Cloning and Functional Analysis of Mammalian Peroxisome Assembly Factors

过氧化物体是一种普遍存在的细胞器，几乎存在于从酵母到人类的所有类型的真核细胞中。它执行重要的代谢功能，包括脂肪酸降解和其他氧化反应。目前已鉴定出20多个Peroxisome组装基因(PEX基因)，并已知由PEX基因缺陷引起的人类过氧化物酶体生物发生障碍(PBDS)。在目前的工作中，我们利用PBD患者的成纤维细胞和过氧酶体缺陷的CHO细胞系来研究过氧酶体的生物发生机制。结果如下：(1)我们克隆了几个新的PEX基因。(2)我们鉴定了几个PBD互补组的随意基因，并检测了相应的突变。(3)在相同的PBD互补组中经常发现不同严重程度的病例。对于几个PBDS的互补组，轻度PBDS患者的成纤维细胞表现出温度敏感的表型，即含过氧化氢酶的过氧化物体在较低的温度下被恢复。在一名患者身上发现了一个因果点突变。这些患者的成纤维细胞有“无过氧化氢酶的过氧化氢酶”，其中含有脂肪酸β氧化酶，但没有过氧化氢酶，这似乎是较温和的表型的原因。(4)许多过氧酶体缺陷细胞含有与过氧酶体相关的膜结构，缺乏过氧酶体基质蛋白，称为“幽灵”。PEX2和PEX5缺陷细胞的遗传互补表明，通过将基质蛋白运输到预先存在的幽灵中，过氧化物体可以被恢复。在编码AAA-ATPase基因PEX6缺陷的突变细胞中，发现与幽灵密切接触但不同于幽灵的膜结构中积累了过氧化物体基质蛋白，提示膜组装过程异常。

Peroxisome is a ubiquitous organelle, present in almost all types of eukaryotic cells from yeast to humans. It carries out important metabolic functions including fatty acid degradation and other oxidative reactions. More than twenty peroxisome assembly gene (PEX genes) have been identified, and human peroxisome biogenesis disorders (PBDs) due to the PEX gene deficiencies are known. In the present work, we studied the mechanism of peroxisome biogenesis, employing the fibroblasts of PBD patients and peroxisome-defective CHO cell lines. The results are :(1) We cloned several new PEX genes.(2) We identified casual genes for several complementation groups of PBDs, and detected the responsible mutations.(3) Cases of different severity are often found in the same complementation groups of PBDs. For several complementation groups of PBDs, fibroblasts from the patients of milder forms of PBDs exhibited a temperature-sensitive phenotype, that is, catalase-containing peroxisomes were recovered at lower temperatures. A causal point mutation was identified in a patient. Fibroblasts form these patients have "catalase-less peroxisomes" that contain fatty acid β-oxidation enzymes but not catalase, which seemed to be the cause of the milder phenotype.(4) Many peroxisome-deficient cells contain peroxisome-related membrane structures lacking the peroxisome matrix proteins, called "ghosts". It was shown by genetic complementation of the PEX2- and PEX5-defective cells, that peroxisomes were recovered by the transport of matrix proteins to the pre-existing ghosts. In the mutant cells defective of PEX6, a gene coding for an AAA-ATPase, peroxisome matrix proteins were found to be accumulated in membrane structures closely contacting with, but different from, the ghosts, suggesting an abnormality in the membrane assembly processes.