RESUMEN
Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314-324) and E2 (residues 412-423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a "scaffolding meta-server" to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a "multivalent scaffolding" approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.
Asunto(s)
Antígenos Virales/química , Epítopos/química , Hepacivirus/química , Proteínas Recombinantes/química , Proteínas del Envoltorio Viral/química , Vacunas contra Hepatitis Viral/genética , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/metabolismo , Antígenos Virales/genética , Antígenos Virales/inmunología , Diseño de Fármacos , Mapeo Epitopo , Epítopos/genética , Epítopos/inmunología , Expresión Génica , Células HEK293 , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
RNA viruses modify intracellular membranes to produce replication scaffolds. In pancreatic cells, coxsackievirus B3 (CVB3) hijacks membranes from the autophagy pathway, and in vivo disruption of acinar cell autophagy dramatically delays CVB3 replication. This is reversed by expression of GFP-LC3, indicating that CVB3 may acquire membranes from an alternative, autophagy-independent, source(s). Herein, using 3 recombinant CVB3s (rCVB3s) encoding different proteins (proLC3, proLC3(G120A), or ATG4B(C74A)), we show that CVB3 is, indeed, flexible in its utilization of cellular membranes. When compared with a control rCVB3, all 3 viruses replicated to high titers in vivo, and caused severe pancreatitis. Most importantly, each virus appeared to subvert membranes in a unique manner. The proLC3 virus produced a large quantity of LC3-I which binds to phosphatidylethanolamine (PE), affording access to the autophagy pathway. The proLC3(G120A) protein cannot attach to PE, and instead binds to the ER-resident protein SEL1L, potentially providing an autophagy-independent source of membranes. Finally, the ATG4B(C74A) protein sequestered host cell LC3-I, causing accumulation of immature phagophores, and massive membrane rearrangement. Taken together, our data indicate that some RNA viruses can exploit a variety of different intracellular membranes, potentially maximizing their replication in each of the diverse cell types that they infect in vivo.
Asunto(s)
Autofagia , Infecciones por Coxsackievirus/virología , Enterovirus/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Proteínas Relacionadas con la Autofagia , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Homocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Páncreas/virología , Fosfatidiletanolaminas/química , Proteínas/metabolismo , Virus ARN/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Ebolavirus NP oligomerizes into helical filaments found at the core of the virion, encapsidates the viral RNA genome, and serves as a scaffold for additional viral proteins within the viral nucleocapsid. We identified a portion of the phosphoprotein homolog VP35 that binds with high affinity to nascent NP and regulates NP assembly and viral genome binding. Removal of the VP35 peptide leads to NP self-assembly via its N-terminal oligomerization arm. NP oligomerization likely causes a conformational change between the NP N- and C-terminal domains, facilitating RNA binding. These functional data are complemented by crystal structures of the NP°-VP35 complex at 2.4 Å resolution. The interactions between NP and VP35 illuminated by these structures are conserved among filoviruses and provide key targets for therapeutic intervention.
Asunto(s)
Nucleoproteínas/química , Multimerización de Proteína , Proteínas del Núcleo Viral/química , Secuencia de Aminoácidos , Sitios de Unión , Datos de Secuencia Molecular , Proteínas de la Nucleocápside , Nucleoproteínas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas del Núcleo Viral/metabolismoRESUMEN
Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable biocompatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multimodal (molecular and anatomical) imaging.
Asunto(s)
Diagnóstico por Imagen/métodos , Oro/química , Nanopartículas del Metal/química , Espectrometría de Masas , Microscopía Electrónica , Nanoestructuras/químicaRESUMEN
Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2(-/-) mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2(-/-) mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2(-/-) brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.
Asunto(s)
Lipasa/metabolismo , Fosfolipasas A1/metabolismo , Paraplejía Espástica Hereditaria/enzimología , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , Cognición , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Marcación de Gen , Células HEK293 , Humanos , Lipasa/antagonistas & inhibidores , Gotas Lipídicas/metabolismo , Gotas Lipídicas/ultraestructura , Locomoción , Ratones Endogámicos C57BL , Neuronas/metabolismo , Fosfolipasas , Fosfolipasas A1/antagonistas & inhibidores , Fosfolipasas A1/deficiencia , Reproducibilidad de los Resultados , Paraplejía Espástica Hereditaria/genética , Triglicéridos/metabolismoRESUMEN
Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.
Asunto(s)
Ebolavirus/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Cristalografía por Rayos X , Dimerización , Ebolavirus/química , Ebolavirus/clasificación , Ebolavirus/genética , Modelos Moleculares , Mutagénesis , Mutación Puntual , Proteínas de la Matriz Viral/genética , Ensamble de Virus , Liberación del VirusRESUMEN
Methamphetamine (Meth) abuse has been shown to induce alterations in mitochondrial function in the brain as well as to induce hyperthermia, which contributes to neurotoxicity and Meth-associated mortality. Brown adipose tissue (BAT), a thermogenic site known to be important in neonates, has recently regained importance since being identified in significant amounts and in correlation with metabolic balance in human adults. Given the high mitochondrial content of BAT and its role in thermogenesis, we aimed to investigate whether BAT plays any role in the development of Meth-induced hyperthermia. By ablating or denervating BAT, we identified a partial contribution of this organ to Meth-induced hyperthermia. BAT ablation decreased temperature by 0.5°C and reduced the length of hyperthermia by 1 h, compared to sham-operated controls. BAT denervation also affected the development of hyperthermia in correlation with decreased the expression of electron transport chain molecules, and increase on PCG1a levels, but without affecting Meth-induced uncoupling protein 1 upregulation. Furthermore, in isolated BAT cells in culture, Meth, but not Norepinephrine, induced H2O2 upregulation. In addition, we found that in vivo Reactive Oxygen Species (ROS) play a role in Meth hyperthermia. Thus, sympathetically mediated mitochondrial activation in the BAT and Meth-induced ROS are key components to the development of hyperthermia in Meth abuse.
RESUMEN
Autophagy protects against many infections by inducing the lysosomal-mediated degradation of invading pathogens. However, previous in vitro studies suggest that some enteroviruses not only evade these protective effects but also exploit autophagy to facilitate their replication. We generated Atg5(f/f)/Cre(+) mice, in which the essential autophagy gene Atg5 is specifically deleted in pancreatic acinar cells, and show that coxsackievirus B3 (CVB3) requires autophagy for optimal infection and pathogenesis. Compared to Cre(-) littermates, Atg5(f/f)/Cre(+) mice had an â¼2,000-fold lower CVB3 titer in the pancreas, and pancreatic pathology was greatly diminished. Both in vivo and in vitro, Atg5(f/f)/Cre(+) acinar cells had reduced intracellular viral RNA and proteins. Furthermore, intracellular structural elements induced upon CVB3 infection, such as compound membrane vesicles and highly geometric paracrystalline arrays, which may represent viral replication platforms, were infrequently observed in infected Atg5(f/f)/Cre(+) cells. Thus, CVB3-induced subversion of autophagy not only benefits the virus but also exacerbates pancreatic pathology.
Asunto(s)
Células Acinares/virología , Autofagia , Infecciones por Coxsackievirus/patología , Enterovirus/fisiología , Páncreas/patología , Replicación Viral , Células Acinares/patología , Células Acinares/fisiología , Animales , Proteína 5 Relacionada con la Autofagia , Infecciones por Coxsackievirus/metabolismo , Interacciones Huésped-Patógeno , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Páncreas/metabolismo , Páncreas/virología , Transducción de SeñalRESUMEN
Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.
Asunto(s)
Cardiomiopatías/dietoterapia , Dieta Cetogénica , Complejo Mediador/genética , Miopatías Mitocondriales/dietoterapia , Mutación Missense , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Expresión Génica , Genes Letales , Estimación de Kaplan-Meier , Masculino , Complejo Mediador/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Electrónica , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/metabolismo , Miocardio/metabolismo , Miocardio/patología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , DesteteRESUMEN
Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas/metabolismo , Hipoxia/metabolismo , Mitocondrias/fisiología , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Adaptación Fisiológica , Animales , Apoptosis , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Dinaminas/genética , Humanos , Hipoxia/genética , Hipoxia/patología , Inmunohistoquímica , Lentivirus , Longevidad , Fusión de Membrana , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Miocitos Cardíacos/citología , Fosforilación , Transducción Genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.
Asunto(s)
Autofagia/fisiología , Infecciones por Coxsackievirus/fisiopatología , Enterovirus Humano B , Páncreas/citología , Fagosomas/virología , Análisis de Varianza , Animales , Western Blotting , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Páncreas/virología , Fagosomas/ultraestructuraRESUMEN
We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (â¼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of â¼60 and â¼45 nm in diameter. The â¼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The â¼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large â¼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.
Asunto(s)
Hepacivirus/ultraestructura , Virión/ultraestructura , Antígenos Virales/análisis , Cápside , Técnicas de Cultivo de Célula , Hepacivirus/patogenicidad , Membrana Dobles de Lípidos , Microscopía Electrónica , Tamaño de la Partícula , ARN Viral/análisis , Virión/patogenicidadRESUMEN
Disruption of autophagy--a key homeostatic process in which cytosolic components are degraded and recycled through lysosomes--can cause neurodegeneration in tissue culture and in vivo. Upregulation of this pathway may be neuroprotective, and much effort is being invested in developing drugs that cross the blood brain barrier and increase neuronal autophagy. One well-recognized way of inducing autophagy is by food restriction, which upregulates autophagy in many organs including the liver; but current dogma holds that the brain escapes this effect, perhaps because it is a metabolically privileged site. Here, we have re-evaluated this tenet using a novel approach that allows us to detect, enumerate and characterize autophagosomes in vivo. We first validate the approach by showing that it allows the identification and characterization of autophagosomes in the livers of food-restricted mice. We use the method to identify constitutive autophagosomes in cortical neurons and Purkinje cells, and we show that short-term fasting leads to a dramatic upregulation in neuronal autophagy. The increased neuronal autophagy is revealed by changes in autophagosome abundance and characteristics, and by diminished neuronal mTOR activity in vivo, demonstrated by a reduction in levels of phosphorylated S6 ribosomal protein in Purkinje cells. The increased abundance of autophagosomes in Purkinje cells was confirmed using transmission electron microscopy. Our data lead us to speculate that sporadic fasting might represent a simple, safe and inexpensive means to promote this potentially therapeutic neuronal response.
Asunto(s)
Autofagia/fisiología , Ayuno/fisiología , Neuronas/citología , Animales , Restricción Calórica , Cerebelo/citología , Cerebelo/ultraestructura , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Fagosomas/metabolismo , Fagosomas/ultraestructura , Células de Purkinje/citología , Células de Purkinje/metabolismo , Células de Purkinje/ultraestructura , Reproducibilidad de los Resultados , Serina-Treonina Quinasas TOR/metabolismo , Factores de TiempoRESUMEN
Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22(phox), creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H(2)O(2) production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22(phox) Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O(2)(-) generation. In contrast, Nox4 released H(2)O(2) and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Secuencias de Aminoácidos , Biocatálisis , Línea Celular , Movimiento Celular , Supervivencia Celular , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Microscopía Electrónica , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/química , NADPH Oxidasas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.
Asunto(s)
Músculo Esquelético/embriología , Sarcómeros/metabolismo , Talina/metabolismo , Animales , Fusión Celular , Citoesqueleto/metabolismo , Integrinas/metabolismo , Ratones , Ratones Noqueados , Enfermedades Musculares/metabolismo , Talina/genéticaRESUMEN
Biophysical studies on amyloidogenic and aggregation-prone peptides often require large quantities of material. However, solid-phase synthesis, handling, and purification of peptides often present challenges on these scales. Recombinant expression is an attractive alternative because of its low cost, the ability to isotopically label the peptides, and access to sequences exceeding approximately 50 residues. However, expression systems that seek to solubilize amyloidogenic peptides suffer from low yields, difficult optimizations, and isolation challenges. We present a general strategy for expressing and isolating amyloidogenic peptides in Escherichia coli by fusion to a polypeptide that drives the expression of attached peptides into bacterial inclusion bodies. This scheme minimizes toxicity during bacterial growth and enables the processing and handling of the peptides in denaturing solutions. Immobilized metal affinity chromatography, reverse phase HPLC, and cyanogen bromide cleavage are used to isolate the peptide, followed by further reverse phase HPLC, which yields milligram quantities of the purified peptide. We demonstrate that driving the peptides into inclusion bodies using fusion to BCL-XL-1/2 is a general strategy for their expression and isolation, as exemplified by the production of 11 peptides species.
Asunto(s)
Amiloide/genética , Amiloide/aislamiento & purificación , Escherichia coli/genética , Péptidos/genética , Péptidos/aislamiento & purificación , Proteína bcl-X/genética , Secuencia de Aminoácidos , Expresión Génica , Cuerpos de Inclusión/química , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Proteína bcl-X/aislamiento & purificaciónRESUMEN
Early apoptosis is defined by stereotypic morphological changes, especially evident in the nucleus, where chromatin condenses and compacts, and assumes a globular, half-moon or crescent-shaped morphology. Accumulating evidence suggests that cytoplasmic organelles such as mitochondria and the Golgi complex are major sites of integration of pro-apoptotic signaling. In this study, cytoplasmic organelles including Golgi complex, mitochondria, endosomes, lysosomes, and peroxisomes were shown to condense at the same unique region adjacent to the crescentic nucleus during a relatively early stage of apoptosis induced by staurosporine or other agents. The co-clustering phenomenon may be caused by shrinkage of cytoplasm during apoptosis although cytoskeletal markers actin and tubulin were not condensed and appeared excluded. These data suggest the co-clustering of cytoplasmic organelles plays an interesting role during the progression of the apoptotic process. It is possible that modification of pro-apoptotic proteins may arise as a result of the interplay of these cytoplasmic organelles.
Asunto(s)
Apoptosis , Nucléolo Celular/ultraestructura , Aparato de Golgi/ultraestructura , Orgánulos/ultraestructura , Línea Celular , Nucléolo Celular/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Humanos , Microscopía Electrónica , Orgánulos/efectos de los fármacos , Estaurosporina/farmacologíaRESUMEN
Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.
Asunto(s)
Desoxirribonucleasa I/metabolismo , Neutrófilos/inmunología , Especies Reactivas de Oxígeno/metabolismo , Desoxirribonucleasa I/inmunología , Escherichia coli/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Listeria monocytogenes/inmunología , Mediciones Luminiscentes , Microscopía Confocal , Microscopía Electrónica de Transmisión , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.