RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Receptores Virales/química , SARS-CoV-2/química , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Mutación/genética , Filogenia , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Electricidad Estática , Homología Estructural de ProteínaRESUMEN
Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.
Asunto(s)
Epigenoma , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Pinus/genética , Aclimatación/genética , Cromosomas de las Plantas/genética , Cycadopsida/genética , Elementos Transponibles de ADN/genética , Bosques , Redes Reguladoras de Genes , Tamaño del Genoma , Genómica/métodos , Intrones , Magnoliopsida/genéticaRESUMEN
Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.
Asunto(s)
COVID-19 , Vacunas , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Ratones , SARS-CoV-2/genéticaRESUMEN
Niemann-Pick disease type C (NPC) is associated with mutations in NPC1 and NPC2, whose gene products are key players in the endosomal/lysosomal egress of low-density lipoprotein-derived cholesterol. NPC1 is also the intracellular receptor for Ebola virus (EBOV). Here, we present a 4.4 Å structure of full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains 13 transmembrane segments (TMs) and three distinct lumenal domains A (also designated NTD), C, and I. TMs 2-13 exhibit a typical resistance-nodulation-cell division fold, among which TMs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through the domain C. Our structural and biochemical characterizations provide an important framework for mechanistic understanding of NPC1-mediated intracellular cholesterol trafficking and Ebola virus infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/ultraestructura , Microscopía por Crioelectrón , Glicoproteínas/química , Glicoproteínas/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestructura , Modelos Moleculares , Proteína Niemann-Pick C1 , Enfermedades de Niemann-Pick/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Proteínas de Transporte Vesicular , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/ultraestructuraRESUMEN
2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Transducción de Señal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , ADN , Inmunidad InnataRESUMEN
The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1-3. The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors4,5. Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG) repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in the inner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.
Asunto(s)
Transporte Activo de Núcleo Celular , Poro Nuclear , Saccharomyces cerevisiae , Microscopía por Crioelectrón , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Fenilalanina , Glicina , Simulación por Computador , SolventesRESUMEN
Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.
Asunto(s)
Arenavirus del Nuevo Mundo/enzimología , Microscopía por Crioelectrón , Virus Lassa/enzimología , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/ultraestructura , Replicación Viral , Apoenzimas/química , Apoenzimas/metabolismo , Apoenzimas/ultraestructura , Arenavirus del Nuevo Mundo/ultraestructura , Dominio Catalítico , Virus Lassa/ultraestructura , Virus de la Coriomeningitis Linfocítica/enzimología , Virus de la Coriomeningitis Linfocítica/ultraestructura , Modelos Moleculares , Regiones Promotoras Genéticas/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.
Asunto(s)
Bacteriófagos , Proteínas Asociadas a CRISPR , Bacteriófagos/genética , Sistemas CRISPR-Cas , ADN , Proteínas Asociadas a CRISPR/metabolismoRESUMEN
SARS-CoV-2 variants with severe immune evasion are a major challenge for COVID-19 prevention, especially the circulating Omicron XBB/BQ.1.1/BF.7 strains. Thus, the next-generation of broad-spectrum vaccines are urgently needed. Previously, we developed a COVID-19 protein subunit vaccine, ZF2001, based on the RBD-homodimer as the immunogen. To adapt SARS-CoV-2 variants, we developed chimeric RBD-heterodimers to induce broad immune responses. In this study, we further explored the concept of tandem RBD homotrimer and heterotrimer. Prototype SARS-CoV-2 RBD-homotrimer, prototype-Delta-BA.1 (PDO) RBD-heterotrimer and Delta-BA.2-BA.5 (DBA2BA5) RBD-heterotrimer were designed. Biochemical and cryo-EM structural characterization demonstrated total epitope exposure of the RBD-trimers. In mouse experiments, PDO and DBA2BA5 elicited broad SARS-CoV-2 neutralization. Potent protection against SARS-CoV-2 variants was observed in challenge assays and was correlated with neutralizing antibody titer. This study validated the design strategy of tandem RBD-heterotrimers as multivalent immunogens and presented a promising vaccine candidate, DBA2BA5, eliciting broad-spectrum immune responses, including against the circulating XBB/BF.7/BQ.1.1.
Asunto(s)
COVID-19 , Vacunas , Animales , Ratones , SARS-CoV-2/genética , COVID-19/prevención & control , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.
Asunto(s)
Antígenos CD55/metabolismo , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/fisiología , Interacciones Huésped-Patógeno , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Enterovirus Humano B/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Virales/química , Relación Estructura-Actividad , Acoplamiento ViralRESUMEN
Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short-form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Tilacoides/metabolismo , Proteínas de Arabidopsis/metabolismo , Péptido Hidrolasas/metabolismo , Proteostasis , Clorofila A/metabolismo , Cloroplastos/metabolismo , Mutación/genética , Metaloendopeptidasas/metabolismoRESUMEN
The optimal timing of the transition from vegetative growth to reproductive growth is critical for plant reproductive success, and the underlying regulatory mechanisms have been well studied in angiosperm model species, but relatively little in gymnosperms. DAL1, a MADS domain transcription factor (TF) gene that shows a conserved age-related expression profile in conifers, may be an age timer. However, how the DAL1 mediates the onset of reproductive growth remains poorly understood. Here, we have shown that the PtDAL1 directly regulates the PtDAL10 transcription by binding to its promoter region in vitro. PtDAL1, forms ternary complexes in vitro and in N. benthamiana with PtDAL10 and PtMADS11, two potential candidate regulators of the vegetative to reproductive transition in Chinese pine (Pinus tabuliformis). The PtDAL10 was progressively induced in new shoots with age and highly accumulated in male and female cones. Overexpression of PtDAL10 rescued the flowering of ft-10 and soc1-1-2 mutants in Arabidopsis. We provide insight into the molecular components associated with the PtDAL1, which integrates the vegetative to reproductive phase transition into age-mediated progressive development of the whole plant in conifers.
RESUMEN
The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 ß-polypeptides sandwiched between 17 α- and 16 γ-polypeptides. Tight packing of the γ-apoproteins between ß-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th γ-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q P, which adopts a compact, export-ready conformation before passage through the pore and eventual diffusion to the cytochrome bc 1 complex.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Hyphomicrobiaceae/química , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/ultraestructura , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Proteínas Bacterianas/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Benzoquinonas/metabolismo , Sitios de Unión , Complejos de Proteína Captadores de Luz/metabolismo , Magnesio/química , Magnesio/metabolismo , Modelos Moleculares , Fotosíntesis , Conformación Proteica , Estabilidad ProteicaRESUMEN
Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.
Asunto(s)
Proteasas de Ácido Aspártico , Fosfolípidos , Solanum tuberosum , Membrana Celular/metabolismo , Liposomas/química , Fusión de Membrana , Fosfatidilserinas/química , Fosfolípidos/química , Fosfolípidos/metabolismo , Dominios Proteicos , Solanum tuberosum/química , Solanum tuberosum/metabolismoRESUMEN
OBJECTIVE: Acculturative stress, often experienced by members of minority groups, is related to problematic eating behaviors. What remains poorly understood is the function of perceived social support in this link. This study evaluated the moderating role of perceived support from family, the significant other, and friends in the association between acculturative stress and problematic eating behaviors, including emotional eating, uncontrolled eating, and cognitive restraint eating. METHOD: An online cross-sectional study was conducted with 497 Asian and Latinx college students using standardized questionnaires. Three hierarchical multivariable models were analyzed, with each eating behavior included as the outcome variable. Demographic variables (Step 1), general perceived stress (Step 2), and predictor variables (i.e., acculturative stress, perceived support; Step 3) were added using Method Enter. Interaction terms were entered using Method Stepwise (Step 4) to test for moderation effects. RESULTS: As perceived family support increased, the magnitude of the link between acculturative stress and higher uncontrolled eating increased (p = .002). When perceived significant other support was higher, the magnitude of the relationship between acculturative stress and higher emotional eating increased (p = .02). Higher perceived friend support was directly related to reduced uncontrolled eating (p = .002). DISCUSSION: This study highlights the heterogeneous effects of various sources of perceived support on the link between acculturative stress and problematic eating behaviors. Findings imply that problematic eating prevention and intervention programs might benefit from considering not only the acculturative stress experienced by racial/ethnic minority young adults but also the sources of support perceived. PUBLIC SIGNIFICANCE: Perceived support from family and the significant other increased the magnitude of the link between higher acculturative stress and increased problematic eating behaviors, whereas support from friends showed a direct relationship with reduced uncontrolled eating. The results highlight the nuanced functions of various sources of perceived social support and contribute to a deeper understanding of potential moderators in the relationship between acculturative stress and problematic eating behaviors among racial/ethnic minorities.
