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Evaluation of mitochondrial beta-oxidation flavoenzymes in a cellular model of Friedreich ataxia
Perspectives of extension of this common project to a more ambitious proposal to be submitted to funding agencies : Long term Added value of the cooperation : Exchange of expertise
Summary of the proposal :
Frataxin deficiency is the cause of Friedreich ataxia, a neurodegenerative disorder. Physiological function of frataxin in mitochondria has not been well understood and several hypotheses on the pathogenic mechanisms have been postulated. The Palau laboratory has described new interactions, by co-immunoprecipation assays, of Saccharomyces cerevisiaefrataxin orthologue Yfh1p with succinate dehydrogenase complex subunits Sdh1p and Sdh2pof the yeast mitochondrial electron transport chainand also with ETF-alpha and ETF-beta subunits from the electron transfer flavoprotein complex.Flavin adenine dinucleotide (FAD) is a prosthetic group that is covalently anchored to the SDHA subunit of Complex II and also acts as a cofactor to the ETF complex. We showed that riboflavin-derived cofactors, especially FAD, rescue cell growth and enzymatic activities of the electron transport chain, except complex II, in yfh1Δyeast. We suggested that improving in yeast growth is not related with complex II but might be related with the electron transfer flavoprotein (ETF) complex. The Gomes lab has been investigating ETF as well as other enzymes in the context of mitochondrial diseases affecting fatty acid metabolism, also in respect to the effects of riboflavin supplementation, and we have optimized procedures for protein and activity analysis in cell extracts.
At this point we are interested to understand how frataxin deficiency affects the activity of mitochondrial fatty acids beta oxidation enzymes. With this purpose we have developed a chronic model of this disease, with RNAi technology, generating stable lines deficient for frataxin in a neuronal human line of neuroblastoma (SH-SY5Y). Up to now we known that frataxin silencing leads to a slower cell growth, and the unique activity of electron transport chain perturbed was at the level of complex IV. This proporal aims at 1) Analyse the expression levels of fatty acid beta oxidation protein levels using immunoblotting and monoclonal antibodies and 2) determine the enzymatic activity of several fatty acid beta oxidation enzymes (e.g MCAD, VLCAD and ETF) in cellular extracts from frataxin deficient human neuroblastoma cell lines (SH-SY5Y).
Antonio PINEDA-LUCENA, Valencia Claudio GOMES, Lisboa
Using metabonomics to understand fatty acid oxidation disease