RESUMO
For cells to obtain inorganic phosphate, ectoenzymes in the plasma membrane, which contain a catalytic site facing the extracellular environment, hydrolyze phosphorylated molecules. In this study, we show that increased Pi levels in the extracellular environment promote a decrease in ecto-phosphatase activity, which is associated with Pi-induced oxidative stress. High levels of Pi inhibit ecto-phosphatase because Pi generates H2 O2 . Ecto-phosphatase activity is inhibited by H2 O2 , and this inhibition is selective for phospho-tyrosine hydrolysis. Additionally, it is shown that the mechanism of inhibition of ecto-phosphatase activity involves lipid peroxidation. In addition, the inhibition of ecto-phosphatase activity by H2 O2 is irreversible. These findings have new implications for understanding ecto-phosphatase regulation in the tumor microenvironment. H2 O2 stimulated by high Pi inhibits ecto-phosphatase activity to prevent excessive accumulation of extracellular Pi, functioning as a regulatory mechanism of Pi variations in the tumor microenvironment.
Assuntos
Neoplasias da Mama , Peróxido de Hidrogênio , Humanos , Feminino , Peróxido de Hidrogênio/farmacologia , Fosfatos/farmacologia , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases , Hidrólise , Microambiente TumoralRESUMO
Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During its life cycle, A. castellanii may present as proliferative and infective trophozoites or resistant cysts. The adhesion of trophozoites to host cells is a key first step in the pathogenesis of infection. A major virulence protein of Acanthamoeba is a mannose-binding protein (MBP) that mediates the adhesion of amoebae to cell surfaces. Ectophosphatases are ecto-enzymes that can dephosphorylate extracellular substrates and have already been described in several microorganisms. Regarding their physiological roles, there is consistent evidence that ectophosphatase activities play an important role in parasite-host interactions. In the present work, we identified and biochemically characterized the ectophosphatase activity of A. castellanii. The ectophosphatase activity is acidic, stimulated by magnesium, cobalt and nickel, and presents the following apparent kinetic parameters: Km = 2.12 ± 0.54 mM p-NPP and Vmax = 26.12 ± 2.53 nmol p-NP × h-1 × 10-6 cells. We observed that sodium orthovanadate, ammonium molybdate, sodium fluoride, and inorganic phosphate are able to inhibit ectophosphatase activity. Comparing the two stages of the A. castellanii lifecycle, ectophosphatase activity is significantly higher in trophozoites than in cysts. The ectophosphatase activity is stimulated by mannose residues and is significantly increased when trophozoites interact with LLC-MK2 cells. The inhibition of ectophosphatase by pretreatment with sodium orthovanadate also inhibits the adhesion of trophozoites to epithelial cells. These results allow us to conclude that the ectophosphatase activity of A. castellanii is somehow important for the adhesion of trophozoites to their host cells. According to our data, we believe that the activation of MBP by mannose residues triggers the stimulation of ectophosphatase activity to facilitate the adhesion process.
Assuntos
Ceratite por Acanthamoeba , Acanthamoeba castellanii , Humanos , Animais , Manose/metabolismo , Vanadatos , Adesão Celular/fisiologia , Sódio , TrofozoítosRESUMO
Breast cancer is one of the most common cancers in the female population worldwide, and its development is thought to be associated with genetic mutations that lead to uncontrolled and accelerated growth of breast cells. This abnormal behavior requires extra energy, and indeed, tumor cells display a rewired energy metabolism compared to normal breast cells. Inorganic phosphate (Pi) is a glycolytic substrate of glyceraldehyde-3-phosphate dehydrogenase and has an important role in cancer cell proliferation. For cells to obtain Pi, ectoenzymes in the plasma membrane with their catalytic site facing the extracellular environment can hydrolyze phosphorylated molecules, and this is an initial and possibly limiting step for the uptake of Pi by carriers that behave as adjuvants in the process of energy harvesting and thus partially contributes to tumor energy requirements. In this study, the activity of an ectophosphatase in MDA-MB-231 cells was biochemically characterized, and the results showed that the activity of this enzyme was higher in the acidic pH range and that the enzyme had a Km = 4.5 ± 0.5 mM para-nitrophenylphosphate and a Vmax = 2280 ± 158 nM × h-1 × mg protein-1 . In addition, classical acid phosphatase inhibitors, including sodium orthovanadate, decreased enzymatic activity. Sodium orthovanadate was able to inhibit ectophosphatase activity while also inhibiting cell proliferation, adhesion, and migration, which are important processes in tumor progression, especially in metastatic breast cancer MDA-MB-231 cells that have higher ectophosphatase activity than MCF-7 and MCF-10 breast cells.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Fosfatos/metabolismo , Neoplasias de Mama Triplo Negativas , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10(8) cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4 °C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows "Michaelis-Menten" kinetics with V = 61.2 U/mg and K0.5 = 60 µM, while ATP hydrolysis showed V = 19.7 U/mg, K0.5 = 110 µM, and nH = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K0.5 = 84 µM, and nH = 2.3. Arsenate and phosphate were competitive inhibitors with K i = 0.6 mM and K i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.
Assuntos
Fosfatase Ácida/metabolismo , Enterobacter/enzimologia , Fósforo/metabolismo , Trifosfato de Adenosina/metabolismo , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Enterobacter/classificação , Enterobacter/genética , Enterobacter/isolamento & purificação , Inibidores Enzimáticos/análise , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Nitrofenóis/metabolismo , Orchidaceae/microbiologia , Compostos Organofosforados/metabolismo , Filogenia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , TemperaturaRESUMO
The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.