Both the familial and sporadic forms of porphyria cutanea tarda (F-PCT and S-PCT) are due to abnormal function of uroporphyrinogen decarboxylase (URO-D). Inherited factors play a role in both forms, but in F-PCT URO-D is mutant whereas in S-PCT it is not. Although URO-D activity is subnormal in the liver in both forms, the enzyme should not be rate-limiting. The mechanism for the accumulation of the oxidized substrate of URO-D is unknown. We propose to establish structure-function relationships in normal and mutant URO-Ds and to test the hypothesis that clinical expression of both forms of PCT is due to an abnormality in the genetic control of cytochrome P45OIA2 activity. We will define the domains of URO-D required for substrate binding, catalytic activity and protein stability using amino acid modifying reagents, site-directed and random mutagenesis, crystallography and characterization of mutations occurring in F-PCT. Amino acid residues required for URO-D function will be determined and conserved domains in human URO-D will be evaluated using site-directed and random mutagenesis. We have identified stability mutants of URO-D in F-PCT. We will determine if ubiquitin-dependent proteolysis mediates degradation of URO-D using antibodies against specific components of the ubiquitin system. Crystallization of human URO-D will be done in collaboration with Dr. James Hogle of Harvard University. URO-D mutations in F-PCT will be characterized in members of 90 families with PCT followed in our porphyria clinic. The P45OIA2 hypothesis will be tested in two ways: First, cell culture experiments will be done employing both yeast and mammalian cells transfected with a human P45OIA2 cDNA in appropriate expression vectors. The ability of transfected cells to oxidize the substrate of URO-D and/or modify URO-D protein will be determined. We will also measure oxidation of substrates of URO-D and uroporphyrinogen III cosynthase by liver microsomes from rats given P450 inducers to determine if substrate oxidation is responsible for the preponderance of uroporphyrin I found in PCT. Second, the metabolism of caffeine will be used to determine P45OIA2 activity in PCT patients in vivo and P45OIA2 cDNAs from liver biopsy samples will be characterized using PCR amplification. Our findings in PCT could serve as a paradigm for the other dominantly transmitted porphyrias where clinical expression cannot be simply-explained by half-normal activity of non-rate-limiting heme biosynthetic enzymes.
| Piel 3rd, Robert B; Shiferaw, Mesafint T; Vashisht, Ajay A et al. (2016) A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase. Biochemistry 55:5204-17 |
| Hanson, W Miachel; Chen, Zhe; Jackson, Laurie K et al. (2016) Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor ? and ? Chain mRNAs. J Am Chem Soc 138:11073-6 |
| Dailey, Harry A; Gerdes, Svetlana; Dailey, Tamara A et al. (2015) Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin. Proc Natl Acad Sci U S A 112:2210-5 |
| Medlock, Amy E; Shiferaw, Mesafint T; Marcero, Jason R et al. (2015) Identification of the Mitochondrial Heme Metabolism Complex. PLoS One 10:e0135896 |
| Yien, Yvette Y; Robledo, Raymond F; Schultz, Iman J et al. (2014) TMEM14C is required for erythroid mitochondrial heme metabolism. J Clin Invest 124:4294-304 |
| Lorenzo 5th, Felipe R; Phillips, John D; Nussenzveig, Roberto et al. (2011) Molecular basis of two novel mutations found in type I methemoglobinemia. Blood Cells Mol Dis 46:277-81 |
| Phillips, John D; Kushner, James P; Bergonia, Hector A et al. (2011) Uroporphyria in the Cyp1a2-/- mouse. Blood Cells Mol Dis 47:249-54 |
| To-Figueras, J; Phillips, J D; Gonzalez-López, J M et al. (2011) Hepatoerythropoietic porphyria due to a novel mutation in the uroporphyrinogen decarboxylase gene. Br J Dermatol 165:499-505 |
| Ajioka, Richard S; Phillips, John D; Weiss, Robert B et al. (2008) Down-regulation of hepcidin in porphyria cutanea tarda. Blood 112:4723-8 |
| Akagi, Reiko; Inoue, Rikako; Muranaka, Shikibu et al. (2006) Dual gene defects involving delta-aminolaevulinate dehydratase and coproporphyrinogen oxidase in a porphyria patient. Br J Haematol 132:237-43 |
Showing the most recent 10 out of 30 publications
