César A. Reyes-López

A single amino acid substitution on the surface of a natural hevein isoform (Hev b 6.0202), confers different IgE recognition

Decreased immune reactivity of isoforms of major allergens has been reported. However, such claims have always been based on experiments with recombinant proteins. This work describes the molecular and physicochemical characterization of a hevein (Hev b 6.0201) natural isoform (Hev b 6.0202), which is present in rubber latex from Hevea brasiliensis. The isoallergen has a single substitution Asn14Asp, which gives rise to local differences in the surface potential, as observed from the crystal structure presented here. Besides, ELISA inhibition using serum pools of adult and pediatric patients showed reduced IgE‐binding capacity (∼27%) with the isoallergen. Overall, these results are relevant to delineate crucial residues involved in this dominant discontinuous epitope.

Chemical unfolding of enolase from Saccharomyces cerevisiae exhibits a three-state model.

Enolase is a multifunctional protein that participates in glycolysis and gluconeogenesis and can act as a plasminogen receptor on the cell surface of several organisms, among other functions. Despite its participation in a variety of biological and pathophysiological processes, its stability and folding/unfolding reaction have not been fully explored. In this paper we present, the urea and GdnHCl-induced denaturation of enolase studied by means of fluorescence and circular dichroism spectroscopies. We found that enolase unfolds through a highly reversible pathway, populating a stable intermediate species in a range of experimental conditions. The refolding reaction also exhibits an intermediate state that might have a slightly more compact conformation compared to the unfolding intermediate. The thermodynamic parameters associated with the unfolding reaction are presented and discussed.

Effects of a buried cysteine-to-serine mutation on yeast triosephosphate isomerase structure and stability.

All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132–138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.

The conserved salt bridge linking two C‐terminal β/α units in homodimeric triosephosphate isomerase determines the folding rate of the monomer

Triosephosphate isomerase (TIM), whose structure is archetypal of dimeric (β/α)8 barrels, has a conserved salt bridge (Arg189–Asp225 in yeast TIM) that connects the two C‐terminal β/α segments to rest of the monomer. We constructed the mutant D225Q, and studied its catalysis and stability in comparison with those of the wild‐type enzyme. Replacement of Asp225 by Gln caused minor drops in kcat and KM, but the catalytic efficiency (kcat/KM) was practically unaffected. Temperature‐induced unfolding–refolding of both TIM samples displayed hysteresis cycles, indicative of processes far from equilibrium. Kinetic studies showed that the rate constant for unfolding was about three‐fold larger in the mutant than in wild‐type TIM. However, more drastic changes were found in the kinetics of refolding: upon mutation, the rate‐limiting step changed from a second‐order (at submicromolar concentrations) to a first‐order reaction. These results thus indicate that renaturation of yTIM occurs through a uni–bimolecular mechanism in which refolding of the monomer most likely begins at the C‐terminal half of its polypeptide chain. From the temperature dependence of the refolding rate, we determined the change in heat capacity for the formation of the transition state from unfolded monomers. The value for the D225Q mutant, which is about 40% of the corresponding value for yTIM, would implicate the folding of only three quarters of a monomer chain in the transition state. Proteins 2008.

Thermodynamic and kinetic destabilization of triosephosphate isomerase resulting from the mutation of conserved and non-conserved cysteines.

Several variants of Saccharomyces cerevisiae triosephosphate isomerase (yTIM) were studied to determine how mutations of conserved and non-conserved Cys residues affect the enzyme. Wild-type yTIM has two buried free cysteines: Cys 41 (non-conserved) and the invariant Cys 126. Single-site mutants, containing substitutions of these cysteines with Ala, Val, or Ser (the three most conservative changes for a buried Cys, according to substitution matrices), were examined for stability and enzymatic activity. Neither of the Cys residues was found to be essential for enzyme catalysis. Determination of the global stability of the mutants indicated that, regardless of which Cys was substituted, individual Cys→Ala and Cys→Val mutations, as well as the C41S substitution, all decrease the unfolding free energy of the dimeric protein by less than 23 kJ mol(-1) (at 37 °C, pH 7.4), as compared to the wild-type enzyme. In contrast, a substantially larger destabilization (37 kJ mol(-1)) was found in the C126S mutant. These results suggest that, with the exception of C126S, all of these mutations can be regarded as neutral (i.e., mutations that do not impair the reproductive success of the organism). Accordingly, Cys 126 has remained invariant across evolution because its neutral substitutions by Ala or Val would require a highly unlikely, concerted double mutation at any of the Cys codons. Furthermore, detrimental effects to a cell expressing the C126S TIM mutant more likely arise from the high unfolding rate of this enzyme.

A continuous peptide epitope reacting with pandemic influenza AH1N1 predicted by bioinformatic approaches

Computational identification of potential epitopes with an immunogenic capacity challenges immunological research. Several methods show considerable success, and together with experimental studies, the efficiency of the algorithms to identify potential peptides with biological activity has improved. Herein, an epitope was designed by combining bioinformatics, docking, and molecular dynamics simulations. The hemagglutinin protein of the H1N1 influenza pandemic strain served as a template, owing to the interest of obtaining a scheme of immunization. Afterward, we performed enzyme‐linked immunosorbent assay (ELISA) using the epitope to analyze if any antibodies in human sera before and after the influenza outbreak in 2009 recognize this peptide. Also, a plaque reduction neutralization test induced by virus‐neutralizing antibodies and the IgG determination showed the biological activity of this computationally designed peptide. The results of the ELISAs demonstrated that the serum of both prepandemic and pandemic recognized the epitope. Moreover, the plaque reduction neutralization test evidenced the capacity of the designed peptide to neutralize influenza virus in Madin‐Darby canine cells. Copyright

Role of α-Dystrobrevin in the differentiation process of HL-60 cells

&NA; The &agr;‐Dystrobrevin gene encodes at least five different protein isoforms, expressed in diverse tissues. The &agr;‐Dystrobrevin‐1 isoform (&agr;‐Db‐1) is a member of the cytoplasmic dystrophin‐associated protein complex, which has a C‐terminal extension comprising at least three tyrosine residues susceptible to phosphorylation in vivo. We previously described &agr;‐Db in stem‐progenitor cells and blood neutrophils as playing a scaffolding role and, in association with kinesin and microtubules, &agr;‐Db promotes platelet‐granule trafficking. Additionally, the microtubules must establish a balanced interaction with the lamina A/C network for appropriate nuclear morphology. Considering that the most outstanding feature during neutrophil differentiation is nuclei lobulation, we hypothesized that &agr;‐Db might possess a pivotal function during the neutrophil differentiation process. Western Blot (WB) and confocal microscope assays evidenced a differential pattern expression and a subcellular redistribution of &agr;‐Db in neutrophils derived from HL‐60 cells. At the end of the differentiation process, we detected an important diminution in the expression of tubulin, kinesin, and &agr;‐Db‐1. Knockdown of &agr;‐Db prevented nuclei lobulation, increased Lamin A/C and syne1 expression and augmented the roughness of derived neutrophil membrane and disturbed filopodia assembly. Our results suggest that HL‐60 cells undergo extensive cytoskeletal reorganization including &agr;‐Db in order to possess lobulated nuclei when they further differentiate into neutrophils. Graphical abstract Figure. No Caption available. Highlights&agr;‐Db is downexpressed in HL‐60 cells induced into neutrophils with DMSO.&agr;‐Db‐1 knockdown prevented nuclei lobulation, increased lamin A/C, and syne1 expression.&agr;‐Db‐1 depletion augmented actin‐F and the roughness of derived neutrophil membrane.