The overall objective of the proposed research is to investigate the molecular and cellular biology, and respective inborn errors, of the four cytosolic heme biosynthetic enzymes, delta- aminolevulinate dehydrase (ALA-D), hydroxylmethylbilane synthase (HMB-S), uroporphyrinogen III synthase (URO-S), and uroporphyrinogen decarboxylase (UROD). To facilitate these studies, we: I) isolated and characterized their full-length cDNA and genomic sequences, 2) identified erythroid-specific promoters for ALA-D and URO-S, 3) developed enzyme purification and assay procedures, 4) identified mutations causing their respective porphyrias, 5) isolated murine URO-S and URO-D sequences, and 6) constructed a novel retroviral vector for erythroid expression of URO-S. We propose to characterize the biochemical properties and 3D structures of each enzyme and to determine if they are associated in a cytosolic complex or """"""""metabolon"""""""" for the efficient, sequential metabolism of delta-aminolevulinate (ALA) to coproporphyrinogen (COPROgen) III. Large amounts of each recombinant enzyme (including erythroid HMB-S) will be produced and purified for characterization, antibody production, and crystallography. To investigate the metabolon hypothesis, functional tests for complex formation will be performed, and each enzyme will be differentially labeled with fluorescent dye-tagged antibodies in cultured HepG2 and K562 cells for simultaneous co-localization by confocal laser scanning microscopy, possibly in association with mitochondria If complexes are identified, their components will be characterized by cross-linking studies with hydrolyzable linkers and/or by immuno- affinity purification of the native complexes. Unknown components will be purified, microsequenced and their cDNAs isolated and expressed to assess their role in substrate metabolism, transport or complex assembly and anchoring. The cis-regulatory sequences and trans-acting proteins which may form a """"""""heme LCR"""""""" responsible for the coordinated up regulation of these enzymes during erythroid differentiation will be investigated, including expression in immortalized GATA-I inducible ES cells. We will continue to identify/characterize the mutations in each gene that cause the respective porphyria using long-range PcR and automated sequencing. To investigate the pathogenesis of congenital erythropoietic porphyria (CEP) and porphyria cutanea tarda (PCT), URO-S and URO-D deficient mice will be generated using murine targeting constructs with null or missense lesions for homologous recombination in Es cells followed by blastocyst implantation. Finally, efforts to treat murine CEP by bone marrow transplantation and stem cell gene therapy will be evaluated, including a novel retroviral vector containing the URO-S erythroid promoter and o'-globin LCR. These studies should provide fundamental understanding of the molecular and cellular biology of these enzymes in health and disease.
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