Navegando por Autor "Machado, Carla Botelho"
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Item Characterization and mRNA expression analysis of PI31, an endogenous proteasome inhibitor from Schistosoma mansoni.(2010) Machado, Carla Botelho; Cabral, Fernanda Janku; Soares, Cláudia Sossai; Moreira, Érika Bueno de Carvalho; Morais, Enyara Rezende; Magalhães, Lizandra Guidi; Gomes, Matheus de Souza; Cota, Renata Guerra de Sá; Rosa, José César; Ruller, R.; Ward, R. J.; Rodrigues, VanderleiThe proline-rich inhibitor of 31 kDa (PI31) is highly conserved through metazoan evolution, and its activity in the proteasome inhibition is well-established although the precise mechanism of inhibition is unclear. The coding DNA sequence of Schistosoma mansoni PI31 (SmPI31) was cloned, and the recombinant protein was expressed in bacterial system. The correct amino acid sequence was confirmed by mass spectrometry and circular dichroism suggests that SmPI31 contains both α-helix and non-structured regions. Inhibition assays, using the SucLeu-Leu-Val-Tyr-4-MCA substrate for proteasome degradation, showed that the S. mansoni proteasome may be regulated by the inhibitory activity of SmPI31. A gene expression assay using qRT-PCR at various stages during the S. mansoni life cycle has shown that SmPI31 transcripts are expressed in all studied stages, suggesting that PI31 plays an important role during the developmental processes of the parasite. In this study first evidence is presented that PI31 has a conserved structure and plays a role as proteasome inhibitor in adult worms and it is expressed through life cycle.Item Uncovering Notch pathway in the parasitic flatworm Schistosoma mansoni.(2016) Magalhães, Lizandra Guidi; Morais, Enyara Rezende; Machado, Carla Botelho; Gomes, Matheus de Souza; Cabral, Fernanda Janku; Souza, Júlia Medeiros; Soares, Cláudia Sossai; Cota, Renata Guerra de Sá; Borges, William de Castro; Rodrigues, VanderleiSeveral signaling molecules that govern development in higher animals have been identified in the parasite Schistosoma mansoni, including the transforming growth factor β, protein tyrosine kinases, nuclear hormone receptors, among others. The Notch pathway is a highly conserved signaling mechanism which is involved in a wide variety of developmental processes including embryogenesis and oogenesis in worms and flies. Here we aimed to provide the molecular reconstitution of the Notch pathway in S. mansoni using the available transcriptome and genome databases. Our results also revealed the presence of the transcripts coded for SmNotch, SmSu(H), SmHes, and the gamma-secretase complex (SmNicastrin, SmAph-1, and SmPen-2), throughout all the life stages analyzed. Besides, it was observed that the viability and separation of adult worm pairs were not affected by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]- S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor. Moreover, DAPT treatment decreased the production of phenotypically normal eggs and arrested their development in culture. Our results also showed a significant decrease in SmHes transcript levels in both adult worms and eggs treated withDAPT. These results provide, for the first time, functional validation of the Notch pathway in S. mansoni and suggest its involvement in parasite oogenesis and embryogenesis. Given the complexity of the Notch pathway, further experiments shall highlight the full repertoire of Notch-mediated cellular processes throughout the S. mansoni life cycle.