Studies on Perxisome Biogenesis and Peroxisomal Disorders

在这个巨大的科学研究援助09044094的支持下，我们获得了两种主要的发现：1）过氧化物酶体生物合成缺陷的新CHO细胞突变体的分离、表征和互补组分析。除了先前分离的，三互补群在过氧化物酶体缺陷型CHO细胞突变体ZP 24、Z65和ZP 92的CGs中，我们分离并分类了CGs新突变体细胞系，包括CG 1的ZP 107（与Z24相同的组）、CG 2、ZP 105/ZP 139、CG 3、ZP 109、ZP 110、ZP 114、CG-J、ZP 119、CG 8、ZP 124、ZP 126、CG-H、ZP 128、CG 11、ZPG 207和ZPG 208，通过P9 OH/UV法测定。发现ZP 110、ZP 114、ZP 126和ZPG 208与来自患有过氧化物酶体生物发生障碍（PBD）如Zellweger综合征的患者的人成纤维细胞的13种CD不同，表明哺乳动物中总共存在17种基因型。2）新过氧化物酶基因的克隆通过对CHO细胞突变株的遗传功能互补分析，我们克隆了几个过氧化物酶基因的cDNA（PEXs），包括ZP 107、ZP 109、ZP 128、ZP 110和ZP 119的PEX 1、PEX 12、PEX 13、PEX 14和PEX 19。然后，我们描述了PEX 1、PEX 12、PEX 13和PEX 19中的基因突变体分别负责CG 1（E）、CG 3、CG-H和CG-J的PBD。此外，我们通过使用酵母基因的表达序列标签（EST）DNA搜索分离了PEX 10和PEX 16，并证明PEX 10和PEX 16的失活分别是CG 7（B）和CG 9（D）PBD的遗传原因。我们还使用CG 2突变体ZP 105和ZP 139发现了过氧化物酶Pex 5 p（PTS 1受体）的两种同种型，证明了更长的Pex 5 p也参与PTS 2输入。此外，我们发现的证据表明，过氧化物酶体膜蛋白Pex 14 p在进口的PTS 1和PTS 2蛋白到过氧化物酶体中起着关键作用。

With the support of this Giant-in Aid for Scientific Research 09044094, we obtained two major types of findings :1) Isolation, characterization, and complementation group analysis of novel CHO cell mutants defective in peroxisome biogenesis.In addition to the previously isolated, three complementation groups (CGs) of peroxisome-deficient CHO cell mutants, ZP24, Z65, and ZP92, we isolated and classified in CGs novel mutant cell lines, including ZP107 of CG1 (the same group as Z24), CG2, ZP105/ZP139, CG3, ZP109, ZP110, ZP114, CG-J, ZP119, CG8, ZP124, ZP126, CG-H, ZP128, CG11, ZPG207, and ZPG208 by the P9OH/UV method. ZP110, ZP114, ZP126, and ZPG208 were found to be distinct from human 13 CDs of fibroblasts from patients with peroxisome biogenesis disorder (PBDs) such as Zellweger syndrome, indicating that totally 17 genotypes are present in mammals. Thus, it is evident that peroxisome assembly requires at least 17 gene-products.2) Cloning of novel peroxin genesBy genetic functional complementation assay of newly isolated CHO cell mutants, we cloned several peroxin cDNAs (PEXs), including PEX1, PEX12, PEX13, PEX14, and PEX19 for ZP107, ZP109, ZP128, ZP110, and ZP119 respectively. We then delineated that gene mutants in PEX1, PEX12, PEX13, and PEX19 are responsible for PBDs of CG1(E), CG3, CG-H, and CG-J, respectively. Moreover, we isolated PEX10 and PEX16 by expressed sequence tag (EST) DNA search using yeast genes and demonstrated that inactivation of PEX10 and PEX16 is the genetic cause of CG7 (B) and CG9 (D) PBDs, respectively. We also found two isoforms of the peroxin Pex5p (PTS1 receptor) using CG2 mutants, ZP105 and ZP139, demonstrating the longer Pex5p to be involved in PTS2 import as well. Moreover, we showed the evidence that peroxisomal membrane protein Pex14p plays a key role in import of PTS1 and PTS2 proteins into peroxisomes.