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The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacological therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the molecular basis of rhodopsin RP in the context of pharmacological rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacological rescue was measured as the fold increase in rhodopsin or opsin levels upon addition of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as determined by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacological rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an analysis of published biochemical and clinical studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacological rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The Halobacterium salinarium purple membrane is a two-dimensional crystalline lattice containing bacteriorhodopsin (BR) and lipid. To test whether molecular packing within the lipid bilayer influences the structural stability of the lattice, BR mutants substituted on the membrane-embedded surface of the protein were expressed in H. salinarium. Lattice stability was assessed by equilibrium density centrifugation of cell lysates containing similar amounts of BR. BR was distributed in low (1.12 to 1.15 g/ml) and high density (1.18 g/ml) membrane fractions. The high density fraction comprised 89% of the total BR in wild-type, but only 19% (G113L), 29% (I117A), 52% (G116L) and 79% (I117F) in the mutants. In each case, this fraction contained BR in a lattice form: its absorption maximum was blue-shifted by < or = 4 nm relative to the wild-type lattice, its light-dark difference spectrum was normal, and its circular dichroism spectrum retained a bilobed feature characteristic of the lattice. Thus, the substitutions do not significantly alter the tertiary structure of the protein. In the low density fraction, the absorption maximum of BR was blue-shifted by 2 to 4 nm relative to the corresponding high density fraction, and the bilobed circular dichroism feature was attenuated (I117F and G116L) or absent (G113L and I117A). This suggests that the substitutions disrupt lattice stability, causing an accumulation of BR monomers or small aggregates. These results support a model in which the BR lattice is stabilized by hydrophobic packing at specific protein-protein and protein-lipid interfaces within the membrane bilayer.

The effect of dam methylation on Tn5 transposition was investigated by analyses of mutations in the host (Escherichia coli) and the element. Wild-type elements transposed at a higher frequency and showed higher levels of transposase expression in a dam-host. Mutations were made in the promoter region of the transcript that codes for the transposase. Transposition and transposase levels from these mutants were independent of the host methylation system. Measurements of the amount of RNA support the hypothesis that dam methylation exerts its effect on Tn5 transposition by modulating the frequency of transcriptional initiation of the transposase gene. Since Tn5 transposition increases when the transposase levels increase, at normal concentrations the amount of transposase is a rate-limiting factor that determines the transposition frequency of Tn5. Transposition of IS50, one of the insertion sequences that constitutes Tn5, is also sensitive to dam methylation by a second mechanism in addition to that of modulating transcriptional initiation. dam methylation, either directly or indirectly, inhibits the usage of IS50 sequences by the transposase. Thus, dam methylation can affect both the expression of the transposase and the DNA substrate upon which it acts.

We have examined transcriptional start sites responsible for expression of the transposase and transposition inhibitor proteins encoded by IS50R, and determined the likely translational start site of transposase. Amino-terminal analysis of a transposase-beta-galactosidase fusion protein gave the sequence Met-Ile-Thr-Ser-Ala, which corresponds to the predicted amino acid sequence starting at position 93 of IS50. S1 nuclease mapping of IS50 RNA produced in vivo indicated that three transcripts, T1, T2 and T3, start near this position. Only T1 starts upstream from the transposase amino terminus. T2 corresponds to an in-vitro transcript described previously. Analysis of the transcripts and proteins produced from deletion derivatives of an IS50-lacZ construct suggested that the three transcripts initiate at independent but overlapping promoters clustered near the end of IS50. This analysis confirmed that only T1 can encode transposase, and that T2 is largely responsible for expression of the inhibitor protein. The coding capacity of T3 was not determined. Finally, transcripts that originate outside of IS50 are prevented from expressing transposase because of a secondary structure that is present in these transcripts only.