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1.
Gene Ther ; 30(3-4): 377-385, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36253453

RESUMEN

The widespread pre-existing αAAV-Abs in humans pose a critical challenge in translation of AAV gene therapy. The IgG degrading enzyme of Streptococci (IdeS) is demonstrated to specifically cleave IgG of humans and other species (not mouse). This study developed a modified new modified IdeS protein product (IdeSop). When incubated in vitro, IdeSop was shown to completely cleave human and rabbit IgGs within 6 h. To test IdeSop in a disease setting, we established a rabbitized αAAV9-Ab+ mouse by an IV infusion of purified acute αAAV9-Ab+ rabbit IgG into MPS IIIA mice, resulting in serum αAAV9-IgG at 1:6,400 and αAAV9-nAbs at 1:800. IdeSop-Ab-cleavage was shown to be dose-dependent. An IV IdeSop infusion at the effective doses resulted in rapid IgG depletion and clearance of pre-existing αAAV9-IgG and αAAV9-nAbs in rabbitized αAAV9-Abs+ MPS IIIA mice. Importantly, an IV injection of a high dose AAV9-hSGSHop vector (5 × 1013vg/kg) at 24 h post IdeSop treatment led to transduction as effective in αAAV9-Abs+ MPS IIIA mice, as in αAAV9-Abs-negative controls. We believe that transient IdeSop administration may offer a great tool to address the pre-existing-αAAV-Abs for the translation of rAAV gene therapy to treat diseases in humans, making effective rAAV gene therapy available to all patients in need.


Asunto(s)
Proteínas Bacterianas , Mucopolisacaridosis III , Conejos , Animales , Ratones , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/uso terapéutico , Mucopolisacaridosis III/tratamiento farmacológico , Inmunoglobulina G , Terapia Genética
2.
Biochem J ; 470(3): 331-42, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26195823

RESUMEN

Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs). During autophagy, aberrant regulation of the lysosomal Ca(2+) efflux channel TRPML1 [transient receptor potential mucolipin 1 (MCOLN1)], also known as MCOLN1, is solely responsible for the human LSD mucolipidosis type IV (MLIV); however, the exact mechanisms involved in the development of the pathology of this LSD are unknown. In the present study, we provide evidence that the target of rapamycin (TOR), a nutrient-sensitive protein kinase that negatively regulates autophagy, directly targets and inactivates the TRPML1 channel and thereby functional autophagy, through phosphorylation. Further, mutating these phosphorylation sites to unphosphorylatable residues proved to block TOR regulation of the TRPML1 channel. These findings suggest a mechanism for how TOR activity may regulate the TRPML1 channel.


Asunto(s)
Mucolipidosis/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Autofagia , Sitios de Unión , Señalización del Calcio , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Genes de Insecto , Células HEK293 , Humanos , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Mucolipidosis/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potencial de Receptor Transitorio/genética
3.
Sci Adv ; 8(13): eabm9718, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35363522

RESUMEN

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.


Asunto(s)
Fibrosis Quística , Animales , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Moco/metabolismo , Sistema Respiratorio/metabolismo , Esputo/metabolismo , Porcinos
4.
Sci Adv ; 7(42): eabg4084, 2021 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-34652943

RESUMEN

Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (<100 pM) and enhance production yield by more than 50-fold. Cross-dimer epitope antibodies bind to the stabilized dimers, and a crystal structure resembles the wild-type (WT) E protein bound to a dimer epitope antibody. Mice immunized with the stabilized dimers developed antibodies that bind to E dimers and not monomers and elicited higher levels of DENV2-neutralizing antibodies compared to mice immunized with WT E antigen. Our findings demonstrate the feasibility of using structure-based design to produce subunit vaccines for dengue and other flaviviruses.

5.
PLoS One ; 12(4): e0176502, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28437447

RESUMEN

Carbonic anhydrases are a family of enzymes that catalyze the reversible condensation of water and carbon dioxide to carbonic acid, which spontaneously dissociates to bicarbonate. Carbonic anhydrase III (Car3) is nutritionally regulated at both the mRNA and protein level. It is highly enriched in tissues that synthesize and/or store fat: liver, white adipose tissue, brown adipose tissue, and skeletal muscle. Previous characterization of Car3 knockout mice focused on mice fed standard diets, not high-fat diets that significantly alter the tissues that highly express Car3. We observed lower protein levels of Car3 in high-fat diet fed mice treated with niclosamide, a drug published to improve fatty liver symptoms in mice. However, it is unknown if Car3 is simply a biomarker reflecting lipid accumulation or whether it has a functional role in regulating lipid metabolism. We focused our in vitro studies toward metabolic pathways that require bicarbonate. To further determine the role of Car3 in metabolism, we measured de novo fatty acid synthesis with in vitro radiolabeled experiments and examined metabolic biomarkers in Car3 knockout and wild type mice fed high-fat diet. Specifically, we analyzed body weight, body composition, metabolic rate, insulin resistance, serum and tissue triglycerides. Our results indicate that Car3 is not required for de novo lipogenesis, and Car3 knockout mice fed high-fat diet do not have significant differences in responses to various diets to wild type mice.


