Antileishmanial activity, cytotoxicity and cellular response of amphotericin B in combination with crotamine derived from Crotalus durissus terrificus venom using in vitro and in silico approaches.

OBJECTIVE: To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis. METHODS: This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated. RESULTS: CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis, decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile. CONCLUSION: Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect.

Molecular Dynamics of CYFIP2 Protein and Its R87C Variant Related to Early Infantile Epileptic Encephalopathy.

The CYFIP2 protein (cytoplasmic FMR1-interacting protein 2) is part of the WAVE regulatory complex (WRC). CYFIP2 was recently correlated to neurological disorders by the association of the R87C variant with early infantile epileptic encephalopathy (EIEE) patients. In this set of syndromes, the epileptic spasms and seizures since early childhood lead to impaired neurological development in children. Inside the WRC, the variant residue is at the CYFIP2 and WAVE1 protein interface. Thus, the hypothesis is that the R87C modification weakens this interaction, allowing the WRC complex's constant activation. This work aimed to investigate the impacts of the mutation on the structure of the WRC complex through molecular dynamics simulation. For that, we constructed WRC models containing WAVE1-NCKAP1 proteins complexed with WT or R87C CYFIP2. Our simulations showed a flexibilization of the loop comprising residues 80-110 due to the loss of contacts between internal residues in the R87C CYFIP2 as well as the key role of residues R/C87, E624, and E689 in structural modification. These data could explain the mechanism by which the mutation impairs the stability and proper regulation of the WRC.

Enhanced xyloglucan-specific endo-ß-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.

Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

Engineering bifunctional laccase-xylanase chimeras for improved catalytic performance.

Two bifunctional enzymes exhibiting combined xylanase and laccase activities were designed, constructed, and characterized by biochemical and biophysical methods. The Bacillus subtilis cotA and xynA genes were used as templates for gene fusion, and the xynA coding sequence was inserted into a surface loop of the cotA. A second chimera was built replacing the wild-type xynA gene by a thermostable variant (xynAG3) previously obtained by in vitro molecular evolution. Kinetic measurements demonstrated that the pH and temperature optima of the catalytic domains in the chimeras were altered by less than 0.5 pH units and 5 °C, respectively, when compared with the parental enzymes. In contrast, the catalytic efficiency (k(cat)/K(m)) of the laccase activity in both chimeras was 2-fold higher than for the parental laccase. Molecular dynamics simulations of the CotA-XynA chimera indicated that the two domains are in close contact, which was confirmed by the low resolution structure obtained by small angle x-ray scattering. The simulation also indicates that the formation of the inter-domain interface causes the dislocation of the loop comprising residues Leu-558 to Lys-573 in the laccase domain, resulting in a more accessible active site and exposing the type I Cu(2+) ion to the solvent. These structural changes are consistent with the results from UV-visible electronic and EPR spectroscopy experiments of the type I copper between the native and chimeric enzymes and are likely to contribute to the observed increase in catalytic turnover number.

Study of the interaction of human defensins with cell membrane models: relationships between structure and biological activity.

The HNP-1, HNP-2, and HNP-3 defensins are human antimicrobial peptides produced in response to microbial invasion. Their properties are distinct, with a more potent action for HNP-3. In this study, the relationship between their structural dissimilarities and their different microbial actions was evaluated by molecular dynamics simulation. Structural determinants related to their intra- and intermolecular interactions were defined for each HNP using a simplified membrane model consisting of a water/n-hexane interface. The hydrophobic portion of the HNPs promotes their diffusion to the interface with a concomitant, slight change in the structure induced by the intermolecular electrostatic interactions between the HPN molecules and the interface. As a consequence, different orientations are probably adopted by the HNPs at the interface, which may explain their different actions. The interaction of HNP-1 and HNP-2 with the surfaces was also studied using Langmuir monolayers as a biomimetic system. It was found that peptides adsorb rapidly at n-hexane/water interfaces as well as at phospholipid Langmuir monolayers but not at the air/liquid interface. This reveals that the presence of an organic phase is required for the exposure of the hydrophobic groups of the peptides. In addition, adsorption kinetics and surface pressure-area isotherms for Langmuir monolayers suggested that the lipid-peptide interaction is strongly influenced by the monolayer electrical charge and packing, depending also on the HPN structure. This study supports a model in which defensins, acting in a dimeric form, are able to disrupt membranes. The model also shows that the individual structures of the HNPs are responsible for their different actions on microbes.