Análise estrutural e funcional de eIF5A selvagem e mutadas
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Data
2010-01-22
Autores
Dias, Camila Arnaldo Olhê [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e é essencial para a viabilidade celular. Este fator tem sido associado com o início da tradução, proliferação celular, transporte nucleocitoplasmático e decaimento de mRNA. Estudos recentes associam eIF5A com a elongação, ao invés do inicio da tradução. eIF5A é a única proteína conhecida que contém o aminoácido essencial hipusina, gerado pelas enzimas desoxihipusina sintase e desoxihipusina hidroxilase. O objetivo deste estudo foi a caracterização estrutural e funcional de eIF5A de S. cerevisiae. Primeiramente, a estrutura terciária de eIF5A foi determinada por cristalografia e foi demonstrada a sua dimerização em solução, independentemente do resíduo hipusina. Foram obtidos e caracterizados 40 mutantes novos de eIF5A, dos quais 19 não complementaram o nocaute do gene selvagem, 13 apresentaram fenótipo de termossensibilidade e 8 não apresentaram nenhuma alteração nos fenótipos investigados. A maioria dos mutantes novos tem seus fenótipos resultantes da degradação da proteína eIF5A. Curiosamente, este é o primeiro estudo que sugere que a α-hélice presente no C-terminal de eIF5A é essencial para a manutenção da sua estrutura. Descrevemos também, que a extensão N-terminal de eIF5A, presente apenas em eucariotos, não é essencial para estrutura e função dessa proteína. Além disso, os mutantes contendo substituições na alça onde está localizado o aminoácido hipusina são inviáveis ou termossensíveis. Embora estes mutantes produzam eIF5A, inclusive na temperatura não permissiva, a proteína produzida não é hipusinada. Finalmente, dois mutantes termossensíveis (tif51AK56A e tif51AQ22H/L93F) produzem a proteína eIF5A estável na temperatura não permissiva, no entanto, apresentam...
The translation initiation factor 5A (eIF5A) is highly conserved from archae to mammals and is essential for cell viability. This factor has been associated with translation initiation, cell proliferation, nucleocytoplasmatic transport and mRNA decay. Recent studies show eIF5A involved in elongation, rather than translation initiation. eIF5A is the only protein known to contain the essential amino acid residue hypusine, generated by the enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase. The main goal of this study was the structural and functional characterization of S. cerevisiae eIF5A. First of all, the tertiary structure of eIF5A was determined by crystallography and this protein was defined as a dimer in solution, independently of the hipusine residue. We obtained and characterized 40 new mutants, which 19 cannot complement tif51A knockout cells, 13 are temperature-sensitive and 8 show no detectable phenotype. The phenotypes of most mutantes are caused by protein folding defects. Interestingly, this is the first study suggesting that the C-terminal -helix present in yeast eIF5A may be an essential structural element. Moreover, we describe that the eIF5A N-terminal extension present only in eukaryotic homologues is not essential in yeast. Furthermore, the mutants containing substitutions surrounding the hypusine modification site showed unviable or temperature-sensitive phenotypes. Although these mutant proteins were stable, they were defective in hypusine modification. Finally, two of the temperature-sensitive mutant strains (tif51AK56A and tif51AQ22H/L93F) produced stable eIF5A protein but showed defects in growth and protein synthesis and these mutants revealed polysome profile defect similar to that described for mutations in factors involved in translation... (Complete abstract click electronic access below)
The translation initiation factor 5A (eIF5A) is highly conserved from archae to mammals and is essential for cell viability. This factor has been associated with translation initiation, cell proliferation, nucleocytoplasmatic transport and mRNA decay. Recent studies show eIF5A involved in elongation, rather than translation initiation. eIF5A is the only protein known to contain the essential amino acid residue hypusine, generated by the enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase. The main goal of this study was the structural and functional characterization of S. cerevisiae eIF5A. First of all, the tertiary structure of eIF5A was determined by crystallography and this protein was defined as a dimer in solution, independently of the hipusine residue. We obtained and characterized 40 new mutants, which 19 cannot complement tif51A knockout cells, 13 are temperature-sensitive and 8 show no detectable phenotype. The phenotypes of most mutantes are caused by protein folding defects. Interestingly, this is the first study suggesting that the C-terminal -helix present in yeast eIF5A may be an essential structural element. Moreover, we describe that the eIF5A N-terminal extension present only in eukaryotic homologues is not essential in yeast. Furthermore, the mutants containing substitutions surrounding the hypusine modification site showed unviable or temperature-sensitive phenotypes. Although these mutant proteins were stable, they were defective in hypusine modification. Finally, two of the temperature-sensitive mutant strains (tif51AK56A and tif51AQ22H/L93F) produced stable eIF5A protein but showed defects in growth and protein synthesis and these mutants revealed polysome profile defect similar to that described for mutations in factors involved in translation... (Complete abstract click electronic access below)
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Biologia molecular, Biotecnologia, Biotechnology, Molecular biology
Como citar
DIAS, Camila Arnaldo Olhê. Análise estrutural e funcional de eIF5A selvagem e mutadas. 2010. 164 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Química, 2010.