Estudo dos efeitos celulares do inibidor de hipusinação GC7 utilizando coleções de mutantes de Saccharomyces cerevisiae
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Data
2018-05-22
Autores
Camacho Iglesias, Luis
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Universidade Estadual Paulista (Unesp)
Resumo
eIF5A é um fator de tradução envolvido com vários processos celulares, como o controle da proliferação celular e inflamação. Esse fator sofre uma modificação pós- traducional totalmente conservada e específica para sua ativação. Essa modificação é chamada de hipusinação e consiste na modificação de uma lisina específica em duas etapas: primeiro, a porção aminobutil da poliamina espermidina é transferida para o grupamento amino da cadeia lateral da lisina, pela ação da enzima desoxi-hipusina sintase; e, em seguida, o radical transferido sofre uma hidroxilação na posição beta, pela ação da desoxi-hipusina hidroxilase. Assim como eIF5A, desoxi-hipusina sintase é altamente conservada em arquea e eucariotos e é essencial em Saccharomyces cerevisiae. A desoxi-hipusina sintase é inibida por N1-Guanyl-1,7-diaminoheptane (GC7), um análogo estrutural da espermidina. No entanto, estudos mais recentes demonstraram que o composto GC7 apresenta efeitos celulares independentes de eIF5A, o que demonstra que este possui outros alvos. Além disso, não é conhecido como se dá o processo de transporte do GC7 para o meio intracelular ou se existem mecanismos de compartimentalização celular ou externalização do GC7. Por fim, também não são conhecidos mecanismos de metabolização celular do GC7. Desta forma, o presente projeto teve como objetivo a busca de novos alvos celulares para o composto GC7 utilizando rastreamento quimio-genético utilizando a coleção haploide de genes nocautes de S. cerevisiae (genes não essenciais). A partir do rastreamento, foram obtidos resultados de sensibilidade ao GC7 para 96 linhagens nocautes e de resistência para 107. As principais vias afetadas por GC7 estão relacionadas ao transporte e biossíntese de aminoácidos e poliaminas, acidificação vacuolar e ribofagia. Além da identificação destes, foram testados outros nocautes relacionados e foram identificados prováveis transportadores de GC7 para fora do citoplasma. Também foi verificado que GC7 reduz os níveis de putrescina, assim como em cultura de células de mamífero, e outros resultados deste trabalho também estão de acordo com um efeito de GC7 de impacto relevante nesta via de poliaminas. Um dos resultados mais importantes deste trabalho foi a resistência de mutantes de ribofagia a GC7, efeito este que se mostrou independente de eIF5A. Por fim, demonstramos que S. cerevisiae, no background genético da coleção de nocautes, é altamente resistente a GC7 e que os efeitos de GC7 na célula não podem ser revertidos pela adição exógena de espermidina, seu análogo estrutural.
eIF5A is a translation factor involved in several cellular processes, as cellular proliferation control and inflammation. This factor undergoes a very conserved and specific posttranslational modification necessary to its activation named hypusination, which consists in the modification of a specific lysine in two steps. First, deoxyhypusine synthase enzyme, transfers 4-aminobutyl moiety from polyamine spermidine to a specific lysine residue in eIF5A precursor; immediately, deoxyhypusine hydroxylase adds a hydroxyl moiety in eIF5A intermediate to generate eIF5A hypusinated. Both, eIF5A and deoxyhypusine synthase are highly conserved from archaea to eukaryotes and are essential in Saccharomyces cerevisiae. Deoxyhypusine synthase is inhibited by N1-Guanyl-1,7-diaminoheptane (GC7), a structural analogue of spermidine. Nevertheless, recent findings have demonstrated that GC7 shows independent cellular effects of eIF5A, pointing out that GC7 has other targets. Besides, it is not known how GC7 is transported into the cell or if there are mechanisms of cellular compartmentalization or externalization of GC7. At last, mechanisms in order to metabolize the compound GC7 are not known. Therefore, the present project aimed to find new cellular targets for the GC7 compound using chemo-genetic screening and the haploid collection of knockouts in S. cerevisiae (nonessential genes). From the screening, GC7 sensitivity results were obtained for 96 knockout strains and 107 knockout strains showed resistance. The main pathways affected by GC7 are related to the transport and biosynthesis of amino acids and polyamines, vacuolar acidification and ribophagy. In addition to identifying these, other related knockout strains were tested and probable GC7 transporters to outside the cytoplasm were identified. It has also been found that GC7 reduces the levels of putrescine as well as in mammalian cell culture, and other results of this work also agree with a relevant impact of GC7 in this polyamine pathway. One of the most important results of this work was the resistance of ribophagy mutants to GC7, which was shown to be independent of eIF5A. Finally, we demonstrate that S. cerevisiae, in the genetic background of the knockout collection, is highly resistant to GC7 and that the effects of GC7 on the cell can not be reversed by the exogenous addition of spermidine, its structural analogue.
eIF5A is a translation factor involved in several cellular processes, as cellular proliferation control and inflammation. This factor undergoes a very conserved and specific posttranslational modification necessary to its activation named hypusination, which consists in the modification of a specific lysine in two steps. First, deoxyhypusine synthase enzyme, transfers 4-aminobutyl moiety from polyamine spermidine to a specific lysine residue in eIF5A precursor; immediately, deoxyhypusine hydroxylase adds a hydroxyl moiety in eIF5A intermediate to generate eIF5A hypusinated. Both, eIF5A and deoxyhypusine synthase are highly conserved from archaea to eukaryotes and are essential in Saccharomyces cerevisiae. Deoxyhypusine synthase is inhibited by N1-Guanyl-1,7-diaminoheptane (GC7), a structural analogue of spermidine. Nevertheless, recent findings have demonstrated that GC7 shows independent cellular effects of eIF5A, pointing out that GC7 has other targets. Besides, it is not known how GC7 is transported into the cell or if there are mechanisms of cellular compartmentalization or externalization of GC7. At last, mechanisms in order to metabolize the compound GC7 are not known. Therefore, the present project aimed to find new cellular targets for the GC7 compound using chemo-genetic screening and the haploid collection of knockouts in S. cerevisiae (nonessential genes). From the screening, GC7 sensitivity results were obtained for 96 knockout strains and 107 knockout strains showed resistance. The main pathways affected by GC7 are related to the transport and biosynthesis of amino acids and polyamines, vacuolar acidification and ribophagy. In addition to identifying these, other related knockout strains were tested and probable GC7 transporters to outside the cytoplasm were identified. It has also been found that GC7 reduces the levels of putrescine as well as in mammalian cell culture, and other results of this work also agree with a relevant impact of GC7 in this polyamine pathway. One of the most important results of this work was the resistance of ribophagy mutants to GC7, which was shown to be independent of eIF5A. Finally, we demonstrate that S. cerevisiae, in the genetic background of the knockout collection, is highly resistant to GC7 and that the effects of GC7 on the cell can not be reversed by the exogenous addition of spermidine, its structural analogue.
Descrição
Palavras-chave
Saccharomyces cerevisiae, eIF5A, Desoxi-hipusina sintase, Rastreamento, GC7, Deoxihipusine synthase, Screening