Abstract
Copper is an essential biometal, and several inherited diseases are directly associated with a disruption to normal copper homeostasis. The best characterized are the copper deficiency and toxicity disorders Menkes and Wilson diseases caused by mutations in the p-type Cu-ATPase genes ATP7A and ATP7B, respectively. Missense mutations in the C-terminal portion of ATP7A have also been shown to cause distal motor neuropathy, whereas polymorphisms in ATP7B are associated with increased risk of Alzheimer's disease. We have generated a single, in vivo model for studying multiple pathogenic mutations in ATP7 proteins using Drosophila melanogaster, which has a single orthologue of ATP7A and ATP7B. Four pathogenic ATP7A mutations and two ATP7B mutations were introduced into a genomic ATP7 rescue construct containing an in-frame C-terminal GFP tag. Analysis of the wild type ATP7-GFP transgene confirmed that ATP7 is expressed at the basolateral membrane of larval midgut copper cells and that the transgene can rescue a normally early lethal ATP7 deletion allele to adulthood. Analysis of the gATP7-GFP transgenes containing pathogenic mutations showed that the function of ATP7 was affected, to varying degrees, by all six of the mutations investigated in this study. Of particular interest, the ATP7BK832R Alzheimer's disease susceptibility allele was found, for the first time, to be a loss of function allele. This in vivo system allows us to assess the severity of individual ATP7A/B mutations in an invariant genetic background and has the potential to be used to screen for therapeutic compounds able to restore function to faulty copper transport proteins.
Highlights
Copper is an essential trace element required as a cofactor for enzymes involved in numerous important cellular functions and pathways [1, 2]
A Full-length Genomic ATP7 Transgene Rescues ⌬ATP7 Flies to Adulthood—A null deletion allele of the Drosophila ATP7 gene, referred to as ⌬ATP7, results in late first instar larval lethality [21], which can be partially rescued to adult viability by expression of UAS-ATP7-FLAG under the control of Selected ATP7A and ATP7B mutations
Selection was based on conservation of mutated residues between Drosophila ATP7 and human ATP7A or ATP7B as well as coverage of a wide variety of human diseases caused by ATP7A and ATP7B mutations
Summary
A Full-length Genomic ATP7 Transgene Rescues ⌬ATP7 Flies to Adulthood—A null deletion allele of the Drosophila ATP7 gene, referred to as ⌬ATP7, results in late first instar larval lethality [21], which can be partially rescued to adult viability by expression of UAS-ATP7-FLAG under the control of TABLE 1. A list of the mutations selected for ATP7 mutagenesis is shown. Selection was based on conservation of mutated residues between Drosophila ATP7 and human ATP7A or ATP7B as well as coverage of a wide variety of human diseases caused by ATP7A and ATP7B mutations
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