Identification of novel PEX gene and functional analysis of Pexlp in peroxisome biogenesis

1)我们研究了CGI过氧化物酶体生物发生障碍(PBDS)的表型-基因型关系。来自IRD的Pex1p，如在G843D上发现最频繁突变的Pex1p，在37℃时在体内大部分被降解，而在允许的温度下可检测到正常水平的Pex1p。相比之下，来自ZS患者的PEX1蛋白，包括L664P突变或残基634-690缺失的蛋白，在两种温度下都稳定存在。Pex1p-G843D在蜂痘时与Pex6p相互作用。正常Pex1p水平的50%，而ZS患者的Pex1p大多表现为对温度不敏感的过氧化物体生物发生，几乎不与Pex6p结合。综上所述，我们认为Pexlp的稳定性很可能反映了IRD成纤维细胞对温度敏感的过氧化物酶的组装。Pex1p-Pex6p相互作用失败会导致更严重的异常，如ZS患者所表现的那样。2)我们发现CG4致病基因PEX6恢复了CG6 PBD成纤维细胞的过氧化物酶组装。该患者为PEX6等位基因复合杂合子。因此，通过将CG6和CG4.3合并，我们将人PBD分为12个CG。)我们最近利用CHO细胞突变体ZP167分离到编码一种新的34 kDa II型过氧体膜蛋白的人PEX26。PEX26的表达仅在CG8PBD患者的成纤维细胞中恢复了过氧化体蛋白的输入。该患者具有一个纯合子、失活的病原点突变R98W。因此，我们可以说，所有与12个CGS PBD有关的致病基因都已被克隆。此外，AAA ATPase家族的Pex6p和Pex1p与Pex26p共沉淀。结合几条形态证据，我们得出结论，Pex26p招募Pex6p-Pex1p复合体为过氧体。

1) We investigated phenotype-genotype relationships of CGI peroxisome biogenesis disorders (PBDs). Pex1p from IRD such as Pex1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37℃, whereas a normal level of Pexlp was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634-690, were stably present at both temperatures. Pex1p-G843D interacted with Pex6p at appox. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of Pexlp reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in Pex1p-Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.2) We showed that PEX6, the CG4 pathogenic gene, restored peroxisome assembly in CG6 PBD fibroblasts. This patient was compound heterozygous for PEX6 alleles. Accordingly, human PBDs are classified into 12 CGs by merging CG6 with CG4.3) We recently isolated human PEX26 encoding a novel, 34-kDa type II peroxisomal membrane protein, using a CHO cell mutant ZP167. PEX26 expression restored peroxisomal protein import in only the CG8 PBD patient's fibroblasts. This patient possessed a homozygous, inactivating pathogenic point mutation, R98W. Accordingly, we can state that all of pathogenic genes responsible for 12 CGs PBDs have been cloned. Moreover, Pex6p and Pex1p of the AAA ATPase family were co-immunoprecipitated with Pex26p. Together with several lines of morphological evidence, we concluded that Pex26p recruits Pex6p-Pex1p complexes to peroxisomes.