RESUMEN
Abnormal accumulation of tau proteins is one pathological hallmark of Alzheimerâ¡s disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 53 phosphorylation sites of tau-0N3R in total, which is about 30% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under a native-like cIEF-MS condition, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.
RESUMEN
Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.
RESUMEN
The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.
Asunto(s)
Adenosina/análogos & derivados , Enfermedad de Alzheimer/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Procesamiento Postranscripcional del ARN , ARN/metabolismo , Proteínas tau/metabolismo , Adenosina/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Masculino , Metilación , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Agregado de Proteínas , Agregación Patológica de Proteínas , ARN/genética , Índice de Severidad de la Enfermedad , Proteínas tau/genéticaRESUMEN
The neurodegenerative Alzheimer's disease (AD) affects more than 30 million people worldwide. There is thus far no cure or prevention for AD. Aggregation of hyperphosphorylated tau in the brain correlates with the cognitive decline of patients of AD and other neurodegenerative tauopathies. Intracerebral injection of tau aggregates isolated from tauopathy brains causes similar pathology in the recipient mice, demonstrating the pathogenic role of abnormally phosphorylated tau. Compounds controlling the aggregation of hyperphosphorylated tau therefore are probable modulators for the disease. Here we report the use of recombinant hyperphosphorylated tau (p-tau) to identify potential tauopathy therapeutics and risk factors. Hyperphosphorylation renders tau prone to aggregate and to impair cell viability. Taking advantage of these two characters of p-tau, we performed a screen of a 1280-compound library, and tested a selective group of prescription drugs in p-tau aggregation and cytotoxicity assays. R-(-)-apomorphine and raloxifene were found to be p-tau aggregation inhibitors that protected p-tau-treated cells. In contrast, a subset of benzodiazepines exacerbated p-tau cytotoxicity apparently via enhancing p-tau aggregation. R-(-)apomorphine and raloxifene have been shown to improve cognition in animals or in humans, whereas benzodiazepines were linked to increased risks of dementia. Our results demonstrate the feasibility and potential of using hyperphosphorylated tau-based assays for AD drug discovery and risk factor identification.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Apomorfina/farmacología , Cognición/efectos de los fármacos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos , Medicamentos bajo Prescripción/farmacología , Agregado de Proteínas/efectos de los fármacos , Clorhidrato de Raloxifeno/farmacología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/psicología , Apomorfina/uso terapéutico , Benzodiazepinas/efectos adversos , Humanos , Fosforilación/efectos de los fármacos , Medicamentos bajo Prescripción/uso terapéutico , Clorhidrato de Raloxifeno/uso terapéutico , Factores de RiesgoRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer's disease mechanistic and drug discovery studies.
Asunto(s)
Agregado de Proteínas , Proteínas tau/metabolismo , Fenómenos Biofísicos , Muerte Celular , Línea Celular , Supervivencia Celular , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Mitocondrias/metabolismo , Oxidación-Reducción , Fosforilación , Unión Proteica , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Superóxidos/metabolismoRESUMEN
The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas de Drosophila/fisiología , Proteínas Mitocondriales/biosíntesis , Biosíntesis de Proteínas/fisiología , Serina-ARNt Ligasa/fisiología , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/fisiología , Animales , Células Cultivadas , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Duplicación de Gen , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Serina-ARNt Ligasa/química , Serina-ARNt Ligasa/genéticaRESUMEN
Branching enzyme (BE) is responsible for the third step in glycogen/starch biosynthesis. It catalyzes the cleavage of α-1,4 glucan linkages and subsequent reattachment to form α-1,6 branch points. These branches are crucial to the final structure of glycogen and starch. The crystal structures of Escherichia coli BE (EcBE) in complex with α-, ß- and γ-cyclodextrin were determined in order to better understand substrate binding. Four cyclodextrin-binding sites were identified in EcBE; they were all located on the surface of the enzyme, with none in the vicinity of the active site. While three of the sites were also identified as linear polysaccharide-binding sites, one of the sites is specific for cyclodextrins. In previous work three additional binding sites were identified as exclusively binding linear malto-oligosaccharides. Comparison of the binding sites shed light on this apparent specificity. Binding site IV is located in the carbohydrate-binding module 48 (CBM48) domain of EcBE and superimposes with the cyclodextrin-binding site found in the CBM48 domain of 5'-AMP-activated protein kinase (AMPK). Comparison of these sites shows the similarities and differences in the two binding modes. While some of the binding sites were found to be conserved between branching enzymes of different organisms, some are quite divergent, indicating both similarities and differences between oligosaccharide binding in branching enzymes from various sources.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/química , Ciclodextrinas/química , Escherichia coli/química , Escherichia coli/enzimología , Sitios de Unión , Cristalografía por Rayos X/métodos , Glucógeno/química , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Almidón/químicaRESUMEN
The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.
