Biosíntese de isoprenos e metabolismo de esteróis no Paracoccidioides brasiliensis
Data
2021-03-01
Autores
Fernandes, Dijiane 
Orientadores
Campos, Claudia Barbosa Ladeira de
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O Paracoccidioides brasiliensis é um fungo patogênico termodimórfico, agente etiológico da micose sistêmica paracoccidioidomicose, endêmica na América do Sul. A calcineurina é uma fosfatase importante no processo de morfogênese e adaptação de fungos tanto sob condições fisiológicas ou em resposta a estresses ambientais. Em estudos anteriores, a qual este trabalho faz parte, avaliou-se o proteoma total de leveduras de P. brasiliensis, cultivadas na presença e ausência da ciclosporina A (CsA), inibidor de calcineurina, como abordagem para revelar processos bioquímicos potencialmente regulados por esta fosfatase. A partir dessas análises proteômicas, se observou que enzimas relacionadas com a via de biossíntese do mevalonato, Isopentenyl-diphosphate delta-isomerase e HMG-CoA synthase, apresentaram níveis reduzidos em 2,15 e 6,71 respectivamente. Essa via é responsável pela síntese de isoprenóides de esterol, com o produto final ergosterol em levedura. O ergosterol está envolvido em diversas funções biológicas, como fluidez, regulação, atividade e distribuição das proteínas integrais da membrana, controle do ciclo celular, crescimento e o processo de infecção. Haja visto a sua importância no metabolismo do fungo, este trabalho teve como objetivo analisar por ferramentas de bioinformática essas enzimas, bem como avaliar os possíveis efeitos do tratamento de leveduras com sinvastatina, na biossíntese de isoprenos e no metabolismo de esteróis, como forma de investigação da importância desta via na proliferação celular. Conclui-se que as enzimas apresentam uma boa similaridade e conservação de sequências entre diferentes espécies de fungos e com humanos. A IPP isomerase tem como domínio conservado Nudix Hydrolase, (cl00447), e a HMG-CoA sintase tem como domínio a superfamília HMG-CoA-S_euk(cl3689). Enquanto em humanos há duas isoformas, mitocondrial e citosólica, a enzima HMG CoA sintase é codificada por um gene cópia única no P. brasiliensis. Acredita-se que a HMG CoA sintase, pode ser bifuncional. O mesmo observa-se na IPP isomerase, em humanos temos a presença de dois genes IDI1 e ID2, enquanto que no P. brasiliensis é codificada por um gene cópia única. Os testes com sinvastatina sugerem uma inibição da proliferação celular nas concentrações de 5 uM e 15 uM da droga após cinco dias do tratado nas duas concentrações celulares analisadas, com valor de p˂0,05 se comparado ao dia zero.
Paracoccidioides brasiliensis is a thermodimorphic pathogenic fungus, etiologic agent of systemic mycosis paracoccidioidomycosis, endemic in South America. Calcineurin is an important phosphatase in the process of morphogenesis and adaptation of fungi both under physiological conditions or in response to environmental stresses. In previous studies, to which this work is part, the total proteome of yeasts of P. brasiliensis, grown in the presence and absence of cyclosporin A (CsA), calcineurin inhibitor, was evaluated as an approach to reveal biochemical processes potentially regulated by this phosphatase. From these proteomic analyzes, it was observed that enzymes related to the mevalonate biosynthesis pathway, Isopentenyl-diphosphate delta-isomerase and HMG-CoA synthase, showed levels reduced by 2.15 and 6.71 respectively. This pathway is responsible for the synthesis of sterol isoprenoids, with the final product ergosterol in yeast. Ergosterol is involved in several biological functions, such as fluidity, regulation, activity and distribution of integral membrane proteins, cell cycle control, growth and the infection process. Having seen its importance in the metabolism of the fungus, this study aimed to analyze these enzymes using bioinformatics tools, as well as to evaluate the possible effects of yeast treatment with simvastatin, on isoprene biosynthesis and on sterol metabolism, as a form of investigation the importance of this pathway in cell proliferation. It is concluded that the enzymes present a good similarity and conservation of sequences between different species of fungi and with humans. IPP isomerase has the conserved domain Nudix Hydrolase, (cl00447), and HMG-CoA synthase has the superfamily HMG-CoA-S_euk (cl3689). While in humans there are two isoforms, mitochondrial and cytosolic, the enzyme HMG CoA synthase is encoded by a copy gene unique in P. brasiliensis. HMG CoA synthase is believed to be bifunctional. The same is observed in IPP isomerase, in humans we have the presence of two genes IDI1 and ID2, whereas in P. brasiliensis it is encoded by a single copy gene. Tests with simvastatin suggest an inhibition of cell proliferation at concentrations of 5 µM and 15 µM of the drug after five days of treatment at the two cell concentrations analyzed, with a value of p0.05 compared to day zero.
Paracoccidioides brasiliensis is a thermodimorphic pathogenic fungus, etiologic agent of systemic mycosis paracoccidioidomycosis, endemic in South America. Calcineurin is an important phosphatase in the process of morphogenesis and adaptation of fungi both under physiological conditions or in response to environmental stresses. In previous studies, to which this work is part, the total proteome of yeasts of P. brasiliensis, grown in the presence and absence of cyclosporin A (CsA), calcineurin inhibitor, was evaluated as an approach to reveal biochemical processes potentially regulated by this phosphatase. From these proteomic analyzes, it was observed that enzymes related to the mevalonate biosynthesis pathway, Isopentenyl-diphosphate delta-isomerase and HMG-CoA synthase, showed levels reduced by 2.15 and 6.71 respectively. This pathway is responsible for the synthesis of sterol isoprenoids, with the final product ergosterol in yeast. Ergosterol is involved in several biological functions, such as fluidity, regulation, activity and distribution of integral membrane proteins, cell cycle control, growth and the infection process. Having seen its importance in the metabolism of the fungus, this study aimed to analyze these enzymes using bioinformatics tools, as well as to evaluate the possible effects of yeast treatment with simvastatin, on isoprene biosynthesis and on sterol metabolism, as a form of investigation the importance of this pathway in cell proliferation. It is concluded that the enzymes present a good similarity and conservation of sequences between different species of fungi and with humans. IPP isomerase has the conserved domain Nudix Hydrolase, (cl00447), and HMG-CoA synthase has the superfamily HMG-CoA-S_euk (cl3689). While in humans there are two isoforms, mitochondrial and cytosolic, the enzyme HMG CoA synthase is encoded by a copy gene unique in P. brasiliensis. HMG CoA synthase is believed to be bifunctional. The same is observed in IPP isomerase, in humans we have the presence of two genes IDI1 and ID2, whereas in P. brasiliensis it is encoded by a single copy gene. Tests with simvastatin suggest an inhibition of cell proliferation at concentrations of 5 µM and 15 µM of the drug after five days of treatment at the two cell concentrations analyzed, with a value of p0.05 compared to day zero.
Descrição
Citação
FERNANDES, Dijiane. Biosíntese de isoprenos e metabolismo de esteróis no Paracoccidioides brasiliensis. 2021. 48 f. Trabalho de conclusão de curso - Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo (UNIFESP), São José dos Campos, 2021.