INBORN ERRORS OF REDOX PROTEINS--GLUTARIC ACIDEMIA II

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The objective of the proposed research is to investigate glutaric acidemia type II, a human inborn error of amino- and fatty-acid metabolism, and the proteins that are deficient in this disorder, namely electron transfer flavoprotein (ETF) and ETF: ubiquinone oxidoreductase (ETF-QO). These proteins catalyze electron transfer from mitochondrial flavoprotein dehydrogenases to the main respiratory chain. These investigations represent a continuation of the research that has been ongoing in our laboratories for more than ten years, and should fill majors gaps in our understanding of how these proteins interact with one another to transfer electrons from primary flavo-protein dehydrogenases of the mitochondrial matrix to ubiquinone (coenzyme Q) in the inner membrane, and how specific defects in these proteins perturb normal function and lead to glutaric acidemia type II. We now have on hand pure preparations of general (medium-chain) acyl-CoA dehydrogenase (GAD), ETF, and ETF-QO, immune serum to all three proteins, monoclonal antibodies against ETF alpha- and beta-subunits, and putative partial human cDNA sequences that encode ETF-QO and one of the two ETF subunits. Our specific objectives are to clone full length cDNA sequences for ETF-QO and the two ETF subunits, and to use these probes to determine the specific mutations that cause glutaric acidamia type II. Monoclonal antibodies will be produced against all three polypeptides to define the domains of ETF that react with primary dehydrogenases and ETF-QO, and to define the domains of ETF-QO that react with ETF and ubiquinone; the amino acid sequences of the domains will then be determined. The information on the functional and structural domains of ETF and ETF-QO developed from these approaches will then be utilized to construct a series of mutant proteins by in vitro mutagenesis and expression in E. coli, in order to address specific hypotheses about how the primary amino acid structure of these proteins influences their function.