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ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , ADN Polimerasa gamma , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Proteínas Mitocondriales/genéticaRESUMEN
Branching enzyme is responsible for all branching of glycogen and starch. It is an unusual member of the α-amylase family because it has both α-1,4-amylase activity and α-1,6-transferase activity [Drummond, G. S., et al. (1972) Eur. J. Biochem. 26, 168-176]. It also does not react with shorter glucans, though it will bind much longer substrates and substrate mimics [Binderup, K., et al. (2002) Arch. Biochem. Biophys. 397, 279-285]. In an effort to better understand how branching enzyme interacts with its polymeric substrate, we have determined the structure of Δ112 Escherichia coli branching enzyme bound to maltoheptaose and maltohexaose. Together, these structures define six distinct oligosaccharide binding sites on the surface of E. coli branching enzyme. Most of these binding sites surround the edge of the ß-barrel domain and are quite far from the active site. Surprisingly, there is no evidence of oligosaccharide binding in the active site of the enzyme. The closest bound oligosaccharide resides almost 18 Å from the active site. Mutations to conserved residues in binding sites I and VI had a debilitating effect on the activity of the enzyme.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Escherichia coli/enzimología , Glucanos/metabolismo , Oligosacáridos/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Conformación Proteica , Especificidad por SustratoRESUMEN
Protein acetylation and phosphorylation are key modifications that regulate both normal and pathological protein functions. The gel systems currently used for analyzing modified proteins require either expensive reagents or time-consuming second dimension electrophoresis. Here we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. The neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system separates proteins based on their charge at pH 7.0 and generates discrete bands from each acetylated and/or phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents and requires only a single dimension of electrophoresis. We demonstrate the effectiveness of this system by analyzing the phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative for resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Histonas/análisis , Procesamiento Proteico-Postraduccional/genética , alfa-Sinucleína/análisis , Acetilación , Células HeLa , Histonas/química , Humanos , Concentración de Iones de Hidrógeno , Octoxinol/química , Fosforilación/genética , Urea/química , alfa-Sinucleína/químicaRESUMEN
The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of â¼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.
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ADN Helicasas/metabolismo , Replicación del ADN , ADN Mitocondrial/metabolismo , Drosophila melanogaster/enzimología , Proteínas Hierro-Azufre/metabolismo , Secuencia de Aminoácidos , Animales , ADN Helicasas/química , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Multiple myeloma (MM) is a malignant disorder of differentiated B-cells for which standard care involves the inhibition of the proteasome. All clinically used proteasome inhibitors, including the chemotherapeutic drug bortezomib, target the catalytic active sites of the proteasome and inhibit protein proteolysis by competing with substrate binding. However, nearly all (~97%) patients become intolerant or resistant to treatments within a few years, after which the average survival time is less than 1 year. We describe herein the inhibition of the human proteasome via a noncompetitive mechanism by the imidazoline scaffold, TCH-13. Consistent with a mechanism distinct from that of competitive inhibitors, TCH-013 acts additively with and overcomes resistance to bortezomib. Importantly, TCH-013 induces apoptosis in a panel of myeloma and leukemia cell lines, but in contrast, normal lymphocytes, primary bone marrow stromal cells (hBMSC), and macrophages are resistant to its cytotoxic effects. TCH-013 was equally effective in blocking MM cell growth in co-cultures of MM cells with hBMSC isolated from CD138 negative bone marrow (BM) samples of MM patients. The cellular activity translated well in vivo where TCH-013 delayed tumor growth in an MM xenograft model to a similar extent as bortezomib.
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Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Resistencia a Antineoplásicos , Imidazoles/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/farmacología , Animales , Bortezomib , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Imidazoles/química , Ratones , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Imidazoles/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Administración Oral , Animales , Artritis Reumatoide/inducido químicamente , Colágeno , Relación Dosis-Respuesta a Droga , Imidazoles/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Imidazolinas/uso terapéutico , FN-kappa B/antagonistas & inhibidores , Administración Oral , Animales , Artritis Experimental/inmunología , Imidazolinas/administración & dosificación , Imidazolinas/síntesis química , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Layer-by-layer polyelectrolyte adsorption is a simple, convenient method for introducing ion-exchange sites in porous membranes. This study demonstrates that adsorption of poly(acrylic acid) (PAA)-containing films at pH 3 rather than pH 5 increases the protein-binding capacity of such polyelectrolyte-modified membranes 3-6-fold. The low adsorption pH generates a high density of -COOH groups that function as either ion-exchange sites or points for covalent immobilization of metal-ion complexes that selectively bind tagged proteins. When functionalized with nitrilotriacetate (NTA)-Ni(2+) complexes, membranes containing PAA/polyethylenimine (PEI)/PAA films bind 93 mg of histidine(6)-tagged (His-tagged) ubiquitin per cm(3) of membrane. Additionally these membranes isolate His-tagged COP9 signalosome complex subunit 8 from cell extracts and show >90% recovery of His-tagged ubiquitin. Although modification with polyelectrolyte films occurs by simply passing polyelectrolyte solutions through the membrane for as little as 5 min, with low-pH deposition the protein binding capacities of such membranes are as high as for membranes modified with polymer brushes and 2-3-fold higher than for commercially available immobilized metal affinity chromatography (IMAC) resins. Moreover, the buffer permeabilities of polyelectrolyte-modified membranes that bind His-tagged protein are ~30% of the corresponding permeabilities of unmodified membranes, so protein capture can occur rapidly with low-pressure drops. Even at a solution linear velocity of 570 cm/h, membranes modified with PAA/PEI/PAA exhibit a lysozyme dynamic binding capacity (capacity at 10% breakthrough) of ~40 mg/cm(3). Preliminary studies suggest that these membranes are stable under depyrogenation conditions (1 M NaOH).
