RESUMEN

Ectophosphatases catalyse the hydrolysis of phosphorylated molecules, such as phospho-amino acids, in the extracellular environment. Nevertheless, the hydrolysis of nucleotides in the extracellular environment is typically catalysed by ectonucleotidases. Studies have shown that acid ectophosphatase, or transmembrane-prostatic acid phosphatase (TM-PAP), a membrane-bound splice variant of prostatic acid phosphatase, has ecto-5'-nucleotidase activity. Furthermore, it was demonstrated that ectophosphatase cannot hydrolyse ATP, ADP, or AMP in triple-negative breast cancer cells. In contrast to previous findings in MDA-MB-231 cells, the ectophosphatase studied in the present work displayed a remarkable capacity to hydrolyse AMP in luminal A breast cancer cells (MCF-7). We showed that AMP dose-dependently inhibited p-nitrophenylphosphate (p-NPP) hydrolysis. The p-NPP and AMP hydrolysis showed similar biochemical behaviours, such as increased hydrolysis under acidic conditions and comparable inhibition by NiCl2, ammonium molybdate, and sodium orthovanadate. In addition, this ectophosphatase with ectonucleotidase activity was essential for the release of adenosine and inorganic phosphate from phosphorylated molecules available in the extracellular microenvironment. This is the first study to show that prostatic acid phosphatase on the membrane surface of breast cancer cells (MCF-7) is correlated with cell adhesion and migration.

Asunto(s)

Fosfatasa Ácida , Neoplasias de la Mama , Humanos , Células MCF-7 , Femenino , Hidrólisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/enzimología , Fosfatasa Ácida/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina Monofosfato/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Nitrofenoles/farmacología , Nitrofenoles/metabolismo , Línea Celular Tumoral , Compuestos Organofosforados

RESUMEN

The heat shock response (HSR) is an ancient and evolutionarily conserved mechanism designed to restore cellular homeostasis following proteotoxic challenges. However, it has become increasingly evident that disruptions in energy metabolism also trigger the HSR. This interplay between proteostasis and energy regulation is rooted in the fundamental need for ATP to fuel protein synthesis and repair, making the HSR an essential component of cellular energy management. Recent findings suggest that the origins of proteostasis-defending systems can be traced back over 3.6 billion years, aligning with the emergence of sugar kinases that optimized glycolysis around 3.594 billion years ago. This evolutionary connection is underscored by the spatial similarities between the nucleotide-binding domain of HSP70, the key player in protein chaperone machinery, and hexokinases. The HSR serves as a hub that integrates energy metabolism and resolution of inflammation, further highlighting its role in maintaining cellular homeostasis. Notably, 5'-adenosine monophosphate-activated protein kinase emerges as a central regulator, promoting the HSR during predominantly proteotoxic stress while suppressing it in response to predominantly metabolic stress. The complex relationship between 5'-adenosine monophosphate-activated protein kinase and the HSR is finely tuned, with paradoxical effects observed under different stress conditions. This delicate equilibrium, known as caloristasis, ensures that cellular homeostasis is maintained despite shifting environmental and intracellular conditions. Understanding the caloristatic controlling switch at the heart of this interplay is crucial. It offers insights into a wide range of conditions, including glycemic control, obesity, type 2 diabetes, cardiovascular and neurodegenerative diseases, reproductive abnormalities, and the optimization of exercise routines. These findings highlight the profound interconnectedness of proteostasis and energy metabolism in cellular function and adaptation.

Asunto(s)

Diabetes Mellitus Tipo 2 , Proteostasis , Humanos , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Adenosina Monofosfato/metabolismo , Proteínas Quinasas/metabolismo

RESUMEN

This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10-6, 10-7 or 10-8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10-8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10-7 or 10-8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10-6 or 10-8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10-8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10-8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.

