Análise do papel biológico dos proteossomos durante a metaciclogênese in vitro do Trypanosoma Cruzi

Abstract

Resumo: Proteossomos sao complexos intracelulares responsaveis pela maior parte da degradacao de proteinas em varios organismos. Neste estudo nos utilizamos diferentes abordagens para determinar a funcao biologica do proteossomos durante a replicacao e metaciclogenese in vitro do Trypanosoma cruzi. Inicialmente utilizamos inibidores especificos de proteossomo e avaliamos o efeito dos mesmos por microscopia optica, microscopia eletronica de transmissao e citometria de fluxo. O crescimento de culturas de T. cruzi foi inibido pela presenca de ate 10 ƒÊM de lactacistina, com CI50 (concentracao que inibe o crescimento em 50%) de 4,35 ƒÊM, apos 24 ou 72 h. A metaciclogenese in vitro foi fortemente (95%) inibida pelo tratamento com 5 ƒÊM lactacistina sendo a fase de adesao ao substrato nao afetada, mas os epimastigotas nao se diferenciaram em tripomastigotas metaciclicos. A maioria dos epimastigotas tratados apresentou inchaco da mitocondria e uma distribuicao alterada do DNA nuclear e do cinetoplasto. Alem disso, tratamento com lactacistina interrompeu o ciclo celular na fase G2. Nossos resultados sugerem que a inibicao da atividade de proteossomos em T. cruzi nao bloqueia a adesao ao substrato, mas interrompe a divisao celular e afeta fatores desencadeadores de diferenciacao. Analisamos tambem as atividades proteoliticas proteossomais durante a metaciclogenese e obtivemos evidencias bioquimicas para a coexistencia de degradacao proteossomal ubiquitina-dependente e ubiquitina-independente. A degradacao proteica nao foi estimulada pela adicao de ubiquitina e ATP. Nao houve picos de degradacao mediada pela ubiquitina, sendo observado um perfil semelhante de conjugados ubiquitinados em todas as fases de diferenciacao analisadas. No entanto, houve uma variacao significativa nos niveis de proteinas oxidadas em diferentes periodos durante o processo de diferenciacao. Esses resultados sugerem que o proteossomo 20S pode estar atuando na degradacao de proteinas oxidadas, uma vez que estas sao os substratos naturais de proteolise ubiquitina-independente. O proteossomo 20S pode ser ativado por diferentes complexos (PA700 e PA26) e a acao coordenada destes permite a coexistencia na celula de proteolise dependente e independente de ubiquitina. Alem disso, verificamos a existencia de outra especie de proteossomo (HslVU) envolvido na degradacao de proteinas sem a participacao de ubiquitina. Estes diferentes complexos estao presentes em todas as formas avaliadas ao longo da metaciclogenese. Todavia, apresentam diferentes localizacoes intracelulares indicando um aumento da proteolise de substratos especificos nestas localizacoes intracelulares.

Abstract: Proteasomes are intracellular complexes that play a major role in degradation of most proteins in several organisms. In this study we have used different approaches to determine the biological function of proteasomes during replication and in vitro metacyclogenesis of the protozoan Trypanosoma cruzi. We have first used specific proteasome inhibitors and then evaluated their effect by light microscopy, transmission electron microscopy and flow cytometry. Culture growth was inhibited by the presence of up to 10 ìM lactacystin, with ICI50 (concentration that inhibits growth in 50%) of 4.35 ìM, after 24 or 72 h. Metacyclogenesis in vitro was strongly (95%) inhibited by treatment with 5 ìM lactacystin: adhesion of parasites to the substrate was not affected, but the epimastigote forms did not differentiate into metacyclic trypomastigotes. Most treated epimastigotes presented mitochondrial swelling and an altered distribution of nuclear and kinetoplast DNA. Furthermore, treatment with lactacystin interrupted the cell cycle at the G2 phase. Our results suggest that inhibition of proteasomal activity in T. cruzi does not block adhesion to the substrate, but blocks cell division and affects differentiation triggering factors. We have also analyzed proteasomal proteolytic activities during metacyclogenesis and we have obtained biochemical evidences for the co-existence of both ubiquitin-dependent and ubiquitin-independent proteasomal degradation. Protein degradation was not stimulated by addiction of ubiquitin and ATP. There were no peaks of ubiquitin-mediated degradation, being observed a similar profile of ubiquitinated conjugates during all phases of differentiation. However, there was a significant variation in the levels of oxidated proteins at different periods during the differentiation process. These results suggest that the 20S proteasome may be acting in degradation of oxidated proteins, since these are the natural substrate of ubiquitin-independent proteolysis. The 20S proteasome could be activated by different complexes (PA700 and PA26) and their coordinated action allowed the co-existence in the cell of both ubiquitin-dependent and -independent proteolysis. Furthermore, we have evidentiated the existence of other proteasome species (HslVU) involved in protein degradation with ubiquitin participation. All these different proteasomal complexes were present during the different cellular forms evaluated during the metacyclogenesis. However, they were found at different cell locations indicating an increase in proteolysis of specific substrates at these intracellular localizations.