Asunto(s)
Resinas Acrílicas/química , Membranas Artificiales , Proteínas/química , Adsorción , Animales , Bovinos , Humanos , Concentración de Iones de Hidrógeno , Hidroxilación , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/química , Nylons/química , Compuestos Organometálicos/química , Permeabilidad , Poliaminas/química , Polietileneimina/químicaRESUMEN
The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Epigénesis Genética , ARN Polimerasa III/metabolismo , ARN Nuclear Pequeño/biosíntesis , Transcripción Genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Sitios Genéticos , Células HeLa , Humanos , ARN Polimerasa III/genética , ARN Interferente Pequeño/farmacología , ARN Nuclear Pequeño/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
DNA damage induced by ionizing radiation activates the ataxia telangiectasia mutated pathway, resulting in apoptosis or DNA repair. The serine/threonine checkpoint kinase (Chk2) is an important transducer of this DNA damage signaling pathway and mediates the ultimate fate of the cell. Chk2 is an advantageous target for the development of adjuvant drugs for cancer therapy, because inhibition of Chk2 allows normal cells to enter cell cycle arrest and DNA repair, whereas many tumors bypass cell cycle checkpoints. Chk2 inhibitors may thus have a radioprotective effect on normal cells. We report herein a class of natural product derived Chk2 inhibitors, exemplified by indoloazepine 1, that elicit a strong ATM-dependent Chk2-mediated radioprotection effect in normal cells and p53 wt cells, but not p53 mutant cells (>50% of all cancers). This study represents the first example of a radioprotective effect in human cells other than T-cells and implicates a functional ATM pathway as a requirement for IR-induced radioprotection by this class of Chk2 inhibitors. Several of the hymenialdisine-derived analogues inhibit Chk2 at nanomolar concentrations, inhibit autophosphorylation of Chk2 at Ser516 in cells, and increase the survival of normal cells following ionizing radiation.
Asunto(s)
Azepinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirroles/farmacología , Protectores contra Radiación/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Azepinas/síntesis química , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quinasa de Punto de Control 2 , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/síntesis química , Radiación Ionizante , Protectores contra Radiación/síntesis química , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiaciónRESUMEN
The mammalian nuclear transcription factor NF-kappaB is responsible for the transcription of multiple cytokines, including the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Elevated levels of pro-inflammatory cytokines play an important role in the pathogenesis of inflammatory disorders such as rheumatoid arthritis (RA). Inhibition of the pro-inflammatory transcription factor NF-kappaB has therefore been identified as a possible therapeutic treatment for RA. We describe herein the synthesis and biological activity of a series of imidazoline-based scaffolds as potent inhibitors of NF-kappaB mediated gene transcription in cell culture as well as inhibitors of TNF-alpha and IL-6 production in interleukin 1 beta (IL-1beta) stimulated human blood.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Imidazolinas/síntesis química , Interleucina-6/antagonistas & inhibidores , FN-kappa B/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Células HeLa , Humanos , Imidazolinas/química , Imidazolinas/farmacología , Técnicas In Vitro , Interleucina-1beta/farmacología , Interleucina-6/biosíntesis , FN-kappa B/genética , Estereoisomerismo , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Human small nuclear RNA gene transcription by RNA polymerases II and III depends upon promoter recognition by the SNAPC general transcription factor. DNA binding by SNAPC involves direct DNA contacts by the SNAP190 subunit in cooperation with SNAP50 and SNAP43. The data presented herein shows that SNAP50 plays an important role in DNA binding by SNAPC through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAPC. However, metal binding studies revealed that SNAPC contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAPC alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.
Asunto(s)
Proteínas de Unión al ADN/química , Factores de Transcripción/química , Factores de Transcripción/genética , Secuencia de Aminoácidos , Cisteína/química , Células HeLa , Histidina/química , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , ARN/química , Homología de Secuencia de Aminoácido , Dedos de ZincRESUMEN
We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.