β-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53

Background: Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: β-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Ações da β-micrustoxina, uma fosfolipase A2 do veneno da serpente Micrurus lemniscatus, sobre a proliferação celular e a apoptose de astrócitos em cultura e os mecanismos envolvidos. / Actions of β-micrustoxin, a phospholipase A2 of the venom of the snake Micrurus lemniscatus, on cell proliferation and apoptosis of astrocytes in culture and the mechanisms involved.

Toxins that exhibit phospholipase A2 (PLA2) activity are present in snake venoms from Elapidae and Viperidae families. They are neurotoxic and act by impairing synaptic transmission from the neuromuscular junction. Although the principal target of these toxins is the neuromuscular junction, there are several studies that have used neuronal cells from central structures in order to characterize their mechanism of action and this model has proved to be very useful. In relation to glial cells, however, there are few studies showing some effect of PLA2 from animal venoms. Glial cells, astrocytes in particular, have important roles in the central and peripheral nervous system, being part of the synapses. The objective of this study was to characterize the action of the PLA2 toxin, β-micrustoxin, isolated from Micrurus lemniscatus snake venom, on cultured astrocytes, addressing cell viability, cell proliferation, cell cycle phases distribution, protein expression of p53, p27 and p21, cell senescence and mitochondrial membrane potential. Gene and protein expression of the phospholipase A2 receptor (PLA2R) was evaluated, as well as the internalization process in cultured astrocytes and hippocampal neurons. The partial toxin primary sequence was also characterized by proteolytic digestion and by comparison of the peptides acquired with data bases. Astrocytes and neurons were obtained from Wistar rats (CEUAIB – Protocolo n°1223/14) and maintained in culture in DMEM medium + 10% SFB or Neurobasal medium + B27, respectively, at 37°C, 5% CO2. Astrocytes were incubated with β–micrustoxin for several times. Cell viability was evaluated by MTT test; cell proliferation, cell cycle phases and p53, p21 and p27 proteins were evaluated by flow cytometry. Cell senescence was analyzed by β-galactosidase assay and mitochondrial potential was also analyzed by using the TMRE probe. In order to investigate the internalization process, β-micrustoxin was conjugated to Alexa 488 fluorophore and the cells observed in confocal microscopy, in normal medium and with the PLA2 activity inhibited. β-micrustoxin reduced cell viability and cell proliferation of astrocytes. The G2/M phase was augmented and does not was verified DNA fragmentation nor mitochondrial potential alteration. β-micrustoxin induced increase in the cell cycle regulatory proteins, p53, p21 and p27. β-micrustoxin conjugated with Alexa 488 internalized in astrocytes and neurons. This process is dependent on PLA2 activity in neurons, but not in astrocytes. PLA2R is expressed very acutely in astrocytes but it is also expressed in hippocampal neurons, even though in minor quantities. The data allowed to conclude that β-micrustoxin interferes in astrocytes cell proliferation, by inducing a cell cycle arrest in G2/M phase and this effect is mediated by p53, p21 and p27, without evidence of cell death. Moreover, there are evidences that β-micrustoxin would induce its effects throughout its internalization in the cell cytosol. This study contributes to a better understanding of the cell events involved in the toxicity induced by the PLA2 β-micrustoxin, isolated from the venom of the snake Micrurus lemniscatus. Besides, it opens a perspective of developing new drugs that could have inhibitory actions on cell proliferation.

Toxinas com atividade de fosfolipase A2 (FLA2) são encontradas em venenos de serpentes das famílias Elapidae e Viperidae e são caracterizadas por sua atividade neurotóxica, ocasionando o bloqueio da junção neuromuscular. Apesar de o alvo dessas toxinas ser a junção neuromuscular, vários estudos utilizaram células neuronais centrais para caracterizar seus mecanismos de ação e estas se mostraram um bom modelo. Pouco se conhece, no entanto, sobre as atividades desempenhadas por essas enzimas em células da glia, como os astrócitos, sendo que este tipo celular desempenha funções importantes tanto no sistema nervoso central quanto periférico. O objetivo deste estudo foi caracterizar as ações da toxina FLA2 β-micrustoxina, isolada do veneno da serpente Micrurus lemniscatus, em astrócitos em cultura, quanto à viabilidade celular, proliferação celular, distribuição das fases do ciclo celular, expressão de proteínas regulatórias p53, p27 e p21, senescência celular e potencial da membrana mitocondrial. Ainda, avaliou-se a expressão proteica e gênica do receptor para FLA2 (FLA2R) e a ocorrência do processo de internalização da toxina em astrócitos e neurônios hipocampais em cultura. A sequência primária parcial da toxina foi identificada pela obtenção de fragmentos peptídicos por digestão proteolítica e comparação com base de dados. Astrócitos e neurônios hipocampais de ratos Wistar (CEUAIB – Protocolo n°1223/14) foram mantidos em cultura em meio DMEM + 10% SFB e meio Neurobasal + B27, respectivamente, a 37°C, 5% CO2. Os astrócitos foram tratados com a β–micrustoxina por diferentes períodos de tempo. A viabilidade celular foi avaliada pelo método do MTT e a proliferação celular, as fases do ciclo celular e as proteínas p53, p21 e p27 foram avaliadas por citometria de fluxo. A senescência celular foi avaliada pelo ensaio de β- galactosidase e o potencial da membrana mitocondrial foram avaliados utilizando a sonda TMRE. Para a análise da internalização celular, a β-micrustoxina foi conjugada ao fluoróforo Alexa 488 e observado o processo de internalização em microscópio confocal, em condições normais ou com a inibição da atividade FLA2. A β-micrustoxina reduziu a viabilidade e a proliferação celular de astrócitos e houve um predomínio da fase G2/M. Não houve fragmentação do DNA nem alteração do potencial mitocondrial. A β-micrustoxina induziu o aumento das proteínas regulatórias do ciclo celular p53, p21 e p27. Observou-se a internalização da β-micrustoxina tanto em astrócitos como em neurônios. Esse processo é dependente da atividade FLA2 em neurônios, mas não em astrócitos. O FLA2R está expresso em astrócitos com muita intensidade, mas também em neurônios hipocampais. Os dados obtidos permitem concluir que a β-micrustoxina interfere na proliferação celular de astrócitos, induzindo a parada do ciclo celular na fase G2/M, mediada pelas proteínas p53, p21 e p27, sem evidências de indução de morte celular. Além disso, os dados evidenciam que a β-micrustoxina pode induzir os seus efeitos através de sua internalização na célula. Este estudo contribui para o melhor entendimento dos eventos celulares que levam à toxicidade induzida pela FLA2 β-micrustoxina isolada do veneno da serpente Micrurus leminiscatus e abre perspectivas de desenvolvimento de novos fármacos com ações inibitórias sobre a proliferação celular.