RESUMO
Lithium therapy received approval during the 1970s, and it has been used for its antidepressant, antimanic, and anti-suicidal effects for acute and long-term prophylaxis and treatment of bipolar disorder (BPD). These properties have been well established; however, the molecular and cellular mechanisms remain controversial. In the past few years, many studies demonstrated that at the cellular level, lithium acts as a regulator of neurogenesis, aging, and Ca2+ homeostasis. At the molecular level, lithium modulates aging by inhibiting glycogen synthase kinase-3ß (GSK-3ß), and the phosphatidylinositol (PI) cycle; latter, lithium specifically inhibits inositol production, acting as a non-competitive inhibitor of inositol monophosphatase (IMPase). Mitochondria and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) have been related to lithium activity, and its regulation is mediated by GSK-3ß degradation and inhibition. Lithium also impacts Ca2+ homeostasis in the mitochondria modulating the function of the lithium-permeable mitochondrial Na+-Ca2+exchanger (NCLX), affecting Ca2+ efflux from the mitochondrial matrix to the endoplasmic reticulum (ER). A close relationship between the protease Omi, GSK-3ß, and PGC-1α has also been established. The purpose of this review is to summarize some of the intracellular mechanisms related to lithium activity and how, through them, neuronal aging could be controlled.
Assuntos
Senescência Celular , Compostos de Lítio , Neurônios , Neurônios/efeitos dos fármacos , Compostos de Lítio/farmacologia , Fármacos Neuroprotetores/farmacologia , Enzimas/metabolismo , Inositol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Cálcio/metabolismo , Humanos , Animais , Senescência Celular/efeitos dos fármacosRESUMO
Biological macromolecules are found in different shapes and sizes. Among these, enzymes catalyze biochemical reactions and are essential in all organisms, but is there a limit size for them to function properly? Large enzymes such as catalases have hundreds of kDa and are formed by multiple subunits, whereas most enzymes are smaller, with molecular weights of 20-60 kDa. Enzymes smaller than 10 kDa could be called microenzymes and the present literature review brings together evidence of their occurrence in nature. Additionally, bioactive peptides could be a natural source for novel microenzymes hidden in larger peptides and molecular downsizing could be useful to engineer artificial enzymes with low molecular weight improving their stability and heterologous expression. An integrative approach is crucial to discover and determine the amino acid sequences of novel microenzymes, together with their genomic identification and their biochemical biological and evolutionary functions.
Assuntos
Enzimas , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Humanos , Peso Molecular , Animais , Peptídeos/química , Peptídeos/metabolismoRESUMO
Feruloyl esterases (FAEs) are versatile enzymes able to release hydroxycinnamic acids or synthesize their ester derivatives, both molecules with interesting biological activities such as: antioxidants, antifungals, antivirals, antifibrotic, anti-inflammatory, among others. The importance of these molecules in medicine, food or cosmetic industries provides FAEs with several biotechnological applications as key industrial biocatalysts. However, FAEs have some operational limitations that must be overcome, which can be addressed through different protein engineering approaches to enhance their thermal stability, catalytic efficiencies, and selectivity. This review aims to present a brief historical tour through the mutagenesis strategies employed to improve enzymes performance and analyze the current protein engineering strategies applied to FAEs as interesting biocatalysts. Finally, an outlook of the future of FAEs protein engineering approaches to achieve successful industrial biocatalysts is given.
Assuntos
Hidrolases de Éster Carboxílico , Engenharia de Proteínas , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ácidos Cumáricos/metabolismo , Biotecnologia , Catálise , Biocatálise , Enzimas/metabolismoRESUMO
Phenytoin (PHE) is an antiepileptic drug that has been widely used in clinical practice for about 80 years. It is mainly used in the treatment of tonic-clonic and partial seizures. The widespread consumption of this drug around the world has led to PHE being introduced into water bodies through municipal, hospital, and industrial effluent discharges. Since the toxic effects of this drug on aquatic species has been scarcely explored, the aim of this work was to investigate the influence of low (25-400 ngL-1) and high (500-1500 ngL-1) environmentally relevant concentrations of PHE on the development and oxidative status of zebrafish (Danio rerio) embryos. The toxicity of PHE was evaluated from 12 to 96 h after fertilization in D. rerio at concentrations between 25 and 1500 ngL-1. In both the control group and the 0.05% DMSO system, no malformations were observed, all embryos developed normally after 96 h. The severity and frequency of malformations increased with increasing PHE concentration compared to embryos in the control group. Malformations observed included developmental delay, hypopigmentation, miscellaneous (more than one malformation in the same embryo), modified chorda structure, tail malformation, and yolk deformation. Concerning the biomarkers of oxidative stress, an increase in the degree of lipid peroxidation, protein carbonylation, and hydroperoxide content was observed (p < 0.05) concerning the control. In addition, a significant increase (p < 0.05) in antioxidant enzymes (SOD, CAT, and GPx) was observed at low exposure concentrations (25-400 ngL-1), with a decrease in enzyme activity at high concentrations (500-1500 ngL-1). Our IBR analysis demonstrated that oxidative damage biomarkers got more influence at 500ngL-1 of PHE. The results demonstrated that PHE may affect the embryonic development of zebrafish and that oxidative stress may be involved in the generation of this embryotoxic process.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fenitoína/toxicidade , Peixe-Zebra/embriologia , Animais , Antioxidantes/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Enzimas/metabolismo , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/metabolismoRESUMO
Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).
