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2.
Mol Biol (Mosk) ; 55(2): 181-193, 2021.
Artículo en Ruso | MEDLINE | ID: mdl-33871434

RESUMEN

The base and nucleotide excision DNA repair (BER and NER) systems are aimed at removing specific types of damaged DNA, i.e., oxidized, alkylated, or deaminated bases in the case of BER and bulky damage caused by UV radiation or chemical carcinogens in the case of NER. In some cases, however, the repair process follows a more complex scenario, which implies that the repair pathways exchange proteins and interact with each other to form a common interactome. This review describes the BER and NER mechanisms and discusses the current data on the involvement of the NER proteins in the repair of DNA lesions caused by oxidative stress and the BER proteins in the removal of bulky DNA adducts. We also discuss the role of poly(ADP-ribose) polymerase 1 in the regulation of the BER and NER processes and their coordination in the repair of complex (cluster) lesions.


Asunto(s)
Daño del ADN , Reparación del ADN , ADN/genética , ADN/metabolismo , Reparación del ADN/genética , Nucleótidos , Estrés Oxidativo/genética
3.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-33805827

RESUMEN

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.


Asunto(s)
Proteína 9 Asociada a CRISPR/química , Sistemas CRISPR-Cas , ADN/química , ARN Guia/química , Ribonucleoproteínas/química , Streptococcus pyogenes/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Dicroismo Circular , ADN/genética , ADN/metabolismo , Edición Génica/métodos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Guia/genética , ARN Guia/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimología
4.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800867

RESUMEN

During tissue injury events, the innate immune system responds immediately to alarms sent from the injured cells, and the adaptive immune system subsequently joins in the inflammatory reaction. The control mechanism of each immune reaction relies on the orchestration of different types of T cells and the activators, antigen-presenting cells, co-stimulatory molecules, and cytokines. Mitochondria are an intracellular signaling organelle and energy plant, which supply the energy requirement of the immune system and maintain the system activation with the production of reactive oxygen species (ROS). Extracellular mitochondria can elicit regenerative effects or serve as an activator of the immune cells to eliminate the damaged cells. Recent clarification of the cytosolic escape of mitochondrial DNA triggering innate immunity underscores the pivotal role of mitochondria in inflammation-related diseases. Human mesenchymal stem cells could transfer mitochondria through nanotubular structures to defective mitochondrial DNA cells. In recent years, mitochondrial therapy has shown promise in treating heart ischemic events, Parkinson's disease, and fulminating hepatitis. Taken together, these results emphasize the emerging role of mitochondria in immune-cell-mediated tissue regeneration and ageing.


Asunto(s)
Envejecimiento/inmunología , Células Presentadoras de Antígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Mitocondrias/fisiología , Regeneración/inmunología , Subgrupos de Linfocitos T/inmunología , Inmunidad Adaptativa , Animales , Citocinas/fisiología , ADN/metabolismo , ADN Mitocondrial/metabolismo , Reposicionamiento de Medicamentos , Péptido 1 Similar al Glucagón/agonistas , Homeostasis , Humanos , Inmunidad Innata , Inflamación , Péptidos y Proteínas de Señalización Intercelular/fisiología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Metformina/farmacología , Metformina/uso terapéutico , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/fisiología , Especies Reactivas de Oxígeno/metabolismo , Inmunología del Trasplante , Heridas y Traumatismos/inmunología , Heridas y Traumatismos/fisiopatología
5.
Nat Commun ; 12(1): 1987, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790284

RESUMEN

A widely regarded model for glucocorticoid receptor (GR) action postulates that dimeric binding to DNA regulates unfavorable metabolic pathways while monomeric receptor binding promotes repressive gene responses related to its anti-inflammatory effects. This model has been built upon the characterization of the GRdim mutant, reported to be incapable of DNA binding and dimerization. Although quantitative live-cell imaging data shows GRdim as mostly dimeric, genomic studies based on recovery of enriched half-site response elements suggest monomeric engagement on DNA. Here, we perform genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding even to open chromatin. We also find that GRdim does not exclusively bind half-response elements. Our results do not support a physiological role for monomeric GR and are consistent with a common mode of receptor binding via higher order structures that drives both the activating and repressive actions of glucocorticoids.


Asunto(s)
ADN/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Multimerización de Proteína , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , ADN/genética , Regulación de la Expresión Génica , Glucocorticoides/metabolismo , Humanos , Ratones , Mutación , Unión Proteica , Receptores de Glucocorticoides/genética , Elementos de Respuesta/genética , Transducción de Señal/genética
6.
Science ; 372(6538)2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33833093

RESUMEN

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.


Asunto(s)
Metilación de ADN , ADN/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Sistemas CRISPR-Cas , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Genoma Humano , Humanos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transcripción Genética
7.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806359

RESUMEN

In systemic mastocytosis (SM), qualitative and serial quantitative assessment of the KIT D816V mutation is of diagnostic and prognostic relevance. We investigated peripheral blood and bone marrow samples of 161 patients (indolent SM (ISM), n = 40; advanced SM, AdvSM, n = 121) at referral and during follow-up for the KIT D816V variant allele frequency (VAF) at the DNA-level and the KIT D816V expressed allele burden (EAB) at the RNA-level. A round robin test with four participating laboratories revealed an excellent correlation (r > 0.99, R2 > 0.98) between three different DNA-assays. VAF and EAB strongly correlated in ISM (r = 0.91, coefficient of determination, R2 = 0.84) but only to a lesser extent in AdvSM (r = 0.71; R2 = 0.5). However, as compared to an EAB/VAF ratio ≤2 (cohort A, 77/121 patients, 64%) receiver operating characteristic (ROC) analysis identified an EAB/VAF ratio of >2 (cohort B, 44/121 patients, 36%) as predictive for an advanced phenotype and a significantly inferior median survival (3.3 vs. 11.7 years; p = 0.005). In terms of overall survival, Cox-regression analysis was only significant for the EAB/VAF ratio >2 (p = 0.006) but not for VAF or EAB individually. This study demonstrates for the first time that the transcriptional activity of KIT D816V may play an important role in the pathophysiology of SM.


Asunto(s)
Mastocitosis Sistémica/genética , Mutación , Proteínas Proto-Oncogénicas c-kit/genética , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Médula Ósea/metabolismo , ADN/sangre , ADN/genética , ADN/metabolismo , Femenino , Frecuencia de los Genes , Humanos , Masculino , Mastocitosis Sistémica/sangre , Mastocitosis Sistémica/metabolismo , Persona de Mediana Edad , Fenotipo , Pronóstico , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN/sangre , ARN/genética , ARN/metabolismo , Transcripción Genética
8.
Molecules ; 26(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671247

RESUMEN

In the present study, naphthyl acetohydrazide (HL) ligand was prepared and used for the synthesis of new six amorphous transition metal (Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II)) complexes. All the compounds were characterized by elemental analysis, UV-vis, FT-IR, 1H- and 13C-NMR, and Matrix-Assisted Laser Desorption Ionization (MALDI). The solubilization study was carried out by estimating the interaction between the metal complexes with surfactants viz. sodium stearate (SS) and Cetyltrimethylammonium bromide (CTAB). UV-Visible spectroscopy was employed to determine partitioning and binding parameters, whereas electrical conductivity measurements were employed to estimate critical micellar concentration (CMC), the extent of dissociation, and free energy of micellization. The CT-DNA interaction of synthesized compounds with DNA represents the major groove binding. The synthesized ligand and metal complexes were also tested against bacterial and fungal strains and it has been observed that Cu(II) complex is active against all the strains except Candida albicans, while Cd(II) complex is active against all bacterial and fungal strains except Pseudomonas. Among all compounds, only the Pd(II) complex shows reasonable activity against cervical cancer HeLa cell lines, representing 97% inhibition.


Asunto(s)
Antiinfecciosos/farmacología , Complejos de Coordinación/síntesis química , ADN/metabolismo , Hidrazonas/síntesis química , Hidrazonas/farmacología , Metales/química , Micelas , Neoplasias/patología , Bacterias/efectos de los fármacos , Espectroscopía de Resonancia Magnética con Carbono-13 , Supervivencia Celular/efectos de los fármacos , Conductividad Eléctrica , Células HeLa , Humanos , Hidrazonas/química , Ligandos , Pruebas de Sensibilidad Microbiana , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/química
9.
Nat Commun ; 12(1): 1395, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654088

RESUMEN

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.


