VERY LONG CHAIN FATTY ACYL-COA SYNTHETASE IN ADRENOLEUKODYSTROPHY

这个项目的长期目标是确定 过氧化物酶超长链脂肪酰辅酶A合成酶 X连锁肾上腺脑白质营养不良(XALD)的缺陷及其原因 这种蛋白质的编码基因存在缺陷。升高的血浆水平 超长链脂肪酸(VLCFA)，由过氧化物体受损引起 分解代谢，是这种疾病的生化标志。已完成的研究 过去5年的研究表明XALD的代谢障碍在 激活VLCFA的酶对其辅酶A衍生物的水平。 因此，这项提案的具体目的是审查这一点的作用 XALD中的VLCFA-CoA合成酶：1)纯化及性质 酶，2)确定它的亚细胞位置，3)检测它的 分子生物学，以及4)进行细胞内靶向研究。 VLCFA-CoA合成酶将纯化至均一，其氨基酸 顺序已确定。针对纯化蛋白产生的抗体将被 用于验证其在培养成纤维细胞中的过氧体亚细胞定位 来自正常对照组。XALD成纤维细胞将被分级，其 过氧核糖体检查这种蛋白质中的异常。基于氨基 酸序列，合成的寡核苷酸探针将被用来克隆 合成酶基因。或者，将根据 VLCFA酶与激活Short的合成酶的相似性 链脂肪酸；后一种酶已被克隆和测序。一次 该cDNA已被克隆，作图研究将确定VLCFA- CoA合成酶基因位于Xq28，该缺陷基因的已知基因座 XALD。如果合成酶映射到Xq28，XALD患者的基因组DNA将 检查有无异常。VLCFA-CoA合成酶靶向性研究 用PULSE/CHASE研究对照和XALD成纤维细胞中的过氧酶体 和显微注射实验。此外，合成酶将是 检查已知的针对过氧化物酶体的目标蛋白质的氨基酸序列。 综上所述，这些研究的结果应该会澄清 XALD的VLCFA-CoA合成酶和/或基因。

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.