Assuntos
Enzimas , Escherichia coli , Proteínas Recombinantes , Cromatografia de Afinidade , Ensaios Enzimáticos , Enzimas/genética , Enzimas/isolamento & purificação , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Tipagem Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transformação BacterianaRESUMO
Enzyme activities (EAs) and the FERTBIO sample concept have been increasingly adopted as a novel approach to estimate the soil quality in Brazil. However, the performance of this strategy in sandy soils of the Cerrado biome remains unclear. During 2 years, in a Cerrado's sandy soil, the short-term effects of ten different cropping systems (conventional tillage or no-tillage associated with monoculture, rotations, and/or successions) on the activities of ß-glucosidase, acid phosphatase, and arylsulfatase were studied. Issues related to annual variability and the feasibility of using the FERTBIO sample concept for soil enzymes activities were also evaluated. Soil samples were collected at three different depths (0-10 cm, 10-20 cm, and 20-40 cm) in March 2017 and February 2018. Five years since the beginning of the experiment, the presence of cover crops and no-till promoted improvements in EAs evidencing the importance of regenerative management practices for the sustainability of agroecosystems in sandy soils. Regardless of the cropping systems and depths evaluated, soil organic carbon and EAs showed low temporal variation during the 2 years of monitoring. Our results also showed that it is possible to use the FERTBIO sample concept for the Quartzipsament soils of Western Bahia, Brazil.
Assuntos
Agricultura , Enzimas , Areia , Microbiologia do Solo , Brasil , Carbono/análise , Enzimas/metabolismo , Areia/microbiologia , Fatores de TempoRESUMO
Plant growth-limiting factors, such as low nutrient availability and weak pathogen resistance, may hinder the production of several crops. Plant growth-promoting bacteria (PGPB) used in agriculture, which stimulate plant growth and development, can serve as a potential tool to mitigate or even circumvent these limitations. The present study evaluated the feasibility of using bacteria isolated from the maize rhizosphere as PGPB for the cultivation of this crop. A total of 282 isolates were collected and clustered into 57 groups based on their genetic similarity using BOX-PCR. A representative isolate from each group was selected and identified at the genus level with 16S rRNA sequencing. The identified genera included Bacillus (61.5% of the isolates), Lysinibacillus (30.52%), Pseudomonas (3.15%), Stenotrophomonas (2.91%), Paenibacillus (1.22%), Enterobacter (0.25%), Rhizobium (0.25%), and Atlantibacter (0.25%). Eleven isolates with the highest performance were selected for analyzing the possible pathways underlying plant growth promotion using biochemical and molecular techniques. Of the selected isolates, 90.9% were positive for indolepyruvate/phenylpyruvate decarboxylase, 54.4% for pyrroloquinoline quinine synthase, 36.4% for nitrogenase reductase, and 27.3% for nitrite reductase. Based on biochemical characterization, 9.1% isolates could fix nitrogen, 36.6% could solubilize phosphate, 54.5% could produce siderophores, and 90.9% could produce indole acetic acid. Enzymatic profiling revealed that the isolates could degrade starch (90.1%), cellulose (72.7%), pectin (81.8%), protein (90.9%), chitin (18.2%), urea (54.5%), and esters (45.4%). Based on the data obtained, we identified three Bacillus spp. (LGMB12, LGMB273, and LGMB426), one Stenotrophomonas sp. (LGMB417), and one Pseudomonas sp. (LGMB456) with the potential to serve as PGPB for maize. Further research is warranted to evaluate the biotechnological potential of these isolates as biofertilizers under field conditions.
