Avaliação da atividade proteolítica intracelular de cisteíno e serino proteases durante o ciclo intraeritrocítico de Plasmodium falciparum
Data
2022-02-23
Autores
Rossetto, Bárbara dos Santos [UNIFESP]
Orientadores
Gazarini, Marcos Leoni [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
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Resumo
A malária é uma doença parasitária tropical ocasionada pelo protozoário do gênero Plasmodium. O ciclo de vida do parasito é complexo e envolve um vetor Anopheles responsável pela transmissão da doença para um hospedeiro vertebrado, o homem. A fase intraeritrocítica do P. falciparum é altamente regulada com participação de diferentes classes de proteases, que atuam essencialmente nos diferentes eventos do ciclo: mecanismo de invasão, metabolismo, diferenciação e egresso dos merozoítos. Em busca de esclarecer alguns mecanismos bioquímicos na célula infectada, avaliamos a atividade proteolítica das classes de cisteíno e serino proteases com a alteração da homeostasia iônica (Ca+2 e H+) durante os estágios assexuados do ciclo intraeritrocítico. Observamos uma atividade significante da classe de cisteíno protease durante a fase de trofozoíto, enquanto, a atividade da classe de serino protease demonstrou ser predominante durante a fase de esquizonte. Além disso, identificamos que a mobilização de Ca2+ e H+ no citoplasma do parasito é importante para a atividade de proteases dependentes destes íons. Observamos um aumento significativo da atividade proteolítica durante a fase de trofozoíto na presença de THG, devido a inibição da bomba de Ca2+ ATPAse localizada no Re favorecendo o aumento rápido e transitório na concentração de Ca+2 intracelular e consequentemente estimulando a ativação de proteases dependentes de Ca+2. Durante a fase de esquizonte, observamos uma redução significativa da atividade proteolítica na presença do iónoforo de MON, alterando o pH do VD do parasito prejudicando a atividade de proteases dependentes de H+. Além disso, devido a necessidade de novos fármacos eficazes para atuarem contra as cepas resistentes de P. falciparum, avaliamos o potencial efeito inibitório de uma série de compostos derivados quinolínicos contra a atividade de cisteíno protease. A molécula de marionoquilina (MQA) isolada pela primeira vez de uma bactéria marinha e sintetizada contendo um núcleo marinoquinolino, apresentou resultados promissores com inibição de 100% da atividade de cisteíno protease durante a fase de trofozoíto na concentração de 10μM. Nossos resultados contribuem para estabelecer um protocolo para o screening de novos compostos contra a proteólise intracelular em eritrócitos infectados de P. falciparum, como também, foi possível identificar algumas condições envolvidas na modulação da proteólise intracelular durante o ciclo do parasita.
Malaria is a tropical parasitic disease caused by the protozoan of the genus Plasmodium. The life cycle of the parasite is complex and involves an Anopheles vector responsible for transmitting the disease to a vertebrate host, man. The intraerythrocytic phase of P. falciparum is highly regulated with the participation of different classes of proteases, which essentially act on different events of the cycle: mechanism of invasion, metabolism, differentiation, and egress of merozoites. In order to clarify some biochemical mechanisms in the infected cell, we evaluated the proteolytic activity of the classes of cysteine and serine proteases with the alteration of ionic homeostasis (Ca+2 and H+ ) during the asexual stages of the intraerythrocytic cycle. We observed significant activity of the cysteine protease class during the trophozoite phase, while the activity of the serine protease class was shown to be predominant during the schizont phase. Furthermore, we identified that the mobilization of Ca2+ and H + in the parasite's cytoplasm is important for the activity of these iondependent proteases. We observed a significant increase in proteolytic activity during the trophozoite phase in the presence of THG, due to the inhibition of the Ca2+ ATPase pump located in the Re, favoring the rapid and transient increase in the intracellular Ca+2 concentration and consequently stimulating the activation of proteases dependent on Ca+2 . During the schizont phase, we observed a significant reduction in the proteolytic activity in the presence of the MON ionophore, altering the parasite's DV pH, impairing the activity of H + dependent proteases. Furthermore, due to the need for new effective drugsto act against resistant strains of P. falciparum, we evaluated the potential inhibitory effect of a series of quinolinederived compounds against cysteine protease activity. The marionoquilin molecule (MQA) isolated for the first time from a marine bacterium and synthesized containing a marinoquinoline nucleus, showed promising results with 100% inhibition of cysteine protease activity during the trophozoite phase at a concentration of 10μM. Our results contribute to establishing a protocol for the screening of new compounds against intracellular proteolysis in P. falciparuminfected erythrocytes, and it was also possible to identify some conditions involved in the modulation of intracellular proteolysis during the parasite cycle
Malaria is a tropical parasitic disease caused by the protozoan of the genus Plasmodium. The life cycle of the parasite is complex and involves an Anopheles vector responsible for transmitting the disease to a vertebrate host, man. The intraerythrocytic phase of P. falciparum is highly regulated with the participation of different classes of proteases, which essentially act on different events of the cycle: mechanism of invasion, metabolism, differentiation, and egress of merozoites. In order to clarify some biochemical mechanisms in the infected cell, we evaluated the proteolytic activity of the classes of cysteine and serine proteases with the alteration of ionic homeostasis (Ca+2 and H+ ) during the asexual stages of the intraerythrocytic cycle. We observed significant activity of the cysteine protease class during the trophozoite phase, while the activity of the serine protease class was shown to be predominant during the schizont phase. Furthermore, we identified that the mobilization of Ca2+ and H + in the parasite's cytoplasm is important for the activity of these iondependent proteases. We observed a significant increase in proteolytic activity during the trophozoite phase in the presence of THG, due to the inhibition of the Ca2+ ATPase pump located in the Re, favoring the rapid and transient increase in the intracellular Ca+2 concentration and consequently stimulating the activation of proteases dependent on Ca+2 . During the schizont phase, we observed a significant reduction in the proteolytic activity in the presence of the MON ionophore, altering the parasite's DV pH, impairing the activity of H + dependent proteases. Furthermore, due to the need for new effective drugsto act against resistant strains of P. falciparum, we evaluated the potential inhibitory effect of a series of quinolinederived compounds against cysteine protease activity. The marionoquilin molecule (MQA) isolated for the first time from a marine bacterium and synthesized containing a marinoquinoline nucleus, showed promising results with 100% inhibition of cysteine protease activity during the trophozoite phase at a concentration of 10μM. Our results contribute to establishing a protocol for the screening of new compounds against intracellular proteolysis in P. falciparuminfected erythrocytes, and it was also possible to identify some conditions involved in the modulation of intracellular proteolysis during the parasite cycle
Descrição
Citação
ROSSETTO, Bárbara dos Santos. Avaliação da atividade proteolítica intracelular de cisteíno e serino proteases durante o ciclo intraeritrocítico de Plasmodium falciparum. 2022. 104 f. Dissertação (Mestrado Interdisciplinar em Ciências da Saúde) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2022.