Identificação e caracterização de uma proteína com motivos zinc finger de Trypanosoma cruzi

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Zinc fingers are compact protein domains composed of a α-helix and a β-sheet held together by a zinc ion. Tandem arrays of zinc fingers are commonly used to recognize nucleic acids. Among other activities, they are involved in the processes of replication, transcription, and DNA repair. The nucleocapsid protein of HIV-1 contains a zinc finger motif CX2CX4HX4C that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. In trypanosomatids, only a few of the proteins that contain such fingers were identified. In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in Trypanosoma brucei and Leishmania major sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Homologous of TcZFP8 in T. brucei is apparently absent, while one candidate in L. major was identified. Molecular cloning of gene TcZFP8 and heterologous expression were performed in Escherichia coli. The purified recombinant protein His6x-TcZFP8 was used to produce antibody in rabbits and GST-TcZFP8 in SELEX and EMSA procedures. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. Analysis using cytoplasm and nuclear cell extracts showed that this protein is present in the nuclear extracts and indirect immunofluorescence microscopy analysis confirmed the nuclear localization of the TcZFP8. SELEX experiment showed four different populations rich in C and/or G nucleotides, but with none consensus sequence. Preliminary EMSA from one population gave evidence that TcZFP8 has affinity to bind to singlestranded DNA

ASSUNTO(S)

trypanosoma cruzi proteína nuclear biologia molecular zing finger cchc

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