RESUMEN

The ubiquitin signaling pathway is crucial for the DNA damage response pathway. More specifically, RNF168 is integral in regulating DNA repair proteins at damaged chromatin. However, the detailed mechanism by which RNF168 is regulated in cells is not fully understood. Here, we identify the ubiquitin-ribosomal fusion proteins UBA80 (also known as RPS27A) and UBA52 (also known as RPL40) as interacting proteins for H2A/H2AX histones and RNF168. Both UBA80 and UBA52 are recruited to laser-induced micro-irradiation DNA damage sites and are required for DNA repair. Ectopic expression of UBA80 and UBA52 inhibits RNF168-mediated H2A/H2AX ubiquitination at K13/15 and impairs 53BP1 recruitment to DNA lesions. Mechanistically, the C-terminal ribosomal fragments of UBA80 and UBA52, S27A and L40, respectively, limit RNF168-nucleosome engagement by masking the regulatory acidic residues at E143/E144 and the nucleosome acidic patch. Together, our results reveal that UBA80 and UBA52 antagonize the ubiquitination signaling pathway and fine-tune the spatiotemporal regulation of DNA repair proteins at DNA damage sites.

Asunto(s)

Reparación del ADN , Histonas , Nucleosomas , Proteínas Ribosómicas , Ubiquitina-Proteína Ligasas , Daño del ADN , Histonas/metabolismo , Nucleosomas/genética , Proteínas Ribosómicas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Humanos

RESUMEN

Correction for 'Enhancing scanning electrochemical microscopy's potential to probe dynamic co-culture systems via hyperspectral assisted-imaging' by Sondrica Goines et al., Analyst, 2022, 147, 2396-2404, https://doi.org/10.1039/D2AN00319H.

RESUMEN

Precise determination of boundaries in co-culture systems is difficult to achieve with scanning electrochemical microscopy alone. Thus, biological scanning electrochemical microscope platforms generally consist of a scanning electrochemical microscope positioner mounted on the stage of an inverted microscope for correlated electrochemical and optical imaging. Use of a fluorescence microscope allows for site-specific fluorescence labeling to obtain more clearly resolved spatial and electrochemical data. Here, we construct a unique hyperspectral assisted-biological scanning electrochemical microscope platform to widen the scope of biological imaging. Specifically, we incorporate a variable fluorescence bandpass source into a biological scanning electrochemical microscope platform for simultaneous optical, spectral, and electrochemical imaging. Not only does this platform serve as a cost-effective alternative to white light laser imaging, but additionally it provides multi-functional analysis of biological samples. Here, we demonstrate the efficacy of our platform to discern the electrochemical contribution of site-specific cells by optically and spectroscopically resolving boundaries as well as cell types within a complex biological system.

Asunto(s)

Rayos Láser , Imagen Óptica , Técnicas de Cocultivo , Microscopía Electroquímica de Rastreo , Microscopía Fluorescente/métodos

RESUMEN

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

Asunto(s)

Proteína BRCA1 , Reparación del ADN , Recombinación Homóloga , Factores de Transcripción , Proteína 1 de Unión al Supresor Tumoral P53 , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Mamíferos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo

RESUMEN

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

Asunto(s)

Replicación del ADN , ADN/química , ADN/metabolismo , G-Cuádruplex , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Línea Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Genes myc , Humanos , Modelos Moleculares , Mutación , Motivos de Nucleótidos , Nucleotidiltransferasas/genética , Unión Proteica

RESUMEN

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B-EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

Asunto(s)

Cromatina , Reparación del ADN/genética , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Transcripción Genética/genética , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Silenciador del Gen , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrK

RESUMEN

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.

Asunto(s)

Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Factores de Transcripción de Tipo Kruppel/genética , Óxido Nítrico Sintasa de Tipo III/genética , Trombomodulina/genética , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Regulación de la Expresión Génica/efectos de la radiación , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Factores de Transcripción MEF2/genética , Proteína Quinasa 7 Activada por Mitógenos/genética , Neoplasias/genética , Neoplasias/patología , Neoplasias/radioterapia , Radiación , Transducción de Señal/efectos de la radiación