Asunto(s)
Anhidrasa Carbónica III/metabolismo , Dieta Alta en Grasa , Ácidos Grasos/biosíntesis , Metabolismo de los Lípidos/fisiología , Lipogénesis/genética , Obesidad/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Composición Corporal/fisiología , Peso Corporal/fisiología , Anhidrasa Carbónica III/genética , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Obesidad/etiología , Obesidad/genética , Triglicéridos/metabolismo
6.
Assay Drug Dev Technol ; 13(9): 558-69, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26505612

RESUMEN

Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. direct and indirect healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Therefore, innovative and efficacious therapies for diabetes are urgently needed. Herein we describe a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic ß-cells with utility for high-content assessment of real-time insulin dynamics. Additionally, we report a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cell-based fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system. We observed that bafilomycin, a vacuolar H(+) ATPase inhibitor and inhibitor of insulin granule formation, significantly increased mCherry FP in INS-1 cells with PPI-mCherry. Partial least squares regression analysis demonstrated that an increase of FP by bafilomycin is significantly correlated with a decrease in granularity of PPI-mCherry signal in the cells. The increased FP by bafilomycin is due to inhibition of self-Förster resonant energy transfer (homo-FRET) caused by the increased mCherry intermolecular distance. FP substantially decreases when insulin is tightly packaged in the granules, and the homo-FRET decreases when insulin granule packaging is inhibited, resulting in increased FP. We performed pilot HTS of 1782 FDA-approved small molecules and natural products from Prestwick and Enzo chemical libraries resulting in an overall Z'-factor of 0.52 ± 0.03, indicating the suitability of this biosensor for HTS. This novel biosensor enables live-cell assessment of protein-protein interaction/protein aggregation in live cells and is compatible with conventional FP plate readers.


Asunto(s)
Técnicas Biosensibles/métodos , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Insulina/análisis , Proteínas Luminiscentes/análisis , Precursores de Proteínas/análisis , Animales , Productos Biológicos/toxicidad , Células Cultivadas , Polarización de Fluorescencia/tendencias , Colorantes Fluorescentes/análisis , Ensayos Analíticos de Alto Rendimiento/tendencias , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Precursores de Proteínas/metabolismo , Ratas , Proteína Fluorescente Roja
7.
Mol Biol Cell ; 23(2): 381-9, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22114351

RESUMEN

AMP-activated protein kinase (AMPK) is a key energy sensor that regulates metabolism to maintain cellular energy balance. AMPK activation has also been proposed to mimic benefits of caloric restriction and exercise. Therefore, identifying downstream AMPK targets could elucidate new mechanisms for maintaining cellular energy homeostasis. We identified the phosphotransferase nucleoside diphosphate kinase (NDPK), which maintains pools of nucleotides, as a direct AMPK target through the use of two-dimensional differential in-gel electrophoresis. Furthermore, we mapped the AMPK/NDPK phosphorylation site (serine 120) as a functionally potent enzymatic "off switch" both in vivo and in vitro. Because ATP is usually the most abundant cellular nucleotide, NDPK would normally consume ATP, whereas AMPK would inhibit NDPK to conserve energy. It is intriguing that serine 120 is mutated in advanced neuroblastoma, which suggests a mechanism by which NDPK in neuroblastoma can no longer be inhibited by AMPK-mediated phosphorylation. This novel placement of AMPK upstream and directly regulating NDPK activity has widespread implications for cellular energy/nucleotide balance, and we demonstrate in vivo that increased NDPK activity leads to susceptibility to energy deprivation-induced death.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Homeostasis , Nucleósido-Difosfato Quinasa/metabolismo , Fosfoserina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Línea Celular Tumoral , Drosophila melanogaster/enzimología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Nucleósido-Difosfato Quinasa/antagonistas & inhibidores , Nucleósido-Difosfato Quinasa/genética , Fosforilación , Electroforesis Bidimensional Diferencial en Gel
8.
Int J High Throughput Screen ; 2010(1): 127-140, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21132080

