Ensaios de Duplo-Híbrido e Pull-down no estudo de interações moleculares da Beta-1,3-glicanosiltransferse 3 de Paracoccidioides brasiliensis
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2010-01-29
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Universidade Federal de Goiás
Resumo
Paracoccidioides brasiliensis is a thermodimorphic fungus that causes
paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In
humans, infection starts by inhalation of fungal propagules that reach the pulmonary
epithelium. The cell wall presents an essential role in the pathobiology of P.
brasiliensis, since it is involved in the morphogenetic changes associated with the life
cycle of this pathogen. The biosynthesis and remodeling of the cell wall polysaccharide
network is required for fungal growth and glycosylphosphatidylinositol (GPI)-anchored
proteins are involved in these processes. The enzymes of the glucan-elongating (GEL)
family are GPI-anchored proteins which have beta-1,3-glucanosyltransferase activity
and play important role in the cross-linking of cell wall components in fungi. The P.
brasiliensis genome possess 3 genes that encode for beta-1,3-glucanosyltransferases
(Gel1p, Gel2p e Gel3p). With the aim of searching for other possible functions for
beta-1,3-glucanosyltransferase 3 (PbGel3p) in P. brasiliensis, the interactions between
this protein and others proteins of the fungus were investigated through the
Saccharomyces cerevisiae two-hybrid system and pull-down assay. The two-hybrid
system enables the study of interactions in vivo and, through this approach, 8 proteins
which interact with PbGel3p were identified. The interaction with phosphatidylinositol-
4-phosphate 5-kinase its3 and protein kinase Dsk1p suggest the participation of
PbGel3p in the cell division cycle. The RNA helicase Dbp5p, whose interaction with
PbGel3p was in vitro confirmed by coimunoprecipitation, and the 5'-3' exoribonuclease
are involved in the mRNA metabolism suggesting a function for PbGel3p in this
process. The interaction between PbGel3p and the lipase/serine esterase, which
participates on the biosynthesis of the GPI-anchor, was also confirmed by
coimunoprecipitation. PbGel3p also interacts with the transcription factor Ctf1Bp and
the 3-oxoacyl reductase enzyme, which suggests its involvement in the lipid
metabolism. The interaction with the leptomycin B resistance protein Pmd1p suggests a
role for PbGel3p in the response to antifungal drugs. The in vitro pull-down assay
resulted in the identification of P. brasiliensis 70 kDa heat shock protein. The
interaction with this protein suggests that PbGel3p may act in the response to stress
conditions, as in the temperature increase. On basis of these data, functional studies may
be carried out in order to confirm the Gel3p multifunctionality in P. brasiliensis.
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Citação
SILVA, Mirelle Garcia. Testing of Double-Hybrid and Pull-down in the study of molecular interactions of beta-1 ,3-glicanosiltransferse three Paracoccidioides brasiliensis. 2010. 95 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2010.