The Role of the Estrogen-Related Receptor Alpha (ERRa) in Hypoxia and Its Implications for Cancer Metabolism.

Under low oxygen conditions (hypoxia), cells activate survival mechanisms including metabolic changes and angiogenesis, which are regulated by HIF-1. The estrogen-related receptor alpha (ERRα) is a transcription factor with important roles in the regulation of cellular metabolism that is overexpressed in hypoxia, suggesting that it plays a role in cell survival in this condition. This review enumerates and analyses the recent evidence that points to the role of ERRα as a regulator of hypoxic genes, both in cooperation with HIF-1 and through HIF-1- independent mechanisms, in invertebrate and vertebrate models and in physiological and pathological scenarios. ERRα's functions during hypoxia include two mechanisms: (1) direct ERRα/HIF-1 interaction, which enhances HIF-1's transcriptional activity; and (2) transcriptional activation by ERRα of genes that are classical HIF-1 targets, such as VEGF or glycolytic enzymes. ERRα is thus gaining recognition for its prominent role in the hypoxia response, both in the presence and absence of HIF-1. In some models, ERRα prepares cells for hypoxia, with important clinical/therapeutic implications.

Docking and Molecular Dynamics Predictions of Pesticide Binding to the Calyx of Bovine ß-Lactoglobulin.

Pesticides are used extensively in agriculture, and their residues in food must be monitored to prevent toxicity. The most abundant protein in cow's milk, ß-lactoglobulin (BLG), shows high affinity for diverse hydrophobic ligands in its central binding pocket, called the calyx. Several of the most frequently used pesticides are hydrophobic. To predict if BLG may be an unintended carrier for pesticides, we tested its ability to bind 555 pesticides and their isomers, for a total of 889 compounds, in a rigid docking screen. We focused on the analysis of 60 unique molecules belonging to the five pesticide classes defined by the World Health Organization, that docked into BLG's calyx with ΔGs ranging from -8.2 to -12 kcal mol-1, chosen by statistical criteria. These "potential ligands" were further analyzed using molecular dynamic simulations, and the binding energies were explored with Molecular Mechanics/Generalized Born/Surface Area (MMGBSA). Hydrophobic pyrethroid insecticides, like cypermethrin, were found to bind as deeply and tightly into the calyx as BLG's natural ligand, palmitate; while polar compounds, like paraquat, were expelled. Our results suggest that BLG could be a carrier for pesticides, in particular for pyrethroid insecticides, allowing for their accumulation in cow's milk beyond their solubility restrictions. This analysis opens possibilities for pesticide biosensor design based on BLG.

Operator recognition by the ROK transcription factor family members, NagC and Mlc.

NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets. These amino acids allow Mlc and NagC to distinguish between a C/G and an A/T bp at positions ±11 of the operators. One linker position, glycine in NagC and arginine in Mlc, corresponds to the major specificity determinant for the two proteins. In certain contexts it is possible to switch repression from Mlc-style to NagC-style, by interchanging this glycine and arginine. Secondary determinants are supplied by other linker positions or the helix-turn-helix motif. A wide genomic survey of unique ROK proteins shows that glycine- and arginine-rich sequences are present in the linkers of nearly all ROK family repressors. Conserved short sequence motifs, within the branches of the ROK evolutionary tree, suggest that these sequences could also be involved in operator recognition in other ROK family members.

Interactions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit α in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase.

Biocatalytic spectrophotometric method to detect paracetamol in water samples.

A biocatalytic methodology based on the quantification of the laccase inhibition during the oxidation of a standard substrate ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) for the indirect determination of paracetamol in drinking water has been developed. The method displayed a fast response time (20 s), and high selectivity to paracetamol in presence of interfering substances such as naproxen, estradiol, ketoprofen, sulfamethoxazole, and diclofenac. The limit of detection (LOD) and limit of quantification (LOQ) were noticed to be 0.55 µM and 8.3 µM, respectively. By comparing the catalytic constants value KM and kcat for ABTS oxidation in the absence and presence of various concentrations of paracetamol, a competitive-type inhibition was disclosed. On the other hand, the close value between Ki and KM indicates similar binding affinity of the enzyme to ABTS and paracetamol corroborated by docking studies. The methodology was successfully applied to real water samples, presenting an interesting potential for further development of a biosensor to paracetamol detection.

