The long term objective of this project is to determine whether the peroxisomal enzyme very long chain fatty acyl-CoA (VLCFA-CoA) synthetase is defective in X-linked adrenoleukodystrophy (XALD) and whether this is due to a defect in the gene coding for this protein. Elevated plasma levels of very long chain fatty acids (VLCFA), resulting from impaired peroxisomal catabolism, are the biochemical hallmark of this disease. Studies done over the last 5 years suggest that the metabolic block in XALD is at the level of the enzyme that activates VLCFA to their coenzyme A derivatives. Thus, the specific aims of this proposal are to examine the role of this enzyme, VLCFA-CoA synthetase, in XALD by 1) purifying and characterizing the enzyme, 2) determining its subcellular location, 3) examining its molecular biology, and 4) performing intracellular targeting studies. VLCFA-CoA synthetase will be purified to homogeneity and its amino acid sequence determined. Antibody raised against the purified protein will be used to verify its peroxisomal subcellular location in cultured fibroblasts from normal controls. XALD fibroblasts will be fractionated and their peroxisomes examined for abnormalities in this protein. Based on the amino acid sequence, synthetic oligonucleotide probes will be used to clone the synthetase cDNA. Alternatively, the cDNA will be cloned based on the similarity of the VLCFA enzyme to the synthetase that activates shorter chain fatty acids; the latter enzyme has been cloned and sequenced. Once the cDNA has been cloned, mapping studies will determine whether the VLCFA- CoA synthetase gene is in Xq28, the known locus of the defective gene in XALD. If the synthetase maps to Xq28, genomic DNA of XALD patients will be examined for abnormalities. Targeting of VLCFA-CoA synthetase to peroxisomes in control and XALD fibroblasts will be studied by pulse/chase and microinjection experiments. In addition, the synthetase will be examined for amino acid sequences known to target proteins to peroxisomes. Taken together, the results of these studies should clarify the role of the VLCFA-CoA synthetase enzyme and/or gene in XALD.
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