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.
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