Asunto(s)

Melatonina , Animales , Bovinos , Femenino , Melatonina/farmacología , Melatonina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Péptido Natriurético Tipo-C/farmacología , Colforsina/farmacología , Colforsina/metabolismo , Oocitos/fisiología , AMP Cíclico/metabolismo , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Células del Cúmulo

RESUMEN

Human ecto-5'-nucleotidase (h-ecto-5'-NT, CD73) is a homodimeric Zn2+-binding metallophosphoesterase that hydrolyzes adenosine 5'-monophosphate (5'-AMP) to adenosine and phosphate. h-Ecto-5'-NT is a key enzyme in purinergic signaling pathways and has been recognized as a promising biological target for several diseases, including cancer and inflammatory, infectious, and autoimmune diseases. Despite its importance as a biological target, little is known about h-ecto-5'-NT dynamics, which poses a considerable challenge to the design of inhibitors of this target enzyme. Here, to explore h-ecto-5'-NT flexibility, all-atom unbiased molecular dynamics (MD) simulations were performed. Remarkable differences in the dynamics of the open (catalytically inactive) and closed (catalytically active) conformations of the apo-h-ecto-5'-NT were observed during the simulations, and the nucleotide analogue inhibitor AMPCP was shown to stabilize the protein structure in the closed conformation. Our results suggest that the large and complex domain motion that enables the h-ecto-5'-NT open/closed conformational switch is slow, and therefore, it could not be completely captured within the time scale of our simulations. Nonetheless, we were able to explore the faster dynamics of the h-ecto-5'-NT substrate binding site, which is mainly located at the C-terminal domain and well conserved among the protein's open and closed conformations. Using the TRAPP ("Transient Pockets in Proteins") approach, we identified transient subpockets close to the substrate binding site. Finally, conformational states of the substrate binding site with higher druggability scores than the crystal structure were identified. In summary, our study provides valuable insights into h-ecto-5'-NT structural flexibility, which can guide the structure-based design of novel h-ecto-5'-NT inhibitors.

Asunto(s)

5'-Nucleotidasa , Simulación de Dinámica Molecular , Humanos , Adenosina Monofosfato/metabolismo , Adenosina/farmacología , Sitios de Unión

RESUMEN

MAIN CONCLUSION: ScHINT1 was identified at sugarcane SAM using subtractive libraries. Here, by bioinformatic tools, two-hybrid approach, and biochemical assays, we proposed that its role might be associated to control redox homeostasis. Such control is important for plant development and flowering transition, and this is ensured with some protein partners such as PAL and SBT that interact with ScHINT1. The shoot apical meristem transition from vegetative to reproductive is a crucial step for plants. In sugarcane (Saccharum spp.), this process is not well known, and it has an important impact on production due to field reduction. In view of this, ScHINT1 (Sugarcane HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN) was identified previously by subtractive cDNA libraries using Shoot Apical Meristem (SAM) by our group. This protein is a member of the HIT superfamily that was composed of hydrolase with an AMP site ligation. To better understand the role of ScHINT1 in sugarcane flowering, here its function in SAM was characterized using different approaches such as bioinformatics, two-hybrid assays, transgenic plants, and biochemical assays. ScHINT1 was conserved in plants, and it was grouped into four clades (HINT1, HINT2, HINT3, and HINT4). The 3D model proposed that ScHINT1 might be active as it was able to ligate to AMP subtract. Moreover, the two-hybrid approach identified two protein interactions: subtilase and phenylalanine ammonia-lyase. The evolutionary tree highlighted the relationships that each sequence has with specific subfamilies and different proteins. The 3D models constructed reveal structure conservation when compared with other PDB-related crystals, which indicates probable functional activity for the sugarcane models assessed. The interactome analysis showed a connection to different proteins that have antioxidative functions in apical meristems. Lastly, the transgenic plants with 35S::ScHINT1_AS (anti-sense orientation) produced more flowers than wild-type or 35S::ScHINT1_S (sense). Alpha-tocopherol and antioxidant enzymes measurement showed that their levels were higher in 35S::ScHINT_S plants than in 35S::ScHINT1_AS or wild-type plants. These results proposed that ScHINT1 might have an important role with other proteins in orchestrating this complex network for plant development and flowering.

