VERY LONG CHAIN FATTY ACYL-COA SYNTHETASE IN ADRENOLEUKODYSTROPHY

肾上腺脑白质营养不良中的极长链脂肪酰基辅酶A合成酶

• 批准号: 5212455
• 负责人: PAUL A WATKINS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5212455
• 关键词: acyl coA; complementary DNA; enzyme deficiency; fatty acid metabolism; genetic disorder; genetic mapping; human tissue; laboratory rabbit; ligase; long chain fatty acid; molecular biology; molecular cloning; neural degeneration; nucleic acid sequence; oligonucleotides; oxidation; peroxisome; sex linked trait

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.

该项目的长期目标是确定是否 过氧化物酶体酶超长链脂肪酰辅酶A (VLCFA-CoA) 合成酶是 X 连锁肾上腺脑白质营养不良 (XALD) 缺陷以及这是否是由于 编码该蛋白质的基因存在缺陷。 血浆水平升高 极长链脂肪酸（VLCFA），由过氧化物酶体受损引起 分解代谢，是这种疾病的生化标志。 研究完成 过去 5 年的研究表明，XALD 中的代谢阻滞处于 将 VLCFA 激活为其辅酶 A 衍生物的酶水平。 因此，本提案的具体目标是研究该机制的作用。 XALD 中的酶，VLCFA-CoA 合成酶，通过 1) 纯化和表征 酶，2) 确定其亚细胞位置，3) 检查其 分子生物学，4) 进行细胞内靶向研究。 VLCFA-CoA合成酶将被纯化至同质及其氨基酸 顺序确定。 针对纯化蛋白质产生的抗体将是 用于验证其在培养的成纤维细胞中的过氧化物酶体亚细胞位置 来自正常控制。 XALD 成纤维细胞将被分级，并且它们的 过氧化物酶体检查了该蛋白质的异常情况。 基于氨基 酸序列，合成寡核苷酸探针将用于克隆 合成酶cDNA。 或者，cDNA 将根据 VLCFA 酶与激活较短的合成酶的相似性 链脂肪酸；后一种酶已被克隆并测序。 一次 cDNA 已被克隆，图谱研究将确定 VLCFA- CoA 合成酶基因位于 Xq28，这是已知的缺陷基因位点 XALD。 如果合成酶定位到 Xq28，则 XALD 患者的基因组 DNA 将是 检查是否有异常。 VLCFA-CoA 合成酶的靶向 将通过脉冲/追踪研究对照和 XALD 成纤维细胞中的过氧化物酶体 和显微注射实验。 此外，合成酶将 检查已知将蛋白质靶向过氧化物酶体的氨基酸序列。 总而言之，这些研究的结果应该阐明了 XALD 中的 VLCFA-CoA 合成酶和/或基因。