Asunto(s)
Aculturación , Etnicidad , Conducta Alimentaria , Humanos , Adulto Joven , Estudios Transversales , Etnicidad/psicología , Amigos , Hispánicos o Latinos/psicología , Grupos Minoritarios/psicología , Estrés Psicológico/psicología , Asiático/psicologíaRESUMEN
CRISPR-Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of both AcrIF14 and its complex with the crRNA-guided surveillance (Csy) complex. Our study demonstrates that apart from interacting with the Csy complex to block the hybridization of target DNA to the crRNA, AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA as AcrIF9 does. Further structural studies of the Csy-AcrIF14-dsDNA complex and biochemical studies uncover that the PAM recognition loop of the Cas8f subunit of the Csy complex and electropositive patches within the N-terminal domain of AcrIF14 are essential for the non-sequence-specific dsDNA binding to the Csy-AcrIF14 complex, which is different from the mechanism of AcrIF9. Our findings highlight the prevalence of Acr-induced non-specific DNA binding and shed light on future studies into the mechanisms of such Acr proteins.
Asunto(s)
Sistemas CRISPR-Cas/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Pseudomonas aeruginosa/genética , Bacteriófagos/genética , Bacteriófagos/crecimiento & desarrollo , Proteínas Asociadas a CRISPR/metabolismo , ADN/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Conformación Proteica , Pseudomonas aeruginosa/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
KREMEN1 (KRM1) has been identified as a functional receptor for Coxsackievirus A10 (CV-A10), a causative agent of hand-foot-and-mouth disease (HFMD), which poses a great threat to infants globally. However, the underlying mechanisms for the viral entry process are not well understood. Here we determined the atomic structures of different forms of CV-A10 viral particles and its complex with KRM1 in both neutral and acidic conditions. These structures reveal that KRM1 selectively binds to the mature viral particle above the canyon of the viral protein 1 (VP1) subunit and contacts across two adjacent asymmetry units. The key residues for receptor binding are conserved among most KRM1-dependent enteroviruses, suggesting a uniform mechanism for receptor binding. Moreover, the binding of KRM1 induces the release of pocket factor, a process accelerated under acidic conditions. Further biochemical studies confirmed that receptor binding at acidic pH enabled CV-A10 virion uncoating in vitro. Taken together, these findings provide high-resolution snapshots of CV-A10 entry and identify KRM1 as a two-in-one receptor for enterovirus infection.
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Proteínas de la Cápside , Enterovirus Humano A , Proteínas de la Membrana , Internalización del Virus , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Enterovirus Humano A/química , Enterovirus Humano A/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Virión/química , Virión/metabolismo , Desencapsidación ViralRESUMEN
A safe, biocompatible, and stimuli-responsive cucurbit[7]uril-mediated supramolecular bactericidal nanoparticle was fabricated by encapsulating a highly bioactive carbazole-decorated imidazolium salt (A1, EC50 = 0.647 µg/mL against phytopathogen Xanthomonas oryzae pv oryzae) into the host cucurbit[7]uril (CB[7]), thereby leading to self-assembled topographies from microsheets (A1) to nanospheroidal architectures (A1@CB[7]). The assembly behaviors were elucidated by acquired single-crystal structures, 1H NMR, ITC, and X-ray powder diffraction experiments. Complex A1@CB[7] displayed lower phytotoxicity and could efficiently switch on its potent antibacterial ability via introducing a simple competitor 1-adamantanamine hydrochloride (AD). In vivo antibacterial trials against rice bacterial blight revealed that A1@CB[7] could relieve the disease symptoms after being triggered by AD and provide a workable control efficiency of 42.6% at 100 µg/mL, which was superior to bismerthiazol (33.4%). These materials can provide a viable platform for fabricating diverse stimuli-responsive supramolecular bactericides for managing bacterial infections with improved safety.
Asunto(s)
Infecciones Bacterianas , Nanopartículas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacterias , Preparaciones de Acción Retardada , Compuestos Heterocíclicos con 2 Anillos , Humanos , Imidazolidinas , Compuestos MacrocíclicosRESUMEN
Imidazole alkaloids, a common class of five-membered aromatic heterocyclic compounds, exist widely in plants, animals and marine organisms. Because of imidazole's extensive and excellent biological and pharmacological activities, it has always been a topic of major interest for researchers and has been widely used as an active moiety in search of bioactive molecules. To find more efficient antibacterial compounds, a series of novel imidazole-fragment-decorated 2-(pyrazol-4-yl)-1,3,4-oxadiazoles were designed and synthesized based on our previous works via the active substructure splicing principle, and their bioactivities were systematically evaluated both in vitro and in vivo. The bioassays showed that some of the target compounds displayed excellent in vitro antibacterial activity toward three virulent phytopathogenic bacteria, including Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac) and Pseudomonas syringae pv. actinidiae (Psa), affording the lowest EC50 values of 7.40 (7c), 5.44 (9a) and 12.85 (9a) µg/mL, respectively. Meanwhile, compound 7c possessed good in vivo protective and curative activities to manage rice bacterial leaf blight at 200 µg/mL, with control efficacies of 47.34% and 41.18%, respectively. Furthermore, compound 9a showed commendable in vivo protective and curative activities to manage kiwifruit bacterial canker at 200 µg/mL, with control efficacies of 46.05% and 32.89%, respectively, which were much better than those of the commercial bactericide TC (31.58% and 17.11%, respectively). In addition, the antibacterial mechanism suggested that these new types of title compounds could negatively impact the cell membranes of phytopathogenic bacteria cells and cause the leakage of the intracellular component, thereby leading to the killing of bacteria. All these findings confirm that novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles containing an imidazole fragment are promising lead compounds for discovering new bactericidal agents.
Asunto(s)
Oryza , Xanthomonas , Animales , Antibacterianos/química , Oxadiazoles/farmacología , Oxadiazoles/química , Pruebas de Sensibilidad Microbiana , Enfermedades de las Plantas/microbiología , Oryza/microbiología , Imidazoles/farmacologíaRESUMEN
Background: The National Health Insurance Administration in Taiwan has promoted the heart failure post-acute care (HF-PAC) program as a means to provide proactive integrated care within the optimal treatment timeframe to enhance functional recovery after acute decompensated heart failure (HF). Objectives: The aim of this program was to reduce HF readmission rates, improved medication prescription rates, and improve the quality of life in HF patients. Methods: Patients who had a reduced left ventricular ejection fraction (LVEF) of ≤ 40% were included and followed up for 6 months after discharge. They underwent cardiac rehabilitation and physiological, and nutritional status evaluations. The main clinical outcomes of the HF-PAC program were guideline-directed medical therapy prescription rate and 6-month readmission rate. Results: A total of 122 patients were recruited from June 2018 to December 2020 at a medical center in southern Taiwan. The patients' activities of daily living, nutritional status, quality of life and LVEF were significantly improved during the HF-PAC program. More than 95% of the patients received guideline-directed medical prescriptions at the end of the HF-PAC program. The cardiovascular-related 6-month re-admission rate after the HF-PAC program ended was 27.7%, and it could be predicted by the New York Health Association functional class [hazard ratio (HR) 95% confidence interval (95% CI) = 4.12 (1.36-12.46)], value of the Mini Nutritional Assessment - Short Form [HR (95% CI) = 0.46 (0.31-0.68)] and LVEF [HR (95% CI) = 0.95 (0.91-0.99)]. Conclusions: By incorporating multidisciplinary healthcare teams, the HF-PAC program improves the guideline- directed medical therapy prescription rate, thus improving patients' cardiac function, physical activity recovery, the quality of life, and also reduces their readmission rate.