RESUMEN
Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The histone H3-H4 modification landscape is restored by parental histone recycling and modification of new histones. How DNA replication impacts on histone H2A-H2B is currently unknown. Here, we measure H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub1, H2BK120ub1, and H2A.Z are recycled accurately during DNA replication. Modified H2A-H2B are segregated symmetrically to daughter strands via POLA1 on the lagging strand, but independent of H3-H4 recycling. Post-replication, H2A-H2B modification and variant landscapes are quickly restored, and H2AK119ub1 guides accurate restoration of H3K27me3. This work reveals epigenetic transmission of parental H2A-H2B during DNA replication and identifies cross talk between H3-H4 and H2A-H2B modifications in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates restoration of stable H3-H4 chromatin states.
Asunto(s)
Cromatina , Memoria a Corto Plazo , Ciclo Celular , Replicación del ADN , Histonas/metabolismo , Nucleosomas , Animales , Ratones , ConejosRESUMEN
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.
Asunto(s)
Gastrulación , Mesodermo , Animales , Conejos , Ratones , Gastrulación/genética , Mesodermo/fisiología , Diferenciación Celular/fisiología , Mamíferos/genética , Trofoblastos , Regulación del Desarrollo de la Expresión GénicaRESUMEN
The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Ratones , Conejos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Macaca mulatta , Macrófagos , Nanovacunas , Fagocitosis , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.
Asunto(s)
Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/inmunología , Macaca fascicularis , Glicoproteína de la Espiga del Coronavirus/química , Animales , Anticuerpos Neutralizantes , Linfocitos B/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Nanopartículas/administración & dosificación , Conejos , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/sangre , Linfocitos T/inmunología , Carga ViralRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.
Asunto(s)
Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Evaluación Preclínica de Medicamentos/métodos , Pandemias/prevención & control , Neumonía Viral/prevención & control , Vacunas de Productos Inactivados/uso terapéutico , Vacunas Virales/uso terapéutico , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/genética , COVID-19 , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Femenino , Cobayas , Inmunogenicidad Vacunal , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Filogenia , Neumonía Viral/virología , Conejos , Ratas , Ratas Wistar , SARS-CoV-2 , Vacunas de Productos Inactivados/efectos adversos , Células Vero , Vacunas Virales/efectos adversosRESUMEN
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/terapia , Mastocitos/enzimología , Mastocitos/inmunología , Triptasas/antagonistas & inhibidores , Triptasas/inmunología , Adolescente , Regulación Alostérica/inmunología , Animales , Línea Celular , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ConejosRESUMEN
The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antiprotozoarios/inmunología , Eritrocitos/parasitología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Adolescente , Adulto , Animales , Sitios de Unión , Proteínas Portadoras/inmunología , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Femenino , Células HEK293 , Voluntarios Sanos , Humanos , Malaria Falciparum/parasitología , Masculino , Merozoítos/fisiología , Persona de Mediana Edad , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/inmunología , Conejos , Ratas , Ratas Sprague-Dawley , Adulto JovenRESUMEN
The L-type voltage-gated Ca2+ (Cav) channels are modulated by various compounds exemplified by 1,4-dihydropyridines (DHP), benzothiazepines (BTZ), and phenylalkylamines (PAA), many of which have been used for characterizing channel properties and for treatment of hypertension and other disorders. Here, we report the cryoelectron microscopy (cryo-EM) structures of Cav1.1 in complex with archetypal antagonistic drugs, nifedipine, diltiazem, and verapamil, at resolutions of 2.9 Å, 3.0 Å, and 2.7 Å, respectively, and with a DHP agonist Bay K 8644 at 2.8 Å. Diltiazem and verapamil traverse the central cavity of the pore domain, directly blocking ion permeation. Although nifedipine and Bay K 8644 occupy the same fenestration site at the interface of repeats III and IV, the coordination details support previous functional observations that Bay K 8644 is less favored in the inactivated state. These structures elucidate the modes of action of different Cav ligands and establish a framework for structure-guided drug discovery.
Asunto(s)
Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/ultraestructura , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Canales de Calcio/ultraestructura , Canales de Calcio Tipo L/fisiología , Microscopía por Crioelectrón , Diltiazem , Ligandos , Masculino , Modelos Moleculares , Nifedipino , Conejos , VerapamiloRESUMEN
The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.
