RESUMEN
Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884 ng/µL and 23.767 ng/µL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.
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Nylons , Saliva , Humanos , ARN Ribosómico 16S , Dermatoglifia del ADN/métodos , ADN RibosómicoRESUMEN
Type I interferons are a widely expressed family of effector cytokines that promote innate antiviral and antibacterial immunity. Paradoxically, they can also suppress immune responses by driving production of anti-inflammatory cytokines, and dysregulation of these cytokines can contribute to host-mediated immunopathology and disease progression. Recent studies describe their anti-inflammatory role in intestinal inflammation and the locus containing IFNAR, a heterodimeric receptor for the type I interferons has been identified as a susceptibility region for human inflammatory bowel disease. This review focuses on the role of type I IFNs in the intestine in health and disease and their emerging role as immune modulators. Clear understanding of type I IFN-mediated immune responses may provide avenues for fine-tuning existing IFN treatment for infection and intestinal inflammation.
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Homeostasis , Inflamación/inmunología , Inflamación/metabolismo , Interferón Tipo I/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/microbiología , Modelos Animales de Enfermedad , Humanos , Inmunomodulación , Inflamación/microbiología , Inflamación/virología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Intestinos/microbiología , Intestinos/patología , Intestinos/virología , Ratones , Virosis/genética , Virosis/inmunología , Virosis/metabolismo , Virosis/virologíaRESUMEN
Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.
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Proteínas Portadoras/metabolismo , Quimiocinas/metabolismo , Leucocitos/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Linfocitos B/citología , Células de la Médula Ósea/citología , Calcio/metabolismo , Quimiotaxis , Células Dendríticas/citología , Femenino , Proteínas de Unión al GTP/metabolismo , Inhibidores de Disociación de Guanina Nucleótido , Sistema Inmunológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Bazo/citología , Linfocitos T/citología , Timocitos/citologíaRESUMEN
If a DNA sample collected in the field is old or degraded, short tandem repeat analysis is difficult to perform, a representative analysis method currently used for individual identification. Given that microorganisms exist everywhere and within the human body, in similar amounts to human cells, microbial analysis could be used to identify individuals even in cases in which human DNA-based identification is difficult. Research has demonstrated that the types of microorganisms within the human body differ depending on various internal or external factors, such as body part or bodily fluid type, lifestyle, geographical area of residence, sex, and age. In this study, we aimed to examine the relationship between lifestyle factors and the composition and diversity of the oral microbiome in individuals living in Korea. We collected 43 saliva samples from Korean individuals and analyzed the oral microbiome and its variations due to external factors, such as coffee consumption, drinking, and smoking. Linear discriminant analysis effect size revealed that Oribacterium, Campylobacter, and Megasphaera were abundant in coffee consumers, whereas Saccharimonadales, Clostridia, and Catonella were abundant in alcohol non-drinkers. We found increased levels of Stomatobaculum in the saliva of smokers, compared with that of non-smokers. Thus, our analysis revealed characteristic microorganisms for each parameter that was evaluated (coffee consumption, smoking, drinking). Consequently, our study provides insight into the oral microbiome in the Korean population and lays the foundation for developing the Korean Forensic Microbiome Database.
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Café , Microbiota , Humanos , Fumar/epidemiología , Estilo de Vida , ADN , República de CoreaRESUMEN
The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.
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COVID-19 , Humanos , SARS-CoV-2 , Etnicidad , Epítopos , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Pruebas de NeutralizaciónRESUMEN
The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were â¼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.
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Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Mitosis , Proteínas de Neoplasias/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina/química , Alanina/química , Animales , Sitios de Unión , Proteína Quinasa CDC2/metabolismo , Ciclina B1/metabolismo , Citometría de Flujo/métodos , Fase G2 , Células HeLa , Humanos , Lisina/química , Modelos Biológicos , Proteínas de Unión a Poli-ADP-Ribosa , Conformación Proteica , Quinasa Tipo Polo 1RESUMEN
Although short-acting and long-acting inhaled ß(2)-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent ß(2)-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to ß(2)-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cß and myosin light chain). In contrast, ß-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5(-/-) mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive ß(2)-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.
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Agonistas de Receptores Adrenérgicos beta 2/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Pulmón/metabolismo , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas RGS/biosíntesis , Transducción de Señal/efectos de los fármacos , Animales , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Proteínas RGS/genética , Transducción de Señal/fisiologíaRESUMEN
At the plasma membrane, heterotrimeric G proteins act as molecular switches to relay signals from G protein-coupled receptors; however, G(alpha) subunits also have receptor-independent functions at intracellular sites. Regulator of G protein signaling (RGS) 14, which enhances the intrinsic GTPase activity of G(ialpha) proteins, localizes in centrosomes, which suggests the coexpression of G(ialpha). We show expression of G(ialpha1), G(ialpha2), and G(ialpha3) in the centrosomes and at the midbody. Fluorescence resonance energy transfer analysis confirms a direct interaction between RGS14 and G(ialpha1) in centrosomes. Expression of GTPase-deficient G(ialpha1) results in defective cytokinesis, whereas that of wild-type or GTPase-deficient G(ialpha3) causes prolonged mitosis. Cells treated with pertussis toxin, with reduced expression of G(ialpha1), G(ialpha2), and G(ialpha3) or with decreased expression of RGS14 also exhibit cytokinesis defects. These results suggest that G(ialpha) proteins and their regulators at these sites may play essential roles during mammalian cell division.
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División Celular/fisiología , Centrosoma/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Animales , Proteínas del Citoesqueleto/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Células HeLa , Humanos , Inmunohistoquímica , Proteínas Luminiscentes/metabolismo , Ratones , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Immunoglobulin (Ig)A antibodies play a critical role in protection against mucosal pathogens. However, the role of serum IgA in immunity to nonmucosal pathogens, such as Plasmodium falciparum, is poorly characterized, despite being the second most abundant isotype in blood after IgG. Here, we investigated the circulating IgA response in humans to P. falciparum sporozoites that are injected into the skin by mosquitoes and migrate to the liver via the bloodstream to initiate malaria infection. We found that circulating IgA was induced in three independent sporozoite-exposed cohorts: individuals living in an endemic region in Mali, malaria-naïve individuals immunized intravenously with three large doses of irradiated sporozoites, and malaria-naïve individuals exposed to a single controlled mosquito bite infection. Mechanistically, we found evidence in an animal model that IgA responses were induced by sporozoites at dermal inoculation sites. From malaria-resistant individuals, we isolated several IgA monoclonal antibodies that reduced liver parasite burden in mice. One antibody, MAD2-6, bound to a conserved epitope in the amino terminus of the P. falciparum circumsporozoite protein, the dominant protein on the sporozoite surface. Crystal structures of this antibody revealed a unique mode of binding whereby two Fabs simultaneously bound either side of the target peptide. This study reveals a role for circulating IgA in malaria and identifies the amino terminus of the circumsporozoite protein as a target of functional antibodies.
Asunto(s)
Anticuerpos Antiprotozoarios , Inmunoglobulina A , Malaria , Animales , Anticuerpos Antiprotozoarios/inmunología , Humanos , Inmunoglobulina A/inmunología , Malaria/inmunología , Ratones , Plasmodium falciparum , Proteínas Protozoarias , EsporozoítosRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domainRBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.
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Anticuerpos Biespecíficos , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Biespecíficos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19 , Humanos , SARS-CoV-2RESUMEN
The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.
RESUMEN
Mononuclear phagocytes are a heterogeneous population of leukocytes essential for immune homeostasis that develop tissue-specific functions due to unique transcriptional programs driven by local microenvironmental cues. Single cell RNA sequencing (scRNA-seq) of colonic myeloid cells from specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice revealed extensive heterogeneity of both colon macrophages (MPs) and dendritic cells (DCs). Modeling of developmental pathways combined with inference of gene regulatory networks indicate two major trajectories from common CCR2+ precursors resulting in colon MP populations with unique transcription factors and downstream target genes. Compared to SPF mice, GF mice had decreased numbers of total colon MPs, as well as selective proportional decreases of two major CD11c+CD206intCD121b+ and CD11c-CD206hiCD121b- colon MP populations, whereas DC numbers and proportions were not different. Importantly, these two major colon MP populations were clearly distinct from other colon MP populations regarding their gene expression profile, localization within the lamina propria (LP) and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations at the molecular level and highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.
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Colon/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Células Mieloides/fisiología , Animales , Antígeno CD11c/metabolismo , Diferenciación Celular , Células Cultivadas , Ontología de Genes , Redes Reguladoras de Genes , Homeostasis , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/genética , Fenotipo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , TranscriptomaRESUMEN
Population pharmacokinetic analysis and modeling procedures typically require estimates of both population and individual pharmacokinetic parameters. However, only some of these parameters are contained in models and only parameters in the model can be estimated. In this paper, we introduce a new R package, PKconverter, to calculate pharmacokinetic parameters using the relationships among them. After fitting the model, other parameters can be calculated from the functional relationship among the parameters. PKconverter provides the functions to calculate whole parameters along with a Shiny application for converting the parameters. With this package, it is also possible to calculate the standard errors of the other parameters that are not in the model and estimate individual parameters simultaneously.