Asunto(s)

Flores , Meristema , Meristema/genética , Plantas Modificadas Genéticamente/genética , Homeostasis , Oxidación-Reducción , Adenosina Monofosfato/metabolismo , Regulación de la Expresión Génica de las Plantas

RESUMEN

Eating behavior is regulated by central and peripheral signals, which interact to modulate the response to nutrient intake. Central control is mediated by the hypothalamus through neuropeptides that activate the orexigenic and anorexigenic pathways. Energy homeostasis depends on the efficiency of these regulatory mechanisms. This neuroendocrine regulation of hunger and appetite can be modulated by nutritional sensors such as adenosine monophosphate-activated protein kinase (AMPK). Thus, this systematic review discusses the literature on correlations between AMPK and hypothalamic neuropeptides regarding control of eating behavior. Lilacs, PubMed/Medline, ScienceDirect, and Web of Science were searched for articles published from 2009 to 2021 containing combinations of the following descriptors: "eating behavior," "hypothalamus," "neuropeptide," and "AMPK." Of the 1330 articles found initially, 27 were selected after application of the inclusion and exclusion criteria. Of the selected articles, 15 reported decreased AMPK activity, due to interventions using angiotensin II infusion, fructose, glucose, cholecystokinin, leptin, or lipopolysaccharide (LPS) injection; dietary control through a low-protein diet or a high-fat diet (60 % fat); induction of hyperthyroidism; or injection of AMPK inhibitors. Seven studies showed a decrease in neuropeptide Y (NPY) through CV4 AICAR administration; fructose, glucose, leptin, or angiotensin II injections; or infusion of LPS from Escherichia coli and liver kinase B1 (LKB1) overexpression. Eleven studies reported a decrease in food consumption due to a decrease in AMPK activity and/or hypothalamic neuropeptides such as NPY. The results indicate that there is a relationship between AMPK and the control of eating behavior: a decrease in AMPK activity due to a dietary or non-dietary stimulus is associated with a consequent decrease in food intake. Furthermore, AMPK activity can be modulated by glucose, thyroid hormones, estradiol, leptin, and ghrelin.

Asunto(s)

Leptina , Neuropéptidos , Leptina/metabolismo , Ghrelina/metabolismo , Neuropéptido Y/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Lipopolisacáridos/metabolismo , Angiotensina II/metabolismo , Hipotálamo/metabolismo , Neuropéptidos/metabolismo , Conducta Alimentaria , Ingestión de Alimentos , Colecistoquinina/metabolismo , Glucosa/metabolismo , Hormonas Tiroideas/metabolismo , Estradiol/metabolismo , Adenosina Monofosfato/metabolismo , Fructosa

RESUMEN

Purinergic signaling is a pathway related to pain underlying mechanisms. Adenosine is a neuromodulator responsible for the regulation of multiple physiological and pathological conditions. Extensive advances have been made to understand the role of adenosine in pain regulation. Here we investigated the effects of purinergic compounds able to modulate adenosine production or catabolism on pain responses induced by Acetic Acid (AA) in zebrafish larvae. We investigated the preventive role of the ecto-5'-nucleotidase inhibitor adenosine 5'-(α,ß-methylene)diphosphate (AMPCP) and adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)-adenine (EHNA) on the AA-pain induced model. The pain responses were evaluated through exploratory and aversive behaviors in zebrafish larvae. The exploratory behavior showed a reduction in the distance covered by animals exposed to 0.0025% and 0.050% AA. The movement and acceleration were reduced when compared to control. The treatment with AMPCP or EHNA followed by AA exposure did not prevent behavioral changes induced by AA for any parameter tested. There were no changes in aversive behavior after the AA-induced pain model. After AA-induced pain, the AMP hydrolysis increased on zebrafish larvae. However, the AMPCP or EHNA exposure did not prevent changes in AMP hydrolysis induced by the AA-induced pain model in zebrafish larvae. Although AMPCP or EHNA did not show differences in the AA-induced pain model, our results revealed changes in AMP hydrolysis, suggesting the involvement of the purinergic system in zebrafish larvae pain responses.