The linker sequence, joining the DNA-binding domain of the homologous transcription factors, Mlc and NagC, to the rest of the protein, determines the specificity of their DNA target recognition in Escherichia coli.

Protein-DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N-acetylglucosamine. Both their helix-turn-helix motifs and their consensus binding sites on DNA are very similar. One distinguishing feature is that most NagC sites have a C/G bp at positions -11 and +11 from the centre of symmetry of the operator, while all Mlc sites have A/T. By constructing Mlc and NagC chimeras, we show that the helix-turn-helix motif per se is not responsible for specific recognition of Mlc or NagC sites, but that a linker, joining the DNA-binding domain to the rest of the protein, is the major determinant. We show that a change of just two amino acids in the NagC linker is sufficient to allow NagC to recognize an A/T bp at positions +/-11 and repress Mlc targets. Modelling of the NagC linker suggests that it forms an extended structure containing two arginines and we suggest that these arginines interact differently with the minor groove at positions +/-11 depending upon the presence of a C/G or A/T bp.

Age and Comorbidities as Risk Factors for Severe COVID-19 in Mexico, before, during and after Massive Vaccination.

During 2020-2023, Mexico had a large COVID-19 emergency with >331,000 adult deaths and one of the highest excess mortalities worldwide. Age at COVID-19 death has been lower in Mexico than in high-income countries, presumably because of the young demographics and high prevalence of chronic metabolic diseases in young and middle-aged adults. SARS-CoV-2 vaccination covered 85% of adults with at least one dose and 50% with booster(s) up to April 2022. No new vaccination efforts or updated boosters were introduced until October 2023; thus, we explored the public health impact of massive SARS-CoV-2 vaccination against ancestral strains and asked whether their real-world protection has persisted through time. We compared three periods with respect to vaccine roll-outs: before, during and after vaccine introduction in a national retrospective cohort of >7.5 million COVID-19 cases. The main findings were that after vaccination, COVID-19 mortality decreased, age at COVID-19 death increased by 5-10 years, both in populations with and without comorbidities; obesity stopped being a significant risk factor for COVID-19 death and protection against severe disease persisted for a year after boosters, including at ages 60-79 and 80+. Middle-aged adults had the highest protection from vaccines/hybrid immunity and they more than halved their proportions in COVID-19 deaths.

Chronic Comorbidities in Middle Aged Patients Contribute to Ineffective Emergency Hematopoiesis in Covid-19 Fatal Outcomes.

BACKGROUND AND AIMS: Mexico is among the countries with the highest estimated excess mortality rates due to the COVID-19 pandemic, with more than half of reported deaths occurring in adults younger than 65 years old. Although this behavior is presumably influenced by the young demographics and the high prevalence of metabolic diseases, the underlying mechanisms have not been determined. METHODS: The age-stratified case fatality rate (CFR) was estimated in a prospective cohort with 245 hospitalized COVID-19 cases, followed through time, for the period October 2020-September 2021. Cellular and inflammatory parameters were exhaustively investigated in blood samples by laboratory test, multiparametric flow cytometry and multiplex immunoassays. RESULTS: The CFR was 35.51%, with 55.2% of deaths recorded in middle-aged adults. On admission, hematological cell differentiation, physiological stress and inflammation parameters, showed distinctive profiles of potential prognostic value in patients under 65 at 7 days follow-up. Pre-existing metabolic conditions were identified as risk factors of poor outcomes. Chronic kidney disease (CKD), as single comorbidity or in combination with diabetes, had the highest risk for COVID-19 fatality. Of note, fatal outcomes in middle-aged patients were marked from admission by an inflammatory landscape and emergency myeloid hematopoiesis at the expense of functional lymphoid innate cells for antiviral immunosurveillance, including NK and dendritic cell subsets. CONCLUSIONS: Comorbidities increased the development of imbalanced myeloid phenotype, rendering middle-aged individuals unable to effectively control SARS-CoV-2. A predictive signature of high-risk outcomes at day 7 of disease evolution as a tool for their early stratification in vulnerable populations is proposed.

Effects of a buried cysteine-to-serine mutation on yeast triosephosphate isomerase structure and stability.

All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132-138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.

Decrease in COVID-19 adverse outcomes in adults during the Delta and Omicron SARS-CoV-2 waves, after vaccination in Mexico.

Mexico, one of the countries severely affected by COVID-19, accumulated more than 5. 1 all-cause excess deaths/1,000 inhabitants and 2.5 COVID-19 confirmed deaths/1,000 inhabitants, in 2 years. In this scenario of high SARS-CoV-2 circulation, we analyzed the effectiveness of the country's vaccination strategy that used 7 different vaccines from around the world, and focused on vaccinating the oldest population first. We analyzed the national dataset published by Mexican health authorities, as a retrospective cohort, separating cases, hospitalizations, deaths and excess deaths by wave and age group. We explored if the vaccination strategy was effective to limit severe COVID-19 during the active outbreaks caused by Delta and Omicron variants. Vaccination of the eldest third of the population reduced COVID-19 hospitalizations, deaths and excess deaths by 46-55% in the third wave driven by Delta SARS-CoV-2. These adverse outcomes dropped 74-85% by the fourth wave driven by Omicron, when all adults had access to vaccines. Vaccine access for the pregnant resulted in 85-90% decrease in COVID-19 fatalities in pregnant individuals and 80% decrease in infants 0 years old by the Omicron wave. In contrast, in the rest of the pediatric population that did not access vaccination before the period analyzed, COVID-19 hospitalizations increased >40% during the Delta and Omicron waves. Our analysis suggests that the vaccination strategy in Mexico has been successful to limit population mortality and decrease severe COVID-19, but children in Mexico still need access to SARS-CoV-2 vaccines to limit severe COVID-19, in particular those 1-4 years old.

In silico testing of flavonoids as potential inhibitors of protease and helicase domains of dengue and Zika viruses.

Background: Dengue and Zika are two major vector-borne diseases. Dengue causes up to 25,000 deaths and nearly a 100 million cases worldwide per year, while the incidence of Zika has increased in recent years. Although Zika has been associated to fetal microcephaly and Guillain-Barré syndrome both it and dengue have common clinical symptoms such as severe headache, retroocular pain, muscle and join pain, nausea, vomiting, and rash. Currently, vaccines have been designed and antivirals have been identified for these diseases but there still need for more options for treatment. Our group previously obtained some fractions from medicinal plants that blocked dengue virus (DENV) infection in vitro. In the present work, we explored the possible targets by molecular docking a group of molecules contained in the plant fractions against DENV and Zika virus (ZIKV) NS3-helicase (NS3-hel) and NS3-protease (NS3-pro) structures. Finally, the best ligands were evaluated by molecular dynamic simulations. Methods: To establish if these molecules could act as wide spectrum inhibitors, we used structures from four DENV serotypes and from ZIKV. ADFR 1.2 rc1 software was used for docking analysis; subsequently molecular dynamics analysis was carried out using AMBER20. Results: Docking suggested that 3,5-dicaffeoylquinic acid (DCA01), quercetin 3-rutinoside (QNR05) and quercetin 3,7-diglucoside (QND10) can tightly bind to both NS3-hel and NS3-pro. However, after a molecular dynamics analysis, tight binding was not maintained for NS3-hel. In contrast, NS3-pro from two dengue serotypes, DENV3 and DENV4, retained both QNR05 and QND10 which converged near the catalytic site. After the molecular dynamics analysis, both ligands presented a stable trajectory over time, in contrast to DCA01. These findings allowed us to work on the design of a molecule called MOD10, using the QND10 skeleton to improve the interaction in the active site of the NS3-pro domain, which was verified through molecular dynamics simulation, turning out to be better than QNR05 and QND10, both in interaction and in the trajectory. Discussion: Our results suggests that NS3-hel RNA empty binding site is not a good target for drug design as the binding site located through docking is too big. However, our results indicate that QNR05 and QND10 could block NS3-pro activity in DENV and ZIKV. In the interaction with these molecules, the sub-pocket-2 remained unoccupied in NS3-pro, leaving opportunity for improvement and drug design using the quercetin scaffold. The analysis of the NS3-pro in complex with MOD10 show a molecule that exerts contact with sub-pockets S1, S1', S2 and S3, increasing its affinity and apparent stability on NS3-pro.

