RESUMO
The Bovine Leukemia Virus (BLV) Envelope (Env) glycoprotein complex is instrumental in viral infectivity and shapes the host's immune response. This study presents the production and characterization of a soluble furin-mutated BLV Env ectodomain (sBLV-EnvFm) expressed in a stable S2 insect cell line. We purified a 63 kDa soluble protein, corresponding to the monomeric sBLV-EnvFm, which predominantly presented oligomannose and paucimannose N-glycans, with a high content of core fucose structures. Our results demonstrate that our recombinant protein can be recognized from specific antibodies in BLV infected cattle, suggesting its potential as a powerful diagnostic tool. Moreover, the robust humoral immune response it elicited in mice shows its potential contribution to the development of subunit-based vaccines against BLV.
Assuntos
Anticorpos Antivirais , Vírus da Leucemia Bovina , Proteínas Recombinantes , Proteínas do Envelope Viral , Animais , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/imunologia , Bovinos , Proteínas Recombinantes/genética , Camundongos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Anticorpos Antivirais/imunologia , Leucose Enzoótica Bovina/virologia , Linhagem Celular , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene env/imunologiaRESUMO
Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1ß, IL-6, IL-12p40, IFN-ß) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.
Assuntos
Echinococcus granulosus , Humanos , Animais , Camundongos , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Ativação de Macrófagos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Macrófagos , Citocinas/metabolismoRESUMO
The COVID-19 pandemic evolves constantly, requiring adaptable solutions to combat emerging SARS-CoV-2 variants. To address this, we created a pentameric scaffold based on a mammalian protein, which can be customized with up to 10 protein binding modules. This molecular scaffold spans roughly 20 nm and can simultaneously neutralize SARS-CoV-2 Spike proteins from one or multiple viral particles. Using only two different modules targeting the Spike's RBD domain, this construct outcompetes human antibodies from vaccinated individuals' serum and blocks in vitro cell attachment and pseudotyped virus entry. Additionally, the multibodies inhibit viral replication at low picomolar concentrations, regardless of the variant. This customizable multibody can be easily produced in procaryote systems, providing a new avenue for therapeutic development and detection devices, and contributing to preparedness against rapidly evolving pathogens.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Pandemias , Junções Célula-Matriz , MamíferosRESUMO
The interaction between the receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 and the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) allows the first specific contact at the virus-cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost-effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep-tag with yields >40 mg L-1 in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep-tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep-Tactin-HRP as a proof-of-concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in-house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Animais , Humanos , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Drosophila/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial ß-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or ß-oxidation activity.
Assuntos
Proteínas de Bactérias , Proteína Mitocondrial Trifuncional , Mycobacterium tuberculosis , Humanos , Metabolismo dos Lipídeos , Filogenia , Ubiquitinas , Proteína Mitocondrial Trifuncional/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase's ATP-binding domain and the regulator's receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.
Assuntos
Bacillus subtilis , Transdução de Sinais , Histidina Quinase/metabolismo , Bacillus subtilis/genética , Fosforilação , Monoéster Fosfórico Hidrolases , Proteínas de Bactérias/metabolismoRESUMO
The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.
Assuntos
Ácido Aspártico Proteases , Vírus da Leucemia Bovina , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Protease de HIV/metabolismo , Cinética , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
BACKGROUND: Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a successful strategy for prevent severe infections. CoronaVac and BNT162b2 are the most used vaccines worldwide, but their use in heterologous vaccination schedules is still subjected to evaluation. METHODS: Fifty healthy individuals who received heterologous prime-boost vaccination with CoronaVac and BNT162b2 were enrolled in a post-vaccination serological follow-up longitudinal prospective study. We evaluated specific serum anti-receptor binding domain (RBD) IgG antibody levels, and their capacity to block RBD-ACE2 interaction with a surrogate neutralization assay. In 20 participants, we assessed antibody binding kinetics by surface plasmon resonance, and Fc-mediated functions by ADCC and ADCP reporter assays. RESULTS: Our baseline seronegative cohort, displayed seroconversion after two doses of CoronaVac and an important decrease in serum anti-RBD IgG antibodies levels 80 days post-second dose. These levels increased significantly early after the third dose with BNT162b2, but 73 days after the booster we found a new fall. Immunoglobulin functionalities showed a similar behavior. CONCLUSIONS: The heterologous prime-boost vaccination with CoronaVac and BNT162b2 generated an impressive increase in serum anti-RBD specific antibody levels followed by a drop. Nevertheless, these titers remained well above those found in individuals only vaccinated with CoronaVac in the same elapsed time. Serum IgG levels showed high correlation with antibody binding analysis, their capacity to block RBD-ACE2 interaction, and Fc-effectors mechanisms. Our work sheds light on the humoral immune response to heterologous vaccination with CoronaVac and BNT162b2, to define a post-vaccination correlate of protection against SARS-CoV-2 infection and to discuss the scheduling of future vaccine boosters in general population.
Assuntos
COVID-19 , Vacinas Virais , Enzima de Conversão de Angiotensina 2 , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade Humoral , Imunoglobulina G , Estudos Prospectivos , SARS-CoV-2 , VacinaçãoRESUMO
Many recombinant proteins are products of great value in biomedical and industrial fields. The use of solubility and affinity tags are commonly used to increase yields and facilitate the purification process. However, it is of paramount importance in several applications to remove the fusion tag from the final product. In this regard, the Tobacco Etch Virus protease (TEV) is one of the most widely used for tag removal. The presence in the TEV of the same tag to be removed facilitates the separation of TEV and the tag from the cleaved recombinant protein in a single purification step. We generated a double-tagged (StrepTagII and HisTag) TEV variant with reported mutations that improve the activity, the expression yield in E.coli, and that decrease the auto-proteolysis. This TEV can be easily purified by two consecutive affinity chromatography steps with high yields and purity. The cleavage reaction can be done to almost completeness in as fast as 15 min at room temperature and the removal of the protease and tags is performed in a single purification step, independent of the previous presence of a StrepTagII or a HisTag on the target.
Assuntos
Endopeptidases , Escherichia coli , Proteínas Recombinantes de Fusão , Endopeptidases/biossíntese , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.