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The conformational change in STIM1 that communicates sensing of ER calcium-store depletion from the STIM ER-luminal domain to the STIM cytoplasmic region and ultimately to ORAI channels in the plasma membrane is broadly understood. However, the structural basis for the STIM luminal-domain dimerization that drives the conformational change has proven elusive. A recently published study has approached this question via molecular dynamics simulations. The report pinpoints STIM residues that may be part of a luminal-domain dimerization interface, and provides unexpected insight into how torsional movements of the STIM luminal domains might trigger release of the cytoplasmic SOAR/CAD domain from its resting tethers to the STIM CC1 segments.
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The Ebola delta peptide is an amphipathic, 40-residue peptide encoded by the Ebola virus, referred to as E40. The membrane-permeabilising activity of the E40 delta peptide has been demonstrated in cells and lipid vesicles suggesting the E40 delta peptide likely acts as a viroporin. The lytic activity of the peptide increases in the presence of anionic lipids and a disulphide bond in the C-terminal part of the peptide. Previous in silico work predicts the peptide to show a partially helical structure, but there is no experimental information on the structure of E40. Here, we use circular dichroism spectroscopy to report the secondary structure propensities of the reduced and oxidised forms of the E40 peptide in water, detergent micelles, and lipid vesicles composed of neutral and anionic lipids (POPC and POPG, respectively). Results indicate that the peptide is predominately a random coil in solution, and the disulphide bond has a small but measurable effect on peptide conformation. Secondary structure analysis shows large uncertainties and dependence on the reference data set and, in our system, cannot be used to accurately determine the secondary structure motifs of the peptide in membrane environments. Nevertheless, the spectra can be used to assess the relative changes in secondary structure propensities of the peptide depending on the solvent environment and disulphide bond. In POPC-POPG vesicles, the peptide transitions from a random coil towards a more structured conformation, which is even more pronounced in negatively charged SDS micelles. In vesicles, the effect depends on the peptide-lipid ratio, likely resulting from vesicle surface saturation. Further experiments with zwitterionic POPC vesicles and DPC micelles show that both curvature and negatively charged lipids can induce a change in conformation, with the two effects being cumulative. Electrostatic screening from Na+ ions reduced this effect. The oxidised form of the peptide shows a slightly lower propensity for secondary structure and retains a more random coil conformation even in the presence of PG-PC vesicles.
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The binding mechanism of molecular interaction between bicalutamide and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using various spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the fluorescence quenching of HSA by bicalutamide was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The thermodynamic parameters, ΔH and ΔS, were calculated to be 4.30 × 104 J·mol-1 and 245 J·mol-1·K-1, respectively, suggesting that the binding of bicalutamide to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies indicated neither Sudlow's site I nor II but subdomain IB as the main binding site for bicalutamide on HSA. The binding distance between bicalutamide and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of circular dichroism, synchronous, and 3D fluorescence spectra demonstrated that HSA conformation was slightly altered in the presence of bicalutamide.
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Anilidas , Nitrilos , Albúmina Sérica Humana , Espectrometría de Fluorescencia , Termodinámica , Compuestos de Tosilo , Compuestos de Tosilo/química , Anilidas/química , Anilidas/metabolismo , Nitrilos/química , Nitrilos/metabolismo , Humanos , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Dicroismo Circular , Sitios de Unión , Modelos Moleculares , Interacciones Hidrofóbicas e Hidrofílicas , Enlace de HidrógenoRESUMEN
Developing new methods to control the size and shape of the helical structures adopted by foldamers is highly important as the secondary structure displayed by these supramolecular scaffolds often dictates their activity and function. Herein, we report on a systematic study demonstrating that the helical pitch of ortho-azobenzene/2,6-pyridyldicarboamide foldamers can be readily controlled through the nature of the terminal functionality. Remarkably, simply through varying the end group of the foldamer, and without modifying any other structural features of the scaffold, the helical pitch can be over doubled in magnitude (from 3.4 Å to 7.3 Å). Additionally, crystallographic analysis of a library ten foldamers has identified general trends in the influence of a range of terminal functionalities, including carboxylbenzyl (Cbz), diphenylcarbamyl (N(Ph)2), ferrocene (Fc) and tert-butyloxycarbonyl (Boc), in controlling the folding behaviour of these supramolecular scaffolds. These studies could prove useful in the future development of functional foldamers which adopt specific sizes and shapes.
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Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~47.4 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.
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BACKGROUND: In vivo measurement of limb stiffness and conformation provides a non-invasive proxy assessment of superficial digital flexor tendon (SDFT) and suspensory ligament (SL) function. Here, we compared it in fore and hindlimbs and after injury. OBJECTIVES: To compare the limb stiffness and conformation in forelimbs and hindlimbs, changes with age, and following injury to the SDFT and SL. STUDY DESIGN: Retrospective cohort study. METHODS: Limb stiffness was calculated using floor scales and an electrogoniometer taped to the dorsal fetlock. The fetlock angle and weight were simultaneously recorded five times with the limb weight-bearing and when the opposite limb was picked up (increased load). Limb stiffness of both limbs was calculated from the gradient of the regression line of angle versus load. Fetlock angle when the weight was zero was extrapolated from the graph and used as a measure of conformation. Limb stiffness was measured in uninjured forelimbs (n = 42 limbs), hindlimbs (n = 19 limbs), forelimbs with SDFT injury (n = 18) and hindlimbs with SL injury (n = 5). RESULTS: Limb stiffness correlated with weight in forelimbs as shown previously (p < 0.001) but also in hindlimbs (p = 0.006). When normalised to the horse's weight (503 kg, IQR 471.5-560), forelimb stiffness was significantly higher (22.3 [±4.5] × 10-3 degree-1) than for the hindlimb (16.4 [±4.0] × 10-3 degree-1; p < 0.001). While there were no significant differences between forelimb and hindlimb conformation in unaffected or SDFT injury, both limb stiffness and conformation was significantly greater in limbs with SL injury (p = 0.009 and p = 0.002, respectively). MAIN LIMITATIONS: Small sample size, lack of clinical data including lameness and quantification of injuries. CONCLUSIONS: Injury to the forelimb SDFT does not alter limb stiffness or conformation in the long-term, while hindlimb SL injury simultaneously increases limb stiffness and fetlock angle, suggesting an increase in SL length following injury.
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Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.
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AIMS/HYPOTHESIS: Genome-wide association studies (GWAS) have identified hundreds of type 2 diabetes loci, with the vast majority of signals located in non-coding regions; as a consequence, it remains largely unclear which 'effector' genes these variants influence. Determining these effector genes has been hampered by the relatively challenging cellular settings in which they are hypothesised to confer their effects. METHODS: To implicate such effector genes, we elected to generate and integrate high-resolution promoter-focused Capture-C, assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq datasets to characterise chromatin and expression profiles in multiple cell lines relevant to type 2 diabetes for subsequent functional follow-up analyses: EndoC-BH1 (pancreatic beta cell), HepG2 (hepatocyte) and Simpson-Golabi-Behmel syndrome (SGBS; adipocyte). RESULTS: The subsequent variant-to-gene analysis implicated 810 candidate effector genes at 370 type 2 diabetes risk loci. Using partitioned linkage disequilibrium score regression, we observed enrichment for type 2 diabetes and fasting glucose GWAS loci in promoter-connected putative cis-regulatory elements in EndoC-BH1 cells as well as fasting insulin GWAS loci in SGBS cells. Moreover, as a proof of principle, when we knocked down expression of the SMCO4 gene in EndoC-BH1 cells, we observed a statistically significant increase in insulin secretion. CONCLUSIONS/INTERPRETATION: These results provide a resource for comparing tissue-specific data in tractable cellular models as opposed to relatively challenging primary cell settings. DATA AVAILABILITY: Raw and processed next-generation sequencing data for EndoC-BH1, HepG2, SGBS_undiff and SGBS_diff cells are deposited in GEO under the Superseries accession GSE262484. Promoter-focused Capture-C data are deposited under accession GSE262496. Hi-C data are deposited under accession GSE262481. Bulk ATAC-seq data are deposited under accession GSE262479. Bulk RNA-seq data are deposited under accession GSE262480.
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The production conditions of exopolysaccharide (EPS) from Leuconostoc mesenteroides XR1 were optimized by response surface methodology (RSM). Maximum EPS yield was 56.59 ± 0.51 g/L under fermentation conditions with 2.6 g/L ammonium citrate, initial pH 6.5 and temperature 23 °C, which was 6.21-fold greater than the EPS yield before optimization. Characterization of the chain conformation using Congo red test and circular dichroism (CD) showed that EPS exhibited a random coil structure in aqueous solution. The CD results revealed that the EPS concentration altered its hydrogen-bond interactions and chirality, but did not change its chain conformation. The average polydispersity index (PDI) of the EPS solution was only 27.16 %, indicating that it was uniformly distributed in the aqueous solution with high stability. The degradation temperature of EPS was 253.11 °C, indicating high thermal stability. EPS possessed the ability to scavenge activities of free radicals and was protective against oxidative stress-induced plasmid DNA damage. In addition, stable hydrogels could be formed at EPS concentrations above 5 % (w/v). These results collectively showed that EPS can be used commercially as an antioxidant and drug delivery carrier.
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Smart materials enabling emission intensity or wavelength tuning by light stimulus attract attention in numerous cutting-edge fields. However, due to the generally dense molecular stacking against photoresponsivity in solid states, especially in crystals, developing rapidly phototunable solid-state luminescent systems remains challenging. Herein, we propose a new luminophore that serves as both a photoresponsive unit and a luminescent group, while possessing enhanced conformational freedom to provide a solution. Namely, photoexcitation-induced molecular conformational change of an ionized persulfurated arene based on weak intermolecular aliphatic C-H···π interaction was employed. On these basis, rapidly enhanced phosphorescence upon irradiation can be observed in a series of phase states, like solution state, crystal, and amorphous state, especially with a high photoresponsive rate of 0.033 s-1 in crystal state that is superior to the relevant reported cases. Moreover, a rapidly phototunable afterglow effect is achieved by extending this molecule into some polymer-based doping systems, endowing the system with unique dynamic imaging and fast photopatterning capabilities. Such a single-luminophore molecular engineering and the underlying mechanism have implications for building different condensed functional materials, principally for those with stimuli responses in solid states.
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Despite recent advancement in 3D molecule conformation generation driven by diffusion models, its high computational cost in iterative diffusion/denoising process limits its application. Here, an equivariant consistency model (EC-Conf) was proposed as a fast diffusion method for low-energy conformation generation. In EC-Conf, a modified SE (3)-equivariant transformer model was directly used to encode the Cartesian molecular conformations and a highly efficient consistency diffusion process was carried out to generate molecular conformations. It was demonstrated that, with only one sampling step, it can already achieve comparable quality to other diffusion-based models running with thousands denoising steps. Its performance can be further improved with a few more sampling iterations. The performance of EC-Conf is evaluated on both GEOM-QM9 and GEOM-Drugs sets. Our results demonstrate that the efficiency of EC-Conf for learning the distribution of low energy molecular conformation is at least two magnitudes higher than current SOTA diffusion models and could potentially become a useful tool for conformation generation and sampling. SCIENTIFIC CONTRIBUTIONS: In this work, we proposed an equivariant consistency model that significantly improves the efficiency of conformation generation in diffusion-based models while maintaining high structural quality. This method serves as a general framework and can be further extended to more complex structure generation and prediction tasks, including those involving proteins, in future steps.
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We report on dual, light-responsive and redox-active foldamers that demonstrate reversible and robust stimuli-induced behaviour. Herein, UV/Vis, 1H NMR and circular dichroism (CD) spectroscopy and cyclic voltammetry have been used to establish the reversibility and highly robust nature of the light- and redox-driven behaviour of these new foldamers with minimal levels of fatigue observed even upon multiple cyclic treatments with irradiative/non-irradiative and oxidative/reductive conditions. This proof-of-concept work paves the way towards the creation of novel stimuli-responsive foldamers of increasing sophistication capable of demonstrating reversible and robust responses to multiple distinct stimuli.
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The formation of amyloid fibrils is a common feature of many protein systems. It has implications in both health, as amyloid fibrils are implicated in over 30 degenerative diseases, and in the biological functions of proteins. Surfaces have long been known to affect the formation of fibrils but the specific effect depends on the details of both the surface and protein. Fully understanding the role of surfaces in fibrillization requires microscopic information on protein conformation on surfaces. In this paper replica exchange molecular dynamics simulation is used to investigate the model fibril forming protein, Aß(10-40) (a 31-residue segment of the amyloid-beta protein) on surfaces of different hydrophobicity. Similar to other proteins Aß(10-40) is found to adsorb strongly onto hydrophobic surfaces. It also adopts significantly different sets of conformations on hydrophobic and polar surfaces, as well as in bulk solution. On hydrophobic surfaces, it adopts partially helical structures, with the helices overlapping with beta-strand regions in the mature fibril. These may be helical intermediates on the fibril formation pathway, suggesting a mechanism for the enhanced fibril formation seen on hydrophobic surfaces.
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Péptidos beta-Amiloides , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Propiedades de Superficie , Péptidos beta-Amiloides/química , Adsorción , Conformación Proteica , Amiloide/química , Humanos , Fragmentos de Péptidos/químicaRESUMEN
The amyloid ß (Aß) peptide has a central role in Alzheimer's disease (AD) pathology. The peptide length can vary between 37 and 49 amino acids, with Aß1-42 being considered the most disease-related length. However, Aß1-40 is also found in Aß plaques and has shown to form intertwined fibrils with Aß1-42. The peptides have previously also shown to form different fibril conformations, proposed to be related to disease phenotype. To conduct more representative in vitro experiments, it is vital to uncover the impact of different fibril conformations on neurons. Hence, we fibrillized different Aß1-40:42 ratios in concentrations of 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100 for either 24 h (early fibrils) or 7 days (aged fibrils). These were then characterized based on fibril width, LCO-staining and antibody-staining. We further challenged differentiated neuronal-like SH-SY5Y human cells with the different fibrils and measured Aß content, cytotoxicity and autophagy function at three different time-points: 3, 24, and 72 h. Our results revealed that both Aß1-40:42 ratio and fibril maturation affect conformation of fibrils. We further show the impact of these conformation changes on the affinity to commonly used Aß antibodies, primarily affecting Aß1-40 rich aggregates. In addition, we demonstrate uptake of the aggregates by neuronally differentiated human cells, where aggregates with higher Aß1-42 ratios generally caused higher cellular levels of Aß. These differences in Aß abundance did not cause changes in cytotoxicity nor in autophagy activation. Our results show the importance to consider conformational differences of Aß fibrils, as this can have fundamental impact on Aß antibody detection. Overall, these insights underline the need for further exploration of the impact of conformationally different fibrils and the need to reliably produce disease relevant Aß aggregates.
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Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.
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Conformación Proteica , Humanos , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Quinasas/química , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Línea Celular TumoralRESUMEN
Insect transient receptor potential vanilloid (TRPV) channels are critical targets for insecticides. In this study, various scaffold-hopping strategies were employed in the rational design of pyridylhydrazono derivatives as potential insect TRPV channels modulators. Insecticidal bioassay demonstrated that the initial target compounds exhibited lower insecticidal activity compared to pymetrozine, with the optimal compound B3 exhibiting a mortality rate of 53.3% against Aphis craccivora at 400 mg·L-1. Conformation analysis indicated that the high energy barrier required for the transition from the lowest-energy conformation to the active conformation may be a key factor contributing to the reduced insecticidal activities of the target compounds. Further structural optimizations aimed at reducing this energy barrier through binding mode-based conformation regulation led to the identification of optimal target 4-(3'-pyridylhydrazono)pyrazol-5-one derivatives C1 and C2. These compounds exhibited reduced transition energy barriers and improved insecticidal activity, with moderate mortality rate of 66.3% and 75.7% against A. craccivora at 400 mg·L-1, respectively. These findings provide valuable insights for future research on the discovery of insect TRPV modulators and have significant implications for the development of more effective agricultural insecticides.
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Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here we investigated whether recently proposed 'open' and 'closed' conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the 'closed' and 'open' conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of wildtype (WT) protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution dSTORM microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of 'closed' CD53 was higher and of 'open' CD81 lower than respective WT protein. In addition, knock-out cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, 'closed' CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19, and 'closed' CD81 interacted less with CD19 than WT CD81, but 'open' CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.
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Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Inmunidad Celular , Inmunidad Humoral , Vacunas Antirrábicas , Virus de la Rabia , Rabia , Vacunas de Partículas Similares a Virus , Animales , Vacunas Antirrábicas/inmunología , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/genética , Rabia/prevención & control , Rabia/inmunología , Virus de la Rabia/inmunología , Virus de la Rabia/genética , Ratones , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Femenino , Vacunas de ARNm/inmunología , Ratones Endogámicos BALB C , Nucleósidos/inmunología , Glicoproteínas/inmunología , Glicoproteínas/genética , Humanos , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/genética , Antígenos Virales/inmunología , Antígenos Virales/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , ARN Mensajero/genética , ARN Mensajero/inmunologíaRESUMEN
This study investigated the impact of high-intensity ultrasound (HIU) treatment on the physiochemical, conformational, and immunomodulatory activity of the OVT-CA complex, emphasizing the structure-function relationship. HIU treatment reduced particle size, improved dispersion, and increased electronegativity of the complex. It facilitated binding between OVT and CA, achieving a maximum degree of 45.22 mg/g CA grafting and reducing interaction time from 2 h to 15 min. HIU-induced cavitation and shear promoted the exposure of -SH and unfolding of OVT, leading to increased surface hydrophobicity of the complex and transformation of its structure from ß-sheet to α-helix. Additionally, CA binds to OVT in the C-lobe region, and HIU treatment modulates the intermolecular forces governing the complex formation, particularly by reinforcing hydrogen bonding, hydrophobic interactions, and introducing electrostatic interactions. Furthermore, HIU treatment increased the immunomodulatory activity of the complex, which was attributed to complex structural changes facilitating enhanced cell membrane affinity, antigen recognition, and B-cell epitope availability. Hierarchical cluster and Pearson correlation analysis confirmed that HIU treatment duration had a greater impact than power on both the structure and activity of the complex, and an optimal HIU treatment duration within 30 min was found to be crucial for activity enhancement. Moreover, structural changes, including ζ-potential, particle size/turbidity, and surface hydrophobicity, were closely correlated with immunomodulatory activity. This study highlights the potential application of HIU in developing protein-polyphenol immunomodulatory agents for public health and food nutrition.
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Serine ß-lactamases inactivate ß-lactam antibiotics in a two-step mechanism comprising acylation and deacylation. For the deacylation step, a water molecule is activated by a conserved glutamate residue to release the adduct from the enzyme. The third-generation cephalosporin ceftazidime is a poor substrate for the class A ß-lactamase BlaC from Mycobacterium tuberculosis but it can be hydrolyzed faster when the active site pocket is enlarged, as was reported for mutant BlaC P167S. The conformational change in the Ω-loop of the P167S mutant displaces the conserved glutamate (Glu166), suggesting it is not required for deacylation of the ceftazidime adduct. Here, we report the characterization of wild type BlaC and BlaC E166A at various pH values. The presence of Glu166 strongly enhances activity against nitrocefin but not ceftazidime, indicating it is indeed not required for deacylation of the adduct of the latter substrate. At high pH wild type BlaC was found to exist in two states, one of which converts ceftazidime much faster, resembling the open state previously reported for the BlaC mutant P167S. The pH-dependent switch between the closed and open states is caused by the loss at high pH of a low-barrier hydrogen bond, a proton shared between Asp172 and Asp179. These results illustrate how readily shifts in substrate specificity can occur as a consequence of subtle changes in protein structure.