Molecular identification of a PGRMC-2 receptor in maturing oocytes of the zoonotic nematode parasite Trichinella spiralis.

We previously reported that the Trichinella nematode showed higher parasite loads in one gender than another, but also the parasite molting rate decreased when it was cultivated in the presence of progesterone. In this study we explored the hypothesis that the direct effect of progesterone on Trichinella spiralis could be mediated by a steroid-binding parasite protein. We sequenced, cloned and amplified the Cyt-domain of the progesterone receptor membrane component-2 of Trichinella spiralis (PGRMC2-Ts). Furthermore, we expressed the protein and developed an antibody to perform confocal microscopy and flow cytometry. The expression of the PGRMC2-Ts protein was exclusively detected at the oocyte and the parasite's cuticle in cross-sections of the parasite, and this expression was confirmed by western blot and flow cytometry. Molecular modeling studies and computer docking for the PGRMC2-Ts protein showed that it is potentially able to bind to progesterone, estradiol, testosterone, and dihydrotestosterone with different affinities. Furthermore, phylogenetic analysis demonstrated that T. spiralis PGRMC2 is related to a steroid-binding protein of another platyhelminth. Progesterone probably acts upon Trichinella spiralis oocytes by binding to PGRMC2-Ts. Our data showed that the PGRMC2-Ts protein is present in the parasite's oocytes, a development step that is crucial for the life cycle of the parasite. Indeed, this research might have implications in the field of host-parasite co-evolution and the sex-associated susceptibility to this infection. In a more practical matter, these results may contribute to the design of new drugs with anti-parasite effects.

Steroidal saponin from Agave marmorata Roezl modulates inflammatory response by inhibiting NF-κB and AP-1.

Agave marmorata Roezl is an endemic succulent specie from the Oaxaca-Puebla area of Mexico. This plant is a medicinal recourse and contain a rich variety of saponins-type compounds with multiples biological effects. Some of them have been shown to be anticancer, antibacterial, or having anti-inflammatory and immunoregulation effects. This paper is the first scientific report to describe the pharmacological activity and chemistry of the saponin smilagenin-3-O-[ß-D-glucopyranosyl (1→2)-ß-D-galactopyranoside] (1), isolated from Agave marmorata Roezl. Saponin (1) displayed immunomodulating activity when assayed on cultured macrophages. It inhibits NO production (EC50 = 5.6 mg/ml, Emax = 101%), as well as NF-κB expression (EC50 = 0.086 mg/ml, Emax = 90%). Using bioinformatic molecular docking, we identified a new smilagenin- PI3K kinase interaction site.

Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data.

Dehydroepiandrosterone Effect on Toxoplasma gondii: Molecular Mechanisms Associated to Parasite Death.

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasite Toxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals. Dehydroepiandrosterone (DHEA) has proved to be an important molecule that could drive resistance against a variety of infections, including intracellular parasites such as Plasmodium falciparum and Trypanozoma cruzi, among others. However, to date, the role of DHEA on T. gondii has not been explored. Here, we demonstrated for the first time the toxoplasmicidal effect of DHEA on extracellular tachyzoites. Ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading to the loss of the organelle structure and organization as well as the loss of the cellular shape. In vitro treatment with DHEA reduces the viability of extracellular tachyzoites and the passive invasion process. Two-dimensional (2D) SDS-PAGE analysis revealed that in the presence of the hormone, a progesterone receptor membrane component (PGRMC) with a cytochrome b5 family heme/steroid binding domain-containing protein was expressed, while the expression of proteins that are essential for motility and virulence was highly reduced. Finally, in vivo DHEA treatment induced a reduction of parasitic load in male, but not in female mice.

Analysis of B-cell epitopes from the allergen Hev b 6.02 revealed by using blocking antibodies.

Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.

Molecular dynamic simulations of the catalytic subunit of calpains 1, 2, 5, and 10: Structural analysis with an aim toward drug design.

Calpains are cysteine proteases involved in the development of several human chronic illnesses such as neurodegenerative diseases, cardiovascular ailments, diabetes, and obesity which constitutes them into possible therapeutic targets. Here, using molecular dynamic simulations and docking, we studied the binding of known inhibitors to representative members of classical and nonclassical calpains. Our aim is to gain better understanding on the inhibition mechanism of calpains and to develop better and more specific inhibitors. Our atomistic models confirmed the importance of calcium ions for the structure of calpains and, as a consequence, their functionality. With these models and their subsequent use in molecular docking, essential structural requirements were identified for the binding of ligands to the calpain catalytic site that provide useful information for the design of new selective calpain inhibitors.

Hydrophobic repacking of the dimer interface of triosephosphate isomerase by in silico design and directed evolution.

Triosephosphate isomerase from Saccharomyces cerevisiae (wt-TIM) is an obligated homodimer. The interface of wt-TIM is formed by 34 residues. In the native dimer, each monomer buries nearly 2600 A(2) of accessible surface area (ASA), and 58.4% of the interface ASA is hydrophobic. We determined the thermodynamic and functional consequences of increasing the hydrophobic character of the wt-TIM interface. Mutations were restricted to a cluster of five nonconserved residues located far from the active site. Two different approaches, in silico design and directed evolution, were employed. In both methodologies, the obtained proteins were soluble, dimeric, and compact. In silico-designed proteins are very stable dimers that bind substrate with a wild-type-like K(m); albeit, they exhibited a very low k cat. Proteins obtained from directed evolution experiments show wild-type-like catalytic activity, while their stability is decreased. Hydrophobic replacements at the interface produced a remarkable shift in the dissociation step. For wt-TIM and for TIMs obtained by directed evolution, dissociation was observed in the first transition, with C(1/2) values ranging from 0.58 to 0.024 M GdnHCl, whereas for TIMs generated by in silico design, dissociation occurred in the last transition, with C(1/2) values ranging form 3.01 to 3.65 M GdnHCl. For the latter mutants, the stabilization of the interface changed the equilibrium transitions to a novel four-state process with two dimeric intermediates. The change in the intermediate nature suggests that the relative stabilities of different folding units are similar so that subtle alterations in their stability produce a total transformation of the folding pathway.

Disulfide bridges in the mesophilic triosephosphate isomerase from Giardia lamblia are related to oligomerization and activity.

Triosephosphate isomerase from the mesophile Giardia lamblia (GlTIM) is the only known TIM with natural disulfide bridges. We previously found that oxidized and reduced thiol states of GlTIM are involved in the interconversion between native dimers and higher oligomeric species, and in the regulation of enzymatic activity. Here, we found that trophozoites and cysts have different oligomeric species of GlTIM and complexes of GlTIM with other proteins. Our data indicate that the internal milieu of G. lamblia is favorable for the formation of disulfide bonds. Enzyme mutants of the three most solvent exposed Cys of GlTIM (C202A, C222A, and C228A) were prepared to ascertain their contribution to oligomerization and activity. The data show that the establishment of a disulfide bridge between two C202 of two dimeric GlTIMs accounts for multimerization. In addition, we found that the establishment of an intramonomeric disulfide bond between C222 and C228 abolishes catalysis. Multimerization and inactivation are both reversed by reducing conditions. The 3D structure of the C202A GlTIM was solved at 2.1 A resolution, showing that the environment of the C202 is prone to hydrophobic interactions. Molecular dynamics of an in silico model of GlTIM when the intramonomeric disulfide bond is formed, showed that S216 is displaced 4.6 A from its original position, causing loss of hydrogen bonds with residues of the active-site loop. This suggests that this change perturb the conformational state that aligns the catalytic center with the substrate, inducing enzyme inactivation.

A novel progesterone receptor membrane component (PGRMC) in the human and swine parasite Taenia solium: implications to the host-parasite relationship.

BACKGROUND: We have previously reported that progesterone (P4) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. METHODS: By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. RESULTS: Our results show that P4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P4-binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P4-binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. CONCLUSION: Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.