RESUMO
A gene encoding the entire highly expressed protein previously identified in the proteome of Paracoccidioides brasiliensis yeast cells as PbY20 has been isolated. The pby20 sequence reveals an open reading frame of 1364bp and a deduced amino acid sequence of 203 residues, which shows high identity to benzoquinone reductase from Phanerochaete chrysosporium (72.0%), Saccharomyces cerevisiae Ycp4 (65%), and Schizosaccharomyces pombe p25 (59%), and to allergens from Alternaria alternata Alt a7 (70%) and from Cladosporium herbarum, Cla h5 (68%). Low levels of the pby20 transcript in the mycelium and highly induced ones in infective yeast cells during the transition of this dimorphic fungus indicate transcriptional control of its expression. PbY20 was immunologically detected only in yeast cell extract, suggesting an important role in cell differentiation or even in the maintenance of the yeast form. Immunoelectron microscopy showed that PbY20 is found inside large granules and vacuoles, in the nucleus, and also in the cytoplasm. Through sequence comparisons analysis and fluorescence emission assay, PbY20 was recognized as a member of the flavin mononucleotide flavodoxin-like WrbA family, which are involved in heat shock and oxidative stress in biological systems. Assuming that PbY20 belongs to this family, a similar role could be attributed to this protein.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Paracoccidioides/genética , Alérgenos/genética , Alternaria/genética , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/química , Cladosporium/genética , Grânulos Citoplasmáticos , Vesículas Citoplasmáticas/química , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Flavodoxina/química , Flavodoxina/genética , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Oxirredutases/genética , Phanerochaete/genética , RNA Fúngico/análise , RNA Mensageiro/análise , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.
Assuntos
Calmodulina/genética , Calmodulina/fisiologia , Paracoccidioides/citologia , Paracoccidioides/genética , Sequência de Aminoácidos , Sequência de Bases , Calmodulina/química , Quelantes/farmacologia , DNA Fúngico/química , Ácido Egtázico/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Dosagem de Genes , Genes Fúngicos/fisiologia , Genoma Fúngico , Imidazóis/farmacologia , Íntrons , Dados de Sequência Molecular , Micélio , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/metabolismo , Alinhamento de Sequência , Homologia de Sequência , Trifluoperazina/farmacologia , LevedurasRESUMO
Kexin-like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms. Kexin-like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins. The kexin-like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi. Disruption of the kex2 gene in C. albicans and Y. lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin-like proteins in dimorphism of human pathogenic fungi. In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2). An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp. The 5' non-coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE-like regulating genes involved in thermo-dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi. In the 3' non-coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation. The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin-like proteinases from A. niger (55%), Emericella nidulans (53%) and C. albicans (48%). Comparative sequence analysis of P. brasiliensis kexin-like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin-like catalytic domain, a P domain, a S/T rich region and a transmembrane domain. A putative Golgi retrieval signal (YEFEMI) has also been found in the cytoplasmic tail. The complete nucleotide sequence of Pbkex2 and its flanking regions have been submitted to GenBank database under Accession No. AF486805.