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

Colegio de Periodistas: el rol vigilante de un gremio debilitado

The low associability around Colegio de Periodistas, states serious doubts about the real democratic vigilance and influence into the public life of a weakened guild. In fact, the journalists' associability is down compared to similar guilds, whit just a 2.9% to the total of graduates in the Eighth Region during last five years, similar quantity in the rest of the country. This reality implicates a responsibility from the Colegio de Periodistas, because the entity don't know the journalists' increasing leading into the organizational and corporative area - in fact, a 64% from the Eighth Region professionals work on these areas- showing an evident unconcern to charm and compromise to those non-linked at the traditional journalism. In summary, the journalists' representatively crisis questions their social role, Colegio de Periodistas' character and more over professional formation itself.La baja asociatividad que presenta el Colegio de Periodistas, plantea dudas respecto a la real vigilancia democrática e injerencia en la vida pública de un gremio debilitado. En efecto, la asociatividad de los profesionales es baja en comparación a gremios similares; registrando sólo un 2,9% del total de egresados en la Octava Región durante los últimos cinco años, cifra similar al resto del país. Esta realidad implica una responsabilidad de la propia entidad, por cuanto el colegio desconoce el creciente protagonismo de los periodistas en el ámbito organizacional y corporativo -de hecho un 64% de los profesionales de la región se desempeña en estas áreas-, existiendo una evidente despreocupación por cautivar y comprometer a aquéllos no vinculados al periodismo tradicional. En síntesis, la crisis de representatividad en que se encuentran los periodistas cuestiona su rol social, el carácter del Colegio y -más aún- la formación profesional misma

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

Chemical composition of Taiwan grass (Pennisetum purpureum Schum.) at different harvesting intervals

Objective: To evaluate the effect of the harvesting interval on the quality of Taiwan grass (Pennisetum purpureum Schum.). Design/Methodology/Approximation: Crude protein (CP), in vitro dry matter digestibility (IVDMD), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, ether extract, and ashes were determined. Samples were collected from the Papaloapan experimental site of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Isla, State of Veracruz (18° 01’ 45’’ N, 95° 31’ 35’’ W). Treatments consisted of five harvesting intervals (30, 60, 90, 120, and 150 days). Data were analyzed under the general linear model and means were separated using Tukey’s test (P<0.05). Results: The nutritional value decreased (P<0.05) as the harvesting interval increased from 30 to 150 days. The following elements decreased: CP (leaves, from 12.3 to 3.7%; stems, from 8.9 to 2.1%), IVDMD (leaves, from 66.5 to 43.5%; stems, from 62.7 to 32.5%), ether extract (leaves, from 2.4 to 1.4%; stems, from 1.4 to 0.6%), and ashes (leaves, from 10.3 to 6.1%; stems, from 10.9 to 2.9%). On the contrary, the following elements increased: CF (leaves, from 28.4 to 41.1%; stems, from 33.4 to 44.5%), NDF (leaves, from 60.4 to 72.5%; stems, from 63.8 to 74.3%), ADF (leaves, from 36.7 to 46.8%; stems, from 34.6 to 50.7%), and lignin (leaves, from 9.7 to 15.3%; stems, from 11.0 to 18.3%). Study Limitations/Implications: Neither ˂30 days harvesting intervals nor yields (tons) per hectare were taken into account. Findings/Conclusions: Taiwan grass should be harvested at 60 days, when its nutritional value has not decreased too much

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

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

Mutational spectrum of PTS gene and in silico pathological assessment of a novel variant in Mexico

BACKGROUND Tetrahydrobiopterin (BH4) is the cofactor for 6-pyruvoyl-tetrahydropterin synthase (PTPS); it is involved in BH4 biosynthesis and is encoded by PTS gene. Its deficiency (PTPSD) is characterized by hyperphenylalaninemia (HPA) and deficit in central monoamine neurotransmitters. We describe the clinical and mutational spectrum of five patients with PTPSD, from four unrelated Mexican families. All patients had symptomatic diagnosis and presented severe early neurological manifestations and HPA. METHODS Clinical and biochemical data from studied patients were recorded. Responsible PTPSD genotypes was determined by direct and bidirectional Sanger DNA sequencing of the six PTS coding exons and their exon-intron borders, and these were directly searched in the available relatives. The novel PTS missense variant [NM_3000317.2:331G > T, p.(Ala111Ser)] was subjected to in silico, to predict a possible deleterious effect. RESULTS Diminished fetal movements were perceived as a uniform characteristic in the studied group. DNA sequencing showed two known p.(Arg25∗) and p.(Val132TyrFs∗19) and the novel missense p.(Ala111Ser) PTS variants, the latter representing potentially a frequent PTPSD-responsible allele (50%, 4/8) in Mexican patients. In silico protein modeling analysis of the p.(Ala111Ser) variant revealed loss of hydrophobic interactions between the alanine and neighboring valines, suggesting that these changes in polarity may be detrimental for enzyme function, structure and/or stability. CONCLUSIONS This work contributes to the knowledge of PTPS molecular spectrum. The delayed diagnosis of these patients emphasizes the importance of considering BH4 metabolism defects in the differential diagnosis of HPA, especially for countries that are beginning their HPA newborn screening programs

Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients