Caracterização da proteina LaRbp38: mapeamento do domínio de interação com ácidos nucléicos e identificação de possíveis proteínas parceiras
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2009-02-19
Autores
Perez, Arina Marina [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho teve por objetivo mapear os domínios de interação da proteína LaRbp38 (RNA binding protein 38 KD) de Leishmania amazonensis com ácidos nucléicos e a identificação de proteínas parceiras que formassem possíveis complexos com ela nos telômeros do parasita. LaRbp38 é uma proteína exclusiva de protozoários tripanosomatídeos, entre os quais estão os agentes etiológicos da leishmaniose, uma doença endêmica presente em diversas regiões do Brasil. Novos casos da doença vêm aumentando progressivamente, principalmente entre indivíduos imunocomprometidos e por isso a Organização Mundial da Saúde tem incentivado a busca de novos alvos para desenvolver drogas que eliminem eficazmente o parasita. LaRbp38 é uma proteína codificada por um gene nuclear, que parece exercer diferentes funções nas maquinarias de replicação nuclear e mitocondrial. Foi primeiramente descrita como proteína estabilizadora de RNA mitocondrial e parece estar envolvida com a replicação de DNA mitocondrial. Em Leishmania, LaRbp38 também interage in vivo com DNA mitocondrial, com seqüências ricas em GT e com DNA telomérico simples e dupla fita. A análise estrutural da proteína não indica a presença de nenhum domínio canônico de interação a ácidos nucléicos. Para dar inicio aos experimentos, a primeira estratégia foi desenhar mutantes delecionais da proteína de L. amazonensis (LaRbp38) de forma que cada um representa uma sobreposição do outro. Cada mutante, nomeados mut1, mut2, mut3, mut4 e mut5, foram subclonados em vetor de expressão bacteriano para produção de polipeptídios recombinantes. Em seguida os polipeptídios foram purificados e testados em western blotting, que mostra tanto LaRbp38 quantos os 5 mutantes sendo reconhecidas pelo soro antLLaRbp38. Ensaios in vitro de interação proteína-DNA (EMSA), usando DNA telomérico simples fita Te11, dupla fita...
Recent results demonstrated that Rbp38 is a protein exclusively expressed in trypanosomatid parasites, which includes the genus Leishmania. Rbp38 is a multifunctional protein exerting functions in the kinetoplast, such as the stabilization of mitochondrial RNAs and in the nucleus, as a protein that binds in vivo kDNA, the double-stranded (LaTel) and the G-rich telomeric DNAs (Tel1). The trypanosomatid Rbp38 does not share sequénce or functional/structural similarities with any other protein deposited in the public data bank. The present work has the aim of mapping the boundaries of the DNA-binding domain of LaRBP38, since using the available in silico tools, we were not able to define any known nucleic acid binding domain in this protein. Therefore, we constructed truncated overlapping mutants of LaRbp38 in order to map the boundaries of its DNA binding domain. These mutant proteins were expressed in a bacterial system and were produced in high amounts as shown by SDS-PAGE and Western blotting analyses. Electrophoretic mobility shift assays (EMSA) were done in order to test the ability of each mutant to bind in vitro the G-rich telomeric DNA, double-stranded telomeric DNA and kDNA. EMSA and competition assays confirmed that LaRbp38 has at least one DNA binding domain (DBD), centrally located, between amino acid residues 141 and 235. This DBD, interacts with distinct affinities with ali DNAs tested. Our results suggested that this binding domain has at least two extensions one towards the N-terminus of mut2 (aa 95 and 141), which showed binding affinity to LaTel and kDNA and other towards the C-terminus of mut3 (aa 235 to283) that strongly interacted with kDNA. However, we can not discard that others parts of the protein may also contribute to these interactions. Other controversial point about LaRbp38 is whether this protein has nuclear and mitochrondrial subcelular localization... (Completo abstract click electronic access below)
Recent results demonstrated that Rbp38 is a protein exclusively expressed in trypanosomatid parasites, which includes the genus Leishmania. Rbp38 is a multifunctional protein exerting functions in the kinetoplast, such as the stabilization of mitochondrial RNAs and in the nucleus, as a protein that binds in vivo kDNA, the double-stranded (LaTel) and the G-rich telomeric DNAs (Tel1). The trypanosomatid Rbp38 does not share sequénce or functional/structural similarities with any other protein deposited in the public data bank. The present work has the aim of mapping the boundaries of the DNA-binding domain of LaRBP38, since using the available in silico tools, we were not able to define any known nucleic acid binding domain in this protein. Therefore, we constructed truncated overlapping mutants of LaRbp38 in order to map the boundaries of its DNA binding domain. These mutant proteins were expressed in a bacterial system and were produced in high amounts as shown by SDS-PAGE and Western blotting analyses. Electrophoretic mobility shift assays (EMSA) were done in order to test the ability of each mutant to bind in vitro the G-rich telomeric DNA, double-stranded telomeric DNA and kDNA. EMSA and competition assays confirmed that LaRbp38 has at least one DNA binding domain (DBD), centrally located, between amino acid residues 141 and 235. This DBD, interacts with distinct affinities with ali DNAs tested. Our results suggested that this binding domain has at least two extensions one towards the N-terminus of mut2 (aa 95 and 141), which showed binding affinity to LaTel and kDNA and other towards the C-terminus of mut3 (aa 235 to283) that strongly interacted with kDNA. However, we can not discard that others parts of the protein may also contribute to these interactions. Other controversial point about LaRbp38 is whether this protein has nuclear and mitochrondrial subcelular localization... (Completo abstract click electronic access below)
Descrição
Palavras-chave
Parasitologia - Aspectos genéticos, Proteínas, Proteins
Como citar
PEREZ, Arina Marina. Caracterização da proteina LaRbp38: mapeamento do domínio de interação com ácidos nucléicos e identificação de possíveis proteínas parceiras. 2009. 80 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências de Botucatu, 2009.