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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Medical Biochemistry Study Section (MEDB)
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Badman, David G
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Mount Sinai School of Medicine
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New York
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Salameh, H; Sarairah, H; Rizwan, M et al. (2018) Relapse of porphyria cutanea tarda after treatment with phlebotomy or 4-aminoquinoline antimalarials: a meta-analysis. Br J Dermatol 179:1351-1357
Clavero, Sonia; Ahuja, Yuri; Bishop, David F et al. (2013) Diagnosis of feline acute intermittent porphyria presenting with erythrodontia requires molecular analyses. Vet J 198:720-2
Balwani, Manisha; Desnick, Robert J (2012) The porphyrias: advances in diagnosis and treatment. Hematology Am Soc Hematol Educ Program 2012:19-27
Balwani, Manisha; Desnick, Robert J (2012) The porphyrias: advances in diagnosis and treatment. Blood 120:4496-504
Hasanoglu, Alev; Balwani, Manisha; Kasapkara, Ci?dem S et al. (2011) Harderoporphyria due to homozygosity for coproporphyrinogen oxidase missense mutation H327R. J Inherit Metab Dis 34:225-31
Machaczka, Maciej; Klimkowska, Monika; Regenthal, Sofie et al. (2011) Gaucher disease with foamy transformed macrophages and erythrophagocytic activity. J Inherit Metab Dis 34:233-5
Clavero, Sonia; Bishop, David F; Giger, Urs et al. (2010) Feline congenital erythropoietic porphyria: two homozygous UROS missense mutations cause the enzyme deficiency and porphyrin accumulation. Mol Med 16:381-8
Cantatore-Francis, Julie L; Cohen-Pfeffer, Jessica; Balwani, Manisha et al. (2010) Hepatoerythropoietic porphyria misdiagnosed as child abuse: cutaneous, arthritic, and hematologic manifestations in siblings with a novel UROD mutation. Arch Dermatol 146:529-33
Yasuda, Makiko; Bishop, David F; Fowkes, Mary et al. (2010) AAV8-mediated gene therapy prevents induced biochemical attacks of acute intermittent porphyria and improves neuromotor function. Mol Ther 18:17-22
Bishop, David F; Schneider-Yin, Xiaoye; Clavero, Sonia et al. (2010) Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively spliced transcripts. Blood 115:1062-9

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