RESUMEN

Peroxisomes are ubiquitous cellular organelles that perform vital functions including fatty acid beta-oxidation, plasmalogen synthesis, and detoxification of harmful oxidative species. In rodents numerous compounds that increase peroxisome biogenesis also alleviate metabolic syndrome (MetS)/type 2 diabetes (T2D) symptoms. However, compounds that increase peroxisome biogenesis in rodents largely do not increase peroxisome biogenesis in humans. We designed a novel genetically encoded high throughput screening (HTS) high content assay to identify small molecule compounds that function as peroxisome proliferators in human cells. From this assay we have confirmed that 4-phenylbutyrate (PBA), a PPAR independent peroxisome proliferator and chemical chaperone, increases peroxisome proliferation in human cells and serves as a positive control for our screen. We performed a small pilot and larger 15,000 compound production screen with an overall Z' factor of 0.74 for 384-well plate format, providing a valuable screening tool for identifying peroxisome modulator compounds. From this screen we have identified 4 existing drugs and 10 novel compounds, some with common scaffolds 1000X more potent than PBA. It is hoped that these novel compounds may serve as scaffolds for testing for efficacy in alleviating MetS/T2D symptoms both in mouse models and ultimately human disease.

9.
Mol Biol Cell ; 21(19): 3433-42, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20685962

RESUMEN

The metabolic regulator AMP-activated protein kinase (AMPK) maintains cellular homeostasis through regulation of proteins involved in energy-producing and -consuming pathways. Although AMPK phosphorylation targets include cytoplasmic and nuclear proteins, the precise mechanisms that regulate AMPK localization, and thus its access to these substrates, are unclear. We identify highly conserved carboxy-terminal hydrophobic amino acids that function as a leptomycin B-sensitive, CRM1-dependent nuclear export sequence (NES) in the AMPK catalytic subunit (AMPKα). When this sequence is modified AMPKα shows increased nuclear localization via a Ran-dependent import pathway. Cytoplasmic localization can be restored by substituting well-defined snurportin-1 or protein kinase A inhibitor (PKIA) CRM1-binding NESs into AMPKα. We demonstrate a functional requirement in vivo for the AMPKα carboxy-terminal NES, as transgenic Drosophila expressing AMPKα lacking this NES fail to rescue lethality of AMPKα null mutant flies and show decreased activation loop phosphorylation under heat-shock stress. Sequestered to the nucleus, this truncated protein shows highly reduced phosphorylation at the key Thr172 activation residue, suggesting that AMPK activation predominantly occurs in the cytoplasm under unstressed conditions. Thus, modulation of CRM1-mediated export of AMPKα via its C-terminal NES provides an additional mechanism for cells to use in the regulation of AMPK activity and localization.


Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Dominio Catalítico , Señales de Exportación Nuclear , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/enzimología , Drosophila melanogaster/metabolismo , Activación Enzimática , Respuesta al Choque Térmico , Humanos , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Fosforilación , Unión Proteica , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Ratas , Relación Estructura-Actividad , Proteína de Unión al GTP ran/metabolismo
10.
PLoS One ; 5(9)2010 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-20862213

RESUMEN

Organisms must utilize multiple mechanisms to maintain energetic homeostasis in the face of limited nutrient availability. One mechanism involves activation of the heterotrimeric AMP-activated protein kinase (AMPK), a cell-autonomous sensor to energetic changes regulated by ATP to AMP ratios. We examined the phenotypic consequences of reduced AMPK function, both through RNAi knockdown of the gamma subunit (AMPKγ) and through expression of a dominant negative alpha (AMPKα) variant in Drosophila melanogaster. Reduced AMPK signaling leads to hypersensitivity to starvation conditions as measured by lifespan and locomotor activity. Locomotor levels in flies with reduced AMPK function were lower during unstressed conditions, but starvation-induced hyperactivity, an adaptive response to encourage foraging, was significantly higher than in wild type. Unexpectedly, total dietary intake was greater in animals with reduced AMPK function yet total triglyceride levels were lower. AMPK mutant animals displayed starvation-like lipid accumulation patterns in metabolically key liver-like cells, oenocytes, even under fed conditions, consistent with a persistent starved state. Measurements of O(2) consumption reveal that metabolic rates are greater in animals with reduced AMPK function. Lastly, rapamycin treatment tempers the starvation sensitivity and lethality associated with reduced AMPK function. Collectively, these results are consistent with models that AMPK shifts energy usage away from expenditures into a conservation mode during nutrient-limited conditions at a cellular level. The highly conserved AMPK subunits throughout the Metazoa, suggest such findings may provide significant insight for pharmaceutical strategies to manipulate AMPK function in humans.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/fisiología , Proteínas Quinasas Activadas por AMP/genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Conducta Alimentaria , Femenino , Metabolismo de los Lípidos , Masculino , Inanición
11.
Autophagy ; 5(8): 1155-65, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19844161

