Expressão, purificação e análise da atividade catalítica das SMases D de Austwickia chelonae, Arcanobacterium haemolyticum, Corynebacterium pseudotuberculosis e Trichophyton rubrum.
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Data
2020-07-03
Autores
Romano, Fernanda Serrano
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Universidade Estadual Paulista (Unesp)
Resumo
A esfingomielinase D (SMase D) é uma hidrolase fosfórica que cliva esfingomielinas em ceramida e fosforilcolina, alterando a fluidez, permeabilidade e cargas da membrana citoplasmática além de estar relacionada à necrose de macrófagos e inflamação pulmonar. Além disso, é conhecida por ser uma enzima dermonecrótica, que permite a disseminação patogênica pelo hospedeiro. Embora muitas vezes classificadas como fosfolipases D, não apresentam a sequência HKD conservada na família dessas enzimas, as quais podem ser encontradas nos organismos Trichophyton rubrum, Arcanobacterium haemolyticum, Austwickia chelonae e Corynebacterium pseudotuberculosis, além de outros organismos. Esses quatro micro-organismos tiveram seus protocolos de expressão e purificação em larga escala estabelecidos e análises de viabilidade celular utilizando linhagens de células HaCaT demonstraram o potencial de toxicidade desses quatro organismos e permitiram encontrar um possível inibidor (doxiciclina) para futuros testes in vivo. Estudos de catálise enzimática por RMN demonstram o padrão de velocidade catalítica dessas proteínas na presença do substrato lisofosfatidilcolina (LPC).
Sphingomyelinase D (SMase D) is a phosphoric hydrolase that cleaves sphingomyelins in ceramide and phosphorylcholine, changing the fluidity, permeability and cytoplasmic membrane loads, in addition to being related to macrophage necrosis and pulmonary inflammation. In addition, it is known to be a dermonecrotic enzyme, which allows pathogenic dissemination by the host. Although they are often classified as phospholipases D, they do not have the HKD sequence conserved in the family of these enzymes, which can be found in the organisms Trichophyton rubrum, Arcanobacterium haemolyticum, Austwickia chelonae and Corynebacterium pseudotuberculosis, in addition to other organisms. These four microorganisms had their large scale expression and purification protocols established and cell viability analyzes using HaCaT cell lines demonstrated the toxicity potential of these four organisms and allowed to find a possible inhibitor (doxycycline) for future in vivo tests. Studies of enzymatic catalysis by NMR demonstrate the pattern of catalytic rate of these proteins in the presence of the substrate lysophosphatidylcholine (LPC).
Sphingomyelinase D (SMase D) is a phosphoric hydrolase that cleaves sphingomyelins in ceramide and phosphorylcholine, changing the fluidity, permeability and cytoplasmic membrane loads, in addition to being related to macrophage necrosis and pulmonary inflammation. In addition, it is known to be a dermonecrotic enzyme, which allows pathogenic dissemination by the host. Although they are often classified as phospholipases D, they do not have the HKD sequence conserved in the family of these enzymes, which can be found in the organisms Trichophyton rubrum, Arcanobacterium haemolyticum, Austwickia chelonae and Corynebacterium pseudotuberculosis, in addition to other organisms. These four microorganisms had their large scale expression and purification protocols established and cell viability analyzes using HaCaT cell lines demonstrated the toxicity potential of these four organisms and allowed to find a possible inhibitor (doxycycline) for future in vivo tests. Studies of enzymatic catalysis by NMR demonstrate the pattern of catalytic rate of these proteins in the presence of the substrate lysophosphatidylcholine (LPC).
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Arcanobacterium haemolyticum, Austwickia chelonae, Corynebacterium pseudotuberculosis, Trichophyton rubrum, SMase D, HaCaT, Catálise enzimática, Enzymatic catalysis