The Giardial Arginine Deiminase Participates in Giardia-Host Immunomodulation in a Structure-Dependent Fashion via Toll-like Receptors

Beyond the problem in public health that protist-generated diseases represent, understanding the variety of mechanisms used by these parasites to interact with the human immune system is of biological and medical relevance. Giardia lamblia is an early divergent eukaryotic microorganism showing remarkable pathogenic strategies for evading the immune system of vertebrates. Among various multifunctional proteins in Giardia, arginine deiminase is considered an enzyme that plays multiple regulatory roles during the life cycle of this parasite. One of its most important roles is the crosstalk between the parasite and host. Such a molecular “chat” is mediated in human cells by membrane receptors called Toll-like receptors (TLRs). Here, we studied the importance of the 3D structure of giardial arginine deiminase (GlADI) to immunomodulate the human immune response through TLRs. We demonstrated the direct effect of GlADI on human TLR signaling. We predicted its mode of interaction with TLRs two and four by using the AlphaFold-predicted structure of GlADI and molecular docking. Furthermore, we showed that the immunomodulatory capacity of this virulent factor of Giardia depends on the maintenance of its 3D structure. Finally, we also showed the influence of this enzyme to exert specific responses on infant-like dendritic cells

Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme’s structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman’s method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis

Naturally occurring deamidated triosephosphate isomerase is a promising target for cell-selective therapy in cancer

Human triosephosphate isomerase (HsTIM) is a central glycolytic enzyme and is overexpressed in cancer cells with accelerated glycolysis. Triple-negative breast cancer is highly dependent on glycolysis and is typically treated with a combination of surgery, radiation therapy, and chemotherapy. Deamidated HsTIM was recently proposed as a druggable target. Although thiol-reactive drugs affect cell growth in deamidated HsTIM-complemented cells, the role of this protein as a selective target has not been demonstrated. To delve into the usefulness of deamidated HsTIM as a selective target, we assessed its natural accumulation in breast cancer cells. We found that deamidated HsTIM accumulates in breast cancer cells but not in noncancerous cells. The cancer cells are selectively programmed to undergo cell death with thiol-reactive drugs that induced the production of methylglyoxal (MGO) and advanced glycation-end products (AGEs). In vivo, a thiol-reactive drug effectively inhibits the growth of xenograft tumors with an underlying mechanism involving deamidated HsTIM. Our findings demonstrate the usefulness of deamidated HsTIM as target to develop new therapeutic strategies for the treatment of cancers and other pathologies in which this post translationally modified protein accumulates

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

Characterization of proteolytic activities of Giardia lamblia with the ability to cleave His-tagged N-terminal sequences

Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to characterize the cysteine protease activity detected in trophozoite lysates. This proteolytic activity showed the ability to cleave NH-terminal sequences with either a recognition sequence for a viral protease or a recognition sequence for thrombin. This cleavage activity was detected in nonencysting trophozoites and increased with the progression of encystation. This activity was also detected in excretion/secretion products of axenic trophozoites and in trophozoites cocultured with differentiated Caco-2 cells. Based on size exclusion chromatography, we obtained a fraction enriched in low- to medium-molecular-weight proteins that was capable of exerting this cleavage activity and aggregating human platelets. Finally, our results suggest that this proteolytic activity is shared with other protozoan parasites.Fil: de la Mora de la Mora, José Ignacio. Instituto Nacional de Pediatría; MéxicoFil: Enríquez Flores, Sergio. Instituto Nacional de Pediatría; MéxicoFil: Fernández Lainez, Cynthia. Instituto Nacional de Pediatría; MéxicoFil: Gutiérrez Castrellón, Pedro. Hospital General Dr. Manuel Gea González; MéxicoFil: Olivos García, Alfonso. Universidad Nacional Autónoma de México; MéxicoFil: González Canto, Augusto. Universidad Nacional Autónoma de México; MéxicoFil: Hernández, Roberto. Universidad Nacional Autónoma de México; MéxicoFil: Lujan, Hugo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas. Universidad Católica de Córdoba. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas; ArgentinaFil: García Torres, Itzhel. Instituto Nacional de Pediatría; MéxicoFil: López Velázquez, Gabriel. Instituto Nacional de Pediatría; Méxic

Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600–1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer

Repurposing of rabeprazole as an anti-Trypanosoma cruzi drug that targets cellular triosephosphate isomerase

AbstractTrypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi, but cellular enzyme inhibition has yet to be established. This study demonstrates that rabeprazole inhibits both cell viability and triosephosphate isomerase activity in T. cruzi epimastigotes. Our results show that rabeprazole has an IC50 of 0.4 µM, which is 14.5 times more effective than benznidazole. Additionally, we observed increased levels of methyl-glyoxal and advanced glycation end products after the inhibition of cellular triosephosphate isomerase by rabeprazole. Finally, we demonstrate that the inactivation mechanisms of rabeprazole on triosephosphate isomerase of T. cruzi can be achieved through the derivatization of three of its four cysteine residues. These results indicate that rabeprazole is a promising candidate against American trypanosomiasis

Structural and Functional Perturbation of <i>Giardia lamblia</i> Triosephosphate Isomerase by Modification of a Non-Catalytic, Non-Conserved Region

<div><p>Background</p><p>We have previously proposed triosephosphate isomerase of <i>Giardia lamblia</i> (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.</p><p>Results</p><p>In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.</p><p>Conclusions</p><p>The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.</p></div