Asunto(s)
ADN/metabolismo , Neoplasias/metabolismo , Polisacáridos/biosíntesis , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Toxina del Cólera/metabolismo , Gangliósido G(M1)/metabolismo , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Análisis de Secuencia por Matrices de Oligonucleótidos , Polisacáridos/química , Subunidades de Proteína/metabolismo
10.
Nat Commun ; 12(1): 1491, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674598

RESUMEN

Abnormally formed FUS/EWS/TAF15 (FET) fusion oncoproteins are essential oncogenic drivers in many human cancers. Interestingly, at the molecular level, they also form biomolecular condensates at specific loci. However, how these condensates lead to gene transcription and how features encoded in the DNA element regulate condensate formation remain unclear. Here, we develop an in vitro single-molecule assay to visualize phase separation on DNA. Using this technique, we observe that FET fusion proteins undergo phase separation at target binding loci and the phase separated condensates recruit RNA polymerase II and enhance gene transcription. Furthermore, we determine a threshold number of fusion-binding DNA elements that can enhance the formation of FET fusion protein condensates. These findings suggest that FET fusion oncoprotein promotes aberrant gene transcription through loci-specific phase separation, which may contribute to their oncogenic transformation ability in relevant cancers, such as sarcomas and leukemia.


Asunto(s)
Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Transcripción Genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , ARN Polimerasa II/metabolismo , Proteína EWS de Unión a ARN , Proteína FUS de Unión a ARN
11.
Nat Struct Mol Biol ; 28(3): 300-309, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33686268

RESUMEN

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core.


Asunto(s)
ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , ADN/química , ADN/ultraestructura , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación F de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/ultraestructura , Células HEK293 , Humanos , Modelos Moleculares , Complejos Multienzimáticos/ultraestructura , Reproducibilidad de los Resultados , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , Ubiquitinación , Ubiquitinas/metabolismo
12.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-33672594

RESUMEN

High alcohol intake results in the accumulation of non-oxidative ethanol metabolites such as fatty acid ethyl esters (FAEEs) in the pancreas. High FAEE concentrations mediate pancreatic acinar cell injury and are associated with alcoholic pancreatitis. Treatment with ethanol and the fatty acid palmitoleic acid (EtOH/POA) increased the levels of palmitoleic acid ethyl ester and induced zymogen activation and cytokine expression in pancreatic acinar cells. EtOH/POA induces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production and pancreatic acinar cell injury. Lycopene, a bright-red carotenoid, is a potent antioxidant due to its high number of conjugated double bands. This study aimed to investigate whether lycopene inhibits the EtOH/POA-induced increase in ROS production, zymogen activation, and expression of the inflammatory cytokine IL-6 in EtOH/POA-stimulated pancreatic acinar AR42J cells. EtOH/POA increased the ROS levels, NADPH oxidase and NF-κB activities, zymogen activation, IL-6 expression, and mitochondrial dysfunction, which were inhibited by lycopene. The antioxidant N-acetylcysteine and NADPH oxidase 1 inhibitor ML171 suppressed the EtOH/POA-induced increases in ROS production, NF-κB activation, zymogen activation, and IL-6 expression. Therefore, lycopene inhibits EtOH/POA-induced mitochondrial dysfunction, zymogen activation, and IL-6 expression by suppressing NADPH oxidase-mediated ROS production in pancreatic acinar cells.


Asunto(s)
Células Acinares/patología , Inflamación/patología , Licopeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Páncreas Exocrino/patología , Acetilcisteína/farmacología , Células Acinares/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/metabolismo , Etanol , Ácidos Grasos Monoinsaturados , Interleucina-6/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo
13.
J Vis Exp ; (168)2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33720126

RESUMEN

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.


Asunto(s)
ADN/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Análisis de la Célula Individual/métodos , Aldehídos/química , Adhesión Celular , Comunicación Celular , Supervivencia Celular , Colesterol/metabolismo , Dimetilpolisiloxanos/química , Compuestos Epoxi/química , Humanos , Hidrogeles/química , Interacciones Hidrofóbicas e Hidrofílicas , Oligonucleótidos/metabolismo , Polímeros/química , Coloración y Etiquetado
14.
Nat Commun ; 12(1): 1897, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772030

RESUMEN

Oxidative damage to DNA generates 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major lesions. Despite the comparable prevalence of these lesions, the biological effects of oxoA remain poorly characterized. Here we report the discovery of a class of DNA interstrand cross-links (ICLs) involving oxidized nucleobases. Under oxidative conditions, oxoA, but not oxoG, readily reacts with an opposite base to produce ICLs, highlighting a latent alkylating nature of oxoA. Reactive halogen species, one-electron oxidants, and the myeloperoxidase/H2O2/Cl- system induce oxoA ICLs, suggesting that oxoA-mediated cross-links may arise endogenously. Nucleobase analog studies suggest C2-oxoA is covalently linked to N2-guanine and N3-adenine for the oxoA-G and oxoA-A ICLs, respectively. The oxoA ICLs presumably form via the oxidative activation of oxoA followed by the nucleophilic attack by an opposite base. Our findings provide insights into oxoA-mediated mutagenesis and contribute towards investigations of oxidative stress-induced ICLs and oxoA-based latent alkylating agents.


Asunto(s)
Adenina/análogos & derivados , Daño del ADN , ADN/química , Estrés Oxidativo , Adenina/química , Cromatografía Liquida/métodos , Reactivos de Enlaces Cruzados/química , ADN/genética , ADN/metabolismo , Reparación del ADN , Guanina/análogos & derivados , Guanina/química , Espectrometría de Masas/métodos , Modelos Químicos , Estructura Molecular , Oxidación-Reducción
15.
Eur J Med Chem ; 216: 113262, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33711764

RESUMEN

The number of cancer cases continues to increase worldwide, and unfortunately the main systemic treatments available have numerous of side effects. Ruthenium complexes have shown to be promising chemotherapeutic agents, since they present low toxicity and are more selective for tumor tissues. We report the synthesis, characterization and biological properties of two new ruthenium (II) complexes containing Lapachol and Lawsone as ligands: (1) [Ru(Law)(dppb)(phen)]PF6 and (2) [Ru(Lap)(dppb)(phen)]PF6, where Law = Lawsone, Lap = Lapachol, dppb = 1,4-bis(diphenylphosphine)butane and phen = 1,10-phenanthroline. The ability of the complexes (1) and (2) to interact with CT-DNA (Calf Thymus) was investigated, and the results indicate that the complexes have shown a weak interaction with this macromolecule. Complexes (1) and (2) showed a moderate interaction with BSA, via a spontaneous process with the involvement of van der Waals and hydrogen bond interactions. Both complexes were tested against human lung cancer cell lines, chronic human myeloid leukemia, murine melanoma and human cervical and non-tumoral murine fibroblast adenocarcinoma, human lung fibroblasts and monkey kidney epithelia. The potential for cytotoxicity was tested out using the MTT assay and the neutral red test, to calculate inhibitory concentrations (IC50) and selectivity indices (IS). Both complexes showed a higher selectivity index of 1.17 and 10.91, respectively, for the HeLa tumor line. Studies of toxicological evaluation, using the micronucleus test and the comet assay against non-tumor cells, as well as an assessment of the potential for acute toxicity and neurotoxicity in zebrafish (Danio rerio). In the in vitro micronucleus test, complex (1) showed the least genotoxic potential, and in the in vitro comet assay both compounds had revealed a genotoxic potential at 0.5 and 1.0 mg L-1, with no difference between 24 h and 48 h exposure times. In the acute toxicity tests on zebrafish embryos, complex (1) showed sublethal effects such as decreased blood circulation and heartbeat rate, which were less pronounced than with complex (2). In contrast to complex 2, which caused lethality even before 48h, complex (1) did not cause the death of the embryos at concentrations up to (2.0 mg L-1). Complex (2) also lead to a delay in the embryo. Cell based in vitro methods thus proved able to provide specific toxicological data, allowing a significant reduction in ∖animal experimentation. Given that in vitro tests cannot completely replace animal tests, the use of less advanced developmental stages such as zebrafish embryos, which - at least in the European Union - are not regarded protected, could be shown to be an excellent alternative for testing with, e.g., mammals.


Asunto(s)
Complejos de Coordinación/química , Rutenio/química , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Bovinos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/metabolismo , Complejos de Coordinación/farmacología , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Humanos , Conformación Molecular , Unión Proteica , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Termodinámica , Pez Cebra/crecimiento & desarrollo
16.
Cell Mol Life Sci ; 78(7): 3709-3724, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33733306

RESUMEN

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.