Assuntos
Bactérias , Rizosfera , Microbiologia do Solo , Zea mays , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodiversidade , Enzimas/metabolismo , Filogenia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Zea mays/microbiologiaRESUMO
Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from ß-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected ß-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.
Assuntos
Enzimas Imobilizadas/química , Enzimas/química , beta-Galactosidase/química , Sítios de Ligação/fisiologia , Biomineralização/fisiologia , Simulação por Computador , Estabilidade Enzimática , Enzimas/metabolismo , Enzimas Imobilizadas/metabolismo , Galactose/química , Concentração de Íons de Hidrogênio , Nanopartículas/química , Oligossacarídeos/química , Temperatura , beta-Galactosidase/metabolismoRESUMO
KEY MESSAGE: The present study showed that the heat stress (40 °C) caused changes in morphophysiological, biochemical, and ultrastructural parameters to the seeds Melanoxylon brauna, ultimately leading to loss of germination capacity. Temperature is an abiotic factor that influences seed germination. In the present study, we investigated morphophysiological, biochemical, and ultrastructural changes during the germination of Melanoxylon brauna seeds under heat stress. Seed germination was evaluated at constant temperatures of 25 and 40 °C. The samples consisted of seeds soaked in distilled and ionized water for 48 and 96 h at both temperatures. For the evaluation of internal morphology, the seeds were radiographed. Ultrastructural parameters were assessed using transmission electron microscopy (TEM). The production of reactive oxygen species (ROS), content of malondialdehyde (MDA) and glucose, carbonylated proteins, and activity of the enzymes (superoxide dismutase-SOD, ascorbate peroxidase-APX, catalase-CAT, peroxidase-POX, glucose-6-phosphate dehydrogenase-G6PDH, lipase, α- and ß-amylase, and protease) were measured by spectrophotometric analysis. An 82% reduction in the germination of M. brauna seeds was observed at 25 °C, and 0% at 40 °C. TEM showed that seeds submitted to heat stress (40 °C) had poorly developed mitochondria and significantly reduced respiration rates. The content of ROS and protein carbonylation in seeds subjected to 40 °C increased compared to that at 25 °C. The activity of antioxidant enzymes, namely SOD, APX, CAT, and POX, was significantly reduced in seeds subjected to heat stress. Glucose content, G6PDH, and lipase activity also decreased when the seeds were exposed to heat stress. Conversely, α- and ß-amylase enzymes and the protease increased due to the increase in temperature. Our data showed that the increase in temperature caused an accumulation of ROS, increasing the oxidative damage to the seeds, which led to mitochondrial dysfunction, ultimately leading to loss of germination.
Assuntos
Fabaceae/fisiologia , Resposta ao Choque Térmico/fisiologia , Proteínas de Plantas/metabolismo , Sementes/fisiologia , Sementes/ultraestrutura , Antioxidantes/metabolismo , Carotenoides/metabolismo , Enzimas/metabolismo , Fabaceae/ultraestrutura , Ácidos Graxos/metabolismo , Germinação , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Estresse Oxidativo , Superóxidos/metabolismoRESUMO
In order to analyze whether the marine macroalga Ulva lactuca can absorb and metabolize anthracene (ANT), the alga was cultivated with 5 µM ANT for 0-72 h, and the level of ANT was detected in the culture medium, and in the alga. The level of ANT rapidly decreased in the culture medium reaching a minimal level at 6 h, and rapidly increased in the alga reaching a maximal level at 12 h and then decreased to reach a minimal level at 48 h of culture. In addition, ANT induced an increase in hydrogen peroxide that remained until 72 h and a higher increase in superoxide anions that reach a maximal level at 24 h and remained unchanged until 72 h, indicating that ANT induced an oxidative stress condition. ANT induced an increase in lipoperoxides that reached a maximal level at 24 h and decreased at 48 h indicating that oxidative stress caused membrane damage. The activity of antioxidant enzymes SOD, CAT, AP, GR and GP increased in the alga treated with ANT whereas DHAR remained unchanged. The level of transcripts encoding these antioxidant enzymes increased and those encoding DHAR did not change. Inhibitors of monooxygenases, dioxygenases, polyphenol oxidases, glutathione-S-transferases and sulfotransferases induced an increase in the level of ANT in the alga cultivated for 24 h. These results strongly suggest that ANT is rapidly absorbed and metabolized in U. lactuca and the latter involves Phase I and II metabolizing enzymes.