Asunto(s)

5'-Nucleotidasa , Pez Cebra , 5'-Nucleotidasa/metabolismo , Adenina , Adenosina/metabolismo , Inhibidores de la Adenosina Desaminasa , Adenosina Monofosfato/metabolismo , Animales , Difosfatos , Larva/metabolismo , Nucleósidos , Dolor/inducido químicamente , Pez Cebra/metabolismo

RESUMEN

Adenosine is a purine nucleoside that, via activation of distinct G protein-coupled receptors, modulates inflammation and immune responses. Under pathological conditions and in response to inflammatory stimuli, extracellular ATP is released from damaged cells and is metabolized to extracellular adenosine. However, studies over the past 30 years provide strong evidence for another source of extracellular adenosine, namely the "cAMP-adenosine pathway." The cAMP-adenosine pathway is a biochemical mechanism mediated by ATP-binding cassette transporters that facilitate cAMP efflux and by specific ectoenzymes that convert cAMP to AMP (ecto-PDEs) and AMP to adenosine (ecto-nucleotidases such as CD73). Importantly, the cAMP-adenosine pathway is operative in many cell types, including those of the airways. In airways, ß2-adrenoceptor agonists, which are used as bronchodilators for treatment of asthma and chronic respiratory diseases, stimulate cAMP efflux and thus trigger the extracellular cAMP-adenosine pathway leading to increased concentrations of extracellular adenosine in airways. In the airways, extracellular adenosine exerts pro-inflammatory effects and induces bronchoconstriction in patients with asthma and chronic obstructive pulmonary diseases. These considerations lead to the hypothesis that the cAMP-adenosine pathway attenuates the efficacy of ß2-adrenoceptor agonists. Indeed, our recent findings support this view. In this mini-review, we will highlight the potential role of the extracellular cAMP-adenosine pathway in chronic respiratory inflammatory disorders, and we will explore how extracellular cAMP could interfere with the regulatory effects of intracellular cAMP on airway smooth muscle and innate immune cell function. Finally, we will discuss therapeutic possibilities targeting the extracellular cAMP-adenosine pathway for treatment of these respiratory diseases.

Asunto(s)

Adenosina , Asma , Adenosina/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/uso terapéutico , Asma/tratamiento farmacológico , Humanos , Receptores Adrenérgicos , Transducción de Señal/fisiología

RESUMEN

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.

Asunto(s)

Antivirales/metabolismo , Tratamiento Farmacológico de COVID-19 , Descubrimiento de Drogas , SARS-CoV-2/efectos de los fármacos , Adenina/efectos adversos , Adenina/análogos & derivados , Adenina/metabolismo , Adenina/farmacología , Adenosina/efectos adversos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Adenosina Monofosfato/efectos adversos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Alanina/efectos adversos , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacología , Amidas/efectos adversos , Amidas/metabolismo , Amidas/farmacología , Antivirales/efectos adversos , Antivirales/farmacología , COVID-19/metabolismo , Cloroquina/efectos adversos , Cloroquina/análogos & derivados , Cloroquina/metabolismo , Cloroquina/farmacología , Diseño de Fármacos , Humanos , Redes y Vías Metabólicas , Simulación del Acoplamiento Molecular , Nitrocompuestos/efectos adversos , Nitrocompuestos/metabolismo , Nitrocompuestos/farmacología , Pirazinas/efectos adversos , Pirazinas/metabolismo , Pirazinas/farmacología , Pirrolidinas/efectos adversos , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Ribavirina/efectos adversos , Ribavirina/metabolismo , Ribavirina/farmacología , SARS-CoV-2/metabolismo , Tiazoles/efectos adversos , Tiazoles/metabolismo , Tiazoles/farmacología

RESUMEN

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.

