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Protein misfolding and cellular responses in metabolic disorders
Publication . Lucas, Tânia; Gomes, Cláudio Emanuel Moreira
This dissertation concerns studies on the electron transfer flavoprotein:ubiquininone oxidoreductase (ETF:QO), a structurally complex membrane protein containing three cofactors organized into three different structural domains. ETF:QO and its partner electron transfer flavoprotein (ETF), constitute an important hub in mitochondrial metabolism mediating electron transfer from several dehydrogenases to the respiratory chain for ATP production. Defects in any of these two proteins result in multiple acyl-CoA dehydrogenase deficiency (MADD), a rare inborn metabolic disorder presenting a broad range of clinical phenotypes, some responding to riboflavin supplementation. Although several MADD cases have been identified, the molecular studies to clarify disease mechanism are scarce, and clinicians have difficulty predicting disease progression and response to riboflavin therapy. Therefore, conformational and structural characterization of ETF:QO is an important step to identify how different mutations impact on clinical phenotypes.
In order to elucidate the molecular aspects behind MADD and riboflavin supplementation, we revisited a riboflavin responsive MADD clinically relevant mutation in ETF:QO (ETF:QO-p.Pro456Leu) and investigated how it affects the enzyme structure and fold. As model, we took advantage of the higher expression levels of the Rhodobacter sphaeroides ETF:QO in Escherichia coli and its 67 % sequence similarity to the mature human protein.
Nevertheless, not all MADD mutations are conserved between these species, and using the recombinant human ETF:QO would have been a better choice. However, human ETF:QO heterologous expression in E. coli has not been a straight forward process. Here, we describe a tour de force approach to better understand the critical steps underlying the folding of human ETF:QO. We investigated possible origins for these difficulties and analysed the possible codon-usage effects, a factor known to influence and regulate protein folding. We also analysed and hypothesized that the codon usage frequency variation might be a possible cause for some MADD related phenotypes. In the last chapter the relationship between fatty acid oxidation and ETF:QO in cancer is discussed.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/EBB-BIO/117793/2010