Caracterização funcional da isoforma A do fator de início de tradução de eucariotos 5A (eIF5A)
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Data
2019-09-16
Autores
Pereira-Thomaz, Karina Danielle
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O fator de início de tradução de eucariotos 5A (eIF5A) é uma proteína altamente conservada em archaeas e eucariotos. Caracteriza-se por ser a única proteína conhecida a apresentar o resíduo de aminoácido hipusina, produzido a partir de uma modificação pós-traducional denominada hipusinação, a qual é essencial para sua atividade. Atualmente eIF5A é correlacionada com o início, elongação e término da tradução, além disso, sua função têm sido associada com diferentes processos celulares, fisiológicos e patológicos. Em humanos, é predito que eIF5A seja gerada a partir de cinco variantes transcricionais (A, B, C, D e X5). As variantes B, C, D e X5 codificam a proteína canônica isoforma B de 17 kDa, a qual vêm sendo estudada nas últimas décadas. Entretanto, é previsto que a variante A codifique uma isoforma alternativa (isoforma A) de 20 kDa, que tem uma sequência adicional de 30 aminoácidos na extremidade N-terminal. Neste contexto, o presente estudo teve como objetivo caracterizar eventos moleculares inéditos envolvendo a isoforma A de eIF5A1 humana. Neste estudo, mostramos que a isoforma A é produzida em células HeLa, a qual foi mostrada ser passível de hipusinação. Análises de bioinformática mostraram que a isoforma A possui alta probabilidade de direcionamento para mitocôndria. Assim, técnicas de fracionamento subcelular mostraram que a isoforma A co-purifica com a mitocôndria, diferentemente da isoforma B. Análises de silenciamento gênico em células HeLa, utilizando moléculas de siRNA específicas para isoforma A de eIF5A1, mostraram que a depleção da proteína é capaz de causar apoptose, além de reduzir a viabilidade e causar alteração no ciclo celular. Ademais, análises moleculares evidenciaram que essa supressão causou disfunção mitocondrial, a qual foi evidenciada pela alteração na expressão de genes envolvidos no metabolismo oxidativo, e pelo aumento na produção de espécies reativas de oxigênio (EROS). Análises de expressão gênica também mostraram que a isoforma A participa do shift metabólico (mudança de metabolismo glicolítico para oxidativo e vice-versa). Ainda, o aumento da capacidade respiratória pela supressão da isoforma A e alterações na morfofisiologia mitocondrial evidenciados pelo aumento da fissão mitocondrial e número de cristas, sugerem uma atividade desordenada da mitocôndria através de um mecanismo compensatório. Coletivamente, os resultados deste estudo indicam que a isoforma A de eIF5A1 é um componente fundamental no controle da função mitocondrial representando uma interface promissora para o estudo do metabolismo oxidativo mitocondrial e associação com o processo de síntese proteica.
The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein in archaeas and eukaryotes. It is characterized by being the only protein known to present the amino acid residue hypusine, produced from a post-translational modification called hypusination, which is essential for its activity. Currently eIF5A is correlated with the beginning, elongation and termination of translation, and its function has been associated with different cellular, physiological and pathological processes. In humans, eIF5A is predicted to be generated from five transcriptional variants (A, B, C, D and X5). Variants B, C, D and X5 encode the 17 kDa canonical protein B, which has been studied in recent decades. However, variant A is expected to encode an alternative 20 kDa isoform (A isoform), which has an additional 30 amino acid sequence in the N-terminal region. In this context, the present study aimed to characterize unpublished molecular events involving the human eIF5A1 isoform A. In this study, we showed that in HeLa cells, isoform A was detected, which was shown to be subject to hypusination. Bioinformatics analysis showed that isoform A has a high probability of targeting mitochondria. Thus, subcellular fractionation techniques have shown that isoform A co-purifies with mitochondria, unlike isoform B. Analysis of gene silencing in HeLa cells using eIF5A1 isoform A-specific siRNA molecules showed that protein depletion is capable of cause apoptosis in cells, as well as decrease viability and cause changes in the cell cycle. In addition, molecular analyzes have shown that this suppression caused mitochondrial dysfunction, which was evidenced by altered expression of genes involved in oxidative metabolism, and increased production of reactive oxygen species (ROS). Gene expression analyzes also showed that isoform A participates in metabolic shift (change from glycolytic to oxidative metabolism and vice versa). Moreover, the increase in respiratory capacity through suppression of isoform A and changes in mitochondrial morphophysiology evidenced by increased mitochondrial fission and number of ridges, suggest a disordered activity of mitochondria through a compensatory mechanism. Collectively, the results of this study indicate that the eIF5A1 isoform A is a key component in mitochondrial function control representing a promising interface for the study of mitochondrial oxidative metabolism and association with the protein synthesis process.
The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein in archaeas and eukaryotes. It is characterized by being the only protein known to present the amino acid residue hypusine, produced from a post-translational modification called hypusination, which is essential for its activity. Currently eIF5A is correlated with the beginning, elongation and termination of translation, and its function has been associated with different cellular, physiological and pathological processes. In humans, eIF5A is predicted to be generated from five transcriptional variants (A, B, C, D and X5). Variants B, C, D and X5 encode the 17 kDa canonical protein B, which has been studied in recent decades. However, variant A is expected to encode an alternative 20 kDa isoform (A isoform), which has an additional 30 amino acid sequence in the N-terminal region. In this context, the present study aimed to characterize unpublished molecular events involving the human eIF5A1 isoform A. In this study, we showed that in HeLa cells, isoform A was detected, which was shown to be subject to hypusination. Bioinformatics analysis showed that isoform A has a high probability of targeting mitochondria. Thus, subcellular fractionation techniques have shown that isoform A co-purifies with mitochondria, unlike isoform B. Analysis of gene silencing in HeLa cells using eIF5A1 isoform A-specific siRNA molecules showed that protein depletion is capable of cause apoptosis in cells, as well as decrease viability and cause changes in the cell cycle. In addition, molecular analyzes have shown that this suppression caused mitochondrial dysfunction, which was evidenced by altered expression of genes involved in oxidative metabolism, and increased production of reactive oxygen species (ROS). Gene expression analyzes also showed that isoform A participates in metabolic shift (change from glycolytic to oxidative metabolism and vice versa). Moreover, the increase in respiratory capacity through suppression of isoform A and changes in mitochondrial morphophysiology evidenced by increased mitochondrial fission and number of ridges, suggest a disordered activity of mitochondria through a compensatory mechanism. Collectively, the results of this study indicate that the eIF5A1 isoform A is a key component in mitochondrial function control representing a promising interface for the study of mitochondrial oxidative metabolism and association with the protein synthesis process.
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Palavras-chave
eIF5A1, Isoforma A, Mitocôndria, Metabolismo oxidativo, Isoform A, Mitochondria, Oxidative metabolism