Identificação proteômica, expressão heteróloga, citolocalização, estudos de regulação transcricional e traducional da Aconitase Mitocondrial de Paracoccidioides brasiliensis
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Data
2009-11-18
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Universidade Federal de Goiás
Resumo
Paracoccidioides brasiliensis is a thermal-dimorphic fungus, the causative agent of
Paracoccidioidomycosis (PCM), an important endemic mycosis in Latin America. A protein
species preferentially expressed in yeast cells with a molecular mass of 80kDa and isoeletric
point (pI) of 7.79 was isolated from the proteome of P. brasiliensis and characterized as an
aconitase (E.C. 4.2.1.3). Aconitase is an enzyme that catalyzes the isomerization of citrate to
isocitrate in both the Krebs cycle (KC) and the glyoxylate cycle (GC). We report the cloning and
characterization of the cDNA encoding the aconitase of P. brasiliensis (PbACO). The cDNA
showed a 2337 bp open reading frame (ORF) and encoded a predicted protein with 779 amino
acids. A polyclonal antibody against the purified recombinant PbACO was obtained in order to
analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in
the extracellular fluid, cell wall, mitochondria, cytosol and peroxisomes of yeast cells as
demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the
Pbaco gene was performed through quantitative real time RT-PCR and results demonstrated
increasing expression during differentiation from mycelium to yeast cells. Real time RT-PCR
assays was also used to evaluate the Pbaco expression when the fungus grows on media with
acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated
that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in highiron
conditions.
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Citação
BRITO, Wesley de Almeida. Identification,characterization and regulation studies of the mitochondrial Aconitase of Paracoccidioides brasiliensis. 2009. 108 f. Tese (Doutorado em Ciencias Biologicas) - Universidade Federal de Goiás, Goiânia, 2009.