Asunto(s)

Enfermedad de Chagas/parasitología , Piruvato Ortofosfato Diquinasa/metabolismo , Trypanosoma cruzi/enzimología , Adenosina Monofosfato/metabolismo , Difosfatos/metabolismo , Glucosa/metabolismo , Glucólisis , Concentración de Iones de Hidrógeno , Cinética , Microcuerpos/enzimología , Fosfoenolpiruvato/metabolismo , Piruvato Ortofosfato Diquinasa/química , Piruvatos/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main factor in the development of cervical cancer (CC). The presence of immunosuppressive factors plays an important role in the development of this type of cancer. To determine whether CD39 and CD73, which participate in the production of immunosuppressive adenosine (Ado), are involved in the progression of CC, we compared the concentrations and hydrolytic activity of these ectonucleotidases in platelet-free plasma (PFP) samples between patients with low-grade squamous intraepithelial lesions (LSILs) (n = 18), high-grade squamous intraepithelial lesions (HSILs) (n = 12), and CC (n = 19) and normal donors (NDs) (n = 15). The concentrations of CD39 and CD73 in PFP increased with disease progression (r = 0.5929, p < 0.001). The PFP of patients with HSILs or CC showed the highest concentrations of CD39 (2.3 and 2.2 times that of the NDs, respectively) and CD73 (1.7 and 2.68 times that of the NDs, respectively), which were associated with a high capacity to generate Ado from the hydrolysis of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of POM-1 and APCP, specific inhibitors of CD39 and CD73, respectively, inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-ß (TGF-ß) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 (r = 0.4432, p < 0.001) and CD73 (r = 0.5786, p < 0.001). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-ß promote the expression of CD39 and CD73 to favor CC progression through Ado generation.

Asunto(s)

5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adulto , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos

RESUMEN

The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-ß than those of CIN-1 patients negative for HPV-16 and ND. These results suggest that persistent infection with HR-HPV, mostly HPV-16, in CIN-1 patients may promote the expression of CD39 and CD73 through the production of TGF-ß in precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.

Asunto(s)

5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Infecciones por Papillomavirus/enzimología , Infecciones por Papillomavirus/metabolismo , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adolescente , Adulto , Estudios Transversales , Femenino , Papillomavirus Humano 16/patogenicidad , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Adulto Joven

RESUMEN

To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.

Asunto(s)

Proteínas Arqueales/metabolismo , Fructosa-Bifosfatasa/metabolismo , Methanosarcina/metabolismo , Fosfofructoquinasa-1/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Pollos , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/aislamiento & purificación , Fructosafosfatos/metabolismo , Genes Arqueales , Cinética , Methanosarcina/genética , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/aislamiento & purificación , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional

RESUMEN

As vascular disease is complex and the various manifestations are influenced by differences in vascular bed architecture, exposure to shear and mechanical forces, cell types involved, and inflammatory responses, in vivo models are necessary to recapitulate the complex physiology and dynamic cellular interactions during pathogenesis. Murine knockout models are commonly used tools for investigators to study the role of a specific gene or pathway in multifaceted disease traits. Although valuable, these models are not perfect, and this is particularly true in regard to CD73 (cluster of differentiation 73), the extracellular enzyme that generates adenosine from AMP. At baseline, CD73-deficient mice do not present with an overt phenotype, whereas CD73-deficient humans present with the complex phenotype of vascular calcification, arteriomegaly and tortuosity, and calcification in small joints. In this review, we highlight the differences between the mouse and human systems and discuss the potential to leverage findings in mice to inform us on the human conditions.

Asunto(s)

5'-Nucleotidasa/fisiología , Enfermedades Vasculares/genética , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/deficiencia , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/inmunología , Adenosina/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/fisiología , Humanos , Inflamación , Artropatías/genética , Artropatías/patología , Ratones Noqueados , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Fenotipo , Especificidad de la Especie , Trombofilia/genética , Túnica Media/patología , Calcificación Vascular/genética , Remodelación Vascular

