PHYTANIC ACID OXIDATION IN DISEASE & PEROXISOME ASSEMBLY

该项目的长期目标是确定为什么植酸 阿尔法氧化在三种先天性代谢性疾病中是有缺陷的：Refsum 疾病、根性点状软骨发育不良和过氧化物酶体紊乱 并研究这一途径中的酶与 过氧化物酶体的组装。阿尔法氧化，一种缩短的途径 将异戊二烯类脂肪酸的链长增加1-碳以使其能够 经过正常的贝塔氧化，在30多年前被发现。 然而，许多关于酶、反应和特定的 疾病方面的缺陷仍有待回答。因此，这一行动的具体目标是 项目有：1)直接演示特定的酶缺陷 Refsum病，并确定该酶或后续的酶是否在 途径在RCDP中有缺陷；2)使用纯化的植酰辅酶Aα- 羟基酶，获得该酶的抗体并获得编码该酶的基因 3)确定植酰辅酶Aα-羟基酶是否如预期的那样 具有氨基末端的过氧化酶体靶向信号(Pts2)，并 研究其在过氧化体组装和相关疾病中的作用；以及 4)阐明植酸α-氧化的后期步骤 路径。这条途径的细节将用纯化的 从大鼠肝脏中提取的过氧化物体和线粒体制剂，并在 人类成纤维细胞。化验将主要使用特定的放射性标记 底物。纯化的α-羟基酶将用于生成 用于亚细胞靶向和定位研究的多克隆抗体 并克隆其cDNAs。CDNAs和氨基酸序列将使 其靶向序列的鉴定，我们认为是PTS2- 型过氧化物酶体靶向信号。利用分子技术， 对pts2的特性进行了研究，并进行了酵母双杂交 系统将被用来识别其受体。代谢缺陷将会是 应用酶分析和免疫学方法对患者成纤维细胞的研究 技巧。这一结果不仅将加深我们对 疾病，但也将澄清确切的机制和 这条途径的细胞器位置。最后，如果α-羟基酶 确实有pts2信号，它将只是第二种动物酶 确定使用此信号；因此，它将增强我们的 了解过氧化体组装的一般现象。

This project's long-term objectives are to determine why phytanic acid alpha-oxidation is defective in three inborn errors of metabolism: Refsum disease, rhizomelic chondrodysplasia punctata, and disorders of peroxisome assembly and to investigate the relationship of enzymes in this pathway to the assembly of peroxisomes. Alpha-Oxidation, a pathway for shortening the chain length of isoprenoid-type fatty acids by 1-carbon to enable them to undergo normal beta-oxidation, was identified more than 30 years ago. Yet, many fundamental questions about the enzymes, reactions, and specific defects in disease remain to be answered. Thus, the specific aims of this project are: 1) to demonstrate directly the specific enzyme defect in Refsum disease and to determine whether this or subsequent enzymes in the pathway are defective in RCDP; 2) to use purified phytenoyl-CoA alpha- hydroxylase to obtain antibody to this enzyme and to obtain cDNA encoding it; 3) to determine whether, as expected, phytanoyl-CoA alpha-hydroxylase has an amino-terminal peroxisome targeting signal (PTS2) and to investigate its role in peroxisome assembly and associated disorders; and 4) to elucidate the latter steps of the phytanic acid alpha-oxidation pathway. Details of the pathway will be investigated using purified peroxisome and mitochondria preparations from rat liver and confirmed in human fibroblasts. Assays will mainly employ specific radiolabeled substrates. Purified alpha-hydroxylase will be used both to generate polyclonal antibodies for subcellular targeting and localization studies and for cloning its cDNA. The cDNA and amino acid sequence will enable the identification of its targeting sequence, which we believe is a PTS2- type peroxisome targeting signal. Using molecular techniques, the characteristics of PTS2 will be investigated and the yeast two-hybrid system will be used to identify its receptor. Metabolic defects will be studied in fibroblasts from patients using enzyme assays and immunologic techniques. The results will not only further our understanding of the diseases, but will also clarify both the exact mechanism and the organellar location of this pathway. Finally, if the alpha-hydroxylase does have a PTS2 signal, it will be only the second animal enzyme identified as employing this signal; thus, it will enhance our understanding of the general phenomenon of peroxisome assembly.