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

The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins

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

Reactivation and stability of TbTIM with various proteins, and reactivation of different TIMs.

<p>In A, 1 µg/ml of TbTIM was allowed to reactivate in presence of 19 µg/ml of the indicated proteins for 24 hours. The bottom of Figure A shows the molecular weights, isoelectric points and the molar ratio of protein:TbTIM of BSA; ADH: alcohol dehydrogenase; DNasa: deoxyribonuclease; Ovo: ovalbumin; Hex: hexokinase; Lyso: lysozyme; CytC: cytocrome c; RNasa: ribonuclease A. B shows the effect of 19 µg/ml of the same proteins on the stability of 1 µg/ml TbTIM dimers after 24 hours of incubation. Note that in the absence of the proteins, TbTIM lost 64% of its activity; in the presence of the proteins, hardly any activity was lost. The activity of native TbTIM was 3322 µmol/min/mg (100%). In C, the effect of 19 µg/ml BSA on the reactivation of 1 µg/ml of TIM from <i>T. cruzi</i> (Tc) <i>Leishmania mexicana</i> (Lm) and <i>Saccharomyces cerevisiae</i> (Sc) is shown. Data with TbTIM are included. The reactivation time was 24 hours. The activities of native TbTIM, TcTIM, LmTIM, and ScTIM were 3422, 3638, 3762, and 7572 µmol/min/mg, respectively. The respective activities were considered as 100%.</p

Effect of BSA on the stability of TbTIM dimers.

<p>In A, the indicated concentrations of native TbTIM dimers were incubated with and without 19 µg/ml BSA; after 24 hours activity was recorded. 100% is the activity at time zero (3401 µmol/min/mg). Note that with 5 µg/ml TbTIM, activity dropped by about 20%; at a concentration of 10 µg/ml, the activity remained unchanged (not shown). In B, 1 µg/ml TbTIM was incubated with the indicated BSA concentrations for 24 hours.</p

Time course of TbTIM reactivation

<p>. The conditions were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000497#pone-0000497-g001" target="_blank">Figure 1</a>. The concentration of TbTIM in the reactivation buffer was 1 µg/ml and that of BSA 19 µg/ml. In A, the appearance of activity is plotted against time; 100% is the activity of native TbTIM, 3391 µmol/min/mg. The inset shows the ln of the appearance of activity versus time, where ln = [(Maximal activity reached−activity at time x)/maximal activity]×100. B shows the effect of adding BSA (19 µg/ml) after the enzyme was allowed to reactivate for 1, 2, 3, 4, and 5 hours; after 24 hours, activity was determined. In the zero time sample, denatured TbTIM was added to media that contained BSA. The activity of native TbTIM was 3121 µmol/min/mg; this was considered 100%.</p

Reactivation of TbTIM with and without BSA.

<p>In A, denatured TbTIM was allowed to reactivate in 100 mM triethanolamine, 10 mM EDTA and 200 mM GdnHCl (pH 7.4) that contained the indicated concentrations of the enzyme without and with 19 µg BSA/ml (open and closed circles, respectively). In B, the reactivation media contained 1 µg/ml TbTIM and the indicated concentrations of BSA. After 24 hours at 25°C, activity was determined. The ordinate shows the extent of reactivation; 100% is the activity of the starting TbTIM which was 3353 and 3488 µmol/min/mg in experiments A and B, respectively.</p

Heat changes of the interaction of TbTIM with lysozyme.

<p>Panel A shows (1) the ITC heat signals observed when 15 µl of buffer were repeatedly injected into a solution of 10 mg/ml lysozyme. (2) The heat changes that occurred when 15 µl of a solution of 17 mg TbTIM per ml were injected into a solution of 10 mg/ml lysozyme. (3) The calorimetric response of repeated injections of a TbTIM solution into buffer alone. Panel B depicts the heat evolved in the interaction between TbTIM and lysozyme as a function of the TbTIM/lysozyme molar ratio. Open squares are heats obtained from the calorimetric traces in A; closed squares are a replica. The average enthalpy change was −0.32 kcal/mole of TbTIM. Inset: heat due to the dissociation of the self-associated lysozyme as function of lysozyme concentration in the main cell.</p

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