Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19.

BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli.

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs. KEY POINTS: • Pre-induction temperature determines inclusion bodies architecture • In pre-induction (above 34 °C), the heat shock response increases recombinant protein production • Inclusion bodies at higher pre-induction temperature show a lower amyloid content.

Energetic and structural effects of the Tanford transition on ligand recognition of bovine ß-lactoglobulin.

Bovine ß-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of ß-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

Integrative overview of antibodies against SARS-CoV-2 and their possible applications in COVID-19 prophylaxis and treatment.

SARS-CoV-2 is a novel ß-coronavirus that caused the COVID-19 pandemic disease, which spread rapidly, infecting more than 134 million people, and killing almost 2.9 million thus far. Based on the urgent need for therapeutic and prophylactic strategies, the identification and characterization of antibodies has been accelerated, since they have been fundamental in treating other viral diseases. Here, we summarized in an integrative manner the present understanding of the immune response and physiopathology caused by SARS-CoV-2, including the activation of the humoral immune response in SARS-CoV-2 infection and therefore, the synthesis of antibodies. Furthermore, we also discussed about the antibodies that can be generated in COVID-19 convalescent sera and their associated clinical studies, including a detailed characterization of a variety of human antibodies and identification of antibodies from other sources, which have powerful neutralizing capacities. Accordingly, the development of effective treatments to mitigate COVID-19 is expected. Finally, we reviewed the challenges faced in producing potential therapeutic antibodies and nanobodies by cell factories at an industrial level while ensuring their quality, efficacy, and safety.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains.

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

Inherent conformational flexibility of F1-ATPase α-subunit.

The core of F1-ATPase consists of three catalytic (ß) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in ß-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3ß3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and ß-subunits show on Mg(II) for recognizing ATP.

Novel "extended sequons" of human N-glycosylation sites improve the precision of qualitative predictions: an alignment-free study of pattern recognition using ProtDCal protein features.

N-Glycosylation is a common post-translational modification that plays an important role in the proper folding and function of many proteins. This modification is largely dependent on the presence of a sequence motif called a "sequon" defined as Asn-Xxx-Ser/Thr. However, evidence has shown that the presence of such a "sequon" is insufficient to determine the occurrence of N-glycosylation with high precision. This study aims to elucidate patterns that can more accurately predict N-glycosylation sites in human proteins. The novel motifs are evaluated using benchmarking data from 188 organisms. Performance is largely sustained compared to the human data, which validates the robustness of the novel extracted "extended sequons". We, therefore, introduce new knowledge about sequence-related factors that control N-glycosylation.

A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.

Isolated noncatalytic and catalytic subunits of F1-ATPase exhibit similar, albeit not identical, energetic strategies for recognizing adenosine nucleotides.

The function of F1-ATPase relies critically on the intrinsic ability of its catalytic and noncatalytic subunits to interact with nucleotides. Therefore, the study of isolated subunits represents an opportunity to dissect elementary energetic contributions that drive the enzyme's rotary mechanism. In this study we have calorimetrically characterized the association of adenosine nucleotides to the isolated noncatalytic α-subunit. The resulting recognition behavior was compared with that previously reported for the isolated catalytic ß-subunit (N.O. Pulido, G. Salcedo, G. Pérez-Hernández, C. José-Núñez, A. Velázquez-Campoy, E. García-Hernández, Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase ß subunit, Biochemistry 49 (2010) 5258-5268). The two subunits exhibit nucleotide-binding thermodynamic signatures similar to each other, characterized by enthalpically-driven affinities in the µM range. Nevertheless, contrary to the catalytic subunit that recognizes MgATP and MgADP with comparable strength, the noncatalytic subunit much prefers the triphosphate nucleotide. Besides, the α-subunit depends more on Mg(II) for stabilizing the interaction with ATP, while both subunits are rather metal-independent for ADP recognition. These binding behaviors are discussed in terms of the properties that the two subunits exhibit in the whole enzyme.

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium.

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

The role of ß2-glycoprotein I (ß2GPI) carbohydrate chains in the reactivity of anti-ß2GPI antibodies from patients with primary antiphospholipid syndrome and in the activation and differentiation of U937 cells.

Several studies have shown that conformational changes of ß(2)-glycoprotein I (ß(2)GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-ß(2)GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of ß(2)GPI in this anti-ß(2)GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-ß(2)GPI antibodies from APS patients with native, partially glycosylated ß(2)GPI (pdß(2)GPI; without sialic acid) and completely deglycosylated ß(2)GPI (cdß(2)GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-ß(2)GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three ß(2)GPI forms were also studied. We found an increased reactivity of anti-ß(2)GPI against pdß(2)GPI and cdß(2)GPI compared to native ß(2)GPI. Both deglycosylated ß(2)GPI isoforms showed higher inhibition of the anti-ß(2)GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/ß(2)GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in ß(2)GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.

Ligand entry into the calyx of ß-lactoglobulin.

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, the detailed process of how a ligand reaches a protein binding site has been an intriguing question over decades. The short time interval between the encounter between a ligand and its receptor to the formation of the stable complex has prevented experimental observations. Bovine ß-lactoglobulin (ßlg) is a lipocalin member that carries fatty acids (FAs) and other lipids in the cellular environment. Βlg accommodates a FA molecule in its highly hydrophobic cavity and exhibits the capability of recognizing a wide variety of hydrophobic ligands. To elucidate the ligand entry process on ßlg, we report molecular dynamics simulations of the encounter between palmitate (PA) or laurate (LA) and ßlg. Our results show that residues localized in loops at the cavity entrance play an important role in the ligand penetration process. Analysis of the short-term interaction energies show that the forces operating on the systems lead to average conformations very close to the crystallographic holo-forms. Whereas the binding free energy analysis using the molecular mechanics Generalized Born surface area method shows that these conformations were thermodynamically favorable.

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli.

BACKGROUND: Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies. RESULTS: Uncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and ß-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented ß-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding. CONCLUSIONS: The manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.

Decoding the mechanism governing the structural stability of wheat germ agglutinin and its isolated domains: A combined calorimetric, NMR, and MD simulation study.

Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.

Ligand binding and self-association cooperativity of ß-lactoglobulin.

Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self-association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine ß-lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled-equilibria revealed that dimerization exerts disparate effects on the ligand-binding capacity of ß-lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of ß-lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution.

Purification and characterization of structural and functional properties of two lectins from a marine sponge Spheciospongia vesparia.

The purification, structural and functional characterization of two different lectins (named Svl-1 and Svl-2) has been reported from the marine sponge Spheciospongia vesparia. Purification procedure includes ammonium sulfate precipitation, combined with chromatography including Octyl-Sepharose-(NH4)SO4 hydrophobic column and DEAE-Toyopearl anion-exchange column using a high performance liquid chromatography. The similarities in function, specificity for saccharides, molecular weight, amino acid content and the N-terminal sequence of two lectins suggest that these proteins are isolectins. Amino acid composition and fluorescence analyses reveal that they contain an intrachain disulfide bridge, which might contribute to their high thermal stability. Furthermore, the purified lectins exhibit antibacterial activity against the gram-negative bacteria Pseudomonas aeruginosa and E. coli, indicating that they may be involved in a recognition strategy and may play a role in the defense response function of the sponge. This is the first report on the isolation of lectins from the S. vesparia. The purified lectins represent a potential possible candidate for future application in the recognition or treatment of cancer cells.

Beta-KTx14.3, a scorpion toxin, blocks the human potassium channel KCNQ1.

Potassium channels play a key role in regulating many physiological processes, thus, alterations in their proper functioning can lead to the development of several diseases. Hence, the search for compounds capable of regulating the activity of these channels constitutes an intense field of investigation. Potassium scorpion toxins are grouped into six subfamilies (α, ß, γ, κ, δ, and λ). However, experimental structures and functional analyses of the long chain ß-KTx subfamily are lacking. In this study, we recombinantly produced the toxins TcoKIK and beta-KTx14.3 present in the venom of Tityus costatus and Lychas mucronatus scorpions, respectively. The 3D structures of these ß-KTx toxins were determined by nuclear magnetic resonance. In both toxins, the N-terminal region is unstructured, while the C-terminal possesses the classic CSα/ß motif. TcoKIK did not show any clear activity against frog Shaker and human KCNQ1 potassium channels; however, beta-KTx14.3 was able to block the KCNQ1 channel. The toxin-channel interaction mode was investigated using molecular dynamics simulations. The results showed that this toxin could form a stable network of polar-to-polar and hydrophobic interactions with KCNQ1, involving key conserved residues in both molecular partners. The discovery and characterization of a toxin capable of inhibiting KCNQ1 pave the way for the future development of novel drugs for the treatment of human diseases caused by the malfunction of this potassium channel. STATEMENT OF SIGNIFICANCE: Scorpion toxins have been shown to rarely block human KCNQ1 channels, which participate in the regulation of cardiac processes. In this study, we obtained recombinant beta-KTx14.3 and TcoKIK toxins and determined their 3D structures by nuclear magnetic resonance. Electrophysiological studies and molecular dynamics models were employed to examine the interactions between these two toxins and the human KCNQ1, which is the major driver channel of cardiac repolarization; beta-KTx14.3 was found to block effectively this channel. Our findings provide insights for the development of novel toxin-based drugs for the treatment of cardiac channelopathies involving KCNQ1-like channels.

[No Hodgkin Linfoma diagnosis with intra-atrial infiltration]. / Diagnóstico de Linfoma no Hodgkin con infiltración intra-auricular.

Cardiac tumors are rare entities in clinical practice, with an incidence of 0.05%. Approximately 75% are benign and 25% malignant. Among these, Lymphomas are uncommon, representing about 0.25%. The non-Hodgkin lymphomas can occur in extranodal tissues in 20% of the cases and 80% of these non-Hodgkin lymphomas are composed of diffuse B cells. The extranodal presentation is most frequent in young adults, with a high degree of malignancy and rapid growth. It can present with primary infiltration of various organs; cardiac involvement occurs in 20 to 28% of cases, usually located in the right chambers and with nonspecific symptoms, depending on the location and extent of the tumor. The diagnostic test in these cases is undoubtedly the biopsy of the lymph node or the affected tissue. We present the case of non-Hodgkin disease of diffuse large cells, with right intra-atrial involvement in a 23-year-old-female patient, who presented with progressive dyspnea. A transesophageal echocardiography was performed and an intra-atrial tumor mass was detected. A biopsy was performed, by femoral venous catheterization, allowing the establishment of the histopathological diagnosis and treatment. At a one year follow up, the patient shows complete remission.

[Successful endovascular treatment of nutcracker's syndrome with self-expanding stent]. / Tratamiento endovascular exitoso del síndrome de cascanueces con stent autoexpandible.

Nutcracker's syndrome is caused by compression of the left renal vein between aorta and superior mesenteric artery, causing extrinsic compression generated functional stenosis. This causes congestion and hypertension of the left renal vein resulting in insufficiency and left gonadal vein varicose, unilateral hematuria and left flank pain, diagnosis is rarely identified by their low frequency and difficulty of suspecting, treatment of nutcracker's syndrome include renal autotransplantation, transposition of superior mesenteric artery revascularization and recently stenting in renal vein, we present the case of a patient, who showed this pathology by diagnostic support by image studies, was performed successfully implant a self-expanding stent with immediate success criteria by angiography, collateral reduction and by disappearance of cava/renal gradient.