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Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ~20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold. [ABSTRACT FROM AUTHOR]

Dendrotoxins are a group of peptide toxins purified from the venom of several mamba snakes. α-Dendrotoxin (α-DTx, from the Eastern green mamba Dendroaspis angusticeps ) is a well-known blocker of voltage-gated K + channels and specifically of K v 1.1, K v 1.2 and K v 1.6. In this work we show that α-DTx inhibited the ASIC currents in DRG neurons (IC 50 = 0.8 μM) when continuously perfused during 25 s (including a 5 s pulse to pH 6.1), but not when co-applied with the pH drop. Additionally, we show that α-DTx abolished a transient component of the outward current that, in some experiments, appeared immediately after the end of the acid pulse. Our data indicate that α-DTx inhibits ASICs in the high nM range while some K v are inhibited in the low nM range. The α-DTx selectivity and its potential interaction with ASICs should be taken in consideration when DTx is used in the high nM range. [ABSTRACT FROM AUTHOR]

• CoronaVac-BNT162b2 scheme shows significant increase in IgG levels after booster. • IgG levels drop ten weeks after booster but remain higher than those after priming. • Specific IgG levels correlate with neutralizing activity and Fc-mediated functions. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives. [ABSTRACT FROM AUTHOR]

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. • An engineered version of a TEV protease with several modifications for increasing its soluble expression and activity was produced. • The engineered variant contains two affinity tags that improves its purification. • The presence of the two affinity tags increase its versatillity for downstream applications. [ABSTRACT FROM AUTHOR]

• We analyzed effects on macrophages of Kunitz inhibitors secreted by E. granulosus. • All effects observed are compatible with roles in immune evasion. • The close paralogs Eg KU-1 and Eg KU-4 had both common and differential effects. • Eg KU-4 inhibited M-CSF-driven macrophage proliferation much more potently than Eg KU-1. • We propose that inhibition of cation channels participates in the effects observed. The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (K v) that are close paralogs. Both inhibitors, Eg KU-1 and Eg KU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, Eg KU-4 but not Eg KU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by Eg KU-3, a family member that is a protease inhibitor without known activity on cation channels. Eg KU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas Eg KU-1 displayed similar activity but with much lower potency, similar to Eg KU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in Eg KU-4 but not in Eg KU-1, that may explain the differential activities of the two close paralogs. [ABSTRACT FROM AUTHOR]