Banca de DEFESA: Renan Pereira Moura

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : Renan Pereira Moura
DATE: 26/04/2024
TIME: 14:00
LOCAL: Auditório da Pós Graduação da Faculdade de Medicina
TITLE:

In silico analysis and production of recombinant apyrase from Rhodnius prolixus

KEY WORDS:

Triatomines; Saliva; Apyrase; Nucleotide metabolism; Bioinformatics analysis; Recombinant protein.

PAGES: 72
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

Chagas Disease (CD) is caused by the flagellated protozoan Trypanosoma cruzi, which is transmitted between hosts by triatomines (Hemiptera: Reduviidae). CD is a neglected and endemic disease in the Americas. Triatomine salivary molecules counteract hemostasis and modulate the host's immune system to facilitate hematophagy. Among them, apyrases catabolize nucleotides and nucleosides and interfere with ADP-mediated platelet aggregation. Rhodnius prolixus is an important vector of CD. The aims of this study were to perform an in silico analysis of R. prolixus apyrases and produce recombinant proteins in a heterologous prokaryotic expression system. Two R. prolixus apyrase sequences (T1H8D6 and T1H852) were characterized in silico, cloned into the expression vector pET17b (Novagen) fused with a 6xHis tail for expression in Escherichia coli BL21(DE3) strain. After transformation, selection and cultivation of bacterial clones, SDS-PAGE and Western blotting were performed to confirm the expression of recombinant R. prolixus apyrases. The apyrase activity was evaluated in solution and by zymography. Of the two R. prolixus apyrases, only the T1H8D6 protein of approximately 40 kDa was expressed. The recombinant protein was observed only in the insoluble fraction in the form of cellular aggregates, forming inclusion bodies. The inclusion bodies were prepared and purified by affinity chromatography. The amino acid sequences of R. prolixus apyrases have sites for N-glycosylation, O-glycosylation, acetylation, phosphorylation and the presence of a signal peptide. Furthermore, the comparative analysis between the R. prolixus apyrase models with members of the CD39, 5'-nucleotidase and Cimex families showed that they are similar to the Cimex family, which are dependent on Ca2+ for their activity in the catabolism of nucleotides. Apyrases facilitate hematophagy through the inhibition of ADP-mediated platelet aggregation in the vertebrate host and their study may assist in the development of approaches to prevent CD transmission.

COMMITTEE MEMBERS:
Interno - 2676451 - CIRO MARTINS GOMES
Interno - 6404963 - JAIME MARTINS DE SANTANA
Externa à Instituição - MILENE APARECIDA ANDRADE - MS
Externa ao Programa - ***.924.081-** - PAULA BEATRIZ DE MEDEIROS SANTIAGO - UnB