title: Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.
abstract: Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.
authors.affiliation: From the Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110 ysasaki@genetics.wustl.edu.
authors.forename: Yo
authors.initials: Y
authors.lastname: Sasaki
authors.affiliation: From the Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Margolin
authors.affiliation: From the Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Borgo
authors.affiliation: From the Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: James J
authors.initials: JJ
authors.lastname: Havranek
authors.affiliation: From the Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Milbrandt
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004795:Enzyme Stability; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D057130:Leber Congenital Amaurosis / Q000201:enzymology* / Q000209:etiology / Q000235:genetics*; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology / Q000473:pathology; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012162:Retinal Degeneration / Q000201:enzymology / Q000209:etiology / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; C484007:NMNAT1 protein, human; D009612:Nicotinamide-Nucleotide Adenylyltransferase
keywords: Leber congenital amaurosis; NMNAT; metabolic disease; neuroprotection; nicotinamide adenine dinucleotide (NAD); retinal degeneration; structural biology