RESUMEN
Despite studies indicating the effects of IL-21 signaling in intestinal inflammation, its roles in intestinal homeostasis and infection are not yet clear. Here, we report potent effects of commensal microbiota on the phenotypic manifestations of IL-21 receptor deficiency. IL-21 is produced highly in the small intestine and appears to be critical for mounting an IgA response against atypical commensals such as segmented filamentous bacteria and Helicobacter, but not to the majority of commensals. In the presence of these atypical commensals, IL-21R-deficient mice exhibit reduced numbers of germinal center and IgA+ B cells and expression of activation-induced cytidine deaminase in Peyer's patches as well as a significant decrease in small intestine IgA+ plasmablasts and plasma cells, leading to higher bacterial burdens and subsequent expansion of Th17 and Treg cells. These microbiota-mediated secondary changes in turn enhance T cell responses to an oral antigen and strikingly dampen Citrobacter rodentium-induced immunopathology, demonstrating a complex interplay between IL-21-mediated mucosal immunity, microbiota, and pathogens.
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Formas Bacterianas Atípicas/fisiología , Linfocitos B/fisiología , Citrobacter rodentium/fisiología , Infecciones por Enterobacteriaceae/inmunología , Helicobacter/fisiología , Inmunoglobulina A/metabolismo , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Receptores de Interleucina-21/genética , Animales , Carga Bacteriana , Diferenciación Celular , Células Cultivadas , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Homeostasis , Humanos , Inmunidad Humoral , Inmunidad Mucosa , Mucosa Intestinal/microbiología , Intestino Delgado/microbiología , Ratones , Ratones Noqueados , Receptores de Interleucina-21/metabolismo , Linfocitos T Reguladores/inmunología , Células Th17/inmunologíaRESUMEN
Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.
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Carcinoma Hepatocelular/metabolismo , Virus de la Hepatitis B/química , Hepatitis B/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Agregado de Proteínas , Proteolisis , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Células HEK293 , Células HeLa , Hepatitis B/complicaciones , Hepatitis B/virología , Humanos , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Agregación Patológica de Proteínas/metabolismo , Tasa de Supervivencia , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), the status of which is also closely associated with genomic instability in liver cancer. Here, we showed that hepatitis B virus X oncoprotein (HBx) expression in Chang cells results in a multinuclear phenotype and an abnormal number of centrosomes (n >or=3). Regulation of centrosome duplication in HBx-expressing ChangX-34 cells was defective and uncoupled from the cell cycle. HBx induced amplification of centrosomes, multipolar spindle formation, and chromosomal missegregation during mitosis and subsequently increased the generation of multinucleated cells and micronuclei formation. Treatment with PD98059, a mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor, significantly reduced the number of cells with hyperamplified centrosomes and decreased the multinucleated cells and micronuclei formation. Consistently, the phospho-ERK level during cell progression was substantially higher in ChangX-34 cells than that of Chang cells. In contrast, neither wortmannin, an inhibitor of phosphoinositide-3 kinase, nor SB203589, an inhibitor of p38 mitogen-activated protein kinase (MAPK), showed any effects. Introduction of Ras dominant-negative (D/N) and MEK2 D/N genes into ChangX-34 cells significantly alleviated centrosome amplification, whereas introduction of the PKC D/N and PKB D/N genes did not. Thus, our results demonstrate that the HBx induced centrosome hyperamplification and mitotic aberration by activation of the Ras-MEK-MAPK. Intervention of this signaling pathway could suppress the centrosome amplification as well as mitotic aberration. These findings may provide a possible mechanism by which HBx promotes phenotypic progression by predisposing chromosomal alteration in HBV-infected liver.
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Centrosoma/metabolismo , Sistema de Señalización de MAP Quinasas , Mitosis/fisiología , Transactivadores/metabolismo , Proteínas ras/metabolismo , Línea Celular , Centrosoma/efectos de los fármacos , Flavonoides/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación/genética , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Transfección , Proteínas Reguladoras y Accesorias Virales , Proteínas ras/genéticaRESUMEN
RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5-E17.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E14.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Gi(alpha) and Gq(alpha) and it attenuated angiotensin II-, endothelin-1-, sphingosine-1-phosphate-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation.
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Arterias/citología , Proteínas Inmediatas-Precoces/metabolismo , Lisofosfolípidos , Pericitos/metabolismo , Proteínas RGS/metabolismo , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Esfingosina/análogos & derivados , Células 3T3 , Angiotensina II/antagonistas & inhibidores , Animales , Arterias/embriología , Arterias/metabolismo , Endotelina-1/antagonistas & inhibidores , Hibridación in Situ , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Biológicos , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Proteínas RGS/genética , Proteínas RGS/fisiología , ARN Mensajero/biosíntesis , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Lisofosfolípidos , Transducción de Señal , Esfingosina/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5(+/-) mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5(H43W)), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Infecciones por Virus ARN/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células HEK293 , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/enzimología , Ubiquitina/metabolismoRESUMEN
Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gαi subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Citocinesis/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN/fisiologíaRESUMEN
Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gα(i) subunits to exchange GDP for GTP. By limiting the duration that Gα(i) subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gα(i2) interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gα(i) activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.