RESUMEN

Histone ubiquitination plays an important role in the DNA damage response (DDR) pathway. RNF168 catalyzes H2A and H2AX ubiquitination on lysine 13/15 (K13/K15) upon DNA damage and promotes the accrual of downstream repair factors at damaged chromatin. Here, we report that RNF168 ubiquitinates the non-canonical H2A variants H2AZ and macroH2A1/2 at the divergent N-terminal tail lysine residue. In addition to their evolutionarily conserved nucleosome acidic patch, we identify the positively charged alpha1-extension helix as essential for RNF168-mediated ubiquitination of H2A variants. Moreover, mutation of the RNF168 UMI (UIM- and MIU-related UBD) hydrophilic acidic residues abolishes RNF168-mediated ubiquitination as well as 53BP1 and BRCA1 ionizing radiation-induced foci formation. Our results reveal a juxtaposed bipartite electrostatic interaction utilized by the nucleosome to direct RNF168 orientation towards the target lysine residues in proximity to the H2A alpha1-extension helix, which plays an important role in the DDR pathway.

Asunto(s)

Histonas/química , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Evolución Molecular , Células HEK293 , Histonas/genética , Humanos , Lisina/metabolismo , Estructura Secundaria de Proteína , Especificidad por Sustrato , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo

RESUMEN

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.

Asunto(s)

Dineínas Citoplasmáticas/fisiología , Roturas del ADN de Doble Cadena , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína BRCA1/genética , Línea Celular , Cromatina/metabolismo , Dineínas Citoplasmáticas/química , Dineínas Citoplasmáticas/metabolismo , Reparación del ADN , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Radiación Ionizante

RESUMEN

Histone H2A variants play important roles in maintaining the integrity of the genome. For example, the histone variant H2AX is phosphorylated on Ser139 (called γH2AX) at DNA double-strand breaks (DSB) and serves as a signal for the initiation of downstream DNA damage response (DDR) factor recruitment and DNA repair activities within damaged chromatin. For decades, genetic studies in human cells involving DNA damage signaling and repair factors have relied mostly on either knockdown by RNA interference (i.e., shRNA and siRNA) or the use of mouse embryonic fibroblasts derived from knockout (KO) mice. Recent advances in gene editing using ZNF nuclease, TALEN, and CRISPR/Cas9 have allowed the generation of human KO cell lines, allowing genetic models for studying the DDR, including histone H2A variants in human cells. Here, we describe a detailed protocol for generating and verifying KO of H2AX and macroH2A histone H2A variants using CRISPR/Cas9 gene editing in human cancer cell lines. This protocol allows the use and development of genetic systems in human cells to study histone variants and their functions, including within the DDR.

Asunto(s)

Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Histonas/metabolismo , Línea Celular Tumoral , Células Clonales , Humanos , Proteínas Mutantes/metabolismo , Isoformas de Proteínas/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Reproducibilidad de los Resultados

RESUMEN

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

Asunto(s)

Proteína BRCA1/metabolismo , Neoplasias Óseas/metabolismo , Cromatina/metabolismo , Reparación del ADN , Proteínas Nucleares/metabolismo , Osteosarcoma/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/genética , Neoplasias Óseas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromatina/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Inestabilidad Genómica , Células HEK293 , Chaperonas de Histonas , Histonas/genética , Histonas/metabolismo , Recombinación Homóloga , Humanos , Proteínas Nucleares/genética , Osteosarcoma/genética , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas

RESUMEN

The Fanconi anemia (FA) core complex provides the essential E3 ligase function for spatially defined FANCD2 ubiquitination and FA pathway activation. Of the seven FA gene products forming the core complex, FANCL possesses a RING domain with demonstrated E3 ligase activity. The other six components do not have clearly defined roles. Through epistasis analyses, we identify three functional modules in the FA core complex: a catalytic module consisting of FANCL, FANCB, and FAAP100 is absolutely required for the E3 ligase function, and the FANCA-FANCG-FAAP20 and the FANCC-FANCE-FANCF modules provide nonredundant and ancillary functions that help the catalytic module bind chromatin or sites of DNA damage. Disruption of the catalytic module causes complete loss of the core complex function, whereas loss of any ancillary module component does not. Our work reveals the roles of several FA gene products with previously undefined functions and a modularized assembly of the FA core complex.

Asunto(s)

Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Técnicas de Cultivo de Célula , Daño del ADN , Células HCT116 , Células HEK293 , Humanos , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

The level of endothelin-1 (ET-1), a potent vasoconstrictor, was associated with retinopathy under ischemia. The effects of endothelial endothelin-1 (ET-1) over-expression in a transgenic mouse model using Tie-1 promoter (TET-1 mice) on pathophysiological changes of retinal ischemia were investigated by intraluminal insertion of a microfilament up to middle cerebral artery (MCA) to transiently block the ophthalmic artery. Two-hour occlusion and twenty-two-hour reperfusion were performed in homozygous (Hm) TET-1 mice and their non-transgenic (NTg) littermates. Presence of pyknotic nuclei in ganglion cell layer (GCL) was investigated in paraffin sections of ipsilateral (ischemic) and contralateral (non-ischemic) retinae, followed by measurement of the thickness of inner retinal layer. Moreover, immunocytochemistry of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS) and aquaporin-4 (AQP4) peptides on retinal sections were performed to study glial cell reactivity, glutamate metabolism and water accumulation, respectively after retinal ischemia. Similar morphology was observed in the contralateral retinae of NTg and Hm TET-1 mice, whereas ipsilateral retina of NTg mice showed slight structural and cellular changes compared with the corresponding contralateral retina. Ipsilateral retinae of Hm TET-1 mice showed more significant changes when compared with ipsilateral retina of NTg mice, including more prominent cell death in GCL characterized by the presence of pyknotic nuclei, elevated GS immunoreactivity in Müller cell bodies and processes, increased AQP-4 immunoreactivity in Müller cell processes, and increased inner retinal thickness. Thus, over-expression of endothelial ET-1 in TET-1 mice may contribute to increased glutamate-induced neurotoxicity on neuronal cells and water accumulation in inner retina leading to edema.

Asunto(s)

Células Endoteliales/metabolismo , Endotelina-1/metabolismo , Isquemia/patología , Papiledema/patología , Retina/patología , Neuronas Retinianas/metabolismo , Neuronas Retinianas/patología , Animales , Acuaporina 4/metabolismo , Muerte Celular , Núcleo Celular/metabolismo , Núcleo Celular/patología , Endotelina-1/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica , Isquemia/enzimología , Masculino , Ratones , Papiledema/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor TIE-1/metabolismo , Retina/enzimología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Neuronas Retinianas/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genética

RESUMEN

Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage.

Asunto(s)

Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Células Cultivadas , Cromatina/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Interferente Pequeño/genética , Elementos de Respuesta , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53

RESUMEN

Mcph1 is mutated in autosomal recessive primary microcephaly and premature chromosome condensation (PCC) syndrome. Increased chromosome condensation is a common feature of cells isolated from patients afflicted with either disease. Normal cells depleted of Mcph1 also exhibit PCC phenotype. Human Mcph1 contains three BRCA1-carboxyl terminal (BRCT) domains, the first of which (Mcph1N) is necessary for the prevention of PCC. The only known disease-associated missense mutation in Mcph1 resides in this domain (T27R). We have determined the X-ray crystal structure of human Mcph1N to 1.6 A resolution. Compared with other BRCT domain structures, the most striking differences are an elongated, ordered beta1-alpha1 loop and an adjacent hydrophobic pocket. This pocket is in the equivalent structural position to the phosphate binding site of BRCT domains that recognize phospho-proteins, although the phosphate-binding residues are absent in Mcph1N. Mutations in the pocket abrogate the ability of full-length Mcph1 to rescue the PCC phenotype of Mcph1(-/-) mouse embryonic fibroblast cells, suggesting that it forms an essential part of a protein-protein interaction site necessary to prevent PCC.

Asunto(s)

Cromosomas Humanos/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas de Ciclo Celular , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/metabolismo , Cristalografía por Rayos X , Proteínas del Citoesqueleto , Genes Recesivos , Humanos , Ratones , Ratones Noqueados , Microcefalia/genética , Microcefalia/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Síndrome

RESUMEN

Endothelin-1 (ET-1) is up-regulated in the endothelial cells and astrocytes under ischemia. Transgenic mice with astrocytic ET-1 over-expression (GET-1) showed more severe neurological deficit and larger infarct after transient middle cerebral artery occlusion (MCAO). Here, the significance of endothelial ET-1 in ischemic brain injury was investigated using transgenic mice with the endothelial ET-1 over-expression (TET-1). Increased ET-1 level was observed in the TET-1 brain infarct core after transient MCAO. ET(A) receptor expression was induced in the penumbra and ET(A) antagonist (A-147627) partially normalized the infarct volume and neurological deficit. In the infarct core of TET-1 brain, superoxide, nitrotyrosine, and gp91(phox) levels were increased. TET-1 brain displayed increased matrix metalloproteinase-2 expression, water content, immunoglobulin leakage and decreased occludin level in the ipsilateral hemisphere indicative of BBB breakdown and hemispheric edema. Interestingly, AQP-4 expression was increased in the penumbra of TET-1 brain following transient MCAO leading to the water accumulation. Taken together, endothelial ET-1 over-expression and ETA receptor activation contributes to the increased oxidative stress, water accumulation and BBB breakdown after transient MCAO leading to more severe neurological deficit and increased infarct.

Asunto(s)

Barrera Hematoencefálica/patología , Permeabilidad Capilar , Endotelina-1/metabolismo , Endotelio Vascular/fisiopatología , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Animales , Acuaporina 4/metabolismo , Atrasentán , Barrera Hematoencefálica/fisiopatología , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encéfalo/fisiopatología , Antagonistas de los Receptores de la Endotelina A , Endotelina-1/genética , Endotelio Vascular/patología , Inmunoglobulinas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Ocludina , Estrés Oxidativo , Regiones Promotoras Genéticas , Pirrolidinas/administración & dosificación , Receptor de Endotelina A/metabolismo , Receptor TIE-1/genética , Superóxidos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Agua/metabolismo

RESUMEN

Previously, we have demonstrated that mRNA expression of endothelin-1 (ET-1), a potent vasoconstrictor, is induced in astrocytes and endothelial cells after ischemic conditions, suggesting that both of these cells synthesize ET-1 under this stress condition. Furthermore, ET-1 protected primary cultured astrocytes from ischemic stress. In order to further investigate the role of endothelial ET-1 in cerebral ischemic injury, transgenic mouse lines (TET) with a transgene that included ET cDNA with SV40 polyA under tyrosine kinase with immunoglobulin and epidermal growth factor homology domain (Tie-1) promoter were used. TET mouse lines were further characterized for ET-1 over-expression in the brain. The reverse transcription-polymerase chain reaction (RT-PCR) analysis using the primers specific for transgene ET-1 showed that transgene ET-1 is only expressed in the brain from TET mice. Total expression of ET- 1 mRNA was also increased in the transgenic brain compared with the non-transgenic brain by semi-quantitative RT-PCR. In situ hybridization and immunocytochemical analyses showed that the increased ET-1 mRNA and peptide expressions were detected in endothelial cells of cerebral vessels of TET mice. Under normal conditions, the TET mice that have a slightly increased blood pressure compared with that of non-transgenic mice showed no gross morphological abnormalities in the brain. However, after transient middle cerebral artery occlusion, TET mice showed a more severe neurological deficit, and larger infarct size and volume, suggesting that over-expressing ET-1 in endothelial cells is deleterious to neuronal survival under ischemic conditions. Our present TET model will serve as an ideal model for studying the role of endothelial ET- 1 in the pathogenesis of ischemic stroke.

Asunto(s)

Encéfalo/irrigación sanguínea , Endotelina-1/metabolismo , Endotelio Vascular/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Degeneración Nerviosa/metabolismo , Animales , Encéfalo/patología , Muerte Celular , Modelos Animales de Enfermedad , Endotelina-1/genética , Endotelio Vascular/patología , Hipocampo/irrigación sanguínea , Hipocampo/patología , Inmunohistoquímica , Hibridación in Situ , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Masculino , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Regulación hacia Arriba

RESUMEN

Classical medications employed to treat depression comprise mostly tricyclic antidepressants (TCAs), specific serotonin reuptake inhibitors (SSRIs) and monoamine oxidase inhibitors (MAOIs), in accord with the recognized involvement of serotonergic and adrenergic systems in depression. Other therapies such as electro-convulsive shock, lithium intake and psychotherapy work via as yet unknown mechanisms. Although GABAergic neurotransmitter systems have not been central to etiological hypotheses for depression, observations are accumulating to suggest that these systems might play an important role in the induction of the disease. Lines of evidence in this regard include interactions between GABAergic and other neurotransmitter systems in depression, GABA levels in patients before and after antidepressant treatments, GABA levels and up/down regulations of GABA receptors in animal models of induced depression, and clinical effects of GABA receptor ligands. Phytomedicines that have a long history of useful applications are drawing increasing attention in pharmaceutical research. Moreover, while drug development is usually focused on single constituent drugs on account of their more accurately predictable physiological responses, complex herbal formulae represent an increasingly important source of drug discovery given the advent of high-throughput screening and specific receptor binding assays. Their active constituents acting on different neurotransmitter systems could be identified, and their therapeutic efficacies tested rigorously. Along with new insights into the underlying mechanisms of depression, the rich abundance of chemical entities from herbs is becoming an inviting resource in the search for effective treatment. This review addresses recent research on the possible role of GABAergic receptors with regard to depression, and potentially antidepressant phytomedicines acting on this class of receptors.

