Cloning and Functional Analysis of Mammalian Peroxisome Assembly Factors

过氧化物酶体是一种普遍存在的细胞器，存在于从酵母到人类的几乎所有真核细胞中。它执行重要的代谢功能，包括脂肪酸降解和其他氧化反应。已经鉴定了二十多个过氧化物酶体组装基因（PEX基因），并且已知由于PEX基因缺陷导致的人类过氧化物酶体生物发生障碍（PBD）。在本工作中，我们研究过氧化物酶体生物合成的机制，采用PBD患者的成纤维细胞和过氧化物酶体缺陷的CHO细胞系。结果表明：（1）克隆了几个新的PEX基因。(2)我们确定了几个PBD互补组的因果基因，并检测了负责的突变。(3)不同严重程度的病例常出现在同一PBD互补群中。对于几个互补组的PBD，成纤维细胞从患者的温和形式的PBD表现出温度敏感的表型，即，过氧化氢酶的过氧化物酶体在较低的温度下恢复。在一名患者中发现了一个致病点突变。来自这些患者的成纤维细胞具有“过氧化氢酶较少的过氧化物酶体”，其含有脂肪酸β-氧化酶但不含过氧化氢酶，这似乎是较温和表型的原因。(4)许多过氧化物酶体缺陷细胞含有缺乏过氧化物酶体基质蛋白的过氧化物酶体相关膜结构，称为“鬼”。PEX 2和PEX 5缺陷细胞的遗传互补表明，过氧化物酶体通过将基质蛋白运输到预先存在的鬼体而恢复。在缺失编码AAA-ATP酶的基因PEX 6的突变细胞中，发现过氧化物酶体基质蛋白在与鬼体紧密接触但与鬼体不同的膜结构中积累，这表明膜组装过程异常。

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.