PHYTANIC ACID OXIDATION IN DISEASE & PEROXISOME ASSEMBLY

该项目的长期目标是确定植烷酸 α-氧化在三种先天性代谢缺陷中是有缺陷的：Refsum 疾病、点状肢根软骨发育不良和过氧化物酶体疾病 组装，并研究该途径中的酶与 过氧化物酶体的组装。 α-氧化，缩短的途径 类异戊二烯型脂肪酸的链长增加1-碳， 进行正常的β-氧化，是在30多年前发现的。 然而，许多关于酶、反应和具体的 疾病的缺陷仍有待解决。 因此，这一具体目标 项目是：1）直接证明特定的酶缺陷， Refsum病，并确定是否这种或随后的酶在 途径在RCDP中有缺陷; 2）使用纯化的植烯酰辅酶A α- 羟化酶以获得该酶抗体，并获得编码 3）确定是否如预期的那样，植烷酰-CoA α-羟化酶 具有氨基末端过氧化物酶体靶向信号（PTS 2）， 研究其在过氧化物酶体组装和相关疾病中的作用; 4)为了阐明植烷酸α-氧化的后面步骤 通路 该途径的细节将使用纯化的 过氧化物酶体和线粒体制备大鼠肝脏和证实， 人成纤维细胞。 检测将主要采用特定的放射性标记 印刷受体. 纯化的α-羟化酶将用于产生 用于亚细胞靶向和定位研究的多克隆抗体 并克隆其cDNA。 cDNA和氨基酸序列将使 其靶向序列的鉴定，我们认为是PTS 2- 型过氧化物酶体靶向信号。 利用分子技术， PTS 2的特性将被研究，酵母双杂交 系统将用于识别其受体。 代谢缺陷将是 使用酶测定和免疫学方法在患者的成纤维细胞中研究 技术. 结果不仅将进一步加深我们对 疾病，但也将澄清确切的机制和 这条通路的细胞器位置。 最后，如果α-羟化酶 有PTS 2信号，它将是第二种动物酶， 确定使用此信号;因此，它将增强我们的 了解过氧化物酶体组装的一般现象。

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.