RESUMEN
Stalled replication forks undergo DNA double-strand breaks (DSBs) under certain conditions. However, the precise mechanism underlying DSB induction and the cellular response to persistent replication fork stalling are not fully understood. Here we show that, in response to hydroxyurea exposure, DSBs are generated in an Artemis nuclease-dependent manner following prolonged stalling with subsequent activation of the ataxia-telangiectasia mutated (ATM) signaling pathway. The kinase activity of the catalytic subunit of the DNA-dependent protein kinase, a prerequisite for stimulation of the endonuclease activity of Artemis, is also required for DSB generation and subsequent ATM activation. Our findings indicate a novel function of Artemis as a molecular switch that converts stalled replication forks harboring single-stranded gap DNA lesions into DSBs, thereby activating the ATM signaling pathway following prolonged replication fork stalling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular Tumoral , Endonucleasas , Técnica del Anticuerpo Fluorescente , Humanos , ImmunoblottingRESUMEN
Inhibition of the early intracellular event that triggers neurodegenerative cascades and reversal of neuronal cell death are essential for effective treatment of Alzheimer's disease (AD). In this study, a novel therapeutic for AD, a transducible humanin with an extended caspase-3 cleavage sequence (tHN-C3), was developed and showed multiple mechanisms of therapeutic action. These included targeted delivery of anti-apoptotic protein humanin through the blood-brain barrier (BBB) to neuronal cells, specific inhibition of caspase-3 activation to inhibit the early triggering of AD progression, and delivery of humanin into the cytoplasm of neuronal cells undergoing apoptosis where it exerts its anti-apoptotic functions effectively. The tHN-C3 prevented neuronal cell death induced by H2O2, or soluble Aß42, via Bax binding. In animal models of AD induced by amyloid beta, in Tg2576 mice, and in the rat middle cerebral artery occlusion model of stroke, tHN-C3 effectively prevented neuronal cell death, inflammatory cell infiltration into the brain, and improved cognitive memory. The therapeutic effectiveness of tHN-C3 was comparable to that of Aricept, a clinically approved drug for AD treatment. Therefore, tHN-C3 may be a new remedy with multiple therapeutic functions targeting the early and late stages of neurodegeneration in AD and other brain injuries.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Citoplasma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/administración & dosificación , Enfermedades Neurodegenerativas/tratamiento farmacológico , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citoprotección , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/farmacocinética , Péptidos y Proteínas de Señalización Intracelular/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/enzimología , Neuronas/patología , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/uso terapéutico , RatasRESUMEN
OBJECTIVE: Vasculitis is characterized by leukocyte infiltration in the vessel walls, with destructive damage to mural structures. Retinoids are compounds that bind to retinoic acid receptors and exert biologic activities similar to those of vitamin A, including modulatory effects on cell proliferation and differentiation. This study was undertaken to examine the therapeutic effects of a synthetic retinoid, Am80, in a murine model of vasculitis induced by Candida albicans water-soluble fraction (CAWS). METHODS: Vasculitis was induced in BALB/c mice by intraperitoneal injection of CAWS. Neutrophils were depleted by injection of antineutrophil antibody-positive serum. Am80 was administered orally once daily. Vasculitis was evaluated histologically. Migration of labeled adoptively transferred cells was quantified. Chemotaxis was assessed by cell mobility analysis. Production of reactive oxygen species (ROS) and phosphorylation of MAPKs were measured by flow cytometry. Concentrations of elastase were measured by enzyme-linked immunosorbent assay. RESULTS: Administration of CAWS induced vasculitis in the coronary arteries and aortic root, with abundant neutrophil infiltration. Depletion of neutrophils reduced CAWS-induced vasculitis. Treatment with Am80 led to a significant attenuation of the vasculitis score and inhibition of the migration of transferred neutrophils into the site of vasculitis. In vitro, Am80 suppressed fMLP-induced chemotaxis of human peripheral blood neutrophils. ROS production and elastase release by stimulated neutrophils were reduced by AM80 treatment, and Am80 also inhibited phosphorylation of ERK-1/2 and p38 in neutrophils stimulated with fMLP plus lipopolysaccharide. CONCLUSION: Am80 significantly suppressed CAWS-induced vasculitis. This effect was presumably exerted via inhibition of neutrophil migration and activation.