RESUMEN

When ATP levels in a cell decrease, various homeostatic intracellular mechanisms initiate attempts to restore ATP levels. As a prominent energy sensor, AMP-activated protein kinase (AMPK) represents one molecular gauge that links energy levels to regulation of anabolic and catabolic processes to restore energy balance. Although pharmacological studies have suggested that an AMPK activator, AIC AR (5-aminoimidazole-4-carboxamide ribonucleoside) may link AMPK activation to autophagy, a process that can provide short-term energy within the cell, AICAR can have AMPK-independent effects. Therefore, using a genetic-based approach we investigated the role of AMPK in cellular energy balance. We demonstrate that genetically altered cells, mouse embryonic fibroblasts (MEFs), lacking functional AMPK, display altered energy balance under basal conditions and die prematurely under low glucose-serum starvation challenge. These AMPK mutant cells appear to be abnormally reliant on autophagy under low glucose basal conditions, and therefore cannot rely further on autophagy like wild-type cells during further energetic stress and instead undergo apoptosis. This data suggests that AMPK helps regulate basal energy levels under low glucose. Further, AMPK mutant cells show increased basal phosphorylation of p53 at serine 15, a residue phosphorylated under glucose deprivation. We propose that cells lacking AMPK function have altered p53 activity that may help sensitize these cells to apoptosis under energetic stress.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Glucosa/farmacología , Proteínas Quinasas Activadas por AMP/deficiencia , Adenina/análogos & derivados , Adenina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Medio de Cultivo Libre de Suero , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Fosfohidrolasa PTEN/metabolismo , Fosfoserina/metabolismo , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo
12.
PLoS One ; 3(8): e3054, 2008 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-18725959

RESUMEN

BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.


Asunto(s)
Actinas/metabolismo , Dendritas/fisiología , Proteínas de Drosophila/genética , Proteínas de Microfilamentos/genética , Microtúbulos/metabolismo , Neuronas/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Animales , Secuencia Conservada , Drosophila , Proteínas de Drosophila/metabolismo , Genes Reporteros , Homeostasis , Cinética , Proteínas de Microfilamentos/metabolismo , Mutación , Fenotipo
13.
Proc Natl Acad Sci U S A ; 103(52): 19701-6, 2006 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-17179038

RESUMEN

Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine O(6) position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O(6)-methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleotides with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C.O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T.O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.


Asunto(s)
Guanina/análogos & derivados , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/química , Guanina/metabolismo , Cinética , Modelos Moleculares , Mutación/genética , Estructura Terciaria de Proteína
14.
EMBO J ; 23(2): 260-71, 2004 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-14739929

RESUMEN

Here we describe 11 crystal structures of nucleosome core particles containing individual point mutations in the structured regions of histones H3 and H4. The mutated residues are located at the two protein-DNA interfaces flanking the nucleosomal dyad. Five of the mutations partially restore the in vivo effects of SWI/SNF inactivation in yeast. We find that even nonconservative mutations of these residues (which exhibit a distinct phenotype in vivo) have only moderate effects on global nucleosome structure. Rather, local protein-DNA interactions are disrupted and weakened in a subtle and complex manner. The number of lost protein-DNA interactions correlates directly with an increased propensity of the histone octamer to reposition with respect to the DNA, and with an overall destabilization of the nucleosome. Thus, the disruption of only two to six of the approximately 120 direct histone-DNA interactions within the nucleosome has a pronounced effect on nucleosome mobility and stability. This has implications for our understanding of how these structures are made accessible to the transcription and replication machinery in vivo.


Asunto(s)
ADN/química , Histonas/química , Modelos Moleculares , Nucleosomas/química , Cromatina/efectos de los fármacos , Cristalografía por Rayos X , ADN/metabolismo , Histonas/genética , Histonas/metabolismo , Calor , Magnesio/farmacología , Movimiento (Física) , Nucleosomas/efectos de los fármacos , Nucleosomas/metabolismo , Mutación Puntual , Conformación Proteica , Cloruro de Sodio/farmacología
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