Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii
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2017-03-10
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Universidade Federal de Goiás
Resumo
Paracoccidioides lutzii alternates between filamentous and yeast growth when exposed to
temperatures of 36ºC. Morphological transition is an essential event for establishment of
paracoccidioidomycosis (PCM), one of the most frequent systemic mycoses in Latin America.
Paracoccidioides brasiliensis and other dimorphic fungi, cAMP-dependent protein kinase (PKA)
is essential in the dimorphic process. However, molecular events responsible for mycelial-yeast
differentiation in P. lutzii remain unknown. The aim of this work was to characterize in silico the
PKA subunits of P. lutzii, and to evaluate the role of this enzyme during the morphological
transition of this fungus. During the in silico approach, we used computational tools for identity,
domains, cell localization prediction, and similarity between P. lutzii and human sequences
analysis. The importance of this enzyme was investigated through the use of specific inhibitor,
H89. The behavior of cells in the presence of the drug was observed and the PKA activity
measured by specific kit. Our in silico results reveal a single regulatory subunit, PbPKA-R located
primarily in the nucleus, and two catalytic subunits, PbPKA-C1 and PbPKA-C2, with localization in the cytoplasm and nucleus respectively, all highly conserved. The PbPKA-R subunits were
identified with two copies of the CNMP domain in the C-terminal, while the catalytic with a single
copy of two domains: protein kinase and AGC kinase. The alignment of the PKA catalytic subunits
of humans and Paracoccidioides shows a large divergence between them, making PKA an
interesting target for antifungal. The results show that PKA activity is phase regulated, being about
five times higher in yeast than in mycelium. We also show that H89 blocks the mycelium to yeast
differentiation when PKA activity decreases. However, when the drug is removal from the culture
medium, the fungus resumes the differentiation to the yeast phase. Our results show that PKA
activity affects the phase transition in P. lutzii.
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SESTARI, Sheila Janaína. Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii. 2017. 54 f. Dissertação (Mestrado em Genética e Biologia Molecular) - Universidade Federal de Goiás, Goiânia, 2017.