Perturbation of the dimer interface of triosephosphate isomerase and its effect on trypanosoma cruzi

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

Multi-anti-parasitic activity of arylidene ketones and thiazolidene hydrazines against trypanosoma cruzi and Leishmania spp.

A series of fifty arylideneketones and thiazolidenehydrazines was evaluated against Leishmania infantum and Leishmania braziliensis. Furthermore, new simplified thiazolidenehydrazine derivatives were evaluated against Trypanosoma cruzi. The cytotoxicity of the active compounds on non-infected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their anti-parasitic effects. Seven thiazolidenehydrazine derivatives and ten arylideneketones had good activity against the three parasites. The IC50 values for T. cruzi and Leishmania spp. ranged from 90 nM–25 μM. Eight compounds had multi-trypanocidal activity against T. cruzi and Leishmania spp. (the etiological agents of cutaneous and visceral forms). The selectivity of these active compounds was better than the three reference drugs: benznidazole, glucantime and miltefosine. They also had low toxicity when tested in vivo on zebrafish. Trying to understand the mechanism of action of these compounds, two possible molecular targets were investigated: triosephosphate isomerase and cruzipain. We also used a molecular stripping approach to elucidate the minimal structural requirements for their anti-T. cruzi activity

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

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

Most of the enzymes of parasites have their counterpart in the host. Throughout evolution, the three-dimensional architecture of enzymes and their catalytic sites are highly conserved. Thus, identifying molecules that act exclusively on the active sites of the enzymes from parasites is a difficult task. However, it is documented that the majority of enzymes consist of various subunits, and that conservation in the interface of the subunits is lower than in the catalytic site. Indeed, we found that there are significant differences in the interface between the two subunits of triosephosphate isomerase from Homo sapiens and Trypanosoma cruzi (TcTIM), which causes Chagas disease in the American continent. In the search for agents that specifically inhibit TcTIM, we found that 2,2′-dithioaniline (DTDA) is far more effective in inactivating TcTIM than the human enzyme, and that its detrimental effect is due to perturbation of the dimer interface. Remarkably, DTDA prevented the growth of Escherichia coli cells that had TcTIM instead of their own TIM and killed T. cruzi epimastigotes in culture. Thus, this study highlights a new approach base of targeting molecular interfaces of dimers

Investigación Educativa en las Ciencias Sociales

El libro se organiza en cinco grandes secciones, cada una con diferentes capítulos: La primera de ellas, rescata dos temas vinculados con aspectos teórico metodológicos de la Investigación Educativa; La segunda sección recoge algunos planteamientos del nivel básico; en tanto que la tercera, concentra dos capítulos de investigación en el nivel superior; la cuarta parte recupera y muestra dos aportes de nivel posgrado y, finalmente, se cierra con una quinta sección donde se concentran, distintos capítulos, de tópicos diversos

Three unrelated and unexpected amino acids determine the susceptibility of the interface cysteine to a sulfhydryl reagent in the triosephosphate isomerases of two trypanosomes.

Proteins with great sequence similarity usually have similar structure, function and other physicochemical properties. But in many cases, one or more of the physicochemical or functional characteristics differ, sometimes very considerably, among these homologous proteins. To better understand how critical amino acids determine quantitative properties of function in proteins, the responsible residues must be located and identified. This can be difficult to achieve, particularly in cases where multiple amino acids are involved. In this work, two triosephosphate isomerases with very high similarity from two related human parasites were used to address one such problem. We demonstrate that a seventy-fold difference in the reactivity of an interface cysteine to the sulfhydryl reagent methylmethane sulfonate in these two enzymes depends on three amino acids located far away from this critical residue and which could not have been predicted using other current methods. Starting from previous observations with chimeric proteins involving these two triosephosphate isomerases, we developed a strategy involving additive mutant enzymes and selected site directed mutants to locate and identify the three amino acids. These three residues seem to induce changes in the interface cysteine in reactivity by increasing (or decreasing) its apparent pKa. Some enzymes with four to seven mutations also exhibited altered reactivity. This study completes a strategy for identifying key residues in the sequences of proteins that can have applications in future protein structure-function studies

The Importance of Polarity in the Evolution of the K+ Binding Site of Pyruvate Kinase

In a previous phylogenetic study of the family of pyruvate kinase, we found one cluster with Glu117 and another with Lys117. Those sequences with Glu117 have Thr113 and are K+-dependent, whereas those with Lys117 have Leu113 and are K+-independent. The carbonyl oxygen of Thr113 is one of the residues that coordinate K+ in the active site. Even though the side chain of Thr113 does not participate in binding K+, the strict co-evolution between position 117 and 113 suggests that T113 may be the result of the evolutionary pressure to maintain the selectivity of pyruvate kinase activity for K+. Thus, we explored if the replacement of Thr113 by Leu alters the characteristics of the K+ binding site. We found that the polarity of the residue 113 is central in the partition of K+ into its site and that the substitution of Thr for Leu changes the ion selectivity for the monovalent cation with minor changes in the binding of the substrates. Therefore, Thr113 is instrumental in the selectivity of pyruvate kinase for K+

The Importance of Polarity in the Evolution of the K+ Binding Site of Pyruvate Kinase

In a previous phylogenetic study of the family of pyruvate kinase, we found one cluster with Glu117 and another with Lys117. Those sequences with Glu117 have Thr113 and are K+-dependent, whereas those with Lys117 have Leu113 and are K+-independent. The carbonyl oxygen of Thr113 is one of the residues that coordinate K+ in the active site. Even though the side chain of Thr113 does not participate in binding K+, the strict co-evolution between position 117 and 113 suggests that T113 may be the result of the evolutionary pressure to maintain the selectivity of pyruvate kinase activity for K+. Thus, we explored if the replacement of Thr113 by Leu alters the characteristics of the K+ binding site. We found that the polarity of the residue 113 is central in the partition of K+ into its site and that the substitution of Thr for Leu changes the ion selectivity for the monovalent cation with minor changes in the binding of the substrates. Therefore, Thr113 is instrumental in the selectivity of pyruvate kinase for K+

Effect of MMTS on WT TbTIM, WT TcTIM and on different site directed mutants of selected amino acids of regions 1 and 4 on the sequence of WT TbTIM.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: F28L, A100L, A115Q. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: A100L, A115Q, TbTIM: F28L, A100L and TbTIM: F28L, A115Q. Panel c) effect of MMTS on WT TcTIM, WT TbTIM and TbTIM: F28L, TbTIM: A100L and TbTIM: A115Q. Assays were performed independently three times.</p

Effect of MMTS on WT TbTIM, WT TcTIM and on different site directed mutants of selected amino acids of regions 1 and 4 of the sequence of WT TbTIM onto WT TcTIM.

<p>Enzymes were incubated at a concentration of 250 μg/mL in 100 mM TEA, 10 mM EDTA pH 7.4 and 5–100 μM MMTS for 2 h at 25°C. At that time, the activity of the samples was determined, including a sample without MMTS to calculate the percentage of remaining activity. Panel a) effect of MMTS on WT TcTIM, WT TbTIM and TcTIM: E26D, T27L, L28F, A30S, T32S, L100A, Q115A, TcTIM: E26D, T27L, L28F, A30S, L100A, Q115A and TcTIM: E26D, T27L, L28F, L100A, Q115A. Panel b) effect of MMTS on WT TcTIM, WT TbTIM and TcTIM: E26D, T27L, L28F, A30S, T32S, L100A; TcTIM: E26D, T27L, L28F, A30S, L100A and TcTIM: E26D, T27L, L28F, L100A. Assays were performed independently three times.</p