Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Patients with non-alcoholic steatohepatitis (NASH) have increased plasmatic and hepatic concentrations of bile acids (BA), suggesting that they can be associated with the progression of the disease. Hepatic nuclear receptors are known to modulate genes controlling BA metabolism; thus, in this work we aimed to compare the expression of liver nuclear receptors -farnesoid X (FXR), small heterodimer partner (SHP) and liver X alpha (LXRα) receptors- and BA transporters -sodium+/taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP)- in liver biopsy samples of patients with simple steatosis (SS) and NASH. Forty patients with biopsy-proven NALFD were enrolled between 2009 and 2012; liver biopsies were classified as SS (N = 20) or NASH (N = 20) according to the NAFLD activity score. Gene expression of nuclear FXR, LXRα, SHP, NTCP and BSEP was analyzed by real-time reverse transcription polymerase chain reaction and protein level was quantified by western blot. Gene expression of FXR, SHP, NTCP and BSEP was significantly up-regulated in the NASH group in comparison with SS patients (P < 0.05). In contrast, protein level for FXR, SHP and NTCP was decreased in the NASH patients vs. the SS group (P < 0.05). Gene and protein profile of LXRα did not show differences between groups. The results suggest that liver nuclear receptors (FXR and SHP) and BA transporters (NTCP and BSEP) are associated with the progression of NAFLD.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The k_cat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T₅₀ (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP⁺ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP⁺, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP⁺, leading to enzyme instability.

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-Mg(2+) 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. V(max) with K(+) was about 400 higher than without K(+). In the presence of K(+), the affinities for phosphoenol-pyruvate, ADP-Mg(2+), oxalate, and ADP-Cr(2+) 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.

Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains ofGiardia lambliawas evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites.G. lambliatriosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effectivein vitroagainst drug-resistant and drug-susceptible strains ofG. lamblia.

Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug.