Identificação proteômica, expressão heteróloga, citolocalização, estudos de regulação transcricional e traducional da Aconitase Mitocondrial de Paracoccidioides brasiliensis / Identification,characterization and regulation studies of the mitochondrial Aconitase of Paracoccidioides brasiliensis
AUTOR(ES)
Wesley de Almeida Brito
DATA DE PUBLICAÇÃO
2009
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.
ASSUNTO(S)
3. proteômica 1. paracoccidioides brasiliensis 2. aconitase 1. paracoccidioides brasiliensis; 2. aconitase; 3.proteômica 1. paracoccidioides brasiliensis biologia molecular 2. aconitase 3. mass spectrometry 4. cellular localization 5. carbon sources
Documentos Relacionados
- Identificação proteômica, expressão heteróloga, citolocalização, estudos de regulação transcricional e traducional da Aconitase Mitocondrial de Paracoccidioides brasiliensis
- CLONAGEM, CARACTERIZAÇÃO E EXPRESSÃO HETERÓLOGA DE UMA CALNEXINA HOMÓLOGA DO FUNGO PATOGÊNICO Paracoccidioides brasiliensis
- Clonagem, expressão heteróloga, purificação e caracterização funcional da endoglicanase A de Aspergillus nidulans
- Bioenergetic characterization of Paracoccidioides brasiliensis yeast form: biochemical and molecular studies of mitochondrial alternative pathways.
- Identification and characterization of involved molecules in the interaction of paracoccidioides brasiliensis with the host