/métodosRESUMEN
The transcription factor E2F activates genes required for S phase, such as cyclin E and cyclin A. We show that, contrary to long term effects of E2F-1 overexpression, short ectopic overexpression of this transcription factor in logarithmically growing cells does neither affect the cell cycle distribution nor the cell size, but heavily induces cyclin E and A expression as well as cyclin E- and A-dependent kinase activities. We further separated logarithmically growing E2F-1-overexpressing cells according to their different cell cycle phases by centrifugal elutriation. These experiments revealed that deregulated E2F-1 expression triggers high levels of cyclin E and A expression and kinase activities in small early G1 cells, normally not exhibiting these activities. These effects on the regulation of cyclin E- and A-associated kinases are not accompanied by any detectable alteration in the rate of progression through the cell cycle, suggesting that these changes are independent of any mitogenic properties of E2F-1.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Proteínas de Unión al ADN , Factores de Transcripción/genética , Animales , Línea Celular , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Regulación de la Expresión Génica , ARN Mensajero/genética , Ratas , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1RESUMEN
The retinoblastoma tumor suppressor protein (pRB) is a paradigm for understanding cell cycle- and proliferation-dependent transcription and how deregulation of this process contributes to the neoplastic process in humans. The ability of pRB to regulate transcription, and consequently cell proliferation and differentiation, is regulated by the activity of cyclin/cdks. In general, phosphorylation of pRB by cyclin/cdks inactivates pRB-mediated transcriptional inhibition and growth suppression. However, it is apparent that pRB is a multi-functional protein that can inhibit transcription through various mechanisms. This review focuses on recent data to suggest that different pRB functions are progressively and cooperatively inactivated by multiple cyclin/cdk complexes during G1- and S-phase. The implications of such a model for pRB-mediated tumor suppression are discussed.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclinas/farmacología , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas , Proteína de Retinoblastoma/genética , Animales , Sitios de Unión , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Nucleares/química , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/farmacología , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/química , Proteína p107 Similar a la del RetinoblastomaRESUMEN
A structural model of pentameric phospholamban (Plb) in a lipid bilayer has been derived using a combination of experimental data, obtained from ATR-FTIR site-directed dichroism, and the implementation of the resulting restraints during a molecular dynamics simulation. Plb (residues 24-52) has been synthesised incorporating a new label, 1-(13)C==(18)O, at residues 42 and 43. We have not only determined the tilt of the helices, 10(+/-6) degrees, but also the relative orientation of the transmembrane segments, with an omega angle of -32(+/-10) degrees for L42. This angle is taken as zero in the direction of the helix tilt. Plb is a simple test case where site-directed dichroism has been applied to resolve the indeterminacy arising from the mutagenesis data available. The results presented point specifically to a single structural model for Plb.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Mutagénesis/genética , Amidas/metabolismo , Proteínas de Unión al Calcio/genética , Isótopos de Carbono , Simulación por Computador , Isótopos de Oxígeno , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , VibraciónRESUMEN
The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.
Asunto(s)
Virus de la Influenza A/metabolismo , Proteínas de la Membrana/química , Proteínas de la Matriz Viral/química , Secuencia de Aminoácidos , Humanos , Canales Iónicos/química , Liposomas/metabolismo , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The product of the retinoblastoma tumor-suppressor gene (RB) is a ubiquitously expressed, 105-kDa nuclear phosphoprotein (pRB). The pRB protein negatively regulates the cellular G1/S phase transition, and it is at this point in the cell cycle that it is thought to play its role as a tumor suppressor. The growth-inhibitory effects of pRB are exerted, at least in part, through the E2F family of transcription factors. This chapter reviews the insights into the mechanism of action of the E2F family members that have been obtained through overexpression studies. Studies in RB-/- SAOS-2 cells have provided evidence in support of the hypothesis that the E2F family members are negatively regulated by pRB and the related protein p130. In particular, the results obtained are consistent with the earlier biochemical data which suggested that E2F1 is regulated primarily by pRB, and E2F4 by p130. Results relating to p107 are also discussed. Consistent with the proposed role of pRB and E2F1 as coregulators of entry into S phase, experiments have demonstrated that overexpression of E2F1 is sufficient to override the cell cycle arrests caused by serum deprivation of fibroblasts or transforming growth factor-beta (TGF-beta) treatment of mink lung epithelial cells. However, at least in the case of the serum deprivation induced arrest, the ultimate result of E2F1 overexpression is death by p53-dependent apoptosis. In light of this and other data, a model is discussed as to how functional inactivation of pRB and p53 might cooperate to promote tumorigenesis. A number of studies have demonstrated the oncogenic potential of E2F family members, at least under certain conditions. This is, again, in keeping with the notion that these proteins play a critical role in controlling proliferation.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Proteínas de Unión al ADN , Factores de Transcripción/fisiología , Animales , Apoptosis/fisiología , Transformación Celular Neoplásica , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F4 , Humanos , Mutación , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Threonine and tyrosine residue phosphorylation of a 42 kDa protein identified as mitogen-activated protein kinase (MAP kinase) was stimulated in extracts from TPA-pretreated cells. It is further shown that TPA pretreatment leads to the enhancement of an activity that will induce reactivation of dephosphorylated/inactivated MAP kinase. This TPA-induced activity induces the threonine and tyrosine phosphorylation of p42 in extracts from unstimulated cells.