Electronic Thesis/Dissertation


Characterization of UBIAD1 and Its Role in Schnyder Corneal Dystrophy Open Access

Schnyder corneal dystrophy (SCD) is an inherited, autosomal dominant eye disease first described in 1924. Mutations in a novel gene, UBIAD1, were recently found to cause SCD. SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea, resulting in progressive corneal opacification and visual loss. We characterized UBIAD1 alterations in ten new SCD families, including five novel mutations, A97T, D112N, V122E, V122G, and L188H. A V122E mutation was observed in a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids that were highly conserved across species. Immunohistochemistry on keratocyte cell lines established after corneal transplant surgery using antibodies specific for UBIAD1 established a subcellular localization to mitochondria of N102S mutant and wild-type protein. Cell line extracts of peripheral blood mononuclear cells immortalized using Epstein-Barr virus were used to examine levels of total cholesterol, cholesteryl ester, and unesterified cholesterol. A fluorometric assay showed no significant alteration in cellular amounts of these molecules relative to non-SCD cell lysates. Molecular modeling was used to examine membrane protein structure, binding of potential ligands, and the effect(s) of SCD mutations. A membrane protein with eight helices was observed, with five residues altered in SCD predicted to lie near the primary active site: A97, N102, D112, V122, and L188. Substrate docking simulations showed active site binding of geranyl- and farnesylpyrophosphates. Prenyl substrates with longer fatty acid tails were docked and predicted to be stabilized by hydrogen bonds with SCD mutant residue, N102, and R235. Though not altered in SCD, R235 appeared to be stabilized by neighboring SCD mutant residues: N232, N233, and D236. A potential secondary substrate binding site for aromatic/phenol molecules was identified based upon homology between UBIAD1 and octaprenyl-transferases. Docking simulations predicted that both prenyl and aromatic molecules could bind to an interior cleft or groove leading to N102 near the primary active site. Substrate docking simulations with in silico mutant UBIAD1 revealed that aromatic substrates were no longer recognized by N102 upon mutation to a serine. Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest protein function is impaired due to SCD mutation. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, in humans, may be involved in cholesterol metabolism in a novel manner.

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