Microbodies, i.e., peroxisomes and microperoxisomes, are ubiquitous catalase-containing subcellular organelles whose exact function in cellular metabolism is not clear. It is generally believed that the major function of these organelles is to metabolize various compounds by oxidation. However, we have shown that in animals these organelles also play a major role in membrane lipid biosynthesis. We demonstrated that the key enzymes of the acyl dihydroxyacetone phosphate pathway for lipid biosynthesis are localized in the membrane of microbodies. Thus pathway is the only route for the biosynthesis of either lipids (glycerol ether lipids and plasmalogens), which are present in all animals. The obligatory role of peroxisomes in ether lipid biosynthesis is manifested by the discovery of a number of genetic diseases (Zellweger cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy, rhizomelic chondrodysplasia punctata, etc.) in which tissues of the patients are deficient in both peroxisomes and ether lipids. We have shown that the absence of ether lipids is due to the deficiency of a key peroxisomal enzyme which catalyzes the synthesis of acyl dihydroxyacetone phosphate. The major objective of this research proposal is to investigate the role of microbodies in the regulation of cellular lipid biogenesis at the molecular level. We will pursue our hypothesis that the main function of microbodies is to compartmentalize the substrates with specific topologic arrangements of the enzymes so that biochemical reactions leading to the formation of membrane lipid intermediates can be effectively regulated. The studies on ate purification and properties of peroxisomal membrane-bound acyl DHAP pathway enzymes and the determination of their molecular structures will be continued. The following specific aims are proposed: 1) To purify the two key enzymes of the acyl DHAP pathway, dihydroxyacetone phosphate (DHAP) acyltransferase and acyl/alkly DHAP reductase, with modified protocols so that sufficient quantities are obtained from structural studies and antibodies production. The molecular properties, regulation and biogenesis of these two enzymes will be investigated by determining their primary structure by cloning and sequencing the corresponding c-DNA's. 2) To generate specific antibodies against these two enzymes and to use these antibodies to study the tissue distribution, subcellular localization and regulation of the enzymes at the molecular level. These antibodies will be also used to detect the presence of mutant enzyme in peroxisomal genetic diseases such as Zellweger syndrome and rhizomelic chondrodysplasta punctata. 3) To study the mechanism of enzymatic generation and transport of metabolites for lipid biosynthesis in microbodies. The biochemical mechanism for the synthesis of specific membrane lipids, such as ethanolamine plasmalogens, from the intermediates of the acyl DHAP pathway will be investigated. 4). To investigate the molecular structure, function and regulation of a peroxisomal lipid-metabolizing enzyme, acyl Coenzyme A thioesterase, a very active constitutive brain enzyme which is also highly induced in rodent liver by peroxisome-proliferating drugs.
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