RESUMEN

Prostate cancer is among the major malignancies that affect men around the world. Adenine nucleotides are important signaling molecules that mediate innumerous biological functions in pathophysiological conditions, including cancer. These molecules are degraded by several ectoenzymes named ectonucleotidases that produce adenosine in the extracellular medium. Some of these ecto-enzymes can be found in soluble in the blood stream. Thus, the present study aimed to evaluate the hydrolysis of adenine nucleotides (ATP, ADP, and AMP) in the plasma blood of patients with prostate cancer. Peripheral blood samples were collected, and questionnaires were filled based on the clinical data of the medical records. The nucleotide hydrolysis was performed by Malachite Green method using ATP, ADP, and AMP as substrates. Plasma from prostate cancer patients presented an elevated hydrolysis of all nucleotides evaluated when compared to healthy individuals. NTPDase inhibitor (ARL67156) and the alkaline phosphatase inhibitor (levamisole) did not alter ATP hydrolysis. However, AMP hydrolysis was reduced by the CD73 inhibitor, APCP, and by levamisole, suggesting the action of a soluble form of CD73 and alkaline phosphatase. On microvesicles, it was observed that there was a low expression and activity of CD39 and almost absent of CD73. The correlation of ATP, ADP, and AMP hydrolysis with clinic pathological data demonstrated that patients who received radiotherapy showed a higher AMP hydrolysis than those who did not, and patients with lower clinical stage (CS-IIA) presented an elevated ATP hydrolysis when compared to those with more advanced clinical stages (CS-IIB and CS-III). Patients of all clinical stages presented an elevated AMPase activity. Therefore, we can suggest that the nucleotide hydrolysis might be attributed to soluble ecto-enzymes present in the plasma, which, in a coordinate manner, produce adenosine in the blood stream, favoring prostate cancer progression.

Asunto(s)

Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Biomarcadores de Tumor/sangre , Neoplasias de la Próstata/sangre , Anciano , Anciano de 80 o más Años , Detección Precoz del Cáncer/métodos , Humanos , Hidrólisis , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/patología

RESUMEN

Sepsis is a potentially lethal condition, and it is associated with platelet alterations. The present study sought to investigate the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA) in the platelets of rats that were induced with sepsis. Male Wistar rats were divided into three groups of ten animals each: a negative control group (normal; NC); a group that underwent surgical procedures (sham); and a group that underwent cecal ligation and perforation (CLP). The induction of sepsis was confirmed by bacteremia, and the causative pathogen identified was Escherichia coli. Hematological parameters showed leukocytosis and thrombocytopenia in animals in the septic group. The results also revealed that there were significant (p < 0.05) increases in adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolyses, and in the deamination of adenosine in the CLP group compared to the sham and control groups. Conversely, ADP hydrolysis was significantly decreased (p < 0.05) in the CLP group compared to the sham and control groups. Purine levels were analyzed by high-performance liquid chromatography (HPLC) in serum samples from control, sham, and CLP groups. Increased concentrations of ATP, adenosine, and inosine were found in the CLP group compared to the sham and control groups. Conversely, the concentrations of ADP and AMP in the CPL group were not significantly altered. We suggest that alterations in hematological parameters, nucleotide hydrolysis in platelets, and nucleotide concentrations in serum samples of rats with induced sepsis may be related to thromboembolic events.

Asunto(s)

5'-Nucleotidasa/metabolismo , Plaquetas/enzimología , Ciego/cirugía , Ligadura/efectos adversos , Complicaciones Posoperatorias/enzimología , Sepsis/enzimología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/metabolismo , Humanos , Masculino , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/metabolismo , Complicaciones Posoperatorias/microbiología , Ratas , Ratas Wistar , Sepsis/etiología , Sepsis/metabolismo , Sepsis/microbiología

RESUMEN

The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNATyr. An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases.

Asunto(s)

Adenosina Monofosfato/análogos & derivados , Dominio Catalítico , Leishmania donovani/enzimología , Modelos Moleculares , Tirosina-ARNt Ligasa/metabolismo , Tirosina/análogos & derivados , Adenosina Monofosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Estructura Terciaria de Proteína , Alineación de Secuencia , Anticuerpos de Cadena Única , Tirosina/metabolismo , Tirosina-ARNt Ligasa/química

RESUMEN

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.

Asunto(s)

Adenilosuccinato Liasa/química , Adenilosuccinato Liasa/metabolismo , Schistosoma mansoni/enzimología , Adenosina Monofosfato/metabolismo , Adenilosuccinato Liasa/genética , Animales , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Fumaratos/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica

RESUMEN

The aim of this study was to evaluate the activity of purinergic enzymes in lymphocytes and cardiac tissue of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. On day 12 post-infection (PI), the animals were anesthetized and after euthanized, and samples were collected for analyses. Infected mice showed reduction in erythrocyte counts, hematocrit and hemoglobin concentration, as well as reduced number of total leukocytes in consequence of neutrophilia (P < 0.01). The number of monocytes increased in infected mice (P < 0.001), however the number of lymphocytes and eosinophils did not differ between groups (P > 0.05). The E-NTPDase (ATP and ADP substrate) and E-ADA activities in lymphocytes increased significantly in mice infected by T. cruzi (P < 0.01). In the heart, multiple pseudocysts containing amastigotes within cardiomyocytes were observed, as well as focally extensive severe necrosis associated with diffuse moderate to severe inflammatory infiltrate of lymphocytes. Although, the NTPDase activity (ATP and ADP substrate) in the cardiac homogenate did not differ between groups, a reduction on 5'-nucleotidase activity (P < 0.001) and an increase in the ADA activity in infected animals (P < 0.05) were observed. Thus, animals infected by T. cruzi experienced the disease, i.e., showed anemia, leucopenia, and heart lesions. Associated with this, purinergic enzymes showed altered activities, which might be related to the modulation of the inflammatory response.

Asunto(s)

Enfermedad de Chagas/enzimología , Linfocitos/enzimología , Miocitos Cardíacos/enzimología , Purinas/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Enfermedad de Chagas/patología , Modelos Animales de Enfermedad , Femenino , Corazón/parasitología , Pruebas Hematológicas , Hidrólisis , Ratones , Miocardio/patología , Parasitemia/parasitología , Trypanosoma cruzi/fisiología

RESUMEN

OBJECTIVES: Empiric treatment with milrinone, a phosphodiesterase (PDE) 3 inhibitor, has become increasingly common in patients with single ventricle heart disease of right ventricular (RV) morphology (SRV); our objective was to characterize the myocardial response to PDE3 inhibition (PDE3i) in the pediatric population with SRV. STUDY DESIGN: Cyclic adenosine monophosphate levels, PDE activity, and phosphorylated phospholamban (PLN) were determined in explanted human ventricular myocardium from nonfailing pediatric donors (n = 10) and pediatric patients transplanted secondary to SRV. Subjects with SRV were further classified by PDE3i treatment (n = 13 with PDE3i and n = 12 without PDE3i). RESULTS: In comparison with nonfailing RV myocardium (n = 8), cyclic adenosine monophosphate levels are lower in patients with SRV treated with PDE3i (n = 12, P = .021). Chronic PDE3i does not alter total PDE or PDE3 activity in SRV myocardium. Compared with nonfailing RV myocardium, SRV myocardium (both with and without PDE3i) demonstrates equivalent phosphorylated PLN at the protein kinase A phosphorylation site. CONCLUSIONS: As evidenced by preserved phosphorylated PLN, the molecular adaptation associated with SRV differs significantly from that demonstrated in pediatric heart failure because of dilated cardiomyopathy. These alterations support a pathophysiologically distinct mechanism of heart failure in pediatric patients with SRV, which has direct implications regarding the presumed response to PDE3i treatment in this population.

Asunto(s)

Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/metabolismo , Ventrículos Cardíacos/anomalías , Milrinona/uso terapéutico , Miocardio/metabolismo , Inhibidores de Fosfodiesterasa 3/uso terapéutico , Adenosina Monofosfato/metabolismo , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatía Dilatada/tratamiento farmacológico , Niño , Preescolar , Femenino , Trasplante de Corazón , Humanos , Lactante , Masculino , Hidrolasas Diéster Fosfóricas/metabolismo