Asunto(s)
Benzoatos/uso terapéutico , Movimiento Celular/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Receptores de Ácido Retinoico/agonistas , Tetrahidronaftalenos/uso terapéutico , Vasculitis/tratamiento farmacológico , Animales , Aorta/efectos de los fármacos , Aorta/inmunología , Aorta/metabolismo , Benzoatos/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/inmunología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tetrahidronaftalenos/farmacología , Vasculitis/inmunología , Vasculitis/metabolismoRESUMEN
The function of the kinase Btk in neutrophil activation is largely unexplored. Here we found that Btk-deficient neutrophils had more production of reactive oxygen species (ROS) after engagement of Toll-like receptors (TLRs) or receptors for tumor-necrosis factor (TNF), which was associated with more apoptosis and was reversed by transduction of recombinant Btk. Btk-deficient neutrophils in the resting state showed hyperphosphorylation and activation of phosphatidylinositol-3-OH kinase (PI(3)K) and protein tyrosine kinases (PTKs) and were in a 'primed' state with plasma membrane-associated GTPase Rac2. In the absence of Btk, the adaptor Mal was associated with PI(3)K and PTKs at the plasma membrane, whereas in control resting neutrophils, Btk interacted with and confined Mal in the cytoplasm. Our data identify Btk as a critical gatekeeper of neutrophil responses.
Asunto(s)
Apoptosis/inmunología , Activación Neutrófila/inmunología , Neutrófilos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología , Agammaglobulinemia Tirosina Quinasa , Separación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Luminiscencia , Neutrófilos/citología , Neutrófilos/inmunología , Proteínas Tirosina Quinasas/inmunología , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASPâ»/â» cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASPâ»/â» Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.
Asunto(s)
Apoptosis/genética , Células Precursoras de Linfocitos B/metabolismo , Transducción de Señal , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígeno CD24/metabolismo , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular/genética , Línea Celular , Polaridad Celular/efectos de los fármacos , Fibronectinas/farmacología , Orden Génico , Marcación de Gen , Vectores Genéticos/genética , Humanos , Complejo Represivo Polycomb 1 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetraspanina 29/antagonistas & inhibidores , Tetraspanina 29/inmunologíaRESUMEN
Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47(phox) and p67(phox) deficiency. The p47(phox) or p67(phox) protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.
Asunto(s)
Enfermedad Granulomatosa Crónica/enzimología , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Fosfoproteínas/metabolismo , Proteínas Recombinantes/metabolismo , Adulto , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Niño , Preescolar , Citoplasma/enzimología , Femenino , Humanos , Masculino , NADPH Oxidasas/genética , Fosfoproteínas/genética , Complejo Represivo Polycomb 1 , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
A nationwide survey was conducted for identifying ataxia-telangiectasia (AT) patients in Japan. Eighty-nine patients were diagnosed between 1971 and 2006. Detailed clinical and laboratory data of 64 patients including affected siblings were collected. Analyses focused on malignancy, therapy-related toxicity, infection, and hematological/immunological parameters. The phenotypic variability of AT was assessed by comparing 26 affected siblings from 13 families. Malignancy developed in 22% of the cases and was associated with a high rate of severe therapy-related complications: chemotherapy-related cardiac toxicity in 2 children, and severe hemorrhagic cystitis requiring surgery in 2 patients. The frequency of serious viral infections correlated with the T cell count. Hypogammaglobulinemia with hyper-IgM (HIGM) was recorded in 5 patients, and 3 patients developed panhypogammaglobulinemia. Differences in immunological parameters were noted in siblings. Four patients showed an HIGM phenotype, in contrast to their siblings with normal IgG and IgM levels. The patients with HIGM phenotype showed reduced levels of TRECs and CD27+CD20+ memory B cells. The findings suggest that hitherto unidentified modifier genes or exogenous environmental factors can influence the overall immune responses. Our data along with future prospective study will lead to better understanding of the hematological/immunological phenotypes and to better care of the patients.