Asunto(s)
ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , G-Cuádruplex , MicroARNs/metabolismo , ARN/metabolismo , Ribonucleasa III/antagonistas & inhibidores , Ribonucleasa III/genética , ARN Helicasas DEAD-box/metabolismo , ADN/química , ADN/genética , Inhibidores Enzimáticos/química , Humanos , MicroARNs/genética , Conformación de Ácido Nucleico , Conformación Proteica , ARN/química , ARN/genética , Ribonucleasa III/metabolismo
17.
Nat Commun ; 12(1): 1965, 2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33785750

RESUMEN

Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. However, it remains methodologically challenging. Herein, we report a molecular recognition mechanism cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments containing diverse chromatin modifications. As an example, we characterize the nanoenvironments of three DNA modifications around one histone posttranslational modification (PTM). These DNA modifications in fixed cells are labeled with respective DNA barcoding probes, and then the PTM site is tethered with a DNA walking probe. Cell-TALKING can continuously produce cleavage records of any barcoding probes nearby the walking probe. New 3'-OH ends are generated on the cleaved barcoding probes to induce DNA amplification for downstream detections. Combining fluorescence imaging, we identify various combinatorial chromatin modifications and investigate their dynamic changes during cell cycles. We also explore the nanoenvironments in different cancer cell lines and clinical specimens. In principle, using high-throughput sequencing instead of fluorescence imaging may allow the detection of complex cellular nanoenvironments containing tens of biomolecules such as transcription factors.


Asunto(s)
Microambiente Celular/genética , Cromatina/genética , ADN/genética , Epigénesis Genética , Cromatina/metabolismo , ADN/metabolismo , Técnicas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/metabolismo , Humanos , Biología Molecular/métodos , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
18.
Int Heart J ; 62(2): 359-366, 2021 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-33678800

RESUMEN

Dilated cardiomyopathy (DCM) is a common cause of heart failure. TTN, which encodes titin protein, is a representative causative gene of DCM, and is presented mainly as a truncation variant. However, TTN truncation variants are also found in healthy individuals, and it is therefore important to evaluate the pathogenicity of each variant. In this study, we analyzed 67 cardiomyopathy-associated genes in a male Japanese patient who was hospitalized for recurrent severe heart failure and identified a novel truncation variant, TTN Ser17456Arg fs*14. This TTN truncation variant was located in the A-band region. Moreover, the patient's mother with heart failure harbored the same variant, whereas the father and brother without heart failure did not harbor the variant. To examine the functional changes associated with the truncation variant, H9c2 cells were subjected to genome editing to generate cells with a homologous truncation variant. The cells were differentiated using all-trans-retinoic acid, and the mRNA expression of skeletal actin and cardiac actin were found to be increased and decreased, respectively, consistent with known changes in patients with DCM or heart failure. In contrast, another cell with the titin truncation variant used as a control showed no changes in heart failure-related genes. In summary, we found a novel TTN truncation variant in familial DCM patients and confirmed its functional changes using a relatively simple cell model. The novel truncation variant was identified as a pathogenic and disease-causing mutation.


Asunto(s)
Cardiomiopatía Dilatada/genética , Conectina/genética , ADN/genética , Mutación , Miocitos Cardíacos/metabolismo , Función Ventricular Izquierda/fisiología , Biopsia , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Conectina/metabolismo , ADN/metabolismo , Análisis Mutacional de ADN , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/patología , Linaje
19.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671470

RESUMEN

G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.


Asunto(s)
Núcleo Celular/metabolismo , G-Cuádruplex , Histonas/metabolismo , Transcripción Genética , Acetilación , Animales , Composición de Base/genética , Línea Celular , Nucléolo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Reparación del ADN , Epigénesis Genética , Humanos , Cuerpos de Inclusión/metabolismo , Metilación , Ratones
20.
J Vis Exp ; (169)2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33749670

RESUMEN

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal cells and in disease progression such as with cancer.


Asunto(s)
Cromosomas/metabolismo , ADN/metabolismo , Sitios Genéticos , Hibridación Fluorescente in Situ , Telómero/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Cromosomas Artificiales Bacterianos/metabolismo , Dermis/citología , Fibroblastos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador
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