Nallely Cabrera

Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana.

Abstract.Leishmania mexicana causes a wide spectrum of clinical diseases. In spite of the variety of clinical forms, no data exist regarding genetic polymorphism of L. mexicana. We analyzed the polymorphism of the internal transcribed spacer (ITS) and the small subunit rRNA genes of 3 reference strains and 24 Mexican isolates of L. mexicana, by means of polymerase chain reaction and subsequent digestion by restriction enzymes.All strains of L. mexicana had invariant patterns for both the ITS and the small subunit of rRNA genes. Leishmania amazonensis and Leishmania venezuelensis displayed polymorphism only in the ITS. The high degree of identity of this region was confirmed by sequencing DNA from three L. mexicana isolates. There was almost complete identity of the sequence for the ITS region of L. venezuelensis and that of strains of Leishmania major, suggesting that these species may be more closely related than previously thought.

Perturbation of the Dimer Interface of Triosephosphate Isomerase and its Effect on Trypanosoma cruzi

Background Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite. Methodology/Principal Findings Dithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 Å resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture. Conclusions/Significance By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.

Pyruvate Kinase Revisited THE ACTIVATING EFFECT OF K

For more than 50 years, it has been known that K+ is an essential activator of pyruvate kinase (Kachmar, J. F., and Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683). However, the role of K+ in the catalysis by pyruvate kinase has not been totally understood. Previous studies without K+ showed that the affinity of ADP-Mg2+ depends on the concentration of phosphoenolpyruvate, although the kinetics of the enzyme at saturating K+ concentrations show independence in the binding of substrates (Reynard, A. M., Hass, L. F., Jacobsen, D. D. & Boyer, P. D. (1961) J. Biol. Chem. 236, 2277-2283). Here, we explored the kinetics of the enzyme with and without K+. The results show that without K+, the kinetic mechanism of pyruvate kinase changes from random to ordered with phosphoenol-pyruvate as first substrate. Vmax with K+ was about 400 higher than without K+. In the presence of K+, the affinities for phosphoenol-pyruvate, ADP-Mg2+, oxalate, and ADP-Cr2+ were 2-6-fold higher than in the absence of K+. This as well as fluorescence data also indicate that K+ is involved in the acquisition of the active conformation of the enzyme, allowing either phosphoenolpyruvate or ADP to bind independently (random mechanism). In the absence of K+, ADP cannot bind to the enzyme until phosphoenolpyruvate forms a competent active site (ordered mechanism). We propose that K+ induces the closure of the active site and the arrangement of the residues involved in the binding of the nucleotide.

Massive screening yields novel and selective Trypanosoma cruzi triosephosphate isomerase dimer-interface-irreversible inhibitors with anti-trypanosomal activity

Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), an enzyme in the glycolytic pathway that exhibits high catalytic rates of glyceraldehyde-3-phosphate- and dihydroxyacetone-phosphate-isomerization only in its dimeric form, was screened against an in-house chemical library containing nearly 230 compounds belonging to different chemotypes. After secondary screening, twenty-six compounds from eight different chemotypes were identified as screening positives. Four compounds displayed selectivity for TcTIM over TIM from Homo sapiens and, concomitantly, in vitro activity against T. cruzi.

An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia

Recombinant triosephosphate isomerase from the parasite Giardia lamblia (GlTIM) was characterized and immunolocalized. The enzyme is distributed uniformly throughout the cytoplasm. Size exclusion chromatography of the purified enzyme showed two peaks with molecular weights of 108 and 55 kDa. Under reducing conditions, only the 55‐kDa protein was detected. In denaturing gel electrophoresis without dithiothreitol, the enzyme showed two bands with molecular weights of 28 and 50 kDa; with dithiotretitol, only the 28‐kDa protein was observed. These data indicate that GlTIM may exist as a tetramer or a dimer and that, in the former, the two dimers are covalently linked by disulfide bonds. The kinetics of the dimer were similar to those of other TIMs. The tetramer exhibited half of the kcat of the dimer without changes in the Km. Studies on the thermal stability and the apparent association constants between monomers showed that the tetramer was slightly more stable than the dimer. This finding suggests the oligomerization is not related to enzyme thermostability as in Thermotoga maritima. Instead, it could be that oligomerization is related to the regulation of catalytic activity in different states of the life cycle of this mesophilic parasite. Proteins 2004.

Proteinases of Entamoeba histolytica associated with different subcellular fractions

Crude lysates of Entamoeba histolytica (strain HM 1:IMSS) analyzed by substrate gel electrophoresis in 12.5% acrylamide separating gels with reducing agents showed six hydrolysis zones with apparent molecular weights of 73,000 (high), 45,000, 36,000 (intermediate), 30,000, 26,000 and 23,000 (low molecular weight proteinases). Amebic lysates fractionated using the procedure of Aley et al. or the procedure of Rosenberg and Gitler and analyzed by the same method show all enzymes in the fractions with the soluble components and only the intermediate and low molecular weight proteinases in the fraction containing internal vesicles or membranes and plasma membrane. Some of these proteinases seem to be integral membrane proteins since they resist treatment with high salt, high urea buffer. All fractions are capable of digesting azocasein. Fractionation of amebic lysates by hydrophobic chromatography using phenyl-Sepharose or phase separation of amebic extracts with Triton X-114 show that proteinases with high, intermediate and low molecular weight behave as hydrophilic proteins while only proteinases of intermediate and low molecular weight behave as hydrophobic proteins. These results suggest that some proteinases are segregated in different compartments of the cell.

Different contribution of conserved amino acids to the global properties of triosephosphate isomerases.

It is generally assumed that the amino acids that exist in all homologous enzymes correspond to residues that participate in catalysis, or that are essential for folding and stability. Although this holds for catalytic residues, the function of conserved noncatalytic residues is not clear. It is not known if such residues are of equal importance and have the same role in different homologous enzymes. In humans, the E104D mutation in triosephosphate isomerase (TIM) is the most frequent mutation in the autosomal diseases named “TPI deficiencies.” We explored if the E104D mutation has the same impact in TIMs from four different organisms (Homo sapiens, Giardia lamblia, Trypanosoma cruzi, and T. brucei). The catalytic properties were not significantly affected by the mutation, but it affected the rate and extent of formation of active dimers from unfolded monomers differently. Scanning calorimetry experiments indicated that the mutation was in all cases destabilizing, but the mutation effect on rates of irreversible denaturation and transition‐state energetics were drastically dependent on the TIM background. For instance, the E104D mutation produce changes in activation energy ranging from 430 kJ mol−1 in HsTIM to −78 kJ mol−1 in TcTIM. Thus, in TIM the role of a conserved noncatalytic residue is drastically dependent on its molecular background. Accordingly, it would seem that because each protein has a particular sequence, and a distinctive set of amino acid interactions, it should be regarded as a unique entity that has evolved for function and stability in the organisms to which it belongs. Proteins 2014; 82:323–335.

Identification of Amino Acids that Account for Long-Range Interactions in Two Triosephosphate Isomerases from Pathogenic Trypanosomes

For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus.

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 μmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.