Asunto(s)

Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/metabolismo , Preparaciones de Plantas/uso terapéutico , Receptores de GABA/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Antidepresivos/uso terapéutico , GABAérgicos/uso terapéutico , Humanos

RESUMEN

Screening of traditional medicines has proven invaluable to drug development and discovery. Utilizing activity-guided purification, we previously reported the isolation of a list of flavonoids from the medicinal herb Scutellaria baicalensis Georgi, one of which manifested an affinity for the benzodiazepine receptor (BDZR) comparable to that of the synthetic anxiolytic diazepam (K(i)=6.4 nM). In the present study, this high-affinity, naturally occurring flavonoid derivative, 5,7,2'-trihydroxy-6,8-dimethoxyflavone (K36), was chosen for further functional and behavioral characterization. K36 inhibited [3H]flunitrazepam binding to native BDZR with a K(i) value of 6.05 nM. In electrophysiological experiments K36 potentiated currents mediated by rat recombinant alpha(1)beta(2)gamma(2) GABA(A) receptors expressed in Xenopus oocytes. This potentiation was characterized by a threshold (1 nM) and half-maximal stimulation (24 nM) similar to diazepam. This enhancement was demonstrated to act via the BDZR, since co-application of 1 microM of the BDZR antagonist Ro15-1788 reversed the potentiation. Oral administration of K36 produced significant BDZR-mediated anxiolysis in the mice elevated plus-maze, which was abolished upon co-administration of Ro15-1788. Sedation, myorelaxation and motor incoordination were not observed in the chosen dosage regimen. Structure-activity relationships utilizing synthetic flavonoids with different 2' substituents on the flavone backbone supported that 2'-hydroxyl-substitution is a critical moiety on flavonoids with regard to BDZR affinities. These results further underlined the potential of flavonoids as therapeutics for the treatment of BDZR-associated syndromes.

Asunto(s)

Flavonoides/farmacología , Moduladores del GABA/farmacología , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Regulación Alostérica , Animales , Benzodiazepinas/metabolismo , Modelos Animales de Enfermedad , Flavonoides/uso terapéutico , Agonistas de Receptores de GABA-A , Ligandos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Dolor/tratamiento farmacológico , Dimensión del Dolor/efectos de los fármacos , Ensayo de Unión Radioligante , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo

RESUMEN

As part of an effort to identify naturally occurring GABA(A) receptor benzodiazepine binding site (BDS) ligands from traditional medicinal herbs, we previously reported that flavonoid derivatives isolated from Scutellaria baicalensis (S. baicalensis) Georgi exhibited significant affinities for the BDS. The present study describes the characterization of 5,7-dihydroxy-6-methoxyflavone (oroxylin A), one of the major components of the herbal extract. Oroxylin A inhibited [3H]flunitrazepam binding to rat cerebral cortical membrane with a IC(50) value of 1.09+/-0.07 microM. A GABA ratio of 1.09+/-0.04 suggests that oroxylin A interacts as an antagonist at the recognition site. In neuropharmacological studies, oral administration of oroxylin A (3.75-60 mg kg(-1)) did not result in significant changes in animal models routinely employed for benzodiazepine (BD) evaluation. However, oroxylin A selectively abolished the anxiolytic, myorelaxant and motor incoordination, but not the sedative and anticonvulsant effects elicited by diazepam, a BDS agonist. These results add oroxylin A to the list of CNS active flavonoids, and as the first naturally occurring member endowed with selective antagonistic actions via the BDS.

Asunto(s)

Ansiolíticos/antagonistas & inhibidores , Benzodiazepinas/antagonistas & inhibidores , Flavonoides/farmacología , Desempeño Psicomotor/efectos de los fármacos , Scutellaria baicalensis/química , Animales , Medicamentos Herbarios Chinos , Flavonoides/aislamiento & purificación , Flavonoides/uso terapéutico , Ligandos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Actividad Motora/efectos de los fármacos , Picrotoxina , Plantas Medicinales/química , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/prevención & control