RESUMO
The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Capacitação Espermática/fisiologia , Proteínas de Ancoragem à Quinase A/metabolismo , Adulto , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Adulto JovemRESUMO
Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Jatropha/enzimologia , Modelos Moleculares , Eletricidade Estática , Sequência de Aminoácidos , Análise de Variância , Hidrolases de Éster Carboxílico/química , Domínio Catalítico , Cátions Bivalentes/farmacologia , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Proteólise/efeitos dos fármacos , Proteômica , Solventes , Estereoisomerismo , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P1' and/or P2' of their substrates (mimicked by the Ala-Ala/P1'-P2' residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.
Assuntos
Metaloendopeptidases/metabolismo , Oligopeptídeos/farmacologia , Cinética , Metaloendopeptidases/antagonistas & inibidores , Mutação/genética , Oligopeptídeos/síntese química , Oligopeptídeos/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Citotoxinas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Neoplasias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Citotoxinas/química , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Humanos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60⯰C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make PeruvianinI an interesting model for further understanding of the action of laticifer fluids in plant defense.
Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteólise , Thevetia/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Filogenia , Inibidores de Proteases/farmacologia , Substâncias Redutoras/química , Análise de Sequência de Proteína , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70⯰C for gill AChE and 75⯰C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32⯱â¯0.20â¯mM for gills and 0.43⯱â¯0.12â¯mM for viscera. The Vmax values for gills and viscera were 53.57⯱â¯1.72 and 27.71⯱â¯1.15â¯mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75â¯kcalâ¯mol-1 (gills) and 11.87â¯kcalâ¯mol-1 (viscera) obtaining rate enhancements of 3.57â¯×â¯105 and 1.01â¯×â¯104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.
Assuntos
Acetilcolinesterase/metabolismo , Crassostrea/enzimologia , Monitoramento Ambiental , Estuários , Temperatura , Animais , Biocatálise/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Praguicidas/toxicidade , Especificidade por Substrato/efeitos dos fármacos , Poluentes Químicos da Água/toxicidadeRESUMO
Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. ß-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for ß-propeller phytase functions could be of great benefit to biotechnology.
Assuntos
6-Fitase/genética , Bactérias/enzimologia , Metagenômica , 6-Fitase/antagonistas & inibidores , 6-Fitase/química , Sequência de Aminoácidos , Bactérias/genética , Meio Ambiente , Estabilidade Enzimática/efeitos dos fármacos , Biblioteca Gênica , Genes Bacterianos , Concentração de Íons de Hidrogênio , Íons , Metais/farmacologia , Modelos Moleculares , Filogenia , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Homologia Estrutural de Proteína , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Glucose and oxygen are vital for the brain, as these molecules provide energy and metabolic intermediates that are necessary for cell function. The glycolysis pathway and mitochondria play a pivotal role in cell energy metabolism, which is closely related to reactive oxygen species (ROS) production. Hexokinase (HK) is a key enzyme involved in glucose metabolism that modulates the level of brain mitochondrial ROS by recycling ADP for oxidative phosphorylation (OxPhos). Here, we hypothesize that the control of mitochondrial metabolism by hexokinase differs in distinct areas of the brain, such as the cortex and hypothalamus, in which ROS might function as signaling molecules. Thus, we investigated mitochondrial metabolism of synaptosomes derived from both brain regions. Cortical synaptosomes (CSy) show a predominance of glutamatergic synapses, while in the hypothalamic synaptosomes (HSy), the GABAergic synapses predominate. Significant differences of oxygen consumption and ROS production were related to higher mitochondrial complex II activity (succinate dehydrogenase-SDH) in CSy rather than to mitochondrial number. Mitochondrial HK (mt-HK) activity was higher in CSy than in HSy regardless the substrate added. Mitochondrial O2 consumption related to mt-HK activation by 2-deoxyglucose was also higher in CSy. In the presence of substrate for complex II, the activation of synaptosomal mt-HK promoted depuration of ROS in both HSy and CSy, while ROS depuration did not occur in HSy when substrate for complex I was used. The impact of the mt-HK inhibition by glucose-6-phosphate (G6P) was the same in synaptosomes from both areas. Together, the differences found between CSy and HSy indicate specific roles of mt-HK and SDH on the metabolism of each brain region, what probably depends on the main metabolic route that is used by the neurons.
Assuntos
Córtex Cerebral/enzimologia , Hexoquinase/metabolismo , Peróxido de Hidrogênio/metabolismo , Hipotálamo/enzimologia , Mitocôndrias/metabolismo , Sinaptossomos/enzimologia , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Glucose-6-Fosfato/farmacologia , Masculino , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacosRESUMO
Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 µg/mouse, icv, PI3K inhibitor), ZnPP (10 µg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 µg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 µg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition.
Assuntos
Antidepressivos/farmacologia , Creatina/farmacologia , Espaço Intracelular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Heme Oxigenase-1/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Abstract L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 μg/mL) and HeLa cells (IC50, 8.3 μg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 μg/mL) and RAW 264.7 cell (IC50, 59.3 μg/mL).