Expressão heteróloga e caracterização bioquímica da nucleosídeo trifosfato difosfohidrolase-2 (NTPDase-2) de Leishmania infantum chagasi

• Ano de defesa: 2010
• Autor(a) principal: Vasconcellos, Raphael de Souza
• Orientador(a): Fietto, Juliana Lopes Rangel
• Banca de defesa: Peternelli, Ethel Fernandes de Oliveira , Marco, Ricardo de , Ramos, Juliana Rocha Lopes Soares , Silva, Eduardo de Almeida Marques da
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Biologia Celular e Estrutural
• Departamento: Análises quantitativas e moleculares do Genoma; Biologia das células e dos tecidos
• País: BR
• Palavras-chave em Português: NTPDase-2; Leishmaniose; Apirase; Caracterização bioquímica
• Palavras-chave em Inglês: (NTPDase-2); Leishmaniasis; Apyrase; Biochemical characterization
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL
• Link de acesso: http://locus.ufv.br/handle/123456789/2326Olivieira

Visceral leishmaniasis is a severe disease, responsible for about 57.000 deaths per year all over the world, having Leishmania chagasi as the etiological agent in the New World. Protozoa of the genus Leishmania belong to the Tripanosomatidae family, wich is incapable of synthetize the purine ring. It is believed that the nucleoside triphosphate diphosphohydrolases (NTPDases) might play a role in purine salvage pathway, and even modulate the host immune system by hydrolysis of nucleotides involved with purinergic signalling. In this work we performed the heterologous expression in pET21b(+) and enzymatic characterization of a NTPDase maped on Leishmania chagasi genome, here named LcNTPDase-2 because a paralog (LcNTPDase-1), more similar to the isoform TcNTPDase-1 de Trypanosoma cruzi. The recombinant enzyme LcNTPDase-2 showed a preference for GTP, UDP e ADP in presence of magnesium, and had no activity in absence of magnesium and presence of calcium. UDPase activity assays in dependence of pH showed that the activity was enhanced at 7,0 e 8,0. The compounds ARL 67156, suramin, ammonium molybdate, sodium azide and gadolinium, were tested as UDPase activity inhibitors and presented 97% inibition in presence of gadolinium at 300μM and 58% in presence of ammonium molybdate at 3μM. We also performed assays to study the activity as a function of the presence of oligomers or monomers, where only the last one was active. Large protein aggregates were observed, but only showed activity in the presence of DTT at 10mM. There is strong evidence that exist a correlation between this family of proteins and the virulence of some parasites, such as with Toxoplasma gondii e Trypanosoma cruzi.