Asunto(s)
Agonistas de los Canales de Calcio/química , Activación del Canal Iónico , Contracción Muscular , Canal Liberador de Calcio Receptor de Rianodina/química , Animales , Sitios de Unión , Cafeína/química , Calcio/química , Microscopía por Crioelectrón , Ligandos , Dominios Proteicos , Conejos , Proteínas de Unión a Tacrolimus/químicaRESUMEN
The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.
Asunto(s)
Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes , Epítopos/química , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1 , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Modelos Moleculares , Mutagénesis , Conformación Proteica , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.
Asunto(s)
Encéfalo , Ojo , Sistema Linfático , Animales , Femenino , Humanos , Masculino , Ratones , Conejos , Bacterias/inmunología , Encéfalo/anatomía & histología , Encéfalo/inmunología , Dependovirus/inmunología , Ojo/anatomía & histología , Ojo/inmunología , Glioblastoma/inmunología , Herpesvirus Humano 2/inmunología , Inyecciones Intravítreas , Sistema Linfático/anatomía & histología , Sistema Linfático/inmunología , Vasos Linfáticos/anatomía & histología , Vasos Linfáticos/inmunología , Macaca mulatta , Meninges/inmunología , Nervio Óptico/inmunología , Porcinos , Pez Cebra , Factor C de Crecimiento Endotelial Vascular/inmunología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1Aâ Val-tRNAâ GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.
Asunto(s)
Mamíferos/metabolismo , Ribosomas/química , Secuencia de Aminoácidos , Animales , Anticodón/metabolismo , Codón/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Extensión de la Cadena Peptídica de Translación , ARN de Transferencia/metabolismo , Conejos , Saccharomyces cerevisiae/metabolismo , Tetrahymena thermophila/metabolismoRESUMEN
About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.
Asunto(s)
Adenosina/análogos & derivados , Epigénesis Genética , Procesamiento Postranscripcional del ARN , Receptor de Adenosina A3/metabolismo , Transducción de Señal , Adenosina/genética , Adenosina/metabolismo , Animales , Femenino , Células HEK293 , Humanos , Masculino , Conejos , Receptor de Adenosina A3/genéticaRESUMEN
The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Liposomas , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD4/química , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Complemento C3/inmunología , Complemento C3/metabolismo , Reactividad Cruzada/inmunología , Epítopos/inmunología , Glicosilación , Infecciones por VIH/virología , Humanos , Inmunoglobulina G/inmunología , Modelos Moleculares , Pruebas de Neutralización , Polisacáridos/inmunología , Polisacáridos/metabolismo , Unión Proteica , Conformación Proteica , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNAi(Met) in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.
Asunto(s)
Mamíferos/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Helicasas/química , ARN Ribosómico/química , Ribonucleoproteínas/química , Animales , Secuencia de Bases , Sistema Libre de Células , Microscopía por Crioelectrón , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Mamíferos/genética , Modelos Moleculares , Datos de Secuencia Molecular , ARN Helicasas/metabolismo , ARN Ribosómico/metabolismo , ARN Ribosómico 18S/química , ARN Ribosómico 18S/metabolismo , Conejos , Ribonucleoproteínas/metabolismoRESUMEN
VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.
Asunto(s)
Actinas/química , Proteínas Bacterianas/química , Vibrio parahaemolyticus/química , Citoesqueleto de Actina , Actinas/genética , Actinas/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , Conejos , Vibrio parahaemolyticus/citología , Vibrio parahaemolyticus/metabolismoRESUMEN
Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Sistema Libre de Células , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Conejos , Subunidades Ribosómicas/genética , Subunidades Ribosómicas/metabolismo , Ribosomas/genética , UbiquitinaciónRESUMEN
The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.
Asunto(s)
Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Sistema Nervioso Simpático/inmunología , Animales , Línea Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Expresión Génica/inmunología , Humanos , Inflamación/genética , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Norepinefrina/inmunología , Norepinefrina/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema Nervioso Simpático/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/inmunología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Mapeo Epitopo/métodos , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Microscopía Electrónica/métodos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Formación de Anticuerpos/inmunología , Línea Celular , Femenino , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Humanos , Inmunización , Fragmentos Fab de Inmunoglobulinas/inmunología , Conejos , Proteínas Recombinantes/inmunologíaRESUMEN
Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.