Ernesto R. Caffarena

Toll-like Receptor 1 N248S Single-Nucleotide Polymorphism Is Associated With Leprosy Risk and Regulates Immune Activation During Mycobacterial Infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Synthesis, antimalarial evaluation and molecular modeling studies of hydroxyethylpiperazines, potential aspartyl protease inhibitors, Part 2.

The antimalarial acitivity of hydroxyethylamines, synthesized from the reaction of intermediated hydroxyethypiperazines with benzenesulfonyl chlorides or benzoyl chlorides, has been evaluated in vitro against a W2 Plasmodium falciparum clone. Some of the nineteen tested derivatives showed a significant activity in vitro, thus turning into a promising new class of antimalarials. In addition, a molecular modeling study of the most active derivative (5l) was performed and its most probable binding modes within plasmepsin II enzyme were identified.

Naturally Occurring Genetic Variants of Human Caspase‐1 Differ Considerably in Structure and the Ability to Activate Interleukin‐1β

Caspase‐1 (Interleukin‐1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. Inflammation is often mediated by enzymatic activation of interleukin (IL)‐1β and IL‐18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase‐1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL‐1β releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase‐1 is compatible with normal life and does not prevent moderate and severe autoinflammation.

Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants

BackgroundHuman TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers.ResultsThe solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain.ConclusionsOur results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances.

In silico predicted epitopes from the COOH-terminal extension of cysteine proteinase B inducing distinct immune responses during Leishmania (Leishmania) amazonensis experimental murine infection

BackgroundLeishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant.ResultsPredicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities.ConclusionsBased on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.

New Parameterization Approaches of the LIE Method to Improve Free Energy Calculations of PlmII-Inhibitors Complexes

The standard parameterization of the Linear Interaction Energy (LIE) method has been applied with quite good results to reproduce the experimental absolute binding free energies for several protein–ligand systems. However, we found that this parameterization failed to reproduce the experimental binding free energy of Plasmepsin II (PlmII) in complexes with inhibitors belonging to four dissimilar scaffolds. To overcome this fact, we developed three approaches of LIE, which combine systematic approaches to predict the inhibitor‐specific values of α, β, and γ parameters, to gauge their ability to calculate the absolute binding free energies for these PlmII‐Inhibitor complexes. Specifically: (i) we modified the linear relationship between the weighted nonpolar desolvation ratio (WNDR) and the α parameter, by introducing two models of the β parameter determined by the free energy perturbation (FEP) method in the absence of the constant term γ, and (ii) we developed a new parameterization model to investigate the linear correlation between WNDR and the correction term γ. Using these parameterizations, we were able to reproduce the experimental binding free energy from these systems with mean absolute errors lower than 1.5 kcal/mol.

Structural and Molecular Modeling Features of P2X Receptors

Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.

Epoxy-α-lapachone has in vitro and in vivo anti-Leishmania (Leishmania) amazonensis effects and inhibits serine proteinase activity in this parasite.

ABSTRACT Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm2), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm2) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm2). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compounds effects on the parasite.

Stochastic molecular dynamics of peanut lectin PNA complex with T-antigen disaccharide

The stochastic boundary molecular dynamics simulation method was applied to investigate the structure of a complex comprised of a tetrameric peanut lectin and the tumour-associated disaccharide, Galbeta1-3GalNAc (T-antigen). Only a small region encompassing the active site was explicitly included in the calculations, but the electrical contribution of most outer atoms was taken into account by adding to the effective potential a term coming from an electrostatic potential grid that was pre-calculated and used to approximate the electrostatic energy and the force at any point in the interacting site. Results of simulating the intermolecular hydrogen bond network agree fairly well with X-ray experiments. An estimation of the direct and water-mediated interaction mean lifetimes and mean water residence times around the T-antigen oxygen atoms was computed over 400 ps. Monitoring the behaviour of water molecules within the active site revealed that there is a constant exchange of water with the bulk, especially in the proximity of ASN41, ASN127 and GLU129. The temporal evolution of the glycosidic linkage was also investigated and compared with simulations of T-antigen in solution. These studies of peanut lectins-sugar complexes clearly emphasize the importance of bound water molecules in generating carbohydrate specificity.

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection

Peptides from the COOH‐terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis‐infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 Db:Ig and H2 Ld:Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep‐derived peptides are important factors in the murine host infection